KR20230118716A - Oligonucleotide compositions and methods thereof - Google Patents

Abstract

The present invention provides in particular oligonucleotides and compositions thereof. In some embodiments, provided oligonucleotides and compositions are useful for adenosine modification. In some embodiments, the present invention provides methods of treating various conditions, disorders or diseases that could benefit from adenosine modification.

Description

Oligonucleotide compositions and methods thereof

CROSS REFERENCES TO RELATED APPLICATIONS

This application is based on U.S. Provisional Application No. 63/111,079, filed on November 8, 2020, No. 63/175036, filed on April 14, 2021, and No. 63/188,415, filed on May 13, 2021, filed on 6/2021. 63/196,178 filed on September 2, 2021 and 63/248,520 filed on September 26, 2021. The entirety of each of the priority applications is incorporated herein by reference.

Oligonucleotides are useful in a variety of applications, such as therapeutic, diagnostic, and/or research applications. For example, oligonucleotides that target various genes may be useful in the treatment of conditions, disorders or diseases associated with such target genes.

In particular, the present invention provides designed oligonucleotides and compositions thereof comprising modifications as described herein (eg, modifications to nucleobase sugars and/or internucleotide linkages, and patterns thereof). In some embodiments, techniques (compounds (eg oligonucleotides), compositions, methods, etc.) of the invention (eg, oligonucleotides, oligonucleotide compositions, methods, etc.) are editing of nucleic acids, e.g., site-directed editing of nucleic acids. (e.g. editing of target adenosine). In some embodiments, as shown herein, provided techniques can significantly improve the efficiency of nucleic acid editing, eg, modification of one or more A residues, such as A to I conversions. In some embodiments, the present invention provides techniques for editing RNA (eg, modification of an A residue, eg, A to I conversion). In some embodiments, the present invention provides techniques for editing (eg, modifying an A residue, eg, converting an A to an I) of a transcript (eg, mRNA). In particular, the provided technology takes advantage of the utilization of endogenous proteins such as Adenosine Deaminases Acting on RNA (ADAR) proteins (eg, ADAR1 and/or ADAR2) for nucleic acid editing, eg, A transformation (eg, G to A mutation). to provide. Those skilled in the art will understand that such utilization of an endogenous protein is an exogenous component (e.g., a protein (e.g., one engineered to bind to an oligonucleotide (and/or duplex with a target nucleic acid) to provide a desired activity), a nucleic acid encoding the protein), , viruses, etc.) can avoid many difficulties and/or provide various advantages compared to techniques that require delivery.

In particular, in some embodiments, oligonucleotides of the provided technology are useful sugar modifications and/or patterns thereof (eg, presence and/or absence of particular modifications), nucleobase modifications and/or patterns thereof (eg, presence and/or absence of particular modifications). / or absence), internucleotidic linkage modifications and / or stereochemistry and / or patterns thereof (eg, types, modifications, and / or arrangements (Rp or Sp) of chiral linking phosphorus, etc.); When combined with one or more other structural elements (eg, additional chemical moieties), high activity and/or a variety of desired properties, such as high efficiency nucleic acid editing, high selectivity, high stability, high cellular uptake, low immune stimulation, may provide lower toxicity, improved distribution, improved affinity, and the like. In some embodiments, provided oligonucleotides provide higher stability compared to oligonucleotides with a high proportion of native RNA sugars used for adenosine editing. In some embodiments, provided oligonucleotides provide high activity, eg, adenosine editing activity. In some embodiments, provided oligonucleotides provide high selectivity. For example, in some embodiments, provided oligonucleotides selectively modify a target adenosine within a target nucleic acid relative to other adenosines within the same target nucleic acid (e.g., by 2, 3 at the target adenosine over other adenosines or all other adenosines within the target nucleic acid). , 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more fold strains).

Among other things, the present invention provides designed oligonucleotides and compositions with improved properties and/or activities compared to reference oligonucleotides and compositions (eg, those described herein or reported in the art). For example, in some embodiments, oligonucleotides and compositions provided as demonstrated herein provide improved stability, pharmacokinetics, pharmacodynamics, and/or improved activity (eg, against AI editing). can do. A variety of designed oligonucleotides and compositions are described herein. For example, in some embodiments, the present invention provides oligonucleotides and compositions thereof, including chirally controlled oligonucleotide compositions, wherein the oligonucleotides have sugar modifications (e.g., 2 '-OR variant (R is an optionally substituted C _1-6 alkyl) (eg, 2'-OMe, 2'-MOE, etc.), bicyclic sugar (eg, LNA sugar, cEt sugar, etc.) and several (eg, 1, 2, 3, 4, or 5 or more; in some embodiments, 3 or more) nucleosides that include independently. In some embodiments, the first few (e.g., 1, 2, 3, 4, or 5 or more; in some embodiments, 3 or more) nucleosides and/or the last few (e.g., 1, 2, 3, 4, or 5 or more; in some embodiments, 3 or more) nucleosides independently comprise sugar modifications. In some embodiments, the first 3 or more and the last 3 or more nucleosides independently comprise sugar modifications. In some embodiments, the one or more internucleotide linkages linked to such nucleosides are non-negatively charged internucleotidic linkages, such as phosphoryl guanidine internucleotide linkages such as n001. In some embodiments, both the first and last internucleotidic linkages are independently non-negatively charged internucleotidic linkages. In some embodiments, both the first and last internucleotide linkages are independently phosphorylguanidine internucleotide linkages. In some embodiments, both the first and last internucleotide linkages are independently n001. In some embodiments, they are all chirally controlled and R p . In some embodiments, the oligonucleotide comprises a nucleoside N ₀ comprising a native DNA sugar (two 2′-H), a native RNA sugar, or a 2′-F modified sugar. In some embodiments, N ₀ is the opposite nucleoside of the target adenosine when the oligonucleotide is used for adenosine editing. In some embodiments, the sugar of N ₀ is a natural DNA sugar. In some embodiments, a per N ₁ ("+" or nothing before a number indicates counting toward the 5'-direction (5'...N ₁ N ₀ N _-1 ...3')) per 2'-F modification, per natural DNA, or per natural RNA. In some embodiments, the sugar of N ₁ is a DNA sugar. In some embodiments, the sugar of N _-1 ("-" indicates counting toward the 3'-direction (5'...N ₁ N ₀ N _-1 ...3')) is a 2'-F variant sugar, natural DNA sugar, or natural RNA sugar. In some embodiments, the sugar of N _-1 is a DNA sugar. In some embodiments, the sugar of N _-3 is a 2'-F modified sugar. In some embodiments, between N ₂ and its 5′-terminal oligonucleotide are multiple 2′-F modified sugars and multiple 2′-modified sugars (eg, 2′-OR modified sugars (R is optionally substituted). C _1-6 alkyl), bicyclic sugars such as LNA sugars, cEt sugars, etc.). In some embodiments, an _{oligonucleotide} comprises one or more (eg, 1-20, 1-15, 1-10, 2-15, 2-10, or 1, 2 , 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more 2'-F blocks and one or more (eg For example, 1-20, 1-15, 1-10, 2-15, 2-10, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 , 14, 15, 16, 17, 18, 19, and 20 separate blocks (for example, when the first subdomain ends with N ₂ and includes N ₂ , the first domain and the second domain are combined) domain), wherein each nucleoside of the 2'-F block independently comprises a 2'-F modification, each nucleoside of the separate block independently contains no 2'-F modification, and each Blocks can be independently one or more (e.g., 1-20, 1-15, 1-10, 2-15, 2-10, or 1, 2, 3, 4, 5, 6, 7, 8, 9 , 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more) nucleosides. In some embodiments, there are two or more such 2'-F blocks and two or more such separating blocks. In some embodiments, one or more or all of these separating blocks are independently linked to the two 2'-F blocks. In some embodiments, each nucleoside of one or more or all separating blocks independently comprises a 2'-OR strain (R is an optionally substituted C _1-6 alkyl) or a bicyclic sugar, such as an LNA sugar, cEt party, etc. In some embodiments, each nucleoside of one or more or all of the separating blocks independently comprises a 2'-OR strain, where R is an optionally substituted C _1-6 alkyl. In some embodiments, each nucleoside of one or more or all of the separating blocks independently comprises a 2'-OMe or 2'-MOE modification. In some embodiments, each of these 2'-F and separating blocks independently comprises 1, 2, 3, 4 or 5 nucleosides. In some embodiments, a nucleoside close to N ₀ , eg, N ₂ , N ₁ , N ₀ , N _-1 , N _{-2 ,} etc., does not contain a large 2'-modification, such as 2'-MOE. . In some embodiments, the sugars of N ₂ , N ₁ , N ₀ , N _-1 and N _-2 are independently natural DNA sugars, 2'-F modified sugars, or 2'-OMe modified sugars. In some embodiments, each of the sugars of N ₁ , N ₀ , and N ₋₁ is a natural DNA sugar. In some embodiments, each chiral internucleotidic linkage is independently chirally controlled.

In some embodiments, the present invention provides an oligonucleotide comprising a first domain and a second domain, wherein the first domain comprises one or more 2'-F modifications and the second domain is free of 2'-F modifications. contains one or more sugars. In some embodiments, provided oligonucleotides include one or more chiral modified internucleotidic linkages. In some embodiments, the present invention

(a) a first domain; and

(b) providing an oligonucleotide comprising a second domain;

wherein the first domain comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 or more sugars and each independently a 2'-OR variant (where R is not -H (e.g., 2'-OMe, 2,-MOE, 2'-OL ^B -4' (where L 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 including optionally substituted -CH ₂ - ^etc , 19, or 20 or more sugars;

The second domains are each independently 2'-OR modification (where R is not -H (eg, 2'-OMe, 2, -MOE, 2'-OL ^B -4', where L ^B is optionally substituted at least 1 _, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 or more sugars.

In some embodiments, the present invention

(a) a first domain; and

(b) providing an oligonucleotide comprising a second domain;

wherein about 20% to 80% of all sugars in the first domain (e.g., about 25% to 80%, 30% to 80%, 35% to 80%, 40% to 80%, 40% to 70%, 40% % to 60%, 50% to 80%, 50% to 75%, 50% to 60%, 55% to 80%, 60 to 80%, or about 50%,55%, 60%, 65%, 70% , 75%, or 80%) contains a 2'-F modification, and about 20% to 70% (e.g., about 20% to 60%, 20% to 50%, 30%) of all sugars in the first domain ~60%, 30%~50%, 40%~50%, or about 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, or 60%) independently of 2 '-OR variant, where R is not -H (e.g., 2'-OMe, 2,-MOE, 2'-OL ^B -4', where L ^B is optionally substituted -CH ₂ -, etc.) contains;

The second domain is at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, not including a 2'-F modification. 19, or 20 or more modified sugars, or at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% of all sugars in the second domain are 2'- Does not include F variants.

In some embodiments, the second domain comprises or consists of a first subdomain, a second subdomain, and a third subdomain as described herein. In some embodiments, each first subdomain is independently a 2'-OR variant (where R is not -H (e.g., 2'-OMe, 2,-MOE, 2'-OL ^B -4'( wherein L ^B is optionally substituted -CH ₂ -, etc.); 8, 9, or 10) sugars. In some embodiments, there are more such sugars in the first subdomain than 2'-F modified sugars. In some embodiments, no sugars in the second subdomain does not contain any 2'-OR modifications, where R is an optionally substituted C _1-6 aliphatic or 2'-OL ^B -4' In some embodiments, each sugar in the second subdomain is independently a natural DNA sugar, natural RNA sugar or 2'-F modified sugar.In some embodiments, each sugar of the second subdomain is independently natural DNA sugar or natural RNA sugar.In some embodiments, each sugar of the second subdomain is independently The sugar is independently a natural DNA sugar or a 2'-F modified sugar.In some embodiments, each sugar in the second subdomain is independently a natural DNA sugar.In some embodiments, the second subdomain has three nucleosomes. There is a seed. In some embodiments, when binding to a target, the second three nucleosides are opposite to the target adenosine. In some embodiments, the sugar of the second nucleoside is any 2' as described herein. -OR modification (eg, 2'-OMe, 2'-MOE, etc.) In some embodiments, this sugar is a natural DNA sugar. In some embodiments, it is a natural RNA sugar. In some embodiments In some embodiments, each third subdomain is independently a 2'-OR modification, wherein R is not -H (e.g., 2'-OMe, 2, one or more (eg ^, 1-10, _1-5 ^, 1-3, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) sugars. In some embodiments, there are more such sugars in the third subdomain than 2'-F modified sugars.

In some embodiments, the second domain comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 or more modified sugars, or at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95% of all sugars in the second domain, or 99% contain the 2'-OR variant, R is an optionally substituted C _1-6 aliphatic. In some embodiments, R is methyl. In some embodiments, R is —CH ₂ CH ₂ OCH ₃ . As described herein, other sugar modifications may also be used in accordance with the present invention, optionally in combination with base modifications and/or internucleotide linkage modifications described herein.

In some embodiments, the oligonucleotide is or comprises a 5'-first domain-second domain-3' structure. In some embodiments, the second domain is or includes a 5′-first subdomain-second subdomain-third subdomain-3′ structure. In some embodiments, the oligonucleotide is or comprises a 5'-first domain-first subdomain-second subdomain-third subdomain-3' structure. In some embodiments, oligonucleotides are conjugated to additional moieties, eg, various additional chemical moieties as described herein. In some embodiments, oligonucleotides include additional moieties, eg, as described herein. In some embodiments, the additional chemical moiety is a small molecule moiety, a carbohydrate moiety (eg, a GalNAc moiety), a nucleic acid moiety (eg, an oligonucleotide moiety, one or more properties and/or activities, etc. Is or comprises a nucleic acid moiety (eg, a moiety of an RNase H dependent oligonucleotide, an RNAi oligonucleotide, an aptamer, a gRNA, etc.), and/or a peptide moiety that can provide and/or modulate.

In some embodiments, the base sequence of a provided oligonucleotide is substantially complementary to the base sequence of a target nucleic acid comprising a target adenosine. In some embodiments, provided oligonucleotides contain one or more mismatches (non-Watson-Crick base pairs) when aligned to a target nucleic acid. In some embodiments, a provided oligonucleotide comprises one or more wobbles (eg G-U, I-A, G-A, I-U, I-C, etc.) when aligned to a target nucleic acid. In some embodiments, mismatches and/or wobbles can help one or more proteins (eg, ADAR1, ADAR2, etc.) recognize a duplex formed by a given oligonucleotide and a target nucleic acid. In some embodiments, a provided oligonucleotide forms a duplex with a target nucleic acid. In some embodiments, an ADAR protein recognizes and binds to such duplexes. In some embodiments, the nucleoside opposite the target adenosine is located in the middle of a provided oligonucleotide (e.g., 5-50 nucleosides on the 5' side and 1-50 nucleosides on the 3' side). has exist). In some embodiments, the 5' side has more nucleosides than the 3' side. In some embodiments, the 5' side has fewer nucleosides than the 3' side. In some embodiments, the 5' side has the same number of nucleosides as the 3' side. In some embodiments, provided oligonucleotides include 15-40 (eg, 15, 20, 25, 30, etc.) contiguous bases of the oligonucleotides listed in the table. In some embodiments, the base sequence of a provided oligonucleotide is or comprises the base sequence of an oligonucleotide listed in the table.

In some embodiments, utilizing a variety of structural elements (e.g., various modifications, stereochemistry, and patterns thereof), the present invention provides short oligonucleotides (about 20-40, 25-40, 25-35, 26-32, 25 , 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 nucleobases in length) to achieve the desired properties and high activity.

In some embodiments, provided oligonucleotides include modified nucleobases. In some embodiments, a modified nucleobase promotes modification of a target adenosine. In some embodiments, the opposite nucleobase of the target adenine interacts with the target adenine less strongly than U (e.g., less strongly than U) while retaining interaction with the enzyme (e.g., ADAR) compared to when U is present. form hydrogen bonds). In some embodiments, the opposing nucleobases and/or their associated sugars provide some flexibility (eg, compared to U) to functional modification of the target adenosine by an enzyme (eg, ADAR1, ADAR2, etc.). In some embodiments, adjacent nucleobases 5' or 3' to opposite nucleobases (of the target adenine), eg, I and derivatives thereof, enhance modification of the target adenine. In particular, the present invention recognizes that such nucleobases are less likely to cause steric hindrance than G when a duplex of a given oligonucleotide and its target nucleic acid interacts with a modification enzyme (eg, ADAR1 or ADAR2). In some embodiments, the base sequence of the oligonucleotide is selected (e.g., where several adenosine residues are suitable targets) and/or designed (e.g., utilizing various nucleobases described herein) to reduce steric hindrance. or can be removed (e.g., no G next to the nucleoside opposite to target A).

In some embodiments, the oligonucleotides of the invention provide modified internucleotidic linkages (ie, internucleotidic linkages that are not natural phosphate linkages). In some embodiments, the linkage phosphorus of a modified internucleotide linkage (eg, a chiral internucleotide linkage) is chiral and can exist in different configurations ( R p and Sp ). In particular, the present invention provides that the incorporation of modified internucleotidic linkages, especially with control of the stereochemistry of the linking phosphorus centers (such that at these controlled centers one sequence is enriched relative to stereorandom oligonucleotide preparations), the properties (e.g. stability) and/or activity (eg, adenosine transforming activity (eg, conversion of adenosine to inosine)) can be significantly improved. In some embodiments, a provided oligonucleotide has a much higher stereochemical purity than a stereorandomized preparation. In some embodiments, provided oligonucleotides are chirally controlled.

In some embodiments, an oligonucleotide of the invention comprises one or more chiral internucleotidic linkages in which the linkage phosphorus is chiral (eg, phosphorothioate internucleotidic linkages). In some embodiments, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 of all internucleotide linkages of an oligonucleotide 19 or 20, or at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% (e.g., 50%- 100%, 60%~100%, 70~100%, 75%~100%, 80%~100%, 90%~100%, 95%~100%, 60%~95%, 70%~95%, 75-95%, 80-95%, 85-95%, 90-95%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or 99%, etc.); Alternatively, all internucleotidic linkages of an oligonucleotide are chiral internucleotidic linkages. In some embodiments, at least one internucleotidic linkage is a chiral internucleotidic linkage. In some embodiments, at least one internucleotidic linkage is a natural phosphate linkage. In some embodiments, each internucleotidic linkage is independently a chiral internucleotidic linkage. In some embodiments, at least one chiral internucleotidic linkage is a phosphorothioate internucleotidic linkage. In some embodiments, each is a phosphorothioate internucleotidic linkage. In some embodiments, the one or more chiral internucleotide linkages are independently non-negatively charged internucleotide linkages or neutral internucleotide linkages. In some embodiments, the one or more chiral internucleotidic linkages are independently phosphorylguanidine internucleotidic linkages. In some embodiments, one or more chiral internucleotidic linkages are independently chirally controlled. In some embodiments, each chiral internucleotidic linkage is independently chirally controlled. In some embodiments, one or more chiral internucleotidic linkages are not chirally controlled. In some embodiments, each phosphorothioate internucleotidic linkage is independently chirally controlled. In some embodiments, each modified internucleotide linkage is independently a phosphorothioate or non-negatively charged internucleotide linkage. In some embodiments, each modified internucleotidic linkage is independently a phosphorothioate or neutral internucleotidic linkage. In some embodiments, each modified internucleotidic linkage is independently a phosphorothioate or neutral internucleotidic linkage. In some embodiments, each modified internucleotide linkage is independently a phosphorothioate or phosphorylguanidine internucleotide linkage. In some embodiments, the phosphoryl guanidine internucleotide linkage is n001. In some embodiments, each phosphorylguanidine internucleotide linkage is n001. In some embodiments, each internucleotide linkage that is not negatively charged is n001. In some embodiments, each neutral internucleotide linkage is n001. In some embodiments, the modified internucleotide linkage is n002. In some embodiments, this is n006. In some embodiments, this is n020. In some embodiments, this is n004. In some embodiments, this is n008. In some embodiments, this is n025. In some embodiments, this is n026. A variety of modified internucleotidic linkages are described herein. The linking phosphorus may be R p or Sp . In some embodiments, at least one linking phosphorus is R p. In some embodiments, at least one linking phosphorus is Sp . In some embodiments, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 of all chiral internucleotide linkages of the oligonucleotide , 19 or 20, or at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% (e.g., 50%) of all chiral internucleotide linkages of the oligonucleotide. %~100%, 60%~100%, 70~100%, 75%~100%, 80%~100%, 90%~100%, 95%~100%, 60%~95%, 70%~95 %, 75-95%, 80-95%, 85-95%, 90-95%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or 99%, etc. ), or all chiral internucleotidic linkages of an oligonucleotide are Sp . In some embodiments, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 of all phosphorothioate internucleotide linkages of the oligonucleotide 17, 18, 19 or 20, or at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% of all phosphorothioate internucleotide linkages of the oligonucleotide (e.g. 50%~100%, 60%~100%, 70~100%, 75%~100%, 80%~100%, 90%~100%, 95%~100%, 60%~95% %, 70%~95%, 75~95%, 80~95%, 85~95%, 90~95%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95 %, or 99%, etc.), or all phosphorothioate internucleotidic linkages of the oligonucleotide are S p. In some embodiments, at least 50% of all phosphorothioate internucleotide linkages are Sp . In some embodiments, at least 60% of all phosphorothioate internucleotide linkages are Sp . In some embodiments, at least 70% of all phosphorothioate internucleotidic linkages are Sp . In some embodiments, at least 75% of all phosphorothioate internucleotide linkages are Sp . In some embodiments, at least 80% of all phosphorothioate internucleotide linkages are Sp . In some embodiments, at least 85% of all phosphorothioate internucleotidic linkages are Sp . In some embodiments, at least 90% of all phosphorothioate internucleotide linkages are Sp . In some embodiments, at least 95% of all phosphorothioate internucleotidic linkages are Sp . In some embodiments, at least 96% of all phosphorothioate internucleotide linkages are Sp . In some embodiments, at least 97% of all phosphorothioate internucleotide linkages are Sp . In some embodiments, at least 98% of all phosphorothioate internucleotide linkages are Sp . In some embodiments, all phosphorothioate internucleotidic linkages are Sp . In some embodiments, no more than 3, 4, 5, 6, 7, 8, 9 or 10 consecutive phosphorothioate internucleotidic linkages are R p . In some embodiments, no more than 3 consecutive phosphorothioate internucleotidic linkages are R p . In some embodiments, no more than 4 consecutive phosphorothioate internucleotidic linkages are R p . In some embodiments, no more than 5 consecutive phosphorothioate internucleotidic linkages are R p . In some embodiments, no more than 6 consecutive phosphorothioate internucleotidic linkages are R p . In some embodiments, no more than 7 consecutive phosphorothioate internucleotidic linkages are R p . In some embodiments, no more than 8 consecutive phosphorothioate internucleotidic linkages are R p . In some embodiments, no more than 9 consecutive phosphorothioate internucleotidic linkages are R p . In some embodiments, no more than 10 consecutive phosphorothioate internucleotidic linkages are R p . In some embodiments, no contiguous R p phosphorothioate internucleotidic linkages are used in part, wherein the majority (e.g., greater than 50%, 60%, 70%, 75%, 80%, 85%, 90% , 95% or more) or all sugars are native DNA and/or RNA and/or 2′-F modified sugars. In some embodiments, when contiguous R pphosphorothioate internucleotidic linkages are used, one or more or most (e.g., greater than 50%, 60%, 70%, 75%, 80%, 85%, 90% , 95% or more) or all of these internucleotidic linkages are independently linked to sugars that can improve stability. In some embodiments, when contiguous R pphosphorothioate internucleotidic linkages are used, one or more or most (e.g., greater than 50%, 60%, 70%, 75%, 80%, 85%, 90% , 95% or more) or all such internucleotidic linkages are independently linked to a bicyclic sugar or a 2′-OR modified sugar (R is an optionally substituted C _1-6 aliphatic). In some embodiments, when contiguous R pphosphorothioate internucleotidic linkages are used, one or more or most (e.g., greater than 50%, 60%, 70%, 75%, 80%, 85%, 90% , 95% or more) or all such internucleotide linkages are independently linked to the 2'-OR modified sugar (R is an optionally substituted C _1-6 aliphatic). In some embodiments, each 2'-OR modified sugar is independently a 2'-OMe modified sugar or a 2'-MOE modified sugar. In some embodiments, each 2'-OR modified sugar is independently a 2'-OMe modified sugar. In some embodiments, each 2'-OR modified sugar is independently a 2'-MOE modified sugar.

In some embodiments, the stereochemistry of one or more chiral linking phosphorus of a provided oligonucleotide is controlled in the composition. In some embodiments, the present invention provides a composition comprising a plurality of oligonucleotides, wherein the plurality of oligonucleotides contain at least one (e.g., about 1-50, 1-40, 1-30 of all chiral internucleotide linkages). , 1~25, 1~20, 1~15, 1~10, 5~50, 5~40, 5~30, 5~25, 5~20, 5~15, 5~10, 1, 2, 3 , 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 or more, or at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or 99%) of chiral internucleotide linkages ("chiral controlled internucleotide linkages"), and share the same configuration of linking phosphorus (eg, all R p or all Sp for chiral linking phosphorus). In some embodiments, they share the same stereochemistry at each chiral linking phosphorus. In some embodiments, a plurality of oligonucleotides share the same composition. In some embodiments, the plurality of oligonucleotides are structurally identical except for internucleotide linkages. In some embodiments, the plurality of oligonucleotides are structurally identical. In some embodiments, at least about 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of all oligonucleotides in the composition, or all oligonucleotides that share a common base sequence % shares a backbone chiral center pattern of a plurality of oligonucleotides. In some embodiments, at least about 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of all oligonucleotides in the composition, or all oligonucleotides that share a common base sequence % is a plurality of oligonucleotides.

In some embodiments, the present invention provides an oligonucleotide composition with chirally controlled oligonucleotides, wherein all oligonucleotides in the composition, or all oligonucleotides having the same base sequence of the oligonucleotides, or all oligonucleotides having the same base sequence and sugar and base modifications At least about 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of all oligonucleotides, or all oligonucleotides of the same composition, are oligonucleotides and one or more (e.g., For example, about 1-50, 1-40, 1-30, 1-25, 1-20, 1-15, 1-10, 5-50, 5-40, 5-30, 5 of all chiral internucleotide linkages ~25, 5~20, 5~15, 5~10, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 , 19, 20, 21, 22, 23, 24, or 25 or more, or at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or 99%) chiral Independently in internucleotidic linkages, they share the same sequence of linking phosphorus (eg, all R p or all S p for chiral linking phosphorus). In some embodiments, the present invention provides an oligonucleotide composition with chirally controlled oligonucleotides, wherein all oligonucleotides in the composition, or all oligonucleotides having the same base sequence of the oligonucleotides, or all oligonucleotides having the same base sequence and sugar and base modifications At least about 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of all oligonucleotides, or all oligonucleotides of the same composition, contain one or more forms of oligonucleotides (e.g., : acid form, salt form (eg, pharmaceutically acceptable salt form; as understood by those skilled in the art, where the oligonucleotide is a salt, another salt form of the corresponding acid or base form of the oligonucleotide), etc.).

In some embodiments, as shown herein, chirally controlled oligonucleotide compositions provide numerous advantages over corresponding stereorandom oligonucleotide compositions, such as higher stability, activity, etc. In some embodiments, a chirally controlled oligonucleotide composition provides high levels of adenosine modification (eg, A to I conversion) activity with various isoforms of ADAR proteins (eg, the p150 and p110 forms of ADAR1), whereas corresponding It has been observed that sterically randomized compositions containing only certain isoforms of ADAR proteins (eg, the p150 isoform of ADAR1) provide high levels of adenosine modification (eg, A to I conversion) activity.

In some embodiments, provided oligonucleotides include additional moieties, such as targeting moieties, carbohydrate moieties, and the like. In some embodiments, the additional moiety is or comprises a ligand for an asialoglycoprotein receptor. In some embodiments, the additional moiety is or comprises GalNAc or a derivative thereof. In particular, the additional moiety can facilitate delivery to a specific target location, eg, a cell, tissue, organ, etc. (eg, a location comprising a receptor that interacts with the additional moiety). In some embodiments, the additional moiety facilitates delivery to the liver.

In some embodiments, the present invention provides techniques for making oligonucleotides and compositions thereof (particularly, chirally controlled oligonucleotide compositions). In some embodiments, provided oligonucleotides and compositions thereof are of high purity. In some embodiments, an oligonucleotide of the invention is at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% stereochemically stereochemically at the linkage phosphorus of the chiral internucleotidic linkage. pure. In some embodiments, oligonucleotides of the invention are prepared stereoselectively and are substantially free of stereoisomers. In some embodiments, a provided composition comprising a plurality of oligonucleotides that share the same base sequence of a stereochemical pattern that is the same chiral linkage (e.g., comprises one or more of R p and/or S p, each chiral linking phosphorus is independently R p or Sp ), at least 50%, 60%, 70%, 75%, 80%, 85% of all oligonucleotides in the composition that share the same base sequence as the plurality of oligonucleotides, 90%, 95%, or 99% are a plurality of oligonucleotides or share a stereochemical pattern that is the same chiral linkage. In some embodiments, in a provided composition comprising a plurality of oligonucleotides that share the same base sequence of a stereochemical pattern that is the same chiral linkage, at least 50% of all oligonucleotides in the composition that share the same configuration as the plurality of oligonucleotides; 60%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% share a stereochemical pattern that is the same chiral linkage or is a plurality of oligonucleotides.

In some embodiments, the present disclosure describes techniques useful for evaluating oligonucleotides and compositions thereof. For example, various techniques of the present invention are useful for evaluating adenosine modifications. As will be appreciated by those skilled in the art, in some embodiments, modification/editing of an adenosine (eg, where adenosine of a target nucleic acid is converted to inosine) optionally occurs in a modification system (eg, an in vitro system, an ex vivo system, Sequencing, mass spectrometry, evaluation (eg level , activity, etc.). Those skilled in the art will understand that oligonucleotides that provide adenosine modification of a target nucleic acid may also provide the modified nucleic acid (eg, where the target adenosine is converted to I) and one or more products thereof (eg, mRNA, protein, etc.) will be. Certain useful techniques are described in the examples.

As described herein, oligonucleotides and compositions of the present invention may be provided/utilized in a variety of forms. In some embodiments, the present invention provides more than one form of an oligonucleotide, e.g., acid form (e.g., when the natural phosphate linkage is -O(P(O)(OH)-O-, phosphorothio Eight internucleotide linkages exist as -O(P(O)(SH)-O-), base forms, salt forms (e.g., natural phosphate linkages exist as salt forms (e.g., sodium salts (-O(P(O) )(O ^- Na ⁺ )-O-), the phosphorothioate internucleotidic linkages are in salt form (e.g. sodium salt (-O(P(O)(S ^- Na ⁺ )-O-)). present), etc. As will be appreciated by those skilled in the art, oligonucleotides may exist in a variety of salt forms, including pharmaceutically acceptable salts, and in solution (e.g., in various aqueous buffer systems), The cation can be separated from the anion In some embodiments, the present invention provides a pharmaceutical composition comprising a provided oligonucleotide and/or one or more pharmaceutically acceptable salts thereof, and a pharmaceutically acceptable carrier. In , the pharmaceutical composition is a chirally controlled oligonucleotide composition.

The provided technology may be utilized for a variety of purposes. For example, one of ordinary skill in the art would appreciate that the techniques provided may be useful for modifying adenosine, e.g., G to A mutation correction, controlling the level of certain nucleic acids and/or products encoded thereby (e.g., proteins by introducing A to G/I modifications). level reduction), control of splicing, control of translation (e.g. control of translation start and/or stop sites by introduction of A to G/I modifications), and the like.

In some embodiments, the present invention provides techniques for preventing or treating conditions, disorders or diseases that are prone to adenosine modifications (eg, A to I or G). As will be appreciated by those skilled in the art, I may perform one or more functions of G, for example in base pairing, translation, and the like. In some embodiments, an A to I conversion can correct a G to A mutation to produce one or more products (eg, proteins) of the G-version nucleic acid. In some embodiments, the present invention is a technique for preventing or treating a condition, disorder or disease associated with a mutation, in which a subject predisposed to or suffering from such condition, disorder or disease is provided with a provided oligonucleotide capable of editing the mutation or A technique comprising administering a composition thereof is provided. In some embodiments, the present invention is a technique for preventing or treating a condition, disorder, or disease associated with a G to A mutation, wherein A is capable of modifying A in a subject predisposed to or suffering from such condition, disorder, or disease. A technique comprising administering a provided oligonucleotide or composition thereof is provided. In some embodiments, provided techniques modify A in a transcript (eg, an RNA transcript). In some embodiments, A is converted to I. In some embodiments, during translation, the protein synthesis machinery reads an I as a G. In some embodiments, the A form encodes one or more proteins that have one or more higher desired activities and/or one or more superior desired properties compared to those encoded by the corresponding G forms. In some embodiments, the A form provides higher levels of one or more proteins having one or more higher desired activities and/or one or more better desired properties compared to the corresponding G form. In some embodiments, the product encoded by the A form is structurally different from that encoded by the corresponding G form (eg, is longer, and in some embodiments is a full-length protein). In some embodiments, the A form provides a structurally identical product (eg, protein) compared to the corresponding G form.

As will be appreciated by those skilled in the art, many conditions, disorders or diseases are associated with mutations that can be modified by, and prevented and/or treated using, the provided techniques. For example, more than 20,000 conditions, disorders or diseases are reported to be associated with G to A mutations and could benefit from A to I editing.

1 . Provided technologies provide editing of mutations associated with conditions, disorders or diseases, and provide products with improved properties and/or functions. The oligonucleotide composition targets the PiZ mutation of SERPINA1 (SA1). Primary mouse hepatocytes expressing the human SA1-PiZ allele were transfected with the indicated oligonucleotide compositions (25 nM oligonucleotides) (WV-38621, WV-38622, WV-38630 and non-targeting (NT) control WV-37317). Infected. Media and RNA were collected 5 days after transfection. RNA editing was quantified by RT-PCR and Sanger sequencing. A1AT protein in the medium was quantified by ELISA assay ("SerpinA1 ng/ml"). All samples were evaluated in N=6 replicates. As confirmed in the data in the figure, the provided technology can provide editing of target human SERPINA1-PIZ mRNA. In addition, the data in the figure confirms that the provided technology increases the level of A1AT protein secretion, indicating that the provided technology can correct mutations at the protein level and correct folding of the A1AT protein can provide an improved protein. (P-values: * <0.05, ** < 0.01, *** <0.005 and **** <0.0005).
2. Provided technology may provide editing. (a) Specific oligonucleotides targeting the SERPINA1-Z allele. The indicated cell lines were stably infected with lentivirus expressing the SERPINA1-Z allelic transcript and transfected with the indicated oligonucleotides. HEK293T cells were also pretransfected with plasmids expressing ADAR1-p110 or ADAR1-p150. RNA was collected after 48 hours and RNA editing was quantified by Sanger sequencing (n=2 biological replicates). (b) The oligonucleotide targets the SERPINA1-Z allele. The indicated cell lines were stably infected with lentivirus expressing the SERPINA1-Z allelic transcript and transfected with the indicated oligonucleotides. HEK 293T cells were also pre-transfected with plasmids expressing ADAR1-p110 or ADAR1-p150. RNA was collected after 48 hours and RNA editing was quantified by Sanger sequencing (n=2 biological replicates).
Figure 3. A provided technique comprising a variety of modifications, including modified bases, can provide editing. The oligonucleotide targets the SERPINA1-Z allele. HEK293T or SF8628 cells stably expressing the SERPINA1-Z allelic transcript were transfected with the indicated oligonucleotides. HEK293T cells were also pre-transfected with human ADAR1-p110 or p150. RNA editing was quantified by Sanger sequencing (n=2 biological replicates).
Figure 4. Provided techniques including a variety of modifications, including modified bases and different types of sugars, can provide editing. The oligonucleotide targets the SERPINA1-Z allele. HEK293T cells stably expressing the SERPINA1-Z allelic transcript were transfected with human ADAR1-p110 or p150 and the indicated oligonucleotides. RNA editing was quantified by Sanger sequencing (n=2 biological replicates).
5. Provided technology including various variations may provide editing. Primary mouse hepatocytes transfected for the human ADAR1-p110 and SERPINA1-Z alleles were treated for 48 hours with the indicated GalNAc conjugated oligonucleotides targeting the SERPINA1-Z alleles. RNA editing was quantified by Sanger sequencing (n=2 biological replicates).
6. Provided technology may provide editing. Primary mouse hepatocytes were treated with the indicated oligonucleotides targeting the SERPINA1-Z allele via gymnotic uptake for 48 hours. RNA editing was quantified by Sanger sequencing (n=2 biological replicates).
7. Provided technology may provide editing. Primary mouse hepatocytes transfected for the human ADAR1-p110 and SERPINA1-Z alleles were treated with the indicated oligonucleotides via zymotic uptake for 48 hours. RNA editing was quantified by Sanger sequencing (n=2 biological replicates).
Figure 8. Provided techniques including various modifications, including base modifications, can provide editing. Primary mouse hepatocytes transfected for the human ADAR1-p110 and SERPINA1-Z alleles were treated for 48 hours with the indicated oligonucleotides targeting the SERPINA1-Z allele via zymotic uptake. RNA editing was quantified by Sanger sequencing (n=2 biological replicates).
9. Provided technology may provide editing. Primary mouse hepatocytes transfected for the human ADAR1-p110 and SERPINA1-Z alleles were treated for 48 hours with the indicated oligonucleotides targeting the SERPINA1-Z allele via zymotic uptake. RNA editing was quantified by Sanger sequencing (n=2 biological replicates).
Figure 10. Provided techniques including various modifications including modified internucleotidic linkages can provide editing. Primary mouse hepatocytes transfected for the human ADAR1-p110 and SERPINA1-Z alleles were treated for 48 hours with the indicated oligonucleotides targeting the SERPINA1-Z allele via zymotic uptake. RNA editing was quantified by Sanger sequencing (n=2 biological replicates).
Figure 11. Provided techniques including various modifications including modified internucleotidic linkages can provide editing. Primary mouse hepatocytes transfected for the human ADAR1-p110 and SERPINA1-Z alleles were treated for 48 hours with the indicated oligonucleotides targeting the SERPINA1-Z allele via zymotic uptake. RNA editing was quantified by Sanger sequencing (n=2 biological replicates).
Figure 12. Provided technology including various modifications including sugar modifications can provide editing. Primary mouse hepatocytes transfected for the human ADAR1-p110 and SERPINA1-Z alleles were treated for 48 hours with the indicated oligonucleotides targeting the SERPINA1-Z allele via zymotic uptake. RNA editing was quantified by Sanger sequencing (n=2 biological replicates).
Figure 13. Provided technology including various modifications, including sugar modifications, can provide editing. Primary mouse hepatocytes transfected for the human ADAR1-p110 and SERPINA1-Z alleles were treated for 48 hours with the indicated oligonucleotides targeting the SERPINA1-Z allele via zymotic uptake. RNA editing was quantified by Sanger sequencing (n=2 biological replicates).
Figure 14. Provided technology comprising oligonucleotides of varying length can provide editing. Primary mouse hepatocytes transfected for the human ADAR1-p110 and SERPINA1-Z alleles were treated for 48 hours with the indicated oligonucleotides targeting the SERPINA1-Z allele via zymotic uptake. RNA editing was quantified by Sanger sequencing (n=2 biological replicates).
Figure 15. Provided techniques including various modifications including various types of internucleotidic linkages can provide for editing. Primary mouse hepatocytes transfected for the human ADAR1-p110 and SERPINA1-Z alleles were treated for 48 hours with the indicated oligonucleotides targeting the SERPINA1-Z allele via zymotic uptake. RNA editing was quantified by Sanger sequencing (n=2 biological replicates).
16. Provided technology may provide editing. Primary mouse hepatocytes transfected for human ADAR1-p110 were treated for 48 hours with the indicated GalNAc conjugated oligonucleotides targeting the SERPINA1-Z allele. RNA editing was quantified by Sanger sequencing (n=2 biological replicates).
17. Provided techniques involving various types of sugars, nucleobases, internucleotidic linkages and/or additional chemical moieties can provide for editing. Primary mouse hepatocytes transfected for human ADAR1-p110 were treated for 48 hours with the indicated GalNAc conjugated oligonucleotides targeting the SERPINA1-Z allele. RNA editing was quantified by Sanger sequencing (n=2 biological replicates).
Figure 18. Provided technology comprising various editing region sequences can provide editing. (a) Oligonucleotides of various editing region sequences containing the nearest neighbor adjacent to the nucleoside opposite the target adenosine were evaluated. Primary mouse hepatocytes transfected for human ADAR1-p110 were treated gymnotically with the indicated oligonucleotides targeting the SERPINA1-Z allele for 48 hours. RNA editing was quantified by Sanger sequencing (n=2 biological replicates). (b) Oligonucleotides of various editing region sequences were evaluated, including the nearest neighbor adjacent to the nucleoside opposite the target adenosine. Primary mouse hepatocytes transfected for human ADAR1-p110 were treated without transfection reagent for 48 hours with the indicated oligonucleotides targeting the SERPINA1-Z allele. RNA editing was quantified by Sanger sequencing (n=2 biological replicates). (c) Oligonucleotides of various editing region sequences containing the nearest neighbor adjacent to the nucleoside opposite the target adenosine were evaluated. Primary mouse hepatocytes transfected for human ADAR1-p110 were treated without transfection reagent for 48 hours with the indicated oligonucleotides targeting the SERPINA1-Z allele. RNA editing was quantified by Sanger sequencing (n=2 biological replicates).
19. Provided techniques involving various types of nucleoside and internucleotide linkages can provide for editing. Primary mouse hepatocytes transfected for human ADAR1-p110 were treated without transfection reagent for 48 hours with the indicated oligonucleotides targeting the SERPINA1-Z allele. RNA editing was quantified by Sanger sequencing (n=2 biological replicates).
Fig. 20. Provided technology may provide editing. Primary mouse hepatocytes transfected for human ADAR1-p110 were treated without transfection reagent for 48 hours with the indicated oligonucleotides targeting the SERPINA1-Z allele. RNA editing was quantified by Sanger sequencing (n=2 biological replicates).
21. Provided techniques involving various types of sugar, nucleoside and internucleotide linkages can provide for editing. Primary mouse hepatocytes transfected for human ADAR1-p110 were treated without transfection reagent for 48 hours with the indicated oligonucleotides targeting the SERPINA1-Z allele. RNA editing was quantified by Sanger sequencing (n=2 biological replicates).
22. Provided techniques involving various types of sugar, nucleoside and internucleotide linkages can provide for editing. Primary mouse hepatocytes transfected for human ADAR1-p110 were treated without transfection reagent for 48 hours with the indicated oligonucleotides targeting the SERPINA1-Z allele. RNA editing was quantified by Sanger sequencing (n=2 biological replicates).
23. Provided techniques involving various types of sugar, nucleoside and internucleotide linkages can provide for editing. Primary mouse hepatocytes transfected for human ADAR1-p110 were treated without transfection reagent for 48 hours with the indicated oligonucleotides targeting the SERPINA1-Z allele. RNA editing was quantified by Sanger sequencing (n=2 biological replicates).
24. Provided techniques involving various types of sugar, nucleoside and internucleoside linkages can provide for editing. Primary mouse hepatocytes transfected for human ADAR1-p110 were treated without transfection reagent for 48 hours with the indicated oligonucleotides targeting the SERPINA1-Z allele. RNA editing was quantified by Sanger sequencing (n=2 biological replicates).
25. Provided techniques involving various types of sugar, nucleoside and internucleotide linkages can provide for editing. Primary mouse hepatocytes transfected for human ADAR1-p110 were treated without transfection reagent for 48 hours with the indicated oligonucleotides targeting the SERPINA1-Z allele. RNA editing was quantified by Sanger sequencing (n=2 biological replicates).
Fig. 26. Provided technology may provide editing. Oligonucleotides comprising wobble base pairs at various positions, such as GU wobble base pairs, can provide editing. Primary mouse hepatocytes transfected for human ADAR1-p110 were treated with the indicated oligonucleotides targeting SERPINA-Z without transfection reagent.
27. Provided techniques including various modifications, including nucleoside modifications, can provide editing. The oligonucleotide targets adenosine in the 3' UTR of beta-actin mRNA. Primary human hepatocytes were treated with the indicated oligonucleotides at the indicated concentrations without transfection reagent. Editing of targets was determined by Sanger sequencing (n=2 biological replicates).
28. Provided techniques including a variety of modifications, including sugar modifications and modified internucleotidic linkages, can provide editing. The oligonucleotide targets adenosine in the 3' UTR of beta-actin mRNA. Primary human hepatocytes were treated with the indicated oligonucleotides at the indicated concentrations without transfection reagent. Editing of targets was determined by Sanger sequencing (n=2 biological replicates).
29. Provided technology may provide editing. The oligonucleotide targets adenosine in the 3' UTR of beta-actin mRNA. Primary human hepatocytes were treated with the indicated oligonucleotides at the indicated concentrations without transfection reagent. Editing of targets was determined by Sanger sequencing (n=2 biological replicates).
Fig. 30. Provided technology may provide editing. Primary mouse hepatocytes transfected for human ADAR1-p110 were treated for 48 hours with the indicated oligonucleotides targeting UGP2 via zymotic uptake. RNA editing was quantified by Sanger sequencing (n=2 biological replicates).
31. Provided technology may provide editing in NHP. (a) Non-human primates (NHP) were administered subcutaneously with the indicated oligonucleotides (50 mg/kg, n=3) or PBS (n=1). After 7 days, animals were necropsied and the indicated tissues were collected. RNA editing was quantified by Sanger sequencing (n=2 biological replicates). (b): Corresponding concentrations of oligonucleotides in the indicated tissues measured by hybridization ELISA.
Fig. 32. Provided technology including various variations may provide editing. (a) Non-human primates (NHP) were administered intrathecally with indicated oligonucleotides (5 mg or 10 mg, n=2 each) or artificial cerebrospinal fluid (aCSF) control (n=1). Animals were necropsied on day 8 (aCSF, 5 mg group, 10 mg group) or day 29 (10 mg group) and indicated tissues were collected. RNA editing was quantified by Sanger sequencing (n=2 biological replicates). (b): Corresponding concentrations of oligonucleotides in the indicated tissues measured by hybridization ELISA.
Figure 33. Provided techniques including duplexization schemes may provide editing. The exemplified oligonucleotide composition includes two oligonucleotides that share 16 bp or 18 bp of complementary sequence, allowing them to associate to create a double-stranded RNA structure capable of recruiting an ADAR. One oligonucleotide (36 bp or 32 bp) also contains a targeting moiety specifically complementary to the target of interest. As shown, the combined oligonucleotide design targets, for example, the premature UAG stop codon in the cLuc coding sequence. HEK293T cells were transfected with a combination of a plasmid encoding human ADAR1-p150, a luciferase reporter construct and the indicated oligonucleotides. cLuc activity was normalized to Gluc expression in mock-treated samples. For each oligonucleotide comprising a duplex region and a target region (WV-42707 to WV-42710 and WV-42715 to WV-42718), the first to last duplexed oligonucleotides are WV-42719 to WV-42730 (i.e. , WV-WV-42719, WV-42720, WV-42721, WV-42722, WV-42723, WV-42724, WV-42725, WV-42726, WV-42727, WV-42728, WV-42729 and WV-42730 )am.
34. Provided techniques including duplexization schemes may provide editing. In some embodiments, a first oligonucleotide (e.g., a duplexed oligonucleotide) comprises a stem loop and can form a duplex, and a second oligonucleotide (e.g., a duplexed oligonucleotide) can be used to target a specific transcript. , an oligonucleotide comprising a duplexed region and a targeting region). In some embodiments, the first and second oligonucleotide complementary sequences (eg, those of 15 nt) allow them to associate. In some embodiments, the formed duplex supplements an ADAR polypeptide such as ADAR1, ADAR2, etc. In Figure 34, the combined oligonucleotide design targets the premature UAG stop codon in the cLuc coding sequence. HEK293T cells were transfected with a combination of plasmid encoding human ADAR1-p110 or p150, a luciferase reporter construct and the indicated oligonucleotides. cLuc activity was normalized to Gluc expression in mock-treated samples. As shown, various combinations provide editing activity.
Figure 35. Specific oligonucleotide design as an example. (a) A duplexed oligonucleotide and an oligonucleotide comprising a duplexed region and a targeting region. (b) a duplexed oligonucleotide comprising a stem loop and an oligonucleotide comprising a duplexed region and a targeting region.
Figure 36. Various oligonucleotide compositions can provide editing. Primary mouse hepatocytes from the transgenic model (expressing human ADARp110 and human SERPINA1-Z alleles) were treated for 48 hours with the indicated GalNAc conjugated oligonucleotides targeting the SERPINA1-Z allele. RNA editing was measured by Sanger sequencing.
Figure 37. Various oligonucleotide compositions can provide in vivo editing. A huADAR/SA1 transgenic mouse model was subcutaneously administered 3x10 mg/kg of the indicated oligonucleotides targeting the SERPINA1-Z allele. Mice were dosed every other day for 3 days (Day 0, Day 2, Day 4) and liver biopsies were collected on Day 7. Percentage editing was determined by Sanger sequencing. One-way ANOVA corrected for multiple comparisons (Dunnett's) was used to test for differences in SERPINA1-Z allelic editing in treated versus PBS groups. ****: P-value is less than 0.0001; ***: P-value is less than 0.001; **: P-value is less than 0.005. P-values were calculated from comparison of pre-dose and day 7 values for each sample.
Figure 38. Various oligonucleotide compositions can increase serum AAT after editing in vivo. Serum was collected from mice before dosing and on day 7 after treatment as described in FIG. 37 . The concentration of total human AAT in serum was determined with a commercially available ELISA kit (AbCam). A matched 2-way ANOVA with correction for multiple comparisons (Bonferroni) was used to test for differences in AAT abundance in treated samples compared to PBS. ****: P-value is less than 0.0001; ***: P-value is less than 0.001; **: P-value is less than 0.005. P-values were calculated from comparison of pre-dose and day 7 values for each sample.
Fig. 39. Provided oligonucleotide compositions are capable of reducing mutant Z-AAT protein levels and increasing wild-type AAT protein levels in serum. Serum was collected from mice before dosing and on day 7 after treatment as described in FIG. 37 . The relative abundance of Z (mutant) versus M (wild type) AAT isoforms was determined by mass spectrometry. Then, the absolute amount of each isoform was calculated by applying the relative abundance to the absolute concentration obtained from ELISA (see FIG. 38).
Figure 40. Editing with various oligonucleotide compositions can generate functional wild-type AAT proteins. Serum was collected from mice before dosing and on day 7 after treatment as described in FIG. 37 . Relative elastase inhibitory activity was determined in serum using a commercially available kit (EnzChek® Elastase Assay Kit (E-12056)). A matched two-way ANOVA with correction for multiple comparisons (Bonferroni) was used to test for differences in elastase inhibitory activity in serum collected before dosing on day 7 for each treatment group. ****: P-value is less than 0.0001; ***: P-value is less than 0.001; **: P-value is less than 0.005. P-values were calculated from comparison of pre-dose and day 7 values for each sample.
41. Provided technology can modulate protein-protein interactions. (a) Provided oligonucleotide compositions edit adenosine in Keap1 and NRF2 transcripts. HEK293T cells were transfected with oligonucleotide compositions targeting Keap1 or NRF2 and plasmids expressing ADAR-p110 (upper bar) or ADAR1-p150 (lower bar). RNA was collected 48 hours after treatment and RNA editing of Keap1 and NRF2 transcripts was measured by Sanger sequencing. “*”: Data unavailable. (b) provided oligonucleotide technology is capable of modulating gene expression. HEK293T cells were transfected with the indicated oligonucleotides targeting NRF2 or Keap1 and plasmids expressing ADAR-p110 or ADAR1-p150. RNA was harvested 48 hours after treatment. Fold changes of various genes regulated by NRF2 were measured by qPCR.
42. Provided technology can provide robust and durable in vivo editing. hADAR mice were treated with a single 100 ug ICV injection of an oligonucleotide composition comprising WV-40590 oligonucleotides targeting UGP2. UGP2 editing was measured between 1 and 16 weeks after dosing.
43. Provided technology may provide editing. Primary human hepatocytes were treated with oligonucleotide compositions targeting UGP2 at 1 uM (left bar) and 0.3 uM (right bar) via zymotic uptake. RNA was collected 48 hours after treatment and RNA editing was measured by Sanger sequencing (n=2 biological replicates).
44. Provided technology may provide editing. Human IPSC-derived neurons (iCells) were treated with oligonucleotide compositions containing the indicated oligonucleotides targeting UGP2 at 3 uM (left bars) and 1 uM (right bars) via zymotic uptake. RNA was collected 6 days after treatment and RNA editing was measured by Sanger sequencing (n=2 biological replicates).
45. Provided technology can provide in vivo editing. Wild-type (Wt) and hADAR mice were treated with PBS (left bars) or an oligonucleotide composition targeting UGP2 via 3 subcutaneous doses of 10 mg/kg (days 0, 2, and 4, respectively) (middle bars). = WV-38702, right bar = WV-48161). Mouse livers were isolated one week after treatment and RNA was collected. RNA editing was determined by Sanger sequencing (n=2 biological replicates).
46. The provided technology can provide editing in a variety of cell populations, including immune cells. ACTB targeting at 10 uM concentration in human PBMC under activating (PHA addition) or inactivating (no PHA addition) conditions (left bar = mock, middle bar = WV-37317 with PHA, right bar = WV-37317 without PHA). treated with the oligonucleotide composition. Cells were treated via zymotic uptake. Cells were isolated 4 days after treatment using a benchtop antibody/bead protocol. RNA was collected and RNA editing was measured by Sanger sequencing (n=2 biological replicates).
47. Provided technology can provide in vivo editing involving the eye. hADAR mice were treated with oligonucleotide compositions targeting UGP2 at indicated doses via intraventricular (ICV) injection into the posterior compartment of the eye. Mouse eyes were isolated at 1 and 4 weeks post treatment and RNA was isolated. RNA editing was measured by PCR and Sanger sequencing.
48. Provided technology can provide persistent in vivo editing. Mice transgenic for the hADAR and SERPINA1-Z alleles were treated with an oligonucleotide composition targeting the SERPINA1-Z allele at a dose of 10 mg/kg via subcutaneous administration on days 0, 2, and 4. did Mouse serum was collected via weekly blood draws on the indicated days after treatment. (a) Levels of human AAT protein were measured by ELISA. . Data are presented as mean ± sem. Statistics: matched 2-way ANOVA; ns: not significant, **: P<0.01, ***: P<0.001. (b) Mass spectrometry and ELISA were used to determine the relative proportions of wild-type (WT/M-AAT) and mutant (Z-AAT/mutant) AAT proteins.
49. Provided technology may provide editing. Primary mouse hepatocytes transfected for the hADARp110 and SERPINA1-Z alleles were treated with oligonucleotide compositions comprising the indicated GalNAc conjugated oligonucleotides targeting the SERPINA1-Z allele at the indicated concentrations. RNA was isolated 48 hours after treatment and RNA editing was measured by Sanger sequencing (n=2 biological replicates).
50. Provided technologies may provide in vivo editing. Mice transgenic for the hADAR and SERPINA1-Z alleles were treated with an oligonucleotide composition targeting the SERPINA1-Z allele at a dose of 5 mg/kg via subcutaneous administration on days 0, 2 and 4. did Liver biopsies of mice were collected on day 7 post treatment. RNA editing was determined by Sanger sequencing in male (left bars) and female (right bars) mice (n=3 mice per sex).
51. Provided technology may provide editing. Primary mouse hepatocytes transfected for the hADARp110 and SERPINA1-Z alleles were treated with oligonucleotide compositions targeting the SERPINA-Z alleles at the indicated concentrations. RNA was isolated 48 hours after treatment and RNA editing was measured by Sanger sequencing (n=3 biological replicates).
52. Provided technologies can provide functionally edited polypeptides in vivo. Mice transgenic for the hADAR and SERPINA1-Z alleles were treated with an oligonucleotide composition targeting the SERPINA1-Z allele at a dose of 10 mg/kg via subcutaneous administration on days 0, 2, and 4. did Mouse serum was collected via weekly blood draws on the indicated days. Levels of human AAT protein were quantified by ELISA and mass spectrometry to assess the relative proportions of wild-type (PiM/WT, left bars) and mutant (PiZ/mutant, right bars) AAT proteins.
53. Provided technology may provide editing. Compositions of oligonucleotides containing various modifications were prepared and evaluated. Editing of the target adenosine of the SERPINA1-Z allele was confirmed in human ADARp110 and primary mouse hepatocytes transfected for the SERPINA1-Z allele (N=2 biological replicates).
54. Provided technology may provide editing. Linkage modifications (eg PS (phosphorothioate), PN (eg phosphoryl guanidine linkages such as n001), etc.), sugar modifications (eg 2'-F, 2'-OMe, etc. ) A composition of oligonucleotides containing various modifications, such as, was prepared and evaluated. Editing of the SERPINA1-Z allele was confirmed in primary mouse hepatocytes transfected for the human ADARp110 and SERPINA1-Z alleles (N=2 biological replicates).
55. Provided technology may provide editing. Linkage modifications (eg PS (phosphorothioate), PN (eg phosphoryl guanidine linkages such as n001), etc.), sugar modifications (eg 2'-F, 2'-OMe, etc. ) A composition of oligonucleotides containing various modifications, such as, was prepared and evaluated. Editing of the SERPINA1-Z allele was confirmed in primary mouse hepatocytes transfected for the human ADARp110 and SERPINA1-Z alleles (N=2 biological replicates).
56. Provided technology may provide editing. base modifications (e.g., b001A, b008U, b010U, b001C, b008C, b011U, b002G, b012U, etc.), linkage modifications (e.g., PS (phosphorothioate), PNs (e.g., phosphorothioates such as n001) Compositions of oligonucleotides containing various modifications, such as polylguanidine linkages), etc.), sugar modifications (eg, 2'-F, 2'-OMe, etc.), were prepared and evaluated. Editing of the targeted adenosine of the SERPINA1-Z allele in human ADARp110 and primary mouse hepatocytes transfected for the SERPINA1-Z allele was confirmed for various oligonucleotide compositions (N=2 biological replicates).
57. Provided technology may provide editing. base modifications (e.g. b008U, b010U, b001C, b008C, b011U, b012U, etc.), linkage modifications (e.g. PS (phosphorothioate), PNs (e.g. phosphorylguanidine linkages such as n001) Compositions of oligonucleotides containing various modifications, such as sugar modifications (eg, 2'-F, 2'-OMe, etc.), were prepared and evaluated. Editing of the targeted adenosine of the SERPINA1-Z allele in human ADARp110 and primary mouse hepatocytes transfected for the SERPINA1-Z allele was confirmed for various oligonucleotide compositions (N=2 biological replicates).
58. Provided technology may provide editing. Compositions of oligonucleotides containing various modifications, such as nucleobase modifications, linkage modifications, sugar modifications, etc., were prepared and evaluated. Editing of the targeted adenosine of the SERPINA1-Z allele in human ADARp110 and primary mouse hepatocytes transfected for the SERPINA1-Z allele was confirmed for various oligonucleotide compositions (N=2 biological replicates).
59. Provided technology may provide editing. Compositions of oligonucleotides containing various modifications, e.g., Csm11, Csm12, b009Csm11, b009Csm12, Gsm11, Gsm12, Tsm11, Tsm12, L010, etc., were prepared and evaluated. Editing of the targeted adenosine of the SERPINA1-Z allele in human ADARp110 and primary mouse hepatocytes transfected for the SERPINA1-Z allele was confirmed for various oligonucleotide compositions (N=2 biological replicates).
60. Provided technology may provide editing. base modifications (eg b008U), linkage modifications (eg PS (phosphorothioate), PN (eg phosphorylguanidine linkages such as n001), etc.), sugar modifications (eg 2 Compositions of oligonucleotides containing various modifications such as '-F, 2'-OMe, sm15, etc.) were prepared and evaluated. Editing of the target adenosine of the SERPINA1-Z allele was confirmed in human ADARp110 and primary mouse hepatocytes transfected for the SERPINA1-Z allele (N=2 biological replicates).
61. Provided technology may provide editing. base modifications (e.g., b008U, b001A, etc.), linkage modifications (e.g., PS (phosphorothioate), PN (e.g., phosphoryl guanidine linkages such as n001), etc.), sugar modifications (e.g., For example, 2'-F, 2'-OMe, etc.), compositions of oligonucleotides containing various modifications were prepared and evaluated. Editing of the target adenosine of the SERPINA1-Z allele was confirmed in human ADARp110 and primary mouse hepatocytes transfected for the SERPINA1-Z allele (N=2 biological replicates).
62. Provided technology may provide editing. First of all, it shows that the 2'-OR variant where R is not -H can be used in a variety of positions. Editing of the target adenosine of the SERPINA1-Z allele was confirmed in human ADARp110 and primary mouse hepatocytes transfected for the SERPINA1-Z allele (N=2 biological replicates).
63. Provided technology may provide editing. base modifications (eg, b008U, etc.), linkage modifications (eg, PS (phosphorothioate), PN (eg, phosphorylguanidine linkages such as n001), etc.), sugar modifications (eg, Compositions of oligonucleotides containing various modifications, such as 2'-F, 2'-OMe, etc.), were prepared and evaluated. Editing of the target adenosine of the SERPINA1-Z allele was confirmed in human ADARp110 and primary mouse hepatocytes transfected for the SERPINA1-Z allele (N=2 biological replicates).
64. Provided technology may provide editing. base modifications (eg, b008U, etc.), linkage modifications (eg, PS (phosphorothioate), PN (eg, phosphorylguanidine linkages such as n001), etc.), sugar modifications (eg, Compositions of oligonucleotides containing various modifications, such as 2'-F, 2'-OMe, etc.), were prepared and evaluated. Editing of the target adenosine of the SERPINA1-Z allele was confirmed in human ADARp110 and primary mouse hepatocytes transfected for the SERPINA1-Z allele (N=2 biological replicates).
65. Provided technology may provide editing. base modifications (eg, b008U, etc.), linkage modifications (eg, PS (phosphorothioate), PN (eg, phosphorylguanidine linkages such as n001), etc.), sugar modifications (eg, Compositions of oligonucleotides containing various modifications, such as 2'-F, 2'-OMe, etc.), were prepared and evaluated. Editing of the target adenosine of the SERPINA1-Z allele was confirmed in human ADARp110 and primary mouse hepatocytes transfected for the SERPINA1-Z allele (N=2 biological replicates).
66. Provided technology may provide editing. base modifications (eg, b008U, etc.), linkage modifications (eg, PS (phosphorothioate), PN (eg, phosphorylguanidine linkages such as n001), etc.), sugar modifications (eg, Compositions of oligonucleotides containing various modifications, such as 2'-F, 2'-OMe, etc.), were prepared and evaluated. Editing of the target adenosine of the SERPINA1-Z allele was confirmed in human ADARp110 and primary mouse hepatocytes transfected for the SERPINA1-Z allele (N=2 biological replicates).
67. Provided technology may provide editing. base modifications (eg, b008U, etc.), linkage modifications (eg, PS (phosphorothioate), PN (eg, phosphorylguanidine linkages such as n001), etc.), sugar modifications (eg, 2'-F, 2'-OMe, 2'-MOE, etc.) were prepared and evaluated for composition of oligonucleotides containing various modifications. Editing of the target adenosine of the SERPINA1-Z allele was confirmed in human ADARp110 and primary mouse hepatocytes transfected for the SERPINA1-Z allele (N=2 biological replicates).
68. Provided technology may provide editing. base modifications (eg, b008U, etc.), linkage modifications (eg, PS (phosphorothioate), PN (eg, phosphorylguanidine linkages such as n001), etc.), sugar modifications (eg, 2'-F, 2'-OMe, 2'-MOE, etc.) were prepared and evaluated for composition of oligonucleotides containing various modifications. Editing of the target adenosine of the SERPINA1-Z allele was confirmed in human ADARp110 and primary mouse hepatocytes transfected for the SERPINA1-Z allele (N=2 biological replicates).
69. Provided technology may provide editing. base modifications (eg, b008U, etc.), linkage modifications (eg, PS (phosphorothioate), PN (eg, phosphorylguanidine linkages such as n001), etc.), sugar modifications (eg, 2'-F, 2'-OMe, 2'-MOE, etc.) were prepared and evaluated for composition of oligonucleotides containing various modifications. Editing of the target adenosine of the SERPINA1-Z allele was confirmed in human ADARp110 and primary mouse hepatocytes transfected for the SERPINA1-Z allele (N=2 biological replicates).
70. Provided technology may provide editing. base modifications (eg, b008U, etc.), linkage modifications (eg, PS (phosphorothioate), PN (eg, phosphorylguanidine linkages such as n001), etc.), sugar modifications (eg, 2'-F, 2'-OMe, 2'-MOE, etc.) were prepared and evaluated for composition of oligonucleotides containing various modifications. Editing of the target adenosine of the SERPINA1-Z allele was confirmed in human ADARp110 and primary mouse hepatocytes transfected for the SERPINA1-Z allele (N=2 biological replicates).
71. Provided technology may provide editing. base modifications (eg, b008U, etc.), linkage modifications (eg, PS (phosphorothioate), PN (eg, phosphorylguanidine linkages such as n001), etc.), sugar modifications (eg, Compositions of oligonucleotides containing various modifications, such as 2'-F, 2'-OMe, sm15, etc.), were prepared and evaluated. Editing of the target adenosine of the SERPINA1-Z allele was confirmed in human ADARp110 and primary mouse hepatocytes transfected for the SERPINA1-Z allele (N=2 biological replicates).
72. Provided technology may provide editing. Compositions of oligonucleotides containing various modifications, e.g., linkages such as n001, n002, n006, n020, etc., were prepared and evaluated. Editing of the target adenosine of the SERPINA1-Z allele was confirmed in human ADARp110 and primary mouse hepatocytes transfected for the SERPINA1-Z allele (N=2 biological replicates).
73. Provided technology may provide editing. Base modifications (eg, b001A, etc.), linkage modifications (eg, PS (phosphorothioate), PN (eg, phosphorylguanidine linkages, such as n001), etc.), sugar modifications (eg, 2'-F, 2'-OMe, morpholine sugars, etc.) were prepared and evaluated for composition of oligonucleotides containing various modifications. Editing of the targeted adenosine of the SERPINA1-Z allele in human ADARp110 and primary mouse hepatocytes transfected for the SERPINA1-Z allele was confirmed for various oligonucleotide compositions (N=2 biological replicates).
74. Provided technology may provide editing. Base modifications (eg, b001A, etc.), linkage modifications (eg, PS (phosphorothioate), PN (eg, phosphorylguanidine linkages, such as n001), etc.), sugar modifications (eg, 2'-F, 2'-OMe, morpholine sugars, etc.) were prepared and evaluated for composition of oligonucleotides containing various modifications. Editing of the targeted adenosine of the SERPINA1-Z allele in human ADARp110 and primary mouse hepatocytes transfected for the SERPINA1-Z allele was confirmed for various oligonucleotide compositions (N=2 biological replicates).
75. Various nearest neighbors can provide editing activity. Editing of the target adenosine of the SERPINA1-Z allele was confirmed in human ADARp110 and primary mouse hepatocytes transfected for the SERPINA1-Z allele (N=2 biological replicates).
76. Provided technology may provide editing. Various variants (e.g. b008U, b012U, b013U, b001A, b002A, b003A, b004I, b002G, b009U, etc.), linking variants (e.g. PS (phosphorothioate), PN (e.g. n001 and Compositions of oligonucleotides containing sugar modifications (e.g., 2'-F, 2'-OMe, etc.), etc.) were prepared and evaluated. Editing of the target adenosine of the SERPINA1-Z allele was confirmed in human ADARp110 and primary mouse hepatocytes transfected for the SERPINA1-Z allele (N=2 biological replicates).
77. Provided technology may provide editing. various sugar and nucleobase modifications (e.g., in b002A, b003A, b008U, b001C, Tsm11, Tsm12, b004C, b007C, 2'-F, 2'-OMe, etc.), linkage modifications (e.g., PS (phosphoro thioate), PNs (eg, phosphoryl guanidine linkages such as n001), etc.), etc. were prepared and evaluated. Editing of the target adenosine of the SERPINA1-Z allele was confirmed in human ADARp110 and primary mouse hepatocytes transfected for the SERPINA1-Z allele (N=2 biological replicates).
78. Provided technology may provide editing. various sugar and nucleobase modifications (e.g. in b003A, b008U, b001C, b008C, Tsm11, Tsm12, b004C, Csm17, etc.), linkage modifications (e.g. PS (phosphorothioate), PN (e.g. A composition of oligonucleotides comprising phosphoryl guanidine linkages such as n001) and the like was prepared and evaluated. Editing of the target adenosine of the SERPINA1-Z allele was confirmed in human ADARp110 and primary mouse hepatocytes transfected for the SERPINA1-Z allele (N=2 biological replicates).
79. Provided technology may provide editing. Various sugars and nucleobases (e.g. in N _-1 to dI, b001A, b003A, b008U, b001C, b008C, Tsm11, Tsm12, b004C, Csm17, etc.), linkage modifications (e.g. PS (phosphorothioate) , PNs (eg, phosphoryl guanidine linkages such as n001), etc.), etc. were prepared and evaluated. Editing of the targeted adenosine of the SERPINA1-Z allele in human ADARp110 and primary mouse hepatocytes transfected for the SERPINA1-Z allele was confirmed for various oligonucleotide compositions (N=2 biological replicates).
80. Provided technology may provide editing. Various sugars and nucleobases (e.g. in N _-1 to dI, b001A, b002A, b003A, b008U, b008C, Tsm11, Tsm12, b004C, Csm17, etc.), linking modifications (e.g. PS (phosphorothioate) , PNs (eg, phosphoryl guanidine linkages such as n001), etc.), etc. were prepared and evaluated. Editing of the targeted adenosine of the SERPINA1-Z allele in human ADARp110 and primary mouse hepatocytes transfected for the SERPINA1-Z allele was confirmed for various oligonucleotide compositions (N=2 biological replicates).
81. Provided technology may provide editing. Various sugars and nucleobases (e.g. in N _-1 to Csm11, Csm12, b009Csm11, b009Csm12, etc.), linking modifications (e.g. PS (phosphorothioate), PN (e.g. phosphoryl like n001) Guanidine linkages), etc.) were prepared and evaluated. Editing of the targeted adenosine of the SERPINA1-Z allele in human ADARp110 and primary mouse hepatocytes transfected for the SERPINA1-Z allele was confirmed for various oligonucleotide compositions (N=2 biological replicates).
82. Oligonucleotides containing various types of internucleotidic linkages can provide editing. base modifications (eg, b008U, b014I, etc.), linkage modifications (eg, PS (phosphorothioate), PN (eg, phosphorylguanidine linkages such as n001, n004, n008, n025, n026, etc.) ), sugar modifications (e.g., 2'-F, 2'-OMe, 2'-MOE, etc.) were prepared and evaluated. Editing of the target adenosine of the SERPINA1-Z allele was confirmed in primary mouse hepatocytes transformed for (N=2 biological replicates) For each oligonucleotide composition, from left to right, 1.0, 0.33, 0.11, respectively. , 0.037 uM.
83. Provided technology may provide editing. Compositions of oligonucleotides containing various modifications and patterns thereof were prepared and evaluated. Editing of the target adenosine of UGP2 in primary human hepatocytes was confirmed for various oligonucleotide compositions (N=2 biological replicates). Concentrations tested were 1 uM, 0.1 uM and 0.01 uM from left to right.
84. Provided technology may provide editing. Compositions of oligonucleotides containing various modifications and their patterns were prepared and evaluated, and editing of the target adenosine in UGP2 was confirmed in primary human hepatocytes at various concentrations.
Figure 85. Provided technology can provide in vivo editing. In vivo editing of the target adenosine of the SERPINA1-Z allele was confirmed in mice transgenic for the human ADAR and SERPINA1-Z alleles. Serum levels of AAT were also increased in treated mice.
86. Provided technology may provide in vivo editing. Various nucleobases (eg, b008U, hypoxanthine, etc.), linkages (eg, PO, PS, PN (eg, phosphorylguanidine linkages such as n001), etc.), sugar modifications (eg, 2 '-F, 2'-OMe, 2'-MOE, etc.), etc., and oligonucleotides containing patterns thereof were prepared. Editing of the target adenosine and an increase in serum AAT were confirmed (N=4 per group). Top: Edit SERPINA1 on day 10. Bottom: Serum AAT fold change.

The present technology may be more readily understood with reference to the following detailed description of specific embodiments.

Justice

As used herein, the following definitions shall apply unless otherwise indicated. For purposes of this invention, chemical elements are identified according to the Periodic Table of the Elements, CAS version, Handbook of Chemistry and Physics, 75th Ed. In addition, general principles of organic chemistry are described in "Organic Chemistry", Thomas Sorrell, University Science Books, Sausalito: 1999, and in "March's Advanced Organic Chemistry", 5th Ed., Ed.: Smith, M.B. and March, J., John Wiley & Sons, New York: 2001.

As used herein herein, unless the context clearly dictates otherwise: (i) a singular term ("a" or "an") may be understood to mean "at least one"; (ii) the term “or” may be understood to mean “and/or”; (iii) "comprising", "includes", "including" (whether used with "not limited to" or not) and "contains" (used with "but not limited to") the term (whether or not) may be understood to include itemized components or steps, whether provided alone or in conjunction with one or more additional components or steps; (iv) the term "another" may be understood to mean at least an additional/second one or more; (v) the terms “about” and “approximately” may be understood to allow for standard variations as understood by those skilled in the art; (vi) endpoints are included where ranges are provided;

Unless otherwise specified, descriptions of oligonucleotides and elements thereof (eg, base sequence, sugar modifications, internucleotidic linkages, linkage phosphorus stereochemistry, patterns thereof, etc.) are in the 5' to 3' direction. As will be appreciated by those of ordinary skill in the art, in some embodiments, oligonucleotides may be provided and/or utilized in salt form, particularly as a pharmaceutically acceptable salt form, such as the sodium salt. As will also be appreciated by those skilled in the art, in some embodiments, individual oligonucleotides within a composition, even though certain such oligonucleotides within such composition (e.g., liquid composition) are in different salt form(s) at a particular moment in time. Even if it can exist (and it can be dissolved and the oligonucleotide chain can exist in anionic form (eg when in a liquid composition)), it can be considered to be of the same composition and/or structure. For example, one skilled in the art will understand that at a given pH the individual internucleotidic linkages along an oligonucleotide chain can be in the acid (H) form, or in the form of a plurality of possible salts (e.g. sodium salt, or salts of different cations (which ions are depending on whether it can be present in the composition))), and its acid form (eg, all cations (if any) are replaced by H ⁺ ) of the same composition and/or structure. So long as they are, these individual oligonucleotides can be considered to be of the same composition and/or structure as appropriate.

Aliphatic: As used herein, “aliphatic” means a straight-chain (i.e., unbranched) or branched, substituted or unsubstituted hydrocarbon chain that is fully saturated or contains one or more units of unsaturation, or a fully saturated or one or more unsaturated (but not a substituted or unsubstituted monocyclic, bicyclic or polycyclic hydrocarbon ring (but not aromatic) containing units that are not aromatic), or combinations thereof. In some embodiments, aliphatic groups contain 1-50 aliphatic carbon atoms. In some embodiments, aliphatic groups contain 1-20 aliphatic carbon atoms. In other embodiments, aliphatic groups contain 1-10 aliphatic carbon atoms. In other embodiments, aliphatic groups contain 1-9 aliphatic carbon atoms. In other embodiments, aliphatic groups contain 1-8 aliphatic carbon atoms. In other embodiments, aliphatic groups contain 1-7 aliphatic carbon atoms. In other embodiments, aliphatic groups contain 1-6 aliphatic carbon atoms. In another embodiment, an aliphatic group contains 1-5 aliphatic carbon atoms, and in still other embodiments, an aliphatic group contains 1, 2, 3, or 4 aliphatic carbon atoms. Suitable aliphatic groups include, but are not limited to, linear or branched, substituted or unsubstituted alkyl, alkenyl, alkynyl groups and hybrids thereof such as (cycloalkyl)alkyl, (cycloalkenyl)alkyl or (cycloalkyl)alkenyl. It doesn't work.

Alkenyl: As used herein, the term “alkenyl” refers to an aliphatic group as defined herein having one or more double bonds.

Alkyl: As used herein, the term "alkyl" is given its conventional meaning in the art and includes straight chain alkyl groups, branched chain alkyl groups, cycloalkyl (alicyclic) groups, alkyl substituted cycloalkyl groups and cycloalkyl groups. saturated aliphatic groups including substituted alkyl groups. In some embodiments, an alkyl has 1-100 carbon atoms. In certain embodiments, a straight chain or branched chain alkyl has about 1 to 20 carbon atoms in its backbone (eg, C ₁ -C ₂₀ for straight chain, C ₂ -C ₂₀ for branched chain), alternatively about It has 1 to 10 carbon atoms. In some embodiments, a cycloalkyl ring has from about 3 to 10 carbon atoms, alternatively about 5, 6 or 7 carbons, within its ring structure (where the ring is monocyclic, bicyclic or polycyclic). In some embodiments, an alkyl group can be a lower alkyl group, wherein the lower alkyl group contains 1-4 carbon atoms (eg, C ₁ -C ₄ for straight chain lower alkyl).

Alkynyl: As used herein, the term “alkynyl” refers to an aliphatic group as defined herein having one or more triple bonds.

Analog: The term “analog” includes any chemical moiety that differs structurally from a reference chemical moiety or class of moieties, but is capable of performing at least one function of the reference chemical moiety or class of moieties. As a non-limiting example, a nucleotide analogue differs structurally from a nucleotide but performs one or more functions of a nucleotide; A nucleobase analog differs structurally from a nucleobase but performs one or more functions of a nucleobase; and so on.

Animal: As used herein, the term “animal” refers to any member of the animal kingdom. In some embodiments, “animal” refers to humans at any stage of development. In some embodiments, “animal” refers to a non-human animal at any stage of development. In certain embodiments, a non-human animal is a mammal (eg, rodent, mouse, rat, rabbit, mouse, dog, cat, sheep, cow, primate, and/or pig). In some embodiments, animals include but are not limited to mammals, birds, reptiles, amphibians, fish, and/or worms. In some embodiments, an animal can be a transgenic animal, genetically engineered animal, and/or clone.

Aryl: As used herein, the term "aryl" used alone or as part of a larger moiety, as in "aralkyl", "aralkoxy", or "aryloxyalkyl", means a total of 5 to Refers to a monocyclic, bicyclic, or polycyclic ring system having 30 ring members, wherein at least one ring in the system is aromatic. In some embodiments, an aryl group is a monocyclic, bicyclic, or polycyclic ring system having a total of 5 to 14 ring members, wherein at least one ring in the system is aromatic and each ring in the system is 3 to 7 ring members. contains two ring members. In some embodiments, each monocyclic ring unit is aromatic. In some embodiments, an aryl group is a biaryl group. The term “aryl” may be used interchangeably with the term “aryl ring”. In certain embodiments of the present invention, “aryl” refers to aromatic ring systems including but not limited to phenyl, biphenyl, naphthyl, binapthyl, anthracyl, and the like, which may have one or more substituents. As used herein, within the scope of the term "aryl" are groups in which an aromatic ring is fused to one or more non-aromatic rings, such as indanyl, phthalimidyl, naphthymidyl, phenanthridinyl, or tetrahydronaphthyl. etc. are also included.

Feature: As used herein, the term "feature" in its broadest sense refers to a portion of a material that correlates with the presence (or absence) of a particular characteristic, property, or activity of the material. do. In some embodiments, a characteristic portion of a material is a portion found in materials and related materials that share a particular feature, property, or activity, but does not share a particular characteristic, property, or activity. In certain embodiments, the feature shares at least one functional characteristic with the intact material. For example, in some embodiments, a “feature” of a protein or polypeptide is one that contains a contiguous stretch of amino acids or a set of contiguous stretches of amino acids that together are characteristic of the protein or polypeptide. In some embodiments, each such contiguous stretch generally contains at least 2, 5, 10, 15, 20, 50 or more amino acids. Typically, a feature of a substance (eg protein, antibody, etc.) is a portion that shares at least one functional characteristic with the relevant intact substance in addition to the sequence and/or structural identity specified above. In some embodiments, a feature can be biologically active.

Chiral Control: As used herein, “chiral control” refers to control of the stereochemical assignment of chiral linkage phosphorus at chiral internucleotidic linkages within an oligonucleotide. As used herein, a chiral internucleotidic linkage is an internucleotidic linkage in which the linkage phosphorus is chiral. In some embodiments, control is achieved through a chiral element that is free of sugar and base moieties of the oligonucleotide, for example, in some embodiments, control is through the use of one or more chiral auxiliaries during oligonucleotide preparation. The chiral auxiliary is often part of a chiral phosphoramidite used during oligonucleotide preparation. In contrast to chiral control, one skilled in the art can control stereochemistry in chiral internucleotidic linkages when conventional oligonucleotide synthesis without the use of chiral auxiliaries is used to form chiral internucleotidic linkages. will understand that there is no In some embodiments, the stereochemical assignment of each chiral linkage phosphorus at each chiral internucleotidic linkage within an oligonucleotide is controlled.

0.90 = 90%). 일부 구현예에서, 조성물 내 복수의 올리고뉴클레오티드의 수준은 올리고뉴클레오티드에 있는 각각의 키랄 제어 뉴클레오티드간 연결의 부분입체순도의 곱으로 표시된다. 일부 구현예에서, 올리고뉴클레오티드(또는 핵산)에 있는 2개의 뉴클레오시드를 연결하는 뉴클레오티드간 연결의 부분입체순도는 상기 2개의 뉴클레오시드를 연결하는 이량체의 뉴클레오티드간 연결의 부분입체순도로 표시되고, 이러한 이량체는 비견되는 조건들, 일부 경우에 동일한 합성 사이클 조건들을 사용하여 제조된다(예를 들어, 올리고뉴클레오티드 …NxNy…에서 Nx와 Ny 사이의 연결의 경우, 이량체는 NxNy임). 일부 구현예에서, 모든 키랄 뉴클레오티드간 연결이 키랄 제어 뉴클레오티드간 연결은 아니며, 조성물은 부분적으로 키랄 제어 올리고뉴클레오티드 조성물이다. 일부 구현예에서, 비-키랄 제어 뉴클레오티드간 연결은 입체무작위 올리고뉴클레오티드 조성물에서 일반적으로 관찰되는 바와 같이(예를 들어, 당업자에 의해 이해되는 바와 같이, 기존의 올리고뉴클레오티드 합성, 예를 들어 포스포아미다이트 방법으로부터), 약 80%, 75%, 70%, 65%, 60%, 55% 미만의, 또는 약 50%의 부분입체순도를 갖는다. 일부 구현예에서, 복수의 올리고뉴클레오티드(또는 핵산)는 동일 유형의 것이다. 일부 구현예에서, 키랄 제어 올리고뉴클레오티드 조성물은 비무작위 또는 제어된 수준의 개별 올리고뉴클레오티드 또는 핵산 유형을 포함한다. 예를 들어 일부 구현예에서, 키랄 제어 올리고뉴클레오티드 조성물은 하나 이하의 올리고뉴클레오티드 유형을 포함한다. 일부 구현예에서, 키랄 제어 올리고뉴클레오티드 조성물은 하나 초과의 올리고뉴클레오티드 유형을 포함한다. 일부 구현예에서, 키랄 제어 올리고뉴클레오티드 조성물은 다수의 올리고뉴클레오티드 유형을 포함한다. 일부 구현예에서, 키랄 제어 올리고뉴클레오티드 조성물은 소정 올리고뉴클레오티드 유형의 올리고뉴클레오티드의 조성물이고, 이 조성물은 상기 올리고뉴클레오티드 유형의 비무작위 또는 제어된 수준의 복수의 올리고뉴클레오티드를 포함한다.Chirally controlled oligonucleotide composition: As used herein, the terms "chirally controlled oligonucleotide composition", "chirally controlled nucleic acid composition", etc. refer to a composition comprising a plurality of oligonucleotides (or nucleic acids) that share a common base sequence. wherein the plurality of oligonucleotides (or nucleic acids) are stereochemically identical linkages in one or more chiral internucleotidic linkages (chiral controlled or stereotypic internucleotidic linkages, in a composition whose chiral linkages are non-chiral controlled internucleotidic linkages). rather than a random Rp and Sp combination as Rp or Sp ("stereodefined")). In some embodiments, a chirally controlled oligonucleotide composition comprises a plurality of oligonucleotides (or nucleic acids) that share 1) a common base sequence, 2) a common backbone linkage pattern, and 3) a common backbone being a modification pattern, wherein the plurality of oligonucleotides Nucleotides (or nucleic acids) are identical in one or more chiral internucleotidic linkages (stereochemistry) (chiral controlled or steric internucleotidic linkages, of which chiral linkages are, in a composition, random Rp and Sp combinations as non-chiral controlled internucleotidic linkages). but not, Rp or Sp (“sterically defined”))). The level of the plurality of oligonucleotides (or nucleic acids) in the chiral controlled oligonucleotide composition is compared to the random level of the non-chiral controlled oligonucleotide composition (e.g., chiral to stereoselectively form one or more chiral internucleotide linkages). pre-determined/controlled or enriched (via control oligonucleotide preparation). In some embodiments, about 1% to 100% (e.g., about 5% to 100%, 10% to 100%, 20% to 100%, 30% to 100%) of all oligonucleotides in the chirally controlled oligonucleotide composition. , 40% to 100%, 50% to 100%, 60% to 100%, 70% to 100%, 80 to 100%, 90 to 100%, 95 to 100%, 50% to 90%, or about 5% , 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97 %, 98%, 99%, or 100%, or at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) are a plurality of oligonucleotides. In some embodiments, about 1% to 100% (e.g., about 5% to 100%) of all oligonucleotides in a chiral controlled oligonucleotide composition share a common base sequence, a common backbone linkage pattern, and a common backbone phosphorus modification pattern. , 10%~100%, 20%~100%, 30%~100%, 40%~100%, 50%~100%, 60%~100%, 70%~100%, 80~100%, 90~ 100%, 95-100%, 50%-90%, or about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) are a plurality of oligonucleotides. In some embodiments, the level is of all oligonucleotides in the composition, or of all oligonucleotides in the composition that share a common base sequence (eg, of a plurality of oligonucleotides or of an oligonucleotide type), or of a common base sequence, common of all oligonucleotides in the composition that share a backbone linkage pattern and a common backbone-in-modification pattern, or a common base sequence, a common base modification pattern, a common sugar modification pattern, a common internucleotidic linkage type pattern, and/or a common internucleotidic linkage variant About 1%-100% (e.g., about 5%-100%, 10%-100%, 20%-100%, 30%-100%, 40%-100%) of all oligonucleotides in the composition that share the pattern %, 50%-100%, 60%-100%, 70%-100%, 80-100%, 90-100%, 95-100%, 50%-90%, or about 5%, 10%, 20 %, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93% , 94%, 95%, 96%, 97%, 98%, or 99%). In some embodiments, the plurality of oligonucleotides is about 1-50 (e.g., about 1-10, 1-20, 5-10, 5-20, 10-15, 10-20, 10-25, 10 ~30, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) chiral internucleotide linkages of the same conformation share chemistry. In some embodiments, the plurality of oligonucleotides is about 1%-100% (e.g., about 5%-100%, 10%-100%, 20%-100%, 30%-100%, 40%-100%). %, 50% to 100%, 60% to 100%, 70% to 100%, 80 to 100%, 90 to 100%, 95 to 100%, 50% to 90%, approximately 5%, 10%, 15% , 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%, or at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99%) of the chiral internucleotidic linkages share the same stereochemistry. In some embodiments, a plurality of oligonucleotides (or nucleic acids) in any case share the same pattern of sugar and/or nucleobase modifications. In some embodiments, the plurality of oligonucleotides (or nucleic acids) are different forms of the same oligonucleotide (eg, acids and/or different salts of the same oligonucleotide). In some embodiments, the plurality of oligonucleotides (or nucleic acids) have the same composition. In some embodiments, the level of the plurality of oligonucleotides (or nucleic acids) is between about 1% and 100% (e.g., , about 5% to 100%, 10% to 100%, 20% to 100%, 30% to 100%, 40% to 100%, 50% to 100%, 60% to 100%, 70% to 100%, 80-100%, 90-100%, 95-100%, 50%-90%, or about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or at least 5%, 10%, 20%, 30% , 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) am. In some embodiments, each chiral internucleotide linkage is a chirally controlled internucleotide linkage and the composition is a fully chirally controlled oligonucleotide composition. In some embodiments, the plurality of oligonucleotides (or nucleic acids) are structurally identical. In some embodiments, the chiral control internucleotide linkages are at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5%. %, typically at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% diastereopurity. In some embodiments, chirally controlled internucleotidic linkages have a diastereopurity of at least 95%. In some embodiments, the chirally controlled internucleotidic linkage has a diastereopurity of at least 96%. In some embodiments, the chirally controlled internucleotidic linkage has a diastereopurity of at least 97%. In some embodiments, the chirally controlled internucleotidic linkage has a diastereopurity of at least 98%. In some embodiments, chirally controlled internucleotidic linkages have a diastereopurity of at least 99%. In some embodiments, the percentage of a level is (DS) ^nc or higher, and DS is a diastereopurity as described herein (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% or more), and nc is the number of chiral controlled internucleotide linkages as described herein (e.g., 1-50, 1-40, 1-30 , 1~25, 1~20, 5~50, 5~40, 5~30, 5~25, 5~20, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more). In some embodiments, the percentage of the level is (DS) ^nc or greater, and the DS is between 95% and 100%. For example, if DS is 99% and nc is 10, the percentage is greater than 90% ((99%) ¹⁰

0.90 = 90%). In some embodiments, the level of the plurality of oligonucleotides in the composition is expressed as the product of the diastereopurity of each chiral control internucleotide linkage in the oligonucleotide. In some embodiments, the diastereopurity of an internucleotide linkage linking two nucleosides in an oligonucleotide (or nucleic acid) is represented by the diastereopurity of an internucleotide linkage of a dimer linking the two nucleosides. and these dimers are prepared using comparable conditions, in some cases identical synthetic cycle conditions (e.g., for a linkage between Nx and Ny in an oligonucleotide...NxNy..., the dimer is NxNy). In some embodiments, not all chiral internucleotide linkages are chirally controlled internucleotide linkages, and the composition is a partially chirally controlled oligonucleotide composition. In some embodiments, non-chiral controlled internucleotidic linkages are formed as commonly observed in sterically random oligonucleotide compositions (e.g., conventional oligonucleotide synthesis, e.g. from the Dite method), less than about 80%, 75%, 70%, 65%, 60%, 55%, or about 50% diastereopurity. In some embodiments, the plurality of oligonucleotides (or nucleic acids) are of the same type. In some embodiments, a chirally controlled oligonucleotide composition comprises nonrandom or controlled levels of individual oligonucleotides or nucleic acid types. For example, in some embodiments, a chirally controlled oligonucleotide composition includes no more than one type of oligonucleotide. In some embodiments, a chirally controlled oligonucleotide composition includes more than one oligonucleotide type. In some embodiments, a chirally controlled oligonucleotide composition includes multiple oligonucleotide types. In some embodiments, a chirally controlled oligonucleotide composition is a composition of oligonucleotides of a given oligonucleotide type, which composition comprises non-random or controlled levels of a plurality of oligonucleotides of said oligonucleotide type.

Comparable: The term "comparable" is used herein to describe two sets (or more sets) of conditions or circumstances that are sufficiently similar to each other to permit comparison of the results obtained or observed phenomena. In some implementations, a set of comparable conditions or circumstances is characterized by a plurality of substantially identical characteristics and no more than one variable characteristic. One of ordinary skill in the art would characterize substantially the same features in a sufficient number and type to warrant a reasonable conclusion that differences in results obtained or observed phenomena under different sets of conditions or circumstances are caused by or represent variations in the various characteristics. It will be appreciated that the sets of conditions are comparable to each other if

Cycloaliphatic: The terms “alicyclic,” “carbocyclic,” “carbocyclyl,” “carbocyclic radical,” and “carbocyclic ring” are used interchangeably and, as used herein, are saturated or partially unsaturated. , non-aromatic, cyclic aliphatic monocyclic, bicyclic, or polycyclic ring systems (as described herein and having 3-30 ring members, unless otherwise specified). Alicyclic groups include, without limitation, cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cycloheptyl, cycloheptenyl, cyclooctyl, cyclooctenyl, norbornyl, adamantyl, and cyclo Includes octadienyl. In some embodiments, cycloaliphatic groups have 3-6 carbons. In some embodiments, a cycloaliphatic group is saturated and is a cycloalkyl. The term "alicyclic" may also include an aliphatic ring fused to one or more aromatic or non-aromatic rings, such as decahydronaphthyl or tetrahydronaphthyl. In some embodiments, a cycloaliphatic group is bicyclic. In some embodiments, an alicyclic group is tricyclic. In some embodiments, cycloaliphatic groups are polycyclic. In some embodiments, “cycloaliphatic” refers to a C ₃ -C ₆ monocyclic hydrocarbon, or a C ₈ -C ₁₀ bicyclic ring that is fully saturated or contains one or more units of unsaturation but is not aromatic and has a single point of attachment to the rest of the molecule; Refers to polycyclic hydrocarbons, or C ₉ -C ₁₆ polycyclic hydrocarbons that are fully saturated or contain one or more units of unsaturation but are not aromatic and have a single point of attachment to the rest of the molecule.

Heteroaliphatic: As used herein, the term "heteroaliphatic" is given its ordinary meaning in the art, wherein one or more carbon atoms are independently one or more heteroatoms (e.g., oxygen, nitrogen, sulfur, silicon , phosphorus, etc.) as described herein. In some embodiments, one or more units selected from C, CH, CH ₂ , and CH ₃ are independently replaced with one or more heteroatoms (including oxidized and/or substituted forms thereof). In some embodiments, a heteroaliphatic group is a heteroalkyl. In some embodiments, a heteroaliphatic group is a heteroalkenyl.

Heteroalkyl: As used herein, the term “heteroalkyl” is given its ordinary meaning in the art, wherein one or more carbon atoms are independently one or more heteroatoms (e.g., oxygen, nitrogen, sulfur, silicon , phosphorus, etc.) as described herein. Examples of heteroalkyl groups include, but are not limited to, alkoxy, poly(ethylene glycol)-, alkyl-substituted amino, tetrahydrofuranyl, piperidinyl, morpholinyl, and the like.

Heteroaryl: As used herein, the terms "heteroaryl" and "heteroar-" are used alone or as part of a larger moiety (e.g., "heteroaralkyl" or "heteroaralkoxy"). The term refers to a monocyclic, bicyclic, or polycyclic ring system having a total of 5 to 30 ring members, wherein at least one ring in the system is aromatic and at least one aromatic ring atom is a heteroatom. In some embodiments, a heteroaryl group is a group having 5 to 10 ring atoms (ie, monocyclic, bicyclic, or polycyclic), and in some embodiments 5, 6, 9, or 10 ring atoms. In some embodiments, each monocyclic ring unit is aromatic. In some embodiments, a heteroaryl group has 6, 10 or 14 π electrons shared in a cyclic array; In addition to carbon atoms, it has 1 to 5 heteroatoms. Heteroaryl groups include, without limitation, thienyl, furanyl, pyrrolyl, imidazolyl, pyrazolyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl, oxadiazolyl, thiazolyl, isothiazolyl, thiadiazolyl, pyri dill, pyridazinyl, pyrimidinyl, pyrazinyl, indolizinyl, purinyl, naphthyridinyl, and pteridinyl. In some embodiments, heteroaryl is a heterobiaryl group, such as bipyridyl and the like. As used herein, the terms "heteroaryl" and "heteroar-" also include groups in which a heteroaromatic ring is fused to one or more aryl, cycloaliphatic, or heterocyclyl rings, wherein the radical or point of attachment is It is on the heteroaromatic ring. Non-limiting examples are indolyl, isoindolyl, benzothienyl, benzofuranil, dibenzofuranil, indazolyl, benzimidazolyl, benzthiazolyl, quinolyl, isoquinolyl, cynnolinyl, phthalazinyl , quinazolinyl, quinoxalinyl, 4H-quinolizinyl, carbazolyl, acridinyl, phenazinyl, phenothiazinyl, phenoxazinyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, and pyrido[ 2,3-b]-1,4-oxazin-3(4H)-one. Heteroaryl groups can be monocyclic, bicyclic, or polycyclic. The term “heteroaryl” may be used interchangeably with the terms “heteroaryl ring,” “heteroaryl group,” or “heteroaromatic,” any of which terms include rings that are optionally substituted. The term "heteroaralkyl" refers to an alkyl group substituted with a heteroaryl group, wherein the alkyl and heteroaryl portions independently are optionally substituted.

Heteroatom: As used herein, the term "heteroatom" refers to an atom that is not carbon or hydrogen. In some embodiments, the heteroatom is boron, oxygen, sulfur, nitrogen, phosphorus, or silicon (oxygenated form of nitrogen, sulfur, phosphorus, or silicon; charged form of nitrogen (e.g., quaternized form, an iminium group). form as in, etc.), including phosphorus, sulfur, oxygen, etc.). In some embodiments, the heteroatom is silicon, phosphorus, oxygen, sulfur or nitrogen. In some embodiments, the heteroatom is silicon, oxygen, sulfur or nitrogen. In some embodiments, the heteroatom is oxygen, sulfur or nitrogen.

Heterocycle: As used herein, the terms "heterocycle", "heterocyclyl", "heterocyclic radical" and "heterocyclic ring" (as used herein) are used interchangeably and are saturated or a monocyclic, bicyclic, or multicyclic ring moiety (eg, 3-30 membered) that is partially unsaturated and has one or more heteroatomic ring atoms. In some embodiments, a heterocyclyl group is a stable 5 to 7 membered monocyclic ring or 7 to 7 membered monocyclic ring, as defined above, that is saturated or partially unsaturated and has one or more, preferably 1 to 4, heteroatoms in addition to carbon atoms. It is a 10-membered bicyclic heterocyclic moiety. When used in reference to a ring atom of a heterocyclic ring, the term "nitrogen" includes substituted nitrogen. As an example, in a saturated or partially unsaturated ring having 0-3 heteroatoms selected from oxygen, sulfur and nitrogen, nitrogen is N (as in 3,4-dihydro-2H-pyrrolyl), NH (p as in rolidinyl), or ⁺ NR (as in N-substituted pyrrolidinyl). A heterocyclic ring may be attached to its pendant group at any heteroatom or carbon atom, which results in a stable structure, and any ring atom may be optionally substituted. Examples of such saturated or partially unsaturated heterocyclic radicals include, without limitation, tetrahydrofuranyl, tetrahydrothienyl, pyrrolidinyl, piperidinyl, pyrrolinyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, decahydro quinolinyl, oxazolidinyl, piperazinil, dioxanil, dioxolanil, diazepinil, oxazepinil, thiazepinil, morpholinil, and quinuclidinyl. The terms "heterocycle", "heterocyclyl", "heterocyclyl ring", "heterocyclic group", "heterocyclic moiety" and "heterocyclic radical" are used interchangeably herein and also refer to heterocycle groups wherein the yl ring is fused to one or more aryl, heteroaryl, or cycloaliphatic rings, such as indolinyl, 3H-indolyl, chromanyl, phenantridinyl, or tetrahydroquinolinyl. Heterocyclyl groups can be monocyclic, bicyclic, or polycyclic. The term "heterocyclylalkyl" refers to an alkyl group substituted with a heterocyclyl, wherein the alkyl and heterocyclyl portions independently are optionally substituted.

Identity: As used herein, the term “identity” refers to the overall relationship between polymer molecules, eg between nucleic acid molecules (eg, oligonucleotides, DNA, RNA, etc.) and/or between polypeptide molecules. In some embodiments, the polymer molecules have their sequence at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85% %, 90%, 95% or 99% identical to each other are considered "substantially identical". For example, calculation of percent identity of two nucleic acid or polypeptide sequences can be performed by aligning the two sequences for the purpose of optimal comparison (e.g., gaps are first and second for optimal alignment). may be introduced into either or both of the 2 sequences, and sequences that are not identical may be disregarded for comparison purposes). In certain embodiments, the length of sequences to be aligned for comparison purposes is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% of the length of the reference sequence. , or substantially 100%. The nucleotides at corresponding positions are then compared. Molecules are identical at that position if a position in the first sequence is occupied by the same residue (eg, nucleotide or amino acid) as the corresponding position in the second sequence. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps and the length of each gap that needs to be introduced for optimal alignment of the two sequences. The comparison of sequences and determination of percent identity between two sequences can be accomplished using mathematical algorithms. For example, the percent identity between two nucleotide sequences can be determined using the algorithm of Meyers and Miller (CABIOS, 1989, 4: 11-17) incorporated into the ALIGN program (version 2.0). In some exemplary embodiments, nucleic acid sequence comparisons made with the ALIGN program use a PAM120 weighted residue table, a gap length penalty of 12 and a gap penalty of 4. Percent identity between two nucleotide sequences can alternatively be determined using the GAP program in the GCG software package using the NWSgapdna.CMP matrix.

Internucleotidic linkage: As used herein, the phrase "internucleotidic linkage" generally refers to a linkage that links nucleoside units of an oligonucleotide or nucleic acid. In some embodiments, the internucleotidic linkages are phosphodiester linkages, as found widely in naturally occurring DNA and RNA molecules (natural phosphate linkages (-OP( =O)(OH)O-)). In some embodiments, the internucleotidic linkages are modified internucleotidic linkages (not natural phosphate linkages). In some embodiments, the internucleotide linkage is a "modified internucleotidic linkage" in which at least one oxygen atom or -OH of the phosphodiester linkage is replaced with a different organic or inorganic moiety. In some embodiments, such organic or inorganic moieties =S, =Se, =NR', -SR', -SeR', -N(R') ₂ , B(R') _3, -S-, - Se-, and -N(R')-, wherein each R' is independently as defined and described herein. In some embodiments, an internucleotidic linkage is a phosphotriester linkage, a phosphorothioate linkage (or a phosphorothioate diester linkage, -OP(=O)(SH)O-(which, as recognized by those skilled in the art, is a salt In some embodiments, the modified internucleotide linkage is a phosphorothioate linkage. In some embodiments, the internucleotide linkage is, for example, a PNA (peptide nucleic acid ) or a PMO (phosphorodiamidate morpholino oligomer) linkage. In some embodiments, the modified internucleotide linkage is a non-negatively charged internucleotidic linkage. In some embodiments, the modified internucleotide linkage is a neutral nucleotide internucleotidic linkages (eg, n001 in certain oligonucleotides provided) Those skilled in the art understand that internucleotidic linkages may exist as anions or cations at a given pH due to the presence of an acid or base moiety in the linkage. In some embodiments, the modified internucleotide linkages are s, s1, s2, s3, s4, s5, s6, s7, s8, s9, s10, s11, s12, s13, s14, s15, as described in WO 2017/210647. These are modified internucleotide linkages denoted by s16, s17 and s18.

In vitro: As used herein, the term “in vitro” refers to an artificial environment rather than within an organism (eg, animal, plant, and/or microorganism), e.g., a test tube or reaction vessel, a cell events that occur in cultivation, etc.

In vivo: As used herein, the term “in vivo” refers to events that occur within an organism (eg, animal, plant, and/or microorganism).

Linking Phosphorus: As defined herein, the phrase “linking phosphorus” is used to indicate that the particular phosphorus atom referred to is a phosphorus atom present in an internucleotidic linkage, which phosphorus atom is a phosphodiester nucleotide found in naturally occurring DNA and RNA. Corresponds to the phosphorus atom of the interlinkage. In some embodiments, the linking phosphorus atom is present in a modified internucleotide linkage, wherein each oxygen atom of the phosphodiester linkage is optionally and independently replaced with an organic or inorganic moiety. In some embodiments, the linking phosphorus atom is chiral (eg, as in a phosphorothioate internucleotidic linkage). In some embodiments, the linking phosphorus atom is an achiral atom (eg, as in natural phosphate linkages).

Modified nucleobase: The terms "modified nucleobase", "modified base" and the like refer to a chemical moiety that is chemically distinct from a nucleobase but is capable of performing one or more functions of a nucleobase. In some embodiments, a modified nucleobase is a nucleobase comprising a modification. In some embodiments, a modified nucleobase is capable of one or more functions of a nucleobase, eg, can form a moiety in a polymer capable of base pairing to a nucleic acid comprising at least a complementary base sequence. In some embodiments, the modified nucleobase is a substituted A, T, C, G, or U, or a substituted tautomer of A, T, C, G, or U. In some embodiments, a modified nucleobase in the context of an oligonucleotide refers to a nucleobase other than A, T, C, G, or U.

Modified nucleoside: The term "modified nucleoside" refers to a moiety derived from or chemically similar to a natural nucleoside, but containing chemical modifications that distinguish it from the natural nucleoside. Non-limiting examples of modified nucleosides include those containing modifications at bases and/or sugars. Non-limiting examples of modified nucleosides include those with 2' modifications at the sugar. Non-limiting examples of modified nucleosides also include free base nucleosides (lacking a nucleobase). In some embodiments, a modified nucleoside is capable of performing at least one function of a nucleoside, e.g., forming a moiety in a polymer capable of base pairing to a nucleic acid comprising at least a complementary base sequence. there is.

Modified nucleotide: The term "modified nucleotide" includes any chemical moiety that differs structurally from a natural nucleotide, but is capable of performing at least one function of a natural nucleotide. In some embodiments, modified nucleotides include modifications in sugars, bases, and/or internucleotidic linkages. In some embodiments, a modified nucleotide comprises a modified sugar, modified nucleobase, and/or modified internucleotide linkage. In some embodiments, a modified nucleotide is capable of at least one function of a nucleotide, eg forming a subunit in a polymer capable of base pairing to a nucleic acid comprising at least a complementary base sequence.

Modified sugar : The term "modified sugar" refers to a moiety that can replace a sugar. Modified sugars mimic the sugar's spatial arrangement, electronic properties or some other physiochemical properties. In some embodiments, as described herein, the modified sugar is substituted ribose or deoxyribose. In some embodiments, a modified sugar comprises a 2'-modification. Examples of useful 2'-modifications are widely used in the art and are described herein. In some embodiments, a 2'-modification is 2'-F. In some embodiments, the 2'-modification is 2'-OR and R is an optionally substituted C _1-10 aliphatic. In some embodiments, the 2'-modification is 2'-OMe. In some embodiments, a 2'-modification is a 2'-MOE. In some embodiments, the modified sugar is a bicyclic sugar (eg, a sugar used in LNA, BNA, etc.). In some embodiments, with respect to oligonucleotides, a modified sugar is a sugar other than ribose or deoxyribose normally found in natural RNA or DNA.

Nucleic acid: As used herein, the term “nucleic acid” includes any nucleotide and polymer thereof. As used herein, the term “polynucleotide” refers to a polymeric form of nucleotides of any length, either ribonucleotides (RNA) or deoxyribonucleotides (DNA) or combinations thereof. These terms refer to the primary structure of the molecule and thus include double-stranded and single-stranded DNA, and double-stranded and single-stranded RNA. These terms include, as equivalents, analogs of RNA or DNA including modified nucleotides and/or modified polynucleotides, such as, but not limited to, methylated, protected and/or capped nucleotides or polynucleotides. The term includes poly- or oligo-ribonucleotides (RNA) and poly- or oligo-deoxyribonucleotides (DNA); RNA or DNA derived from N-glycosides or C-glycosides of nucleobases and/or modified nucleobases; nucleic acids derived from sugars and/or modified sugars; and nucleic acids derived from phosphate bridges and/or modified internucleotide linkages. The term includes nucleic acids containing any combination of nucleobases, modified nucleobases, sugars, modified sugars, phosphate bridges or modified internucleotide linkages. Examples include, but are not limited to, nucleic acids containing ribose moieties, nucleic acids containing deoxy-ribose moieties, nucleic acids containing both ribose and deoxyribose moieties, nucleic acids containing ribose and modified ribose moieties. don't Unless otherwise specified, the prefix poly- refers to nucleic acids containing from 2 to about 10,000 nucleotide monomer units, and the prefix oligo- refers to nucleic acids containing from 2 to about 200 nucleotide monomer units.

Nucleobase: The term "nucleobase" refers to a portion of a nucleic acid that participates in hydrogen bonds that join one nucleic acid strand to another complementary strand in a sequence-specific manner. The most common naturally occurring nucleobases are adenine (A), guanine (G), uracil (U), cytosine (C), and thymine (T). In some embodiments, the naturally occurring nucleobase is a modified adenine, guanine, uracil, cytosine, or thymine. In some embodiments, the naturally occurring nucleobase is methylated adenine, guanine, uracil, cytosine, or thymine. In some embodiments, the nucleobases include heteroaryl rings in which the ring atoms are nitrogen, and when present in the nucleoside, the nitrogen is attached to the sugar moiety. In some embodiments, the nucleobase comprises a heterocyclic ring in which the ring atom is nitrogen, and when present in a nucleoside, the nitrogen is attached to the sugar moiety. In some embodiments, the nucleobases are "modified nucleobases" that are nucleobases other than adenine (A), guanine (G), uracil (U), cytosine (C), and thymine (T). In some embodiments, a modified nucleobase is a substituted A, T, C, G or U. In some embodiments, a modified nucleobase is a substituted tautomer of A, T, C, G, or U. In some embodiments, the modified nucleobases are methylated adenine, guanine, uracil, cytosine, or thymine. In some embodiments, a modified nucleobase mimics the spatial arrangement, electronic properties, or some other physicochemical property of the nucleobases, and retains the property of hydrogen bonding to bind one nucleic acid strand to another in a sequence-specific manner. . In some embodiments, the modified nucleobase is combined with all five naturally occurring bases (uracil, thymine, adenine, cytosine, or guanine) without substantially affecting the melting behavior, recognition by intracellular enzymes, or activity of the oligonucleotide duplex. can form pairs. As used herein, the term “nucleobase” also includes structural analogs used in place of naturally or naturally occurring nucleotides, such as modified nucleobases and nucleobase analogs. In some embodiments, the nucleobase is an optionally substituted A, T, C, G, or U, or an optionally substituted tautomer of A, T, C, G, or U. In some embodiments, "nucleobase" refers to a nucleobase unit in an oligonucleotide or nucleic acid (eg, A, T, C, G or U as in an oligonucleotide or nucleic acid).

Nucleoside: The term “nucleoside” refers to a moiety in which a nucleobase or modified nucleobase is covalently linked to a sugar or modified sugar. In some embodiments, the nucleoside is a natural nucleoside, eg, adenosine, deoxyadenosine, guanosine, deoxyguanosine, thymidine, uridine, cytidine, or deoxycytidine. In some embodiments, nucleosides are modified nucleosides, e.g., substituted natural nucleosides (adenosine, deoxyadenosine, guanosine, deoxyguanosine, thymidine, uridine, cytidine and deoxy selected from cytidine). In some embodiments, the nucleoside is a modified nucleoside, e.g., a natural selected from adenosine, deoxyadenosine, guanosine, deoxyguanosine, thymidine, uridine, cytidine, and deoxycytidine. It is a substituted tautomer of a nucleoside. In some embodiments, “nucleoside” refers to a nucleoside unit in an oligonucleotide or nucleic acid.

Nucleotide: As used herein, the term “nucleotide” refers to a monomeric unit of a polynucleotide consisting of a nucleobase, a sugar, and one or more internucleotidic linkages (eg, phosphate linkages in native DNA and RNA). The naturally occurring bases [guanine (G), adenine (A), cytosine (C), thymine (T), and uracil (U)] are derivatives of purines or pyrimidines, but naturally occurring and non-naturally occurring base analogs are also included. this should be understood Naturally occurring sugars are pentose (pentose sugar) deoxyribose (which forms DNA) or ribose (which forms RNA), but it should be understood that naturally occurring and naturally non-occurring sugar analogs are included. Nucleotides are linked via internucleotidic linkages to form nucleic acids or polynucleotides. Many internucleotide linkages (such as, but not limited to, phosphate, phosphorothioate, boranophosphate, etc.) are known in the art. Artificial nucleic acids include peptide nucleic acids (PNAs), phosphotriesters, phosphorothionates, H-phosphonates, phosphoramidates, boranophosphates, methylphosphonates, phosphonoacetates, thiophosphonoacetates, and natural nucleic acids. other variants of the phosphate backbone, such as those described herein. In some embodiments, natural nucleotides include naturally occurring bases, sugars, and internucleotidic linkages. As used herein, the term "nucleotide" also includes structural analogs used in place of natural or naturally occurring nucleotides, such as modified nucleotides and nucleotide analogs. In some embodiments, “nucleotide” refers to a unit of nucleotide in an oligonucleotide or nucleic acid.

Oligonucleotide: The term “oligonucleotide” refers to a polymer or oligomer of nucleotides and may include any combination of natural and non-natural nucleobases, sugars, and internucleotidic linkages.

Oligonucleotides can be single-stranded or double-stranded. A single-stranded oligonucleotide may have a double-stranded region (formed by two parts of the single-stranded oligonucleotide), and a double-stranded oligonucleotide comprising two oligonucleotide chains is such that, for example, the two oligonucleotide chains are complementary to each other. It may have single-stranded regions in regions that are not. Exemplary oligonucleotides include structural genes, genes containing control and termination regions, self-replicating systems such as viral or plasmid DNA, single-stranded and double-stranded RNAi agents and other RNA interference reagents (RNAi agents or iRNA agents), shRNA, antisense Oligonucleotides, ribozymes, microRNAs, microRNA mimics, supermirs, aptamers, antimirs, antagomirs, Ul adapters, triplex-forming oligonucleotides , G-quadruplex oligonucleotides, RNA activators, immunostimulatory oligonucleotides and decoy oligonucleotides.

Oligonucleotides of the present invention can be of various lengths. In certain embodiments, oligonucleotides may be in the range of about 2 to about 200 nucleosides in length. In various related embodiments, the single-stranded, double-stranded, or triple-stranded oligonucleotide is about 4 to about 10 nucleosides, about 10 to about 50 nucleosides, about 20 to about 50 nucleosides, about 15 to about 30 nucleosides, or about 20 to about 30 nucleosides in length. In some embodiments, oligonucleotides are from about 9 to about 39 nucleosides in length. In some embodiments, oligonucleotides are about 25 to about 70 nucleosides in length. In some embodiments, oligonucleotides are about 26 to about 70 nucleosides in length. In some embodiments, oligonucleotides are about 27 to about 70 nucleosides in length. In some embodiments, oligonucleotides are about 28 to about 70 nucleosides in length. In some embodiments, oligonucleotides are about 29 to about 70 nucleosides in length. In some embodiments, oligonucleotides are about 30 to about 70 nucleosides in length. In some embodiments, oligonucleotides are from about 31 to about 70 nucleosides in length. In some embodiments, oligonucleotides are about 32 to about 70 nucleosides in length. In some embodiments, oligonucleotides are about 25 to about 60 nucleosides in length. In some embodiments, oligonucleotides are about 25 to about 50 nucleosides in length. In some embodiments, oligonucleotides are about 25 to about 40 nucleosides in length. In some embodiments, oligonucleotides are about 30 to about 40 nucleosides in length. In some embodiments, the oligonucleotide is at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 , or 25 nucleosides in length. In some embodiments, an oligonucleotide is at least 4 nucleosides in length. In some embodiments, an oligonucleotide is at least 5 nucleosides in length. In some embodiments, an oligonucleotide is at least 6 nucleosides in length. In some embodiments, an oligonucleotide is at least 7 nucleosides in length. In some embodiments, an oligonucleotide is at least 8 nucleosides in length. In some embodiments, an oligonucleotide is at least 9 nucleosides in length. In some embodiments, an oligonucleotide is at least 10 nucleosides in length. In some embodiments, an oligonucleotide is at least 11 nucleosides in length. In some embodiments, an oligonucleotide is at least 12 nucleosides in length. In some embodiments, an oligonucleotide is at least 15 nucleosides in length. In some embodiments, an oligonucleotide is at least 15 nucleosides in length. In some embodiments, an oligonucleotide is at least 16 nucleosides in length. In some embodiments, an oligonucleotide is at least 17 nucleosides in length. In some embodiments, an oligonucleotide is at least 18 nucleosides in length. In some embodiments, an oligonucleotide is at least 19 nucleosides in length. In some embodiments, an oligonucleotide is at least 20 nucleosides in length. In some embodiments, an oligonucleotide is at least 25 nucleosides in length. In some embodiments, an oligonucleotide is at least 26 nucleosides in length. In some embodiments, an oligonucleotide is at least 27 nucleosides in length. In some embodiments, an oligonucleotide is at least 28 nucleosides in length. In some embodiments, an oligonucleotide is at least 29 nucleosides in length. In some embodiments, an oligonucleotide is at least 30 nucleosides in length. In some embodiments, an oligonucleotide is at least 31 nucleosides in length. In some embodiments, an oligonucleotide is at least 32 nucleosides in length. In some embodiments, an oligonucleotide is at least 33 nucleosides in length. In some embodiments, an oligonucleotide is at least 34 nucleosides in length. In some embodiments, an oligonucleotide is at least 35 nucleosides in length. In some embodiments, an oligonucleotide is at least 36 nucleosides in length. In some embodiments, an oligonucleotide is at least 37 nucleosides in length. In some embodiments, an oligonucleotide is at least 38 nucleosides in length. In some embodiments, an oligonucleotide is at least 39 nucleosides in length. In some embodiments, an oligonucleotide is at least 40 nucleosides in length. In some embodiments, an oligonucleotide is 25 nucleosides in length. In some embodiments, an oligonucleotide is 26 nucleosides in length. In some embodiments, an oligonucleotide is 27 nucleosides in length. In some embodiments, an oligonucleotide is 28 nucleosides in length. In some embodiments, an oligonucleotide is 29 nucleosides in length. In some embodiments, an oligonucleotide is 30 nucleosides in length. In some embodiments, an oligonucleotide is 31 nucleosides in length. In some embodiments, an oligonucleotide is 32 nucleosides in length. In some embodiments, an oligonucleotide is 33 nucleosides in length. In some embodiments, an oligonucleotide is 34 nucleosides in length. In some embodiments, an oligonucleotide is 35 nucleosides in length. In some embodiments, an oligonucleotide is 36 nucleosides in length. In some embodiments, an oligonucleotide is 37 nucleosides in length. In some embodiments, an oligonucleotide is 38 nucleosides in length. In some embodiments, an oligonucleotide is 39 nucleosides in length. In some embodiments, an oligonucleotide is 40 nucleosides in length. In some embodiments, each nucleoside counted by oligonucleotide length independently comprises a nucleobase comprising a ring having at least one nitrogen ring atom. In some embodiments, each nucleoside counted in oligonucleotide length is independently A, T, C, G, or U, or optionally substituted A, T, C, G, or U, or A, T, C, optionally substituted tautomers of G or U are included.

Oligonucleotide type: As used herein, the phrase “oligonucleotide type” refers to a specific base sequence, backbone linkage pattern (i.e., type of internucleotide linkages such as phosphate, phosphorothioate, phosphorothioate triester, etc.) pattern), the pattern of backbone chiral centers [i.e., the pattern of linkage phosphorus stereochemistry (Rp/Sp)], and the pattern of backbone phosphorus modifications. In some embodiments, oligonucleotides of a common designation “type” are structurally identical to each other.

One skilled in the art will recognize that the synthetic methods of the present invention provide a degree of control during the synthesis of oligonucleotide strands so that each nucleotide unit of an oligonucleotide strand has a specific stereochemistry at linking phosphorus and/or a specific modification and/or specific modification at linking phosphorus. It will be appreciated that it may be pre-designed and/or selected to have bases and/or specific sugars. In some embodiments, oligonucleotide strands are pre-designed and/or selected to have a specific combination of stereocenters at linkages. In some embodiments, an oligonucleotide strand is designed and/or determined to have a specific combination of modifications at linking phosphorus. In some embodiments, an oligonucleotide strand is designed and/or selected to have a specific combination of bases. In some embodiments, an oligonucleotide strand is designed and/or selected to have a specific combination of one or more of the above structural features. In some embodiments, the present invention provides a composition comprising or consisting of a plurality of oligonucleotide molecules (eg, a chirally controlled oligonucleotide composition). In some embodiments, all such molecules are of the same type (ie, structurally identical to each other). However, in some embodiments, a provided composition comprises a plurality of oligonucleotides of different types, typically in predetermined relative amounts.

Optionally Substituted: As described herein, the compounds of the invention, e.g., oligonucleotides, may include optionally substituted moieties and/or substituted moieties. In general, the term "substituted", whether preceded by the term "optionally" or not, means that one or more hydrogens of the indicated moiety are replaced with a suitable substituent. Unless otherwise indicated, an "optionally substituted" group may have a suitable substituent at each substitutable position of the group, and in any given structure more than one position may be substituted with more than one substituent selected from a particular group. In this case, the substituents may be the same or different at all positions. In some embodiments, an optionally substituted group is unsubstituted. Combinations of substituents envisioned by the present invention are preferably those that result in stable or chemically feasible compounds. As used herein, the term "stable" refers to a compound that is produced, detected, and, in certain embodiments, recovered when subjected to conditions that permit its use for one or more of the purposes disclosed herein. refers to compounds that are not substantially altered. Specific substituents are described below.

Suitable monovalent substituents on substitutable atoms, such as suitable carbon atoms, are independently halogen; -(CH ₂ ) _0-4 R°; -(CH2) _0-4 OR°; -O(CH ₂ ) _0-4 R°, -O-(CH ₂ ) _0-4 C(O)OR°; -(CH ₂ ) _0-4 CH(OR°)2; -(CH ₂ ) _0-4 Ph which may be substituted by R°; -(CH ₂ ) _0-4 O(CH ₂ ) _0-1 Ph which may be substituted by R°; -CH=CHPh which may be substituted by R°; -(CH ₂ ) _0-4 O(CH ₂ ) _0-1 -pyridyl which may be substituted by R°; -NO ₂ ; -CN; -N ₃ ; -(CH ₂ ) _0-4 N(R°) ₂ ; -(CH ₂ ) _0-4 N(R°)C(O)R°; -N(R°)C(S)R°; -(CH2) _0-4 N(R°)C(O)NR° ₂ ; -N(R°)C(S)NR° ₂ ; -(CH ₂ ) _0-4 N(R°)C(O)OR°; -N(R°)N(R°)C(O)R°; -N(R°)N(R°)C(O)NR° ₂ ; -N(R°)N(R°)C(O)OR°; -(CH2) _0-4 C(O)R°; -C(S)R°; -(CH ₂ ) _0-4 C(O)OR°; -(CH ₂ ) _0-4 C(O)SR°; -(CH ₂ ) _0-4 C(O)OSiR° ₃ ; -(CH ₂ ) _0-4 OC(O)R°; -OC(O)(CH ₂ ) _0-4 SR°, -SC(S)SR°; -(CH ₂ ) _0-4 SC(O)R°; -(CH ₂ ) _0-4 C(O)NR° ₂ ; -C(S)NR° ₂ ; -C(S)SR°; -(CH ₂ ) _0-4 OC(O)NR° ₂ ; -C(O)N(OR°)R°; -C(O)C(O)R°; -C(O)CH ₂ C(O)R°; -C(NOR°)R°; -(CH ₂ ) _0-4 SSR°; -(CH ₂ ) _0-4 S(O) ₂ R°; -(CH ₂ ) _0-4 S(O) ₂ OR°; -(CH ₂ ) _0-4 OS(O) ₂ R°; -S(O) ₂ NR° ₂ ; -(CH ₂ ) _0-4 S(O)R°; -N(R°)S(O) ₂ NR° ₂ ; -N(R°)S(O) ₂ R°; -N(OR°)R°; -C(NH)NR° ₂ ; -Si(R°) ₃ ; -OSi(R°) ₃ ; -B(R°) ₂ ; -OB(R°) ₂ ; -OB(OR°) ₂ ; -P(R°) ₂ ; -P(OR°) ₂ ; -P(R°)(OR°); -OP(R°) ₂ ; -OP(OR°) ₂ ; -OP(R°)(OR°); -P(O)(R°) ₂ ; -P(O)(OR°) ₂ ; -OP(O)(R°) ₂ ; -OP(O)(OR°) ₂ ; -OP(O)(OR°)(SR°); -SP(O)(R°) ₂ ; -SP(O)(OR°) ₂ ; -N(R°)P(O)(R°) ₂ ; -N(R°)P(O)(OR°) ₂ ; -P(R°) ₂ [B(R°) ₃ ]; -P(OR°) ₂ [B(R°) ₃ ]; -OP(R°) ₂ [B(R°) ₃ ]; -OP(OR°) ₂ [B(R°) ₃ ]; -(C _1-4 straight-chain or branched alkylene)ON(R°) ₂ ; or -(C _1-4 straight chain or branched alkylene)C(O)ON(R°) ₂ , each R° may be substituted as defined herein, independently hydrogen, C _1-20 C _1-20 heteroaliphatic having 1-5 heteroatoms independently selected from aliphatic, nitrogen, oxygen, sulfur, silicon, and phosphorus, -CH ₂ -(C _6-14 aryl), -O(CH ₂ ) _0-1 (C _6-14 aryl), -CH ₂ -(5-14 membered heteroaryl ring), 5-20 membered, monocyclic, bicyclic or polycyclic, saturated, partially unsaturated or aryl ring (nitrogen, oxygen, sulfur , silicon, and phosphorus; -20 membered, monocyclic, bicyclic, or polycyclic, saturated, partially unsaturated, or aryl ring having 0 to 5 heteroatoms independently selected from nitrogen, oxygen, sulfur, silicon, and phosphorus, which is May be substituted as defined.

Suitable monovalent substituents on R° (or a ring formed by taking together two independently present R° with their intervening atoms) are independently halogen, -(CH ₂ ) _0-2 R ^● , -(haloR ^● ), -(CH ₂ ) _0-2 OH, -(CH ₂ ) _0-2 OR ^● , -(CH ₂ ) _0-2 CH(OR ^● ) ₂ ; -O(haloR ^● ), -CN, -N ₃ , -(CH ₂ ) _0-2 C(O)R ^● , -(CH ₂ ) _0-2 C(O)OH, -(CH ₂ ) _{0 -2} C(O)OR ^● , -(CH ₂ ) _0-2 SR ^● , -(CH ₂ ) _0-2 SH, -(CH ₂ ) _0-2 NH ₂ , -(CH ₂ ) _0-2 NHR ^● , -(CH ₂ ) _0-2 NR ^● ₂ , -NO ₂ , -SiR ^● ₃ , -OSiR ^● ₃ , -C(O)SR ^● , -(C _1-4 straight chain or branched alkylene)C (O)OR ^● , or -SSR ^● , wherein each R ^● is unsubstituted or, when preceded by "halo", substituted with only one or more halogens, independently C _1-4 aliphatic, -CH ₂ Ph, -O(CH ₂ ) _0-1 Ph, and a 5-6 membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur. do. Suitable divalent substituents on the saturated carbon atom of R° include =O and =S.

For example, suitable divalent substituents on suitable carbon atoms are independently: =O, =S, =NNR* ₂ , =NNHC(O)R*, =NNHC(O)OR*, =NNHS(O) ₂ R*, =NR*, =NOR*, -O(C(R* ₂ )) _2-3 O-, or -S(C(R* ₂ )) _2-3 S- (where each independently R* present is hydrogen, C _1-6 aliphatic which may be substituted as defined below, and an unsubstituted 5-6 membered saturated, partially unsaturated, or aryl ring (independently selected from nitrogen, oxygen, and sulfur). having 0-4 heteroatoms). Suitable divalent substituents attached to adjacent substitutable carbons of an “optionally substituted” group include: -O(CR ^* ₂ ) _2-3 O-, wherein each independently present R ^* is hydrogen, C _1-6 aliphatic, which may be substituted as defined in, and unsubstituted 5-6 membered saturated, partially unsaturated, and aryl rings having 0-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur ) is selected from

Suitable substituents on an aliphatic group of R ^* are independently halogen, -R ^● , -(haloR ^● ), -OH, -OR ^● , -O(haloR ^● ), -CN, -C(O)OH, - C(O)OR ^● , -NH ₂ , -NHR ^● , -NR ^● ₂ , or -NO ₂ , each R ^● is unsubstituted or, when preceded by "halo", is substituted only with one or more halogens. and, independently, C _1-4 aliphatic, -CH ₂ Ph, -O(CH ₂ ) _0-1 Ph, or a 5-6 membered saturated, partially unsaturated, or aryl ring (independently selected from nitrogen, oxygen, and sulfur) has 0 to 4 heteroatoms).

In some embodiments, suitable substituents on substitutable nitrogens are independently -R ^† , -NR ^† ₂ , -C(O)R ^† , -C(O)OR ^† , -C(O)C(O)R ^† , -C(O)CH ₂ C(O)R ^† , -S(O) ₂ R ^† , -S(O) ₂ NR ^† ₂ , -C(S)NR ^† ₂ , -C(NH)NR ^† ₂ , or -N(R ^† )S(O) ₂ R ^† , where each R ^† is independently hydrogen, C _1-6 aliphatic which may be substituted as defined below, unsubstituted -OPh, or unsubstituted -OPh Ring A 5-6 membered saturated, partially unsaturated, or aryl ring (having 0-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur), or, notwithstanding the above definition, two independently occurring R ^† is an unsubstituted 3-12 membered saturated, partially unsaturated, or aryl monocyclic or bicyclic ring (0-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur), together with the atom(s) intervening therebetween. have) form.

Suitable substituents on the aliphatic groups of R† are independently halogen, -R ^● , -(haloR ^● ), -OH, -OR ^● , -O(haloR ^● ), -CN, -C(O)OH, - C(O)OR ^● , -NH ₂ , -NHR ^● , -NR ^● ₂ , or -NO ₂ , each R ^● is unsubstituted or, when preceded by "halo", is substituted only with one or more halogens. and, independently, C _1-4 aliphatic, -CH ₂ Ph, -O(CH ₂ ) _0-1 Ph, or a 5-6 membered saturated, partially unsaturated, or aryl ring (independently selected from nitrogen, oxygen, and sulfur) has 0 to 4 heteroatoms).

P-modification: As used herein, the term "P-modification" refers to any modification at the linking phosphorus other than a stereochemical modification. In some embodiments, the P-modification includes the addition, substitution, or removal of a pendant moiety covalently attached to the linking phosphorus.

Partially unsaturated: As used herein, the term “partially unsaturated” refers to a ring moiety that includes at least one double or triple bond. The term "partially unsaturated" is intended to include rings having multiple sites of unsaturation, but is not intended to include aryl or heteroaryl moieties as defined herein.

Pharmaceutical Composition: As used herein, the term “pharmaceutical composition” refers to an active agent formulated with one or more pharmaceutically acceptable carriers. In some embodiments, the active agent is present in a unit dose in an amount appropriate for administration in a treatment regimen that, when administered to a relevant population, exhibits a statistically significant probability of achieving a desired therapeutic effect. In some embodiments, pharmaceutical compositions may be formulated specifically for administration in solid or liquid form, including those modified for: e.g., drenches (aqueous or non-aqueous solutions or suspensions), tablets, for oral administration, such as boluses, powders, granules, pastes for application to the tongue, eg those targeted for buccal, sublingual, and systemic absorption; for parenteral administration, eg by subcutaneous, intramuscular, intravenous or epidural injection, eg as a sterile solution or suspension, or as a sustained release formulation; for topical application, for example as a cream, ointment or controlled release patch or spray applied to the skin, lungs or oral cavity; for vaginal or intrarectal administration, eg as a pessary, cream or foam; for sublingual administration; for ocular administration; for transdermal administration; or for nasal, pulmonary and other mucosal surface administration.

Pharmaceutically Acceptable: As used herein, the phrase "pharmaceutically acceptable" means that within the scope of sound medical judgment, commensurate with a reasonable benefit/risk ratio, excessive toxicity, irritation, allergic reaction, or other problem or Refers to compounds, materials, compositions and/or dosage forms suitable for use in contact with human and animal tissues without complications.

Pharmaceutically acceptable carrier: As used herein, the term “pharmaceutically acceptable carrier” refers to a pharmaceutically acceptable carrier involved in carrying or transporting a subject compound from one organ, or part of the body, to another organ or part of the body. A substance, composition or vehicle such as a liquid or solid filler, diluent, excipient, or solvent encapsulating material. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient. Some examples of materials that can serve as pharmaceutically acceptable carriers include: sugars such as lactose, glucose and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients such as cocoa butter and suppository wax; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols such as propylene glycol; polyols such as glycerin, sorbitol, mannitol and polyethylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffers such as magnesium hydroxide and aluminum hydroxide; alginic acid; heat-free water; isotonic brine; Ringer's solution; ethyl alcohol; pH buffer solution; polyesters, polycarbonates and/or polyanhydrides; and other non-toxic compatible substances used in pharmaceutical formulations.

Pharmaceutically acceptable salt: As used herein, the term "pharmaceutically acceptable salt" refers to a salt of a compound suitable for use in a pharmaceutical context, i.e., excessive toxicity, irritation, allergic reaction, etc. within the scope of sound medical judgment. salts which are suitable for use in contact with the tissues of humans and lower animals without them, and commensurate with a reasonable benefit/risk ratio. Pharmaceutically acceptable salts are well known in the art. For example, SM Berge et al. prepare pharmaceutically acceptable salts [J. Pharmaceutical Sciences, 66: 1-19 (1977)]. In some embodiments, pharmaceutically acceptable salts are formed with inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid and perchloric acid or organic acids such as acetic acid, maleic acid, tartaric acid, citric acid, succinic acid or malonic acid, or with ion exchange non-toxic acid addition salts, which are salts of amino groups formed using other methods used in the art, such as, but are not limited to. In some embodiments, the pharmaceutically acceptable salts are adipates, alginates, ascorbates, aspartates, benzenesulfonates, benzoates, bisulfates, borates, butyrates, camphorates, camphorsulfonates, citrates, Cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide , 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, maleate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, Oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate nates, p-toluenesulfonates, undecanoates, valerate salts, and the like. In some embodiments, provided compounds include one or more acidic groups, eg, oligonucleotides, and pharmaceutically acceptable salts are alkali, alkaline earth metal, or ammonium (eg, ammonium salts of N(R) _{3 )} . wherein each R is independently defined and described herein)) is a salt. Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium and the like. In some embodiments, the pharmaceutically acceptable salt is the sodium salt. In some embodiments, the pharmaceutically acceptable salt is a potassium salt. In some embodiments, the pharmaceutically acceptable salt is a calcium salt. In some embodiments, pharmaceutically acceptable salts are, where appropriate, opposite salts such as halides, hydroxides, carboxylates, sulfates, phosphates, nitrates, alkyls having 1 to 6 carbon atoms, sulfonates and aryl sulfonates. It includes non-toxic ammonium, quaternary ammonium and amine cations formed using ions. In some embodiments, a provided compound contains more than one acid group, for example an oligonucleotide may contain two or more acid groups (eg, in natural phosphate linkages and/or modified internucleotide linkages). In some embodiments, pharmaceutically acceptable salts, or salts generally, of such compounds contain two or more cations, which may be the same or different. In some embodiments, in a pharmaceutically acceptable salt (or salt in general), all ionizable hydrogens in the acidic group (e.g., about 11, 10, 9, 8, 7, 6, 5, 4, 3 or 2 or less; in some embodiments about 7 or less; in some embodiments about 6 or less; in some embodiments about 5 or less; in some embodiments about 4 or less; in some embodiments in an aqueous solution having a pKa of about 3 or less) replaced by cations. In some embodiments, each phosphorothioate and phosphate group is independently present in its salt form (e.g., -OP(O)(SNa)-O- and -OP(O)(ONa, respectively, when sodium salt). )-O-). In some embodiments, each phosphorothioate and phosphate internucleotidic linkage is independently present in its salt form (e.g., -OP(O)(SNa)-O- and -OP(O- when sodium salt, respectively). )(ONa)-O-). In some embodiments, the pharmaceutically acceptable salt is the sodium salt of an oligonucleotide. In some embodiments, a pharmaceutically acceptable salt is an oligonucleotide in which each acidic phosphate and modified phosphate group (e.g., phosphorothioate, phosphate, etc.) is present in salt form, if any, (all sodium salts). It is the sodium salt of a nucleotide.

Predetermined: Predetermined (or pre-determined) means deliberately selected or non-random or controlled, as opposed to, for example, occurring randomly, randomly, or without control. Those of ordinary skill in the art will, reading this specification, understand that the present disclosure provides techniques that make it possible to select and incorporate specific chemical and/or stereochemical characteristics into an oligonucleotide composition, and further oligonucleotides having such chemical and/or stereochemical characteristics. It will be appreciated that this allows for the controlled preparation of nucleotide compositions. Such provided compositions are "predetermined" as described herein. A composition that may contain a particular oligonucleotide is not a "predetermined" composition because it was produced by chance through a process that is not controlled to intentionally create a particular chemical and/or stereochemical characteristic. In some embodiments, a predetermined composition is a composition that can be intentionally reproducible (eg, through controlled repetition of a process). In some embodiments, a predetermined level of a plurality of oligonucleotides in a composition means that the absolute and/or relative amounts (ratio, percentage, etc.) of the plurality of oligonucleotides in the composition are controlled. In some embodiments, predetermined levels of a plurality of oligonucleotides in a composition are achieved through chirally controlled oligonucleotide preparation.

Protecting Group: As used herein, the term "protecting group" is well known in the art and is described in detail in Protecting Groups in Organic Synthesis, TW Greene and PGM Wuts, 3 ^rd edition, John Wiley & Sons, 1999. Including what has been described, the entirety of which is incorporated herein by reference. See Current Protocols in Nucleic Acid Chemistry, edited by Serge L. Beaucage et al. 06/2012] (Chapter 2 in its entirety incorporated herein by reference), specifically modified protecting groups for nucleoside and nucleotide chemistry are also included. Suitable amino protecting groups are methyl carbamate, ethyl carbamate, 9-fluorenylmethyl carbamate (Fmoc), 9-(2-sulfo)fluorenylmethyl carbamate, 9-(2,7-dibromo)fluoro Roenylmethyl carbamate, 2,7-di-t-butyl-[9-(10,10-dioxo-10,10,10,10-tetrahydrothioxantyl)]methyl carbamate (DBD-Tmoc ), 4-methoxyphenacyl carbamate (Phenoc), 2,2,2-trichloroethyl carbamate (Troc), 2-trimethylsilylethyl carbamate (Teoc), 2-phenylethyl carbamate (hZ), 1-(1-adamantyl)-1-methylethyl carbamate (Adpoc), 1,1-dimethyl-2-haloethyl carbamate, 1,1-dimethyl-2,2-dibromoethyl carbamate ( DB-t-BOC), 1,1-dimethyl-2,2,2-trichloroethyl carbamate (TCBOC), 1-methyl-1-(4-biphenylyl)ethyl carbamate (Bpoc), 1- (3,5-di-t-butylphenyl)-1-methylethyl carbamate (t-Bumeoc), 2-(2'- and 4'-pyridyl)ethyl carbamate (Pyoc), 2-(N, N-dicyclohexylcarboxamido)ethyl carbamate, t-butyl carbamate (BOC), 1-adamantyl carbamate (Adoc), vinyl carbamate (Voc), allyl carbamate (Alloc), 1-iso propyl allyl carbamate (Ipaoc), cinnamyl carbamate (Coc), 4-nitrocinnamyl carbamate (Noc), 8-quinolyl carbamate, N-hydroxypiperidinyl carbamate, alkyldithio carbamate, Benzyl Carbamate (Cbz), p-Methoxybenzyl Carbamate (Moz), p-Nitrobenzyl Carbamate, p-Bromobenzyl Carbamate, p-Chlorobenzyl Carbamate, 2,4-Dichlorobenzyl Carbamate, 4 -Methylsulfinylbenzyl carbamate (Msz), 9-antrylmethyl carbamate, diphenylmethyl carbamate, 2-methylthioethyl carbamate, 2-methylsulfonylethyl carbamate, 2-(p-toluenesulfonyl )ethyl carbamate, [2-(1,3-dithianyl)]methyl carbamate (Dmoc), 4-methylthiophenyl carbamate (Mtpc), 2,4-dimethylthiophenyl carbamate (Bmpc), 2- Phosphonioethyl carbamate (Peoc), 2-triphenylphosphonioisopropyl carbamate (Ppoc), 1,1-dimethyl-2-cyanoethyl carbamate, m-chloro-p-acyloxybenzyl carbamate, p-(dihydroxyboryl)benzyl carbamate, 5-benzisoxazolylmethyl carbamate, 2-(trifluoromethyl)-6-chromonylmethyl carbamate (Tcroc), m-nitrophenyl carbamate, 3, 5-dimethoxybenzyl carbamate, o-nitrobenzyl carbamate, 3,4-dimethoxy-6-nitrobenzyl carbamate, phenyl (o-nitrophenyl)methyl carbamate, phenothiazinyl-(10)-carbonyl Derivative, N'-p-toluenesulfonylaminocarbonyl derivative, N'-phenylaminothiocarbonyl derivative, t-amyl carbamate, S-benzyl thiocarbamate, p-cyanobenzyl carbamate, cyclobutyl carbamate , cyclohexyl carbamate, cyclopentyl carbamate, cyclopropylmethyl carbamate, p-decyloxybenzyl carbamate, 2,2-dimethoxycarbonylvinyl carbamate, o-(N,N-dimethylcarboxamido)benzyl Carbamate, 1,1-dimethyl-3-(N,N-dimethylcarboxamido)propyl carbamate, 1,1-dimethylpropynyl carbamate, di(2-pyridyl)methyl carbamate, 2-furanyl Methyl carbamate, 2-iodoethyl carbamate, isobornyl carbamate, isobutyl carbamate, isonicotinyl carbamate, p-(p'-methoxyphenylazo)benzyl carbamate, 1-methylcyclobutyl carbamate Mate, 1-methylcyclohexyl carbamate, 1-methyl-1-cyclopropylmethyl carbamate, 1-methyl-1-(3,5-dimethoxyphenyl)ethyl carbamate, 1-methyl-1-(p- Phenylazophenyl)ethyl carbamate, 1-methyl-1-phenylethyl carbamate, 1-methyl-1-(4-pyridyl)ethyl carbamate, phenyl carbamate, p-(phenylazo)benzyl carbamate, 2 ,4,6-tri-t-butylphenyl carbamate, 4-(trimethylammonium)benzyl carbamate, 2,4,6-trimethylbenzyl carbamate, formamide, acetamide, chloroacetamide, trichloroacetamide, Trifluoroacetamide, phenylacetamide, 3-phenylpropanamide, picolinamide, 3-pyridylcarboxamide, N-benzoylphenylalanyl derivative, benzamide, p-phenylbenzamide, o-nitrophenylacetamide , o-nitrophenoxyacetamide, acetoacetamide, (N'-dithiobenzyloxycarbonylamino)acetamide, 3-(p-hydroxyphenyl)propanamide, 3-(o-nitrophenyl)propanamide , 2-methyl-2- (o-nitrophenoxy) propanamide, 2-methyl-2- (o-phenylazophenoxy) propanamide, 4-chlorobutanamide, 3-methyl-3-nitrobutanamide , o-nitrocinamide, N-acetylmethionine derivatives, o-nitrobenzamide, o-(benzoyloxymethyl)benzamide, 4,5-diphenyl-3-oxazolin-2-one, N-phthalimide , N-dithiasuccinimide (Dts), N-2,3-diphenylmaleimide, N-2,5-dimethylpyrrole, N-1,1,4,4-tetramethyldisilylazacyclopentane addition Water (STABASE), 5-substituted 1,3-dimethyl-1,3,5-triazacyclohexan-2-one, 5-substituted 1,3-dibenzyl-1,3,5-triazacyclohexane- 2-one, 1-substituted 3,5-dinitro-4-pyridone, N-methylamine, N-allylamine, N-[2-(trimethylsilyl)ethoxy]methylamine (SEM), N-3 -Acetoxypropylamine, N-(1-isopropyl-4-nitro-2-oxo-3-pyrrolin-3-yl)amine, quaternary ammonium salt, N-benzylamine, N-di(4-methoxy Phenyl) methylamine, N-5-dibenzosuberylamine, N-triphenylmethylamine (Tr), N-[(4-methoxyphenyl)diphenylmethyl]amine (MMTr), N-9-phenylflu Orenylamine (PhF), N-2,7-dichloro-9-fluorenylmethyleneamine, N-ferrocenylmethylamino (Fcm), N-2-picolylamino N'-oxide, N-1,1 -Dimethylthiomethyleneamine, N-benzylideneamine, Np-methoxybenzylideneamine, N-diphenylmethyleneamine, N-[(2-pyridyl)mesityl]methyleneamine, N-(N',N' -Dimethylaminomethylene)amine, N,N'-isopropylidenediamine, Np-nitrobenzylideneamine, N-salicylideneamine, N-5-chlorosalicylideneamine, N-(5-chloro-2 -Hydroxyphenyl)phenylmethyleneamine, N-cyclohexylideneamine, N-(5,5-dimethyl-3-oxo-1-cyclohexenyl)amine, N-borane derivatives, N-diphenylboric acid derivatives , N-[phenyl(pentacarbonylchromium- or tungsten)carbonyl]amine, N-copper chelate, N-zinc chelate, N-nitroamine, N-nitrosoamine, amine N-oxide, diphenylphosphinamide (Dpp), dimethylthiophosphinamide (Mpt), diphenylthiophosphinamide (Ppt), dialkyl phosphoramidate, dibenzyl phosphoramidate, diphenyl phosphoramidate, benzenesulfenamide, o- Nitrobenzenesulfenamide (Nps), 2,4-dinitrobenzenesulfenamide, pentachlorobenzenesulfenamide, 2-nitro-4-methoxybenzenesulfenamide, triphenylmethylsulfenamide, 3-nitropyridinesulfenamide (Npys ), p-toluenesulfonamide (Ts), benzenesulfonamide, 2,3,6,-trimethyl-4-methoxybenzenesulfonamide (Mtr), 2,4,6-trimethoxybenzenesulfonamide (Mtb) , 2,6-dimethyl-4-methoxybenzenesulfonamide (Pme), 2,3,5,6-tetramethyl-4-methoxybenzenesulfonamide (Mte), 4-methoxybenzenesulfonamide (Mbs) , 2,4,6-trimethylbenzenesulfonamide (Mts), 2,6-dimethoxy-4-methylbenzenesulfonamide (iMds), 2,2,5,7,8-pentamethylchroman-6-sulfone Amide (Pmc), methanesulfonamide (Ms), β-trimethylsilylethanesulfonamide (SES), 9-anthracenesulfonamide, 4-(4',8'-dimethoxynaphthylmethyl)benzenesulfonamide (DNMBS) , benzylsulfonamide, trifluoromethylsulfonamide, and phenacylsulfonamide.

Suitably protected carboxylic acids further include, but are not limited to, silyl-, alkyl-, alkenyl-, aryl-, and arylalkyl-protected carboxylic acids. Examples of suitable silyl groups include trimethylsilyl, triethylsilyl, t-butyldimethylsilyl, t-butyldiphenylsilyl, triisopropylsilyl, and the like. Examples of suitable alkyl groups include methyl, benzyl, p-methoxybenzyl, 3,4-dimethoxybenzyl, trityl, t-butyl, tetrahydropyran-2-yl. Examples of suitable alkenyl groups include allyl. Examples of suitable aryl groups include optionally substituted phenyl, biphenyl, or naphthyl. Examples of suitable arylalkyl groups include optionally substituted benzyl (e.g., p-methoxybenzyl (MPM), 3,4-dimethoxybenzyl, O-nitrobenzyl, p-nitrobenzyl, p-halobenzyl, 2, 6-dichlorobenzyl, p-cyanobenzyl), and 2- and 4-picolyl.

Suitable hydroxyl protecting groups are methyl, methoxylmethyl (MOM), methylthiomethyl (MTM), t-butylthiomethyl, (phenyldimethylsilyl)methoxymethyl (SMOM), benzyloxymethyl (BOM), p-methoxy Benzyloxymethyl (PMBM), (4-methoxyphenoxy)methyl (p-AOM), Guairolmethyl (GUM), t-butoxymethyl, 4-pentenyloxymethyl (POM), siloxymethyl, 2-methoxyethoxymethyl (MEM), 2,2,2-trichloroethoxymethyl, bis(2-chloroethoxy)methyl, 2-(trimethylsilyl)ethoxymethyl (SEMOR), tetrahydropyranyl (THP), 3-bromotetrahydropyranyl, tetrahydrothiopyranyl, 1-methoxycyclohexyl, 4-methoxytetrahydropyranyl (MTHP), 4-methoxytetrahydrothiopyranyl, 4- Methoxytetrahydrothiopyranyl S,S-dioxide, 1-[(2-chloro-4-methyl)phenyl]-4-methoxypiperidin-4-yl (CTMP), 1,4-dioxane-2 -yl, tetrahydrofuranyl, tetrahydrothiofuranyl, 2,3,3a,4,5,6,7,7a-octahydro-7,8,8-trimethyl-4,7-methanobenzofuran- 2-yl, 1-ethoxyethyl, 1-(2-chloroethoxy)ethyl, 1-methyl-1-methoxyethyl, 1-methyl-1-benzyloxyethyl, 1-methyl-1-benzyloxy- 2-fluoroethyl, 2,2,2-trichloroethyl, 2-trimethylsilylethyl, 2-(phenylselenyl)ethyl, t-butyl, allyl, p-chlorophenyl, p-methoxyphenyl, 2, 4-dinitrophenyl, benzyl, p-methoxybenzyl, 3,4-dimethoxybenzyl, o-nitrobenzyl, p-nitrobenzyl, p-halobenzyl, 2,6-dichlorobenzyl, p-cyanobenzyl, p-phenylbenzyl, 2-picolyl, 4-picolyl, 3-methyl-2-picolyl N-oxido, diphenylmethyl, p,p'-dinitrobenzhydryl, 5-dibenzosuberyl, Triphenylmethyl, α-naphthyldiphenylmethyl, p-methoxyphenyldiphenylmethyl, di(p-methoxyphenyl)phenylmethyl, tri(p-methoxyphenyl)methyl, 4-(4'-bromophena Siloxyphenyl) diphenylmethyl, 4,4', 4''-tris (4,5-dichlorophthalimidophenyl) methyl, 4,4', 4''-tris (levulinoyloxyphenyl) methyl, 4 ,4',4''-tris(benzoyloxyphenyl)methyl, 3-(imidazol-1-yl)bis(4',4''-dimethoxyphenyl)methyl, 1,1-bis(4-methyl Toxyphenyl) -1'-pyrenylmethyl, 9-anthryl, 9- (9-phenyl) xanthenyl, 9- (9-phenyl-10-oxo) anthryl, 1,3-benzodithiolane-2- 1, benzisothiazolyl S,S-dioxido, trimethylsilyl (TMS), triethylsilyl (TES), triisopropylsilyl (TIPS), dimethylisopropylsilyl (IPDMS), diethylisopropylsilyl (DEIPS) ), dimethylthexylsilyl, t-butyldimethylsilyl (TBDMS), t-butyldiphenylsilyl (TBDPS), tribenzylsilyl, tri-p-xylylsilyl, triphenylsilyl, diphenylmethylsilyl (DPMS), t -Butylmethoxyphenylsilyl (TBMPS), formate, benzoylformate, acetate, chloroacetate, dichloroacetate, trichloroacetate, trifluoroacetate, methoxyacetate, triphenylmethoxyacetate, phenoxyacetate, p- Chlorophenoxyacetate, 3-phenylpropionate, 4-oxopentanoate (levulinate), 4,4-(ethylenedithio)pentanoate (levulinoyldithioacetal), pivaloate, a Damantoate, crotonate, 4-methoxycrotonate, benzoate, p-phenylbenzoate, 2,4,6-trimethylbenzoate (mesitoate), alkyl methyl carbonate, 9-fluorenylmethyl carbonate (Fmoc), alkyl ethyl carbonate, alkyl 2,2,2-trichloroethyl carbonate (Troc), 2-(trimethylsilyl)ethyl carbonate (TMSEC), 2-(phenylsulfonyl)ethyl carbonate (Psec), 2 -(triphenylphosphonio) ethyl carbonate (Peoc), alkyl isobutyl carbonate, alkyl vinyl carbonate, alkyl allyl carbonate, alkyl p-nitrophenyl carbonate, alkyl benzyl carbonate, alkyl p-methoxybenzyl carbonate, alkyl 3,4- dimethoxybenzyl carbonate, alkyl o-nitrobenzyl carbonate, alkyl p-nitrobenzyl carbonate, alkyl S-benzyl thiocarbonate, 4-ethoxy-1-naphthyl carbonate, methyl dithiocarbonate, 2-iodobenzoate, 4-azidobutyrate, 4-nitro-4-methylpentanoate, o-(dibromomethyl)benzoate, 2-formylbenzenesulfonate, 2-(methylthiomethoxy)ethyl, 4-(methyl Thiomethoxy)butyrate, 2-(methylthiomethoxymethyl)benzoate, 2,6-dichloro-4-methylphenoxyacetate, 2,6-dichloro-4-(1,1,3,3-tetramethyl Butyl)phenoxyacetate, 2,4-bis(1,1-dimethylpropyl)phenoxyacetate, chlorodiphenylacetate, isobutyrate, monosuccinoate, (E)-2-methyl-2-butenoate, o-(methoxycarbonyl)benzoate, α-naphthoate, nitrate, alkyl N,N,N',N'-tetramethylphosphorodiamidate, alkyl N-phenylcarbamate, borate, dimethylphos pinothioil, alkyl 2,4-dinitrophenylsulfonates, sulfates, methanesulfonates (mesylates), benzylsulfonates, and tosylates (Ts). For 1,2- or 1,3-diol protection, the protecting group is methylene acetal, ethylidene acetal, 1-t-butylethylidene ketal, 1-phenylethylidene ketal, (4-methoxyphenyl)ethylidene Acetal, 2,2,2-trichloroethylidene acetal, acetonide, cyclopentylidene ketal, cyclohexylidene ketal, cycloheptylidene ketal, benzylidene acetal, p-methoxybenzylidene acetal, 2, 4-dimethoxybenzylidene ketal, 3,4-dimethoxybenzylidene acetal, 2-nitrobenzylidene acetal, methoxymethylene acetal, ethoxymethylene acetal, dimethoxymethylene ortho ester, 1-methoxyethylidene ortho Esters, 1-ethoxyethylidine ortho esters, 1,2-dimethoxyethylidene ortho esters, α-methoxybenzylidene ortho esters, 1-(N,N-dimethylamino)ethylidene derivatives, α-( N,N'-dimethylamino)benzylidene derivative, 2-oxacyclopentylidene ortho ester, di-t-butylsilylene group (DTBS), 1,3-(1,1,3,3-tetraisopropyl disiloxanilidene) derivatives (TIPDS), tetra-t-butoxydisiloxane-1,3-diylidene derivatives (TBDS), cyclic carbonates, cyclic boronates, ethyl boronates, and phenyl boronates.

In some embodiments, the hydroxyl protecting group is acetyl, t-butyl, t-butoxymethyl, methoxymethyl, tetrahydropyranyl, 1-ethoxyethyl, 1-(2-chloroethoxy)ethyl, 2-trimethylsilyl Ethyl, p-chlorophenyl, 2,4-dinitrophenyl, benzyl, benzoyl, p-phenylbenzoyl, 2,6-dichlorobenzyl, diphenylmethyl, p-nitrobenzyl, triphenylmethyl (trityl), 4, 4'-dimethoxytrityl, trimethylsilyl, triethylsilyl, t-butyldimethylsilyl, t-butyldiphenylsilyl, triphenylsilyl, triisopropylsilyl, benzoyl formate, chloroacetyl, trichloroacetyl, trifluoro Roacetyl, pivaloyl, 9-fluorenylmethyl carbonate, mesylate, tosylate, triflate, trityl, monomethoxytrityl (MMTr), 4,4'-dimethoxytrityl (DMTr) and 4 ,4',4''-trimethoxytrityl (TMTr), 2-cyanoethyl (CE or Cne), 2-(trimethylsilyl)ethyl (TSE), 2-(2-nitrophenyl)ethyl, 2 -(4-cyanophenyl)ethyl 2-(4-nitrophenyl)ethyl (NPE), 2-(4-nitrophenylsulfonyl)ethyl, 3,5-dichlorophenyl, 2,4-dimethylphenyl, 2- Nitrophenyl, 4-nitrophenyl, 2,4,6-trimethylphenyl, 2-(2-nitrophenyl)ethyl, butylthiocarbonyl, 4,4',4''-tris(benzoyloxy)trityl, di phenylcarbamoyl, levulinyl, 2-(dibromomethyl)benzoyl (Dbmb), 2-(isopropylthiomethoxymethyl)benzoyl (Ptmt), 9-phenylxanthen-9-yl (pixyl) or 9- (p-methoxyphenyl)xanthin-9-yl (MOX). In some embodiments, each hydroxyl protecting group is independently selected from acetyl, benzyl, t-butyldimethylsilyl, t-butyldiphenylsilyl, and 4,4'-dimethoxytrityl. In some embodiments, the hydroxyl protecting group is selected from the group consisting of trityl, monomethoxytrityl, and 4,4'-dimethoxytrityl groups. In some embodiments, a phosphorus linkage protecting group is a group that is attached to a phosphorus linkage (eg, an internucleotide linkage) throughout oligonucleotide synthesis. In some embodiments, the protecting group is attached to the sulfur atom of the phosphorothioate group. In some embodiments, the protecting group is attached to an oxygen atom of an internucleotidic phosphorothioate linkage. In some embodiments, the protecting group is attached to an oxygen atom of an internucleotidic phosphate linkage. In some embodiments, the protecting group is 2-cyanoethyl (CE or Cne), 2-trimethylsilylethyl, 2-nitroethyl, 2-sulfonylethyl, methyl, benzyl, o-nitrobenzyl, 2-(p-nitro Phenyl)ethyl (NPE or Npe), 2-phenylethyl, 3-(N-tert-butylcarboxamido)-1-propyl, 4-oxopentyl, 4-methylthio-1-butyl, 2-cyano- 1,1-dimethylethyl, 4-N-methylaminobutyl, 3-(2-pyridyl)-1-propyl, 2-[N-methyl-N-(2-pyridyl)]aminoethyl, 2-( N-formyl,N-methyl)aminoethyl, or 4-[N-methyl-N-(2,2,2-trifluoroacetyl)amino]butyl.

Subject: As used herein, the term “subject” or “test subject” refers to a compound (e.g., an oligonucleotide) or composition of the present invention, e.g., for experimental, diagnostic, prophylactic and/or therapeutic purposes. refers to any organism administered according to Typical subjects include animals (eg, mammals such as mice, rats, rabbits, non-human primates, and humans; insects; insects, etc.) and plants. In some embodiments, the subject is a human. In some embodiments, the subject may be suffering from and/or predisposed to a disease, disorder and/or condition.

Substantially: As used herein, the term “substantially” refers to the qualitative state of exhibiting all or nearly all extent or extent of a characteristic or characteristic of interest. A base sequence that is substantially identical or complementary to the second sequence is not completely identical or complementary to the second sequence, but is substantially identical or nearly identical or complementary to the second sequence. In some embodiments, an oligonucleotide having a sequence substantially complementary to another oligonucleotide or nucleic acid forms a duplex with the oligonucleotide or nucleic acid in a manner similar to an oligonucleotide having a fully complementary sequence. Moreover, those skilled in the biological and/or chemical arts will understand that biological and chemical phenomena seldom complete and/or achieve perfection or achieve or avoid absolute results. Accordingly, the term “substantially” is used herein to capture the potential lack of integrity inherent in many biological and/or chemical phenomena.

Sugar: The term "sugar" refers to a monosaccharide or polysaccharide in closed and/or open form. In some embodiments, the sugar is a monosaccharide. In some embodiments, the sugar is a polysaccharide. Sugars include, but are not limited to, ribose, deoxyribose, pentofuranose, pentopyranose and hexopyranose moieties. As used herein, the term “sugar” also includes structural analogs used in place of conventional sugar molecules, such as glycols (polymers of which form the backbone of nucleic acid analogs), glycol nucleic acids (“GNAs”), and the like. As used herein, the term “sugar” also includes structural analogs used in place of naturally or naturally occurring nucleotides, such as modified sugars and nucleotide sugars. In some embodiments, the sugar is an RNA or DNA sugar (ribose or deoxyribose). In some embodiments, the sugar is a modified ribose or deoxyribose sugar, e.g., 2'-modified, 5'-modified, and the like. As described herein, in some embodiments, when used in oligonucleotides and/or nucleic acids, modified sugars may provide one or more desired properties, activities, etc. In some embodiments, the sugar is optionally substituted ribose or deoxyribose. In some embodiments, "sugar" refers to a sugar unit in an oligonucleotide or nucleic acid.

Susceptibility: An individual who is “susceptible to” a disease, disorder, and/or condition is an individual at higher risk of developing that disease, disorder, and/or condition than members of the general public. In some embodiments, an individual predisposed to a disease, disorder and/or condition is predisposed to having that disease, disorder and/or condition. In some embodiments, an individual predisposed to a disease, disorder and/or condition may not have been diagnosed with the disease, disorder and/or condition. In some embodiments, an individual predisposed to a disease, disorder and/or condition may exhibit symptoms of that disease, disorder and/or condition. In some embodiments, an individual predisposed to a disease, disorder and/or condition may not display symptoms of the disease, disorder and/or condition. In some embodiments, an individual predisposed to a disease, disorder and/or condition will develop that disease, disorder and/or condition. In some embodiments, an individual predisposed to a disease, disorder and/or condition will not develop that disease, disorder and/or condition.

Therapeutic agent: As used herein, the term “therapeutic agent” generally refers to any agent that, when administered to a subject, causes a desired effect (eg, a desired biological, clinical or pharmacological effect). In some embodiments, an agent is considered therapeutic if it exhibits a statistically significant effect across an appropriate population. In some embodiments, a suitable population is a population of subjects suffering from and/or susceptible to a disease, disorder or condition. In some embodiments, a suitable population is a population of model organisms. In some embodiments, a suitable population may be defined by one or more criteria, such as age group, gender, genetic background, pre-existing clinical condition, prior exposure to therapy. In some embodiments, a therapeutic agent, when administered to a subject in an effective amount, relieves, ameliorates, alleviates and/or inhibits one or more symptoms or characteristics of a disease, disorder and/or condition in a subject; and/or prevents, delays its onset, reduces its severity, and/or reduces its incidence. In some embodiments, a “therapeutic agent” is a drug that has been approved or requires approval by a government agency before it can be marketed for human administration. In some embodiments, a “therapeutic agent” is an agent that requires a medical prescription for administration to humans. In some embodiments, a therapeutic agent is a provided compound, eg, a provided oligonucleotide.

Therapeutically effective amount: As used herein, the term “therapeutically effective amount” refers to an amount of a substance (eg, a therapeutic agent, composition, and/or formulation) that elicits a desired biological response when administered as part of a treatment regimen. do. In some embodiments, a therapeutically effective amount of a substance, when administered to a subject suffering from or susceptible to a disease, disorder and/or condition, is effective in treating, diagnosing, preventing and/or delaying the onset of that disease, disorder and/or condition. It is sufficient. As will be appreciated by those skilled in the art, the effective amount of an agent may vary depending on such factors as the desired biological endpoint, the agent being delivered, the target cell or tissue, and the like. An effective amount of a compound in a formulation, for example for treating a disease, disorder and/or condition, relieves, ameliorates, alleviates, suppresses, prevents, delays the onset of, or reduces the severity of one or more symptoms or characteristics of the disease, disorder and/or condition. and/or an amount that reduces occurrence. In some embodiments, the therapeutically effective amount is administered as a single dose; In some embodiments, multiple unit doses are required to deliver a therapeutically effective amount.

Treat: As used herein, the terms “treat,” “treatment,” or “treating” refer to partial or complete relief, amelioration, alleviation, inhibition of one or more symptoms or characteristics of a disease, disorder, and/or condition. , refers to any method used to prevent, delay onset, reduce severity, and/or reduce incidence. Therapeutic agents can be administered to subjects who do not exhibit symptoms of the disease, disorder, and/or condition. In some embodiments, a therapeutic agent may be administered to a subject exhibiting only the early signs of a disease, disorder, and/or condition, for example to reduce the risk of developing a condition associated with the disease, disorder, and/or condition.

Unsaturated: As used herein, the term “unsaturated” means that a moiety has one or more units of unsaturation.

Wild type: As used herein, the term “wild type” refers to an entity that has structures and/or activities found in nature in its “normal” state or context (as opposed to mutated, diseased, altered, etc.) has a meaning understood in the art. One skilled in the art will understand that wild-type genes and polypeptides often exist in a number of different forms (eg, alleles).

As will be appreciated by those skilled in the art, the methods and compositions described herein with respect to provided compounds (eg oligonucleotides) also apply generally to pharmaceutically acceptable salts of such compounds.

Description of Specific Embodiments

Oligonucleotides are useful in a variety of therapeutic, diagnostic, and research applications. The use of naturally occurring nucleic acids is limited due to, for example, sensitivity to endo- and exo-nucleases. Accordingly, various synthetic counterparts have been developed to avoid these drawbacks and/or to further improve various properties and activities. Such counterparts include synthetic oligonucleotides that contain chemical modifications, e.g., base modifications, sugar modifications, backbone modifications, etc., in particular those that make these molecules less susceptible to degradation and improve other properties and/or activities. do.

From a structural point of view, modifications to the internucleotidic linkages can introduce chirality, and certain properties and activities can be influenced by the arrangement of the linking phosphorus atoms of the oligonucleotide. For example, binding affinity, sequence specific binding to complementary RNA, stability to nucleases, cleavage, delivery of active nucleic acids, pharmacokinetics, etc. may be influenced, among other things, by the chirality of the backbone linking phosphorus atoms. there is.

In particular, the present invention relates to various structural elements in oligonucleotides, such as sugar modifications and their patterns, nucleobase modifications and their patterns, linkages between modified nucleotides and their patterns, linkage phosphorus stereochemistry and their patterns, additional chemical moieties ( moieties that are not usually in the oligonucleotide chain) and their patterns, etc. are used. Taking advantage of the ability to fully control the structural elements of oligonucleotides, the present invention provides oligonucleotides with improved and/or new properties and/or activities for a variety of applications, eg, as therapeutics, probes, and the like. For example, as shown herein, provided oligonucleotides and compositions thereof are particularly powerful at editing a target adenosine in a target nucleic acid to correct a G to A mutation by converting A to I in some embodiments.

In some embodiments, an oligonucleotide is 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, typically 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60 or more contiguous bases identical to, or completely or substantially complementary to, a sequence. In some embodiments, the nucleic acid is a target nucleic acid comprising one or more target adenosines. In some embodiments, a target nucleic acid comprises no more than one target adenosine. In some embodiments, an oligonucleotide is capable of hybridizing to a target nucleic acid. In some embodiments, such hybridization facilitates modification of A (eg, conversion of A to I) in a nucleic acid or product thereof, eg, by ADAR1, ADAR2, etc.

In some embodiments, the present invention provides oligonucleotides, which oligonucleotides contain about 10-40, about 15-40, about 20-40, or at least about 10-40 of the oligonucleotides or nucleic acids disclosed herein (e.g., Tables). 27, at least 28, at least 29, at least 30, at least 31, at least 32, at least 33, at least 34 contiguous bases or a base sequence comprising them, or a target RNA sequence disclosed herein Has a sequence complementary to a gene, transcript, etc. Each T may optionally and independently be replaced by U and vice versa. In some embodiments, the present invention provides an oligonucleotide or oligonucleotide composition as disclosed herein (eg, in a table).

In some embodiments, the oligonucleotide is a single-stranded oligonucleotide for site-directed editing of a nucleoside (eg, targeting adenosine) within a target nucleic acid (eg, RNA).

As described herein, an oligonucleotide may include one or more modified internucleotidic linkages (non-natural phosphate linkages). In some embodiments, a modified internucleotide linkage is a chiral internucleotide linkage in which the linkage phosphorus is chiral. In some embodiments, the modified internucleotidic linkage is a phosphorothioate internucleotidic linkage. In some embodiments, the oligonucleotide comprises one or more negatively charged internucleotidic linkages (eg, phosphorothioate internucleotidic linkages, natural phosphate linkages, etc.). In some embodiments, an oligonucleotide comprises one or more internucleotidic linkages that are not negatively charged. In some embodiments, an oligonucleotide comprises one or more neutral internucleotidic linkages.

In some embodiments, oligonucleotides are chirally controlled. In some embodiments, an oligonucleotide is chirally pure (or "stereopure", "stereochemically pure"), and the oligonucleotide is in a single stereoisomeric form (in many cases, an oligonucleotide, e.g., a linking phosphorus, a sugar carbon Since multiple chiral centers can exist in the back, it exists as a single diastereomer (diastereoisomeric or diastereomeric form). As will be appreciated by those skilled in the art, chirally pure oligonucleotides are separated from other stereoisomeric forms (to the extent that some impurities may be present since chemical and biological processes, selectivity and/or purification, etc., are rarely completely complete). In chirally pure oligonucleotides, each chiral center is independently defined for configuration (for chirally pure oligonucleotides, each internucleotide linkage is independently stereodefined or chirally controlled). Racemic (or "stereorandom", "non-chiral controlled") oligonucleotides (e.g., conventional Derived from conventional phosphoramidite oligonucleotide synthesis (generating stereorandom phosphorothioate internucleotidic linkages) with no stereochemical control during the coupling step combined with sulfurization, various stereoisomers (oligonucleotides contain multiple Since there is a chiral center, it is usually a diastereoisomer (or diastereomer); for example, those derived from conventional oligonucleotide preparations using reagents that contain no chiral elements other than those at the nucleoside and linking phosphorus). refers to a random mixture. For example, for A*A*A, where * is a phosphorothioate internucleotidic linkage (which includes a chiral linking phosphorus), the racemic oligonucleotide preparation contains the following four diastereomers: [2 ² = 4, considering the two chiral linkages above, each of which can exist in one of two configurations ( S p or R p)]: A *SA *SA, A *SA *RA, A * RA *SA, and A *RA *RA, where *S represents an S p phosphorothioate internucleotidic linkage and *R represents a R p phosphorothioate internucleotidic linkage. For chirally pure oligonucleotides (e.g., A *SA *SA), they exist in a single stereoisomeric form and can form other stereoisomers (e.g., diastereomers A *SA *RA, A *RA *SA, and A *RA *RA).

In some embodiments, an oligonucleotide comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more sterically random internucleotide linkages (e.g., derived from conventional non-chiral controlled oligonucleotide synthesis). , a mixture of R p and Sp linked phosphorus in internucleotidic linkages). In some embodiments, the oligonucleotide is one or more (e.g., 1-60, 1-50, 1-40, 1-30, 1-25, 1-20, 1, 2, 3, 4, 5, 6 , 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56 , 57, 58, 59, 60 or more) of chirally controlled internucleotide linkages (e.g., R p or Sp linkages in an internucleotide derived from chirally controlled oligonucleotide synthesis). In some embodiments, the internucleotidic linkages are phosphorothioate internucleotidic linkages. In some embodiments, the internucleotidic linkages are stereorandom phosphorothioate internucleotidic linkages. In some embodiments, the internucleotidic linkage is a chirally controlled phosphorothioate internucleotidic linkage.

In particular, the present invention provides techniques for preparing chirally controlled (in some embodiments, stereochemically pure) oligonucleotides. In some embodiments, oligonucleotides are stereochemically pure. In some embodiments, an oligonucleotide of the invention is stereochemically about 5%-100%, 10%-100%, 20%-100%, 30%-100%, 40%-100%, 50%-100% , 60% to 100%, 70% to 100%, 80 to 100%, 90 to 100%, 95 to 100%, 50% to 90%, or about 5%, 10%, 15%, 20%, 25% , 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%, or at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85% , 90%, 95%, or 99% pure.

In some embodiments, the present invention provides various oligonucleotide compositions. In some embodiments, the oligonucleotide composition is sterically random or not chirally controlled. In some embodiments, oligonucleotides of provided compositions are free of chirally controlled internucleotide linkages. In some embodiments, the internucleotide linkages of the oligonucleotides in the composition include one or more chirally controlled internucleotide linkages (eg, a chirally controlled oligonucleotide composition).

In some embodiments, an oligonucleotide composition comprises a plurality of oligonucleotides that share a common base sequence, wherein at least one internucleotide linkage in the oligonucleotide is chirally controlled and at least one internucleotide linkage is sterically random (non-chirally controlled ). In some embodiments, an oligonucleotide composition comprises a plurality of oligonucleotides that share a common base sequence, and each internucleotidic linkage comprising a chiral linkage phosphorus in the oligonucleotide is independently a chiral controlled internucleotidic linkage. In some embodiments, the plurality of oligonucleotides share the same base sequence and the same base and sugar modifications. In some embodiments, the plurality of oligonucleotides share the same base sequence and the same base, sugar, and internucleotide linkage modifications. In some embodiments, an oligonucleotide composition comprises oligonucleotides of identical composition, wherein one or more internucleotidic linkages are chirally controlled and one or more internucleotidic linkages are sterically random (not chirally controlled). In some embodiments, an oligonucleotide composition comprises oligonucleotides of identical composition, and each internucleotidic linkage comprising a chiral linkage phosphorus is independently a chiral controlled internucleotidic linkage. In some embodiments, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90% of all oligonucleotides, or all oligonucleotides of a consensus sequence. %, or 95% is a plurality of oligonucleotides.

In some embodiments, the present invention provides techniques for making, evaluating, and/or utilizing provided oligonucleotides and compositions thereof.

As used herein, in some embodiments, "one or more" is 1-200, 1-150, 1-100, 1-90, 1-80, 1-70, 1-60, 1-50, 1-40, 1-30, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46 , 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60. In some embodiments, “one or more” is 1. In some embodiments, “one or more” is two. In some embodiments, “one or more” is three. In some embodiments, “one or more” is four. In some embodiments, “one or more” is five. In some embodiments, “one or more” is six. In some embodiments, “one or more” is seven. In some embodiments, “one or more” is eight. In some embodiments, “one or more” is nine. In some embodiments, “one or more” is ten. In some embodiments, “one or more” is at least one. In some embodiments, “one or more” is at least two. In some embodiments, “one or more” is at least three. In some embodiments, “one or more” is at least four. In some embodiments, “one or more” is at least five. In some embodiments, “one or more” is at least six. In some embodiments, “one or more” is at least seven. In some embodiments, “one or more” is at least eight. In some embodiments, “one or more” is at least nine. In some embodiments, “one or more” is at least 10.

As used herein, in some embodiments, "at least one" is one or more.

Various implementations for variables such as R, R ^L , L, etc. are described as examples. Embodiments described for a variable, eg R, are generally applicable to any variable that can be such a variable (eg, R′, R″, R ^L , R ^L1 , etc.).

oligonucleotide

In particular, the present invention relates to oligonucleotides of various designs that may include various nucleobases and patterns thereof, sugars and patterns thereof, internucleotide linkages and patterns thereof, and/or additional chemical moieties and patterns thereof, as described herein. provide nucleotides. In some embodiments, provided oligonucleotides are capable of directing A to I editing in a target nucleic acid. In some embodiments, the oligonucleotides of the invention are single-stranded oligonucleotides capable of site-directed editing (A to I conversion) of adenosine in a target RNA sequence.

In some embodiments, oligonucleotides have suitable length and sequence complementarity to specifically hybridize with a target nucleic acid. In some embodiments, the oligonucleotide is long enough and sufficiently complementary to the target nucleic acid to distinguish it from other nucleic acids to reduce off-target effects. In some embodiments, oligonucleotides are short enough to facilitate delivery and reduce manufacturing complexity and/or cost of maintaining desired properties and activities (eg, editing of adenosine).

In some embodiments, the oligonucleotide is about 10-200 (e.g., about 10-20, 10-30, 10-40, 10-50, 10-60, 10-70, 10-80, 10-90 , 10~100, 10~120, 10~150, 20~30, 20~40, 20~50, 20~60, 20~70, 20~80, 20~90, 20~100, 20~120, 20 ~150, 20~200, 25~30, 25~40, 25~50, 25~60, 25~70, 25~80, 25~90, 25~100, 25~120, 25~150, 25~200 , 30~40, 30~50, 30~60, 30~70, 30~80, 30~90, 30~100, 30~120, 30~150, 30~200, 10, 20, 25, 26, 27 , 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 45, 50, 60, etc.) nucleobases in length. In some embodiments, the base sequence of an oligonucleotide is about 10-60 nucleobases in length. In some embodiments, the base sequence is about 15-50 nucleobases in length. In some embodiments, the base sequence is about 15 to about 35 nucleobases in length. In some embodiments, the base sequence is about 25 to about 34 nucleobases in length. In some embodiments, the base sequence is about 26 to about 35 nucleobases in length. In some embodiments, the base sequence is about 27 to about 32 nucleobases in length. In some embodiments, the base sequence is about 29 to about 35 nucleobases in length. In some embodiments, the base sequence is about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 , 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55 , 56, 57, 58, 59, or 60 nucleobases in length. In some other embodiments, the base sequence is 35 or more nucleobases in length. In some other embodiments, the base sequence is at least 34 nucleobases in length. In some other embodiments, the base sequence is 33 or more nucleobases in length. In some other embodiments, the base sequence is at least 32 nucleobases in length. In some other embodiments, the base sequence is 31 or more nucleobases in length. In some other embodiments, the base sequence is 30 or more nucleobases in length. In some other embodiments, the base sequence is at least 29 nucleobases in length. In some other embodiments, the base sequence is at least 28 nucleobases in length. In some other embodiments, the base sequence is at least 27 nucleobases in length. In some other embodiments, the base sequence is at least 26 nucleobases in length. In some other embodiments, the base sequence of the complementary portion in the duplex is at least 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 16, 27, 28, 29, 30, 31, 32, 33, 34, 35 or more nucleobases in length. In some other embodiments, it is at least 18 nucleobases in length. In some other embodiments, it is at least 19 nucleobases in length. In some other embodiments, it is at least 20 nucleobases in length. In some other embodiments, it is at least 21 nucleobases in length. In some other embodiments, it is at least 22 nucleobases in length. In some other embodiments, it is at least 23 nucleobases in length. In some other embodiments, it is at least 24 nucleobases in length. In some other embodiments, it is at least 25 nucleobases in length. In particular, the present invention provides an oligonucleotide of shorter length but with comparable or better properties and/or comparable or higher activity compared to previously reported adenosine editing oligonucleotides.

In some embodiments, the base sequence of the oligonucleotide is complementary to the base sequence of the target nucleic acid (e.g., complementary to a portion of the target nucleic acid that includes the target adenosine) and is a Watson-Crick base pair (AT, AU, and CG). ), there are 0 to 10 mismatches (e.g., 0 to 1, 0 to 2, 0 to 3, 0 to 4, 0 to 5, 0 to 6, 0 to 7, 0 to 8, 0 to 9, 0~10, 1~2, 1~3, 1~4, 1~5, 1~6, 1~7, 1~8, 1~9, 1~10, 2~3, 2~4, 2~ 5, 2~6, 2~7, 2~8, 2~9, 2~10, 3~4, 3~5, 3~6, 3~7, 3~8, 3~9, 3~10, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, etc.). In some implementations, there is no discrepancy. In some implementations, there is 1 mismatch. In some implementations, there are two mismatches. In some implementations, there are three mismatches. In some implementations, there are 4 mismatches. In some implementations, there are 5 mismatches. In some implementations, there are 6 mismatches. In some implementations, there are 7 mismatches. In some implementations, there are 8 mismatches. In some implementations, there are 9 mismatches. In some implementations, there are 10 mismatches. In some embodiments, oligonucleotides may include portions not designed for complementarity (eg, loops for replenishment of proteins (eg, ADARs), protein binding sequences, etc.). As will be appreciated by those of ordinary skill in the art, when calculating mismatches and/or complementarity, such portions may be excluded as appropriate. In some embodiments, for example, complementarity between an oligonucleotide and a target nucleic acid is about 50%-100% (e.g., about 50%-80%, 50%-85%, 50%-90%, 50%-95%). %, 60%~80%, 60%~85%, 60%~90%, 60%~95%, 60%~100%, 65%~80%, 65%~85%, 65%~90%, 65%~95%, 65%~100%, 70%~80%, 70%~85%, 70%~90%, 70%~95%, 70%~100%, 75%~80%, 75% ~85%, 75%~90%, 75%~95%, 75%~100%, 80%~85%, 80%~90%, 80%~95%, 80%~100%, 85%~90 %, 85%~95%, 85%~100%, 90%~95%, 90%~100%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%, etc.). In some embodiments, complementarity is at least about 60%. In some embodiments, complementarity is at least about 65%. In some embodiments, complementarity is at least about 70%. In some embodiments, complementarity is at least about 75%. In some embodiments, the complementarity is at least about 80%. In some embodiments, complementarity is at least about 85%. In some embodiments, complementarity is at least about 90%. In some embodiments, complementarity is at least about 95%. In some embodiments, complementarity is 100% over the length of the oligonucleotide. In some embodiments, complementarity is 100% over the length of the oligonucleotide, except for the nucleoside opposite the target nucleoside (eg, adenosine). In general, complementarity is based on the Watson-Crick base pairs AT, AU, and CG. One skilled in the art will understand that when evaluating the complementarity of two sequences of different lengths (e.g., a provided oligonucleotide and a target nucleic acid), complementarity can be evaluated based appropriately on the length of the shorter sequence and/or the maximum complementarity between the two sequences. will be. In many embodiments, the oligonucleotide and target nucleic acid have sufficient complementarity such that modifications are directed selectively to target adenosine sites.

In some implementations, one or more mismatches are independently wobbles. In some implementations, each mismatch is a wobble. In some embodiments, 0-10 (e.g., 0-1, 0-2, 0-3, 0-4, 0-5, 0-6, 0-7, 0-8, 0-9, 0~10, 1~2, 1~3, 1~4, 1~5, 1~6, 1~7, 1~8, 1~9, 1~10, 2~3, 2~4, 2~ 5, 2~6, 2~7, 2~8, 2~9, 2~10, 3~4, 3~5, 3~6, 3~7, 3~8, 3~9, 3~10, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, etc.) wobbles. In some embodiments, the number is zero. In some embodiments, the number is one. In some embodiments, the number is two. In some embodiments, the number is three. In some embodiments, the number is four. In some embodiments, the number is five. In some embodiments, the wobble is G-U, I-A, G-A, I-U, I-C, I-T, A-A, or inverted A-T. In some embodiments, the wobble is G-U, I-A, G-A, I-U, or I-C. In some embodiments, I-C can be considered a match if I is the 3' adjacent nucleoside to the opposite nucleoside of the target nucleoside. In some embodiments, a base forming a wobble pair (e.g., a U capable of forming a G-U wobble) may replace a base forming a consensus pair (e.g., a C matching a G) and have editing activity It can provide an oligonucleotide having.

In some embodiments, a duplex of an oligonucleotide and a target nucleic acid comprises one or more bulges, each independently comprising one or more mismatches that are not wobbles. In some embodiments, 0-10 (e.g., 0-1, 0-2, 0-3, 0-4, 0-5, 0-6, 0-7, 0-8, 0-9, 0~10, 1~2, 1~3, 1~4, 1~5, 1~6, 1~7, 1~8, 1~9, 1~10, 2~3, 2~4, 2~ 5, 2~6, 2~7, 2~8, 2~9, 2~10, 3~4, 3~5, 3~6, 3~7, 3~8, 3~9, 3~10, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, etc.) In some embodiments, the number is zero. In some embodiments, the number is one. In some embodiments, the number is two. In some embodiments, the number is three. In some embodiments, the number is four. In some embodiments, the number is five.

In some embodiments, between two mismatches, between one or both ends of an oligonucleotide (or portion thereof, eg, a first domain, a second domain, a first subdomain, a second subdomain, a third subdomain) and a mismatch. , and/or the distance between the mismatch and the nucleoside opposite the target adenosine is independently 0-50, 0-40, 0-30, 0-25, 0-20, 0-15, 0-10 (e.g. , 0~1, 0~2, 0~3, 0~4, 0~5, 0~6, 0~7, 0~8, 0~9, 0~10, 1~2, 1~3, 1 ~4, 1~5, 1~6, 1~7, 1~8, 1~9, 1~10, 2~3, 2~4, 2~5, 2~6, 2~7, 2~8 , 2~9, 2~10, 3~4, 3~5, 3~6, 3~7, 3~8, 3~9, 3~10, 1, 2, 3, 4, 5, 6, 7 , 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32 , 33, 34, or 35 nucleobases (excluding mismatches, terminal nucleosides, and nucleosides opposite to target adenosine). In some embodiments, the number is 0-30. In some embodiments, the number is 0 to 20. In some embodiments, the number is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 , or 20. In some embodiments, the distance between two mismatches is 0 to 20. In some embodiments, the distance between two mismatches is 1 to 10. In some embodiments, the 5'-terminal nucleoside of the oligonucleotide and the distance between the mismatch is 0 to 20. In some embodiments, the distance between the 5'-terminal nucleoside of the oligonucleotide and the mismatch is 5 to 20. In some embodiments, the 3'-terminal nucleoside of the oligonucleotide is and the distance between the mismatch is 0 to 40. In some embodiments, the distance between the 3'-terminal nucleoside of the oligonucleotide and the mismatch is 5 to 20. In some embodiments, between the mismatch and the nucleoside opposite the target adenosine. The distance of is from 0 to 20. In some embodiments, the distance between the mismatch and the nucleoside opposite the target adenosine is 1-10. In some embodiments, the number of nucleobases for distance is zero. In some embodiments, the number is one. In some embodiments, the number is two. In some embodiments, the number is three. In some embodiments, the number is four. In some embodiments, the number is five. In some embodiments, the number is six. In some embodiments, the number is 7. In some embodiments, the number is eight. In some embodiments, the number is 9. In some embodiments, the number is 10. In some embodiments, the number is 11. In some embodiments, the number is 12. In some embodiments, the number is 13. In some embodiments, the number is 14. In some embodiments, the number is 15. In some embodiments, the number is 16. In some embodiments, the number is 17. In some embodiments, the number is 18. In some embodiments, the number is 19. In some embodiments, the number is 20. In some embodiments, the mismatch is terminal, eg, at the 5'-end or 3'-end of the first domain, second domain, first subdomain, second subdomain, or third subdomain. In some embodiments, the mismatch is on the opposite nucleoside of the target adenosine.

In some embodiments, provided oligonucleotides are capable of directing adenosine editing (e.g., A to I conversion) in a target nucleic acid, and consist of, include, or part of (e.g., a sequence of bases of an oligonucleotide disclosed herein) an oligonucleotide disclosed herein. For example, spans of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or more contiguous bases), each T may be independently replaced by U and vice versa Also possible, oligonucleotides contain at least one non-naturally occurring modification of bases, sugars, and/or internucleotidic linkages.

In some embodiments, provided oligonucleotides include one or more carbohydrate moieties. In some embodiments, provided oligonucleotides include one or more GalNAc moieties. In some embodiments, provided oligonucleotides include one or more targeting moieties. Non-limiting examples of such additional chemical moieties that can be conjugated to oligonucleotide chains are described herein.

In some embodiments, a provided oligonucleotide is capable of directing correction of a G to A mutation in a target sequence or product thereof. In some embodiments, the correction of a G to A mutation is or comprises a conversion of A to I, wherein I can be read as G during translation or other biological process. In some embodiments, a provided oligonucleotide is capable of directing correction of a G to A mutation in a target sequence or product thereof via ADAR-mediated deamination. In some embodiments, provided oligonucleotides recruit endogenous ADAR (e.g., in a target cell) and promote ADAR-mediated deamination, resulting in the generation of a G to A mutation in a target sequence or product thereof via ADAR-mediated deamination. correction can be ordered. However, nevertheless, the present invention is not limited to any particular mechanism. In some embodiments, the present invention provides oligonucleotides capable of operating via double-stranded RNA interference, single-stranded RNA interference, RNase H-mediated knockdown, steric hindrance of translation, ADAR-mediated deamination, or a combination of two or more of these mechanisms. , compositions, methods, and the like.

In some embodiments, an oligonucleotide comprises a structural element or portion thereof described herein (eg, in a table). In some embodiments, an oligonucleotide is a sequence of bases comprising the base sequence (or portion thereof) of an oligonucleotide disclosed herein, eg, in a table or figure, or elsewhere (where each T is independently substituted with a U). may), chemical modification pattern (or part thereof), and/or format. In some embodiments, such oligonucleotides are capable of directing the correction of a G to A mutation in a target sequence or product thereof.

In particular, a provided oligonucleotide can hybridize to its target nucleic acid (eg, pre-mRNA, mature mRNA, etc.). In some embodiments, an oligonucleotide can hybridize to a target RNA sequence nucleic acid at any stage of RNA processing, including but not limited to pre-mRNA or mature mRNA. In some embodiments, an oligonucleotide may hybridize to any element of an oligonucleotide nucleic acid or its complement, including but not limited to: promoter region, enhancer region, transcription termination region, translation start signal, translation Termination signal, coding region, noncoding region, exon, intron, intron/exon or exon/intron junction, 5' UTR, or 3' UTR.

In some embodiments, an oligonucleotide hybridizes to two or more variants of a transcript derived from the sense strand of a target site (eg, a target sequence).

In some embodiments, provided oligonucleotides contain increased levels of one or more isotopes. In some embodiments, provided oligonucleotides are labeled with, for example, one or more isotopes of one or more elements, such as hydrogen, carbon, nitrogen, and the like. In some embodiments, provided oligonucleotides in a provided composition, e.g., oligonucleotides of a plurality of compositions, contain base modifications, sugar modifications, and/or internucleotide linkage modifications, and the oligonucleotides contain enriched levels of deuterium. . In some embodiments, provided oligonucleotides are labeled with deuterium at one or more positions (replace - ¹ H with - ² H). In some embodiments, one or more ¹ H of an oligonucleotide chain or any moiety conjugated to an oligonucleotide chain (eg, a targeting moiety, etc.) is replaced with ² H. Such oligonucleotides can be used in the compositions and methods described herein.

In some embodiments, an oligonucleotide comprises one or more modified nucleobases, one or more modified sugars, and/or one or more modified internucleotide linkages as described herein. In some embodiments, an oligonucleotide contains a certain level of modified nucleobases, modified sugars, and/or modified internucleotide linkages (e.g., between about 5% and 100% of all nucleobases, sugars, and internucleotide linkages in the oligonucleotide, respectively). , about 10% to 100%, 20 to 100%, 30% to 100%, 40% to 100%, 50% to 80%, 50% to 85%, 50% to 90%, 50% to 95%, 60 %~80%, 60%~85%, 60%~90%, 60%~95%, 60%~100%, 65%~80%, 65%~85%, 65%~90%, 65%~ 95%, 65% to 100%, 70% to 80%, 70% to 85%, 70% to 90%, 70% to 95%, 70% to 100%, 75% to 80%, 75% to 85% , 75%~90%, 75%~95%, 75%~100%, 80%~85%, 80%~90%, 80%~95%, 80%~100%, 85%~90%, 85 %~95%, 85%~100%, 90%~95%, 90%~100%, 10%, 20%, 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%, etc.).

In some embodiments, oligonucleotides include one or more modified sugars. In some embodiments, the oligonucleotide is about 1-50 (e.g., about 5, 6, 7, 8, 9, or 10 to about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 40 or 50, or about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 , 18, 19, 20, 21, 22, 23, 24, 25, 30, 40 or 50, etc., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, etc.) modified sugars. In some embodiments, the oligonucleotide contains about 1-50 (e.g., about 5, 6, 7, 8, 9, or 10 to about 10, 11, 12, 13, 14) with 2'-F modifications. , 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 40 or 50, or about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 40 or 50 etc, about 1, 2, 3, 4, 5, 6, 7, 8, 9 , 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, etc.) modified sugars. In some embodiments, the oligonucleotide contains about 2-50 (e.g., about 2, 3, 4, 5, 6, 7, 8, 9, or 10 to about 10, 11) 2'-F modifications. , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 40 or 50, or about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 40 or 50, etc., 2 to 40, 2 to 30, 2 to 25, 2~20, 2~15, 2~10, 3~40, 3~30, 3~25, 3~20, 3~15, 3~10, 4~40, 4~30, 4~25, 4~ 20, 4~15, 4~10, 5~40, 5~30, 5~25, 5~20, 5~15, 5~10, 6~40, 6~30, 6~25, 6~20, 6~15, 6~10, 7~40, 7~30, 7~25, 7~20, 7~15, 7~10, 8~40, 8~30, 8~25, 8~20, 8~ 15, 8~10, 9~40, 9~30, 9~25, 9~20, 9~15, 9~10, 10~40, 10~30, 10~25, 10~20, 10~15 , about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, etc.) . In some embodiments, an oligonucleotide comprises two consecutive 2'-F modified sugars. In some embodiments, an oligonucleotide comprises three consecutive 2'-F modified sugars. In some embodiments, an oligonucleotide comprises four consecutive 2'-F modified sugars. In some embodiments, an oligonucleotide comprises 5 contiguous 2'-F modified sugars. In some embodiments, an oligonucleotide comprises six consecutive 2'-F modified sugars. In some embodiments, the oligonucleotide comprises 7 contiguous 2'-F modified sugars. In some embodiments, an oligonucleotide comprises 8 contiguous 2'-F modified sugars. In some embodiments, an oligonucleotide comprises 9 contiguous 2'-F modified sugars. In some embodiments, an oligonucleotide comprises 10 contiguous 2'-F modified sugars. In some embodiments, an oligonucleotide comprises two or more 2'-F strain sugar blocks, wherein each sugar in the 2'-F strain sugar block is independently a 2'-F strain sugar. In some embodiments, each block per 2'-F variant independently comprises 2, 3, 4, 5, 6, 7, 8, 9 or 10 contiguous 2'-F variant sugars as described herein; consists of this In some embodiments, two contiguous 2'-F variant sugar blocks are independently separated by a separating block comprising one or more sugars that are not 2'-F variant sugars. In some embodiments, the oligonucleotide is one or more (e.g., 1-20, 1-15, 1-14, 1-13, 1-12, 1-11, 1-10, 2-20, 3-15 , 4~15, 5~15, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, etc. ) and one or more (eg, 1-20, 1-15, 1-14, 1-13, 1-12, 1-11, 1-10, 2-20, 3-15 , 4~15, 5~15, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, etc. ) contains a separate block of In some embodiments, the first domain is one or more (e.g., 1-20, 1-15, 1-14, 1-13, 1-12, 1-11, 1-10, 2-20, 3- 15, 4~15, 5~15, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 2'-F blocks of (e.g., 1 to 20, 1 to 15, 1 to 14, 1 to 13, 1 to 12, 1 to 11, 1 to 10, 2 to 20, 3 to 15, 4~15, 5~15, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 etc.) In some embodiments, each first domain block coupled to the first domain 2'-F block is a separate block. In some embodiments, each first domain block coupled to the first domain isolation block is a first domain 2'-F block. In some embodiments, each sugar in the separation block is independently not 2'-F modified. In some embodiments, two or more (eg, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more) or all sugars in the separation block are independently 2'-F modified. In some embodiments, the separating block comprises one or more bicyclic sugars (eg, LNA sugars, cEt sugars, etc.) and/or one or more 2'-OR modified sugars (R is optionally substituted C _1-6 aliphatic) (eg For example, 2'-OMe, 2'-MOE, etc.). In some embodiments, the separating block comprises one or more 2'-OR modified sugars, where R is an optionally substituted C _1-6 aliphatic (eg, 2'-OMe, 2'-MOE, etc.). In some embodiments, two or more non-2'-F modified sugars are contiguous. In some embodiments, two or more 2'-OR modified sugars (R is an optionally substituted C _1-6 aliphatic (eg, 2'-OMe, 2'-MOE, etc.)) are contiguous. In some embodiments, the separating block comprises two or more (eg, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more) 2'-OR modified sugars, wherein R is optionally substituted C _1-6 aliphatic (eg, 2'-OMe, 2'-MOE, etc.). In some embodiments, the split block comprises two or more (eg, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more) consecutive 2'-OR modified sugars, wherein R is an optionally substituted C _1-6 aliphatic (eg, 2'-OMe, 2'-MOE, etc.). In some embodiments, each 2'-OR modified sugar is independently a 2'-OMe or 2'-MOE sugar. In some embodiments, each 2'-OR modified sugar is independently a 2'-OMe sugar. In some embodiments, each 2'-OR modified sugar is independently a 2'-MOE sugar. In some embodiments, the separating block comprises one or more 2'-F modified sugars. In some embodiments, none of the 2'-F strain sugars in the separating block are next to each other. In some embodiments, the separating block does not contain 2'-F modified sugars. In some embodiments, each sugar in the separating block is independently a 2′-OR modified sugar (R is an optionally substituted C _1-6 aliphatic) or a bicyclic sugar. In some embodiments, each sugar in each separating block is independently a 2′-OR modified sugar (R is an optionally substituted C _1-6 aliphatic) or a bicyclic sugar. In some embodiments, each sugar in the separating block is independently a 2'-OR modified sugar, where R is an optionally substituted C _1-6 aliphatic. In some embodiments, each sugar in each separating block is independently a 2'-OR modified sugar, where R is an optionally substituted C _1-6 aliphatic. In some embodiments, each sugar in the separation block is independently a 2'-OMe or 2'-MOE modified sugar. In some embodiments, each sugar in each separation block is independently a 2'-OMe or 2'-MOE modified sugar. In some embodiments, each sugar in the separation block is independently a 2'-OMe modified sugar. In some embodiments, each sugar in the separation block is independently a 2'-MOE modified sugar. In some embodiments, the separating block comprises a 2'-OMe sugar and a 2'-MOE modified sugar. In some embodiments, each 2'-F block and each separating block independently contains 1, 2, 3, 4 or 5 nucleosides. In some embodiments, each 2'-F block and each separating block independently contains 1, 2 or 3 nucleosides.

In some embodiments, about 10%-100%, 20-100%, 30%-100%, 40%-100%, 50%-80%, 50%-85%, 50%-90%, 50% of all sugars %~95%, 60%~80%, 60%~85%, 60%~90%, 60%~95%, 60%~100%, 65%~80%, 65%~85%, 65%~ 90%, 65% to 95%, 65% to 100%, 70% to 80%, 70% to 85%, 70% to 90%, 70% to 95%, 70% to 100%, 75% to 80% , 75%~85%, 75%~90%, 75%~95%, 75%~100%, 80%~85%, 80%~90%, 80%~95%, 80%~100%, 85 %~90%, 85%~95%, 85%~100%, 90%~95%, 90%~100%, 10%, 20%, 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%, etc. is a modified sugar. In some embodiments, about 10%-100%, 20-100%, 30%-100%, 40%-100%, 50%-80%, 50%-85%, 50%-90%, 50% of all sugars %~95%, 60%~80%, 60%~85%, 60%~90%, 60%~95%, 60%~100%, 65%~80%, 65%~85%, 65%~ 90%, 65% to 95%, 65% to 100%, 70% to 80%, 70% to 85%, 70% to 90%, 70% to 95%, 70% to 100%, 75% to 80% , 75%~85%, 75%~90%, 75%~95%, 75%~100%, 80%~85%, 80%~90%, 80%~95%, 80%~100%, 85 %~90%, 85%~95%, 85%~100%, 90%~95%, 90%~100%, 10%, 20%, 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%, etc., per 2'-F variant, per 2'-OR variant (R is an optionally substituted C _1-6 aliphatic), and It is a modified sugar independently selected from bicyclic sugars (eg, LNA sugars, cEt sugars, etc.). In some embodiments, the percentage is about or at least about 30%. In some embodiments, the percentage is about or at least about 40%. In some embodiments, the percentage is about or at least about 50%. In some embodiments, the percentage is about or at least about 60%. In some embodiments, the percentage is about or at least about 70%. In some embodiments, the percentage is about or at least about 80%. In some embodiments, the percentage is about or at least about 90%. In some embodiments, the percentage is about or at least about 95%.

In some embodiments, about 10%-100%, 20-100%, 30%-100%, 40%-100%, 50%-80%, 50%-85%, 50%-90%, 50% of all sugars %~95%, 60%~80%, 60%~85%, 60%~90%, 60%~95%, 60%~100%, 65%~80%, 65%~85%, 65%~ 90%, 65% to 95%, 65% to 100%, 70% to 80%, 70% to 85%, 70% to 90%, 70% to 95%, 70% to 100%, 75% to 80% , 75%~85%, 75%~90%, 75%~95%, 75%~100%, 80%~85%, 80%~90%, 80%~95%, 80%~100%, 85 %~90%, 85%~95%, 85%~100%, 90%~95%, 90%~100%, 10%, 20%, 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%, etc., from the 2'-F variant sugar and the 2'-OR variant sugar (R is an optionally substituted C _1-6 aliphatic) is an independently selected modified sugar. In some embodiments, about 10%-100%, 20-100%, 30%-100%, 40%-100%, 50%-80%, 50%-85%, 50%-90%, 50% of all sugars %~95%, 60%~80%, 60%~85%, 60%~90%, 60%~95%, 60%~100%, 65%~80%, 65%~85%, 65%~ 90%, 65% to 95%, 65% to 100%, 70% to 80%, 70% to 85%, 70% to 90%, 70% to 95%, 70% to 100%, 75% to 80% , 75%~85%, 75%~90%, 75%~95%, 75%~100%, 80%~85%, 80%~90%, 80%~95%, 80%~100%, 85 %~90%, 85%~95%, 85%~100%, 90%~95%, 90%~100%, 10%, 20%, 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%, etc. is a modified sugar independently selected from 2'-F modified sugars, 2'-OMe modified sugars and 2'-MOE modified sugars. . In some embodiments, the percentage is about or at least about 30%. In some embodiments, the percentage is about or at least about 40%. In some embodiments, the percentage is about or at least about 50%. In some embodiments, the percentage is about or at least about 60%. In some embodiments, the percentage is about or at least about 70%. In some embodiments, the percentage is about or at least about 80%. In some embodiments, the percentage is about or at least about 90%. In some embodiments, the percentage is about or at least about 95%.

In some embodiments, about 10%-100%, 20-100%, 30%-100%, 40%-100%, 50%-80%, 50%-85%, 50%-90%, 50% of all sugars %~95%, 60%~80%, 60%~85%, 60%~90%, 60%~95%, 60%~100%, 65%~80%, 65%~85%, 65%~ 90%, 65% to 95%, 65% to 100%, 70% to 80%, 70% to 85%, 70% to 90%, 70% to 95%, 70% to 100%, 75% to 80% , 75%~85%, 75%~90%, 75%~95%, 75%~100%, 80%~85%, 80%~90%, 80%~95%, 80%~100%, 85 %~90%, 85%~95%, 85%~100%, 90%~95%, 90%~100%, 10%, 20%, 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% and the like are modified sugars independently selected from 2'-F modified sugars and 2'-OMe modified sugars. In some embodiments, the percentage is about or at least about 30%. In some embodiments, the percentage is about or at least about 40%. In some embodiments, the percentage is about or at least about 50%. In some embodiments, the percentage is about or at least about 60%. In some embodiments, the percentage is about or at least about 70%. In some embodiments, the percentage is about or at least about 80%. In some embodiments, the percentage is about or at least about 90%. In some embodiments, the percentage is about or at least about 95%.

In some embodiments, about 10%-100%, 20-100%, 30%-100%, 40%-100%, 50%-80%, 50%-85%, 50%-90%, 50% of all sugars %~95%, 60%~80%, 60%~85%, 60%~90%, 60%~95%, 60%~100%, 65%~80%, 65%~85%, 65%~ 90%, 65% to 95%, 65% to 100%, 70% to 80%, 70% to 85%, 70% to 90%, 70% to 95%, 70% to 100%, 75% to 80% , 75%~85%, 75%~90%, 75%~95%, 75%~100%, 80%~85%, 80%~90%, 80%~95%, 80%~100%, 85 %~90%, 85%~95%, 85%~100%, 90%~95%, 90%~100%, 10%, 20%, 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%, etc. is a 2'-F modified sugar. In some embodiments, the percentage is about or at least about 30%. In some embodiments, the percentage is about or at least about 40%. In some embodiments, the percentage is about or at least about 50%. In some embodiments, the percentage is about or at least about 60%. In some embodiments, the percentage is about or at least about 70%. In some embodiments, the percentage is about or at least about 80%. In some embodiments, the percentage is about or at least about 90%. In some embodiments, the percentage is about or at least about 95%. In some embodiments, 10 or more (e.g., about or at least about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26 , 27, 28, 29 or 30 or more, 10-50, 10-40, 10-30, 10-25, 15-50, 15-40, 15-30, 15-25, 20-50, 20-40 , 20-30, 20-25, etc.) are 2'-F modified sugars. In some embodiments, the oligonucleotide is 2 or more (e.g., 2-30, 2-25, 2-20, 2-15, 3-10, 3-30, 3-25, 3-20, 3-20, 15, 3-10, 4-30, 4-25, 4-20, 4-15, 4-10, 5-30, 5-25, 5-20, 5-15, 5-10, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) consecutive 2'-F modified sugars. In some embodiments, the oligonucleotides are each independently two or more (e.g., 2-30, 2-25, 2-20, 2-15, 3-10, 3-30, 3-25, 3-20 , 3~15, 3~10, 4~30, 4~25, 4~20, 4~15, 4~10, 5~30, 5~25, 5~20, 5~15, 5~10, 2 , 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) consecutive 2'-F variants one or more 2'-F blocks. In some embodiments, an oligonucleotide comprises two or more 2′-F blocks as described herein separated by one or more separating blocks as described herein. In some embodiments, a 2'-F block has 2, 3, 4, 5, 6, 7, 8, 9, or 10 2'-F modified sugars. In some embodiments, a 2'-F block has no more than 2, 3, 4, 5, 6, 7, 8, 9, or 10 2'-F modified sugars. In some embodiments, each sugar of each 2'-F block is a 2'-F modification sugar, and each 2'-F block is independently 2, 3, 4, 5, 6, 7, 8, 9 or 10 2'-F modified sugars. In some embodiments, each sugar of each 2'-F block is a 2'-F modification sugar, and each 2'-F block is independently 2, 3, 4, 5, 6, 7, 8, 9 or less than 10 2'-F modified sugars. In some embodiments, each sugar of each 2'-F block is a 2'-F modification sugar, and each 2'-F block independently has no more than 10 2'-F modification sugars. In some embodiments, each sugar of each 2'-F block is a 2'-F modified sugar, and each 2'-F block independently has no more than 9 2'-F modified sugars. In some embodiments, each sugar of each 2'-F block is a 2'-F modified sugar, and each 2'-F block independently has no more than 8 2'-F modified sugars. In some embodiments, each sugar of each 2'-F block is a 2'-F modified sugar, and each 2'-F block independently has no more than 7 2'-F modified sugars. In some embodiments, each sugar of each 2'-F block is a 2'-F modified sugar, and each 2'-F block independently has 6 or fewer 2'-F modified sugars. In some embodiments, each sugar of each 2'-F block is a 2'-F modified sugar, and each 2'-F block independently has no more than 5 2'-F modified sugars. In some embodiments, each sugar of each 2'-F block is a 2'-F modified sugar, and each 2'-F block independently has no more than 4 2'-F modified sugars. In some embodiments, each block linked to a 2'-F block is independently a block that does not contain a 2'-F modified sugar. In some embodiments, each block linked to the 2'-F block independently comprises a natural DNA or RNA sugar, a 2'-OR modified sugar (R is an optionally substituted C _1-6 aliphatic), or a bicyclic sugar. It is a block. In some embodiments, each block linked to a 2'-F block is a block that independently comprises a native DNA or RNA sugar, a 2'-OMe modified sugar, a 2'-MOE modified sugar, or a bicyclic sugar. In some embodiments, each block linked to a 2'-F block is independently a block comprising a native DNA or RNA sugar, a 2'-OMe modified sugar, or a 2'-MOE modified sugar. In some embodiments, each nucleoside of the first domain linked to the 2′-F block of the first domain is independently a 2′-OR modified sugar (R is an optionally substituted C _1-6 aliphatic) or bicyclic sugar. am. In some embodiments, each nucleoside of the first domain linked to the 2′-F block of the first domain is independently a 2′-OR modified sugar, where R is an optionally substituted C _1-6 aliphatic. In some embodiments, each nucleoside of the first domain linked to the 2'-F block of the first domain is independently a 2'-OMe or 2'-MOE modified sugar. In some embodiments, each nucleoside of the second domain linked to the 2′-F block of the second domain is independently a 2′-OR modified sugar (R is an optionally substituted C _1-6 aliphatic) or bicyclic sugar. am. In some embodiments, each nucleoside of the second domain linked to the 2'-F block of the second domain is independently a 2'-OR modified sugar, where R is an optionally substituted C _1-6 aliphatic. In some embodiments, each nucleoside of the second domain linked to the 2'-F block of the second domain is independently a 2'-OMe or 2'-MOE modified sugar.

In some embodiments, about 10%-100%, 20-100%, 30%-100%, 40%-100%, 50%-80%, 50%-85%, 50%-90%, 50% of all sugars %~95%, 60%~80%, 60%~85%, 60%~90%, 60%~95%, 60%~100%, 65%~80%, 65%~85%, 65%~ 90%, 65% to 95%, 65% to 100%, 70% to 80%, 70% to 85%, 70% to 90%, 70% to 95%, 70% to 100%, 75% to 80% , 75%~85%, 75%~90%, 75%~95%, 75%~100%, 80%~85%, 80%~90%, 80%~95%, 80%~100%, 85 %~90%, 85%~95%, 85%~100%, 90%~95%, 90%~100%, 10%, 20%, 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%, etc. is a 2'-OR modified sugar (R is an optionally substituted C _1-6 aliphatic). In some embodiments, about 10%-100%, 20-100%, 30%-100%, 40%-100%, 50%-80%, 50%-85%, 50%-90%, 50% of all sugars %~95%, 60%~80%, 60%~85%, 60%~90%, 60%~95%, 60%~100%, 65%~80%, 65%~85%, 65%~ 90%, 65% to 95%, 65% to 100%, 70% to 80%, 70% to 85%, 70% to 90%, 70% to 95%, 70% to 100%, 75% to 80% , 75%~85%, 75%~90%, 75%~95%, 75%~100%, 80%~85%, 80%~90%, 80%~95%, 80%~100%, 85 %~90%, 85%~95%, 85%~100%, 90%~95%, 90%~100%, 10%, 20%, 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%, etc. are 2'-OMe or 2'-MOE modified sugars. In some embodiments, the percentage is about or at least about 30%. In some embodiments, the percentage is about or at least about 40%. In some embodiments, the percentage is about or at least about 50%. In some embodiments, the percentage is about or at least about 60%. In some embodiments, the percentage is about or at least about 70%. In some embodiments, the percentage is about or at least about 80%. In some embodiments, the percentage is about or at least about 90%. In some embodiments, the percentage is about or at least about 95%.

In some embodiments, about 10%-100%, 20-100%, 30%-100%, 40%-100%, 50%-80%, 50%-85%, 50%-90%, 50% of all sugars %~95%, 60%~80%, 60%~85%, 60%~90%, 60%~95%, 60%~100%, 65%~80%, 65%~85%, 65%~ 90%, 65% to 95%, 65% to 100%, 70% to 80%, 70% to 85%, 70% to 90%, 70% to 95%, 70% to 100%, 75% to 80% , 75%~85%, 75%~90%, 75%~95%, 75%~100%, 80%~85%, 80%~90%, 80%~95%, 80%~100%, 85 %~90%, 85%~95%, 85%~100%, 90%~95%, 90%~100%, 10%, 20%, 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%, etc. is per 2'-OMe modification. In some embodiments, the percentage is about or at least about 30%. In some embodiments, the percentage is about or at least about 40%. In some embodiments, the percentage is about or at least about 50%. In some embodiments, the percentage is about or at least about 60%. In some embodiments, the percentage is about or at least about 70%. In some embodiments, the percentage is about or at least about 80%. In some embodiments, the percentage is about or at least about 90%. In some embodiments, the percentage is about or at least about 95%.

In some embodiments, about 10%-100%, 20-100%, 30%-100%, 40%-100%, 50%-80%, 50%-85%, 50%-90%, 50% of all sugars %~95%, 60%~80%, 60%~85%, 60%~90%, 60%~95%, 60%~100%, 65%~80%, 65%~85%, 65%~ 90%, 65% to 95%, 65% to 100%, 70% to 80%, 70% to 85%, 70% to 90%, 70% to 95%, 70% to 100%, 75% to 80% , 75%~85%, 75%~90%, 75%~95%, 75%~100%, 80%~85%, 80%~90%, 80%~95%, 80%~100%, 85 %~90%, 85%~95%, 85%~100%, 90%~95%, 90%~100%, 10%, 20%, 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%, etc. is a 2'-OR modified sugar (R is an optionally substituted C _1-6 aliphatic). In some embodiments, about 10%-100%, 20-100%, 30%-100%, 40%-100%, 50%-80%, 50%-85%, 50%-90%, 50% of all sugars %~95%, 60%~80%, 60%~85%, 60%~90%, 60%~95%, 60%~100%, 65%~80%, 65%~85%, 65%~ 90%, 65% to 95%, 65% to 100%, 70% to 80%, 70% to 85%, 70% to 90%, 70% to 95%, 70% to 100%, 75% to 80% , 75%~85%, 75%~90%, 75%~95%, 75%~100%, 80%~85%, 80%~90%, 80%~95%, 80%~100%, 85 %~90%, 85%~95%, 85%~100%, 90%~95%, 90%~100%, 10%, 20%, 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%, etc. are 2'-MOE modified sugars.

In some embodiments, one or several (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 1-10, 1-10) of the first (5'-end) 9, 1~8, 1~7, 1~6, 1~5, 1~4, 1~3, 2~10, 2~9, 2~8, 2~7, 2~6, 2~5, 2-4, 2-3, 3-10, 3-9, 3-8, 3-7, 3-6, 3-5, 3-4, etc.) and/or one of the last (3'-end) or several (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 1-10, 1-9, 1-8, 1-7, 1-6, 1~5, 1~4, 1~3, 2~10, 2~9, 2~8, 2~7, 2~6, 2~5, 2~4, 2~3, 3~10, 3~ 9, 3-8, 3-7, 3-6, 3-5, 3-4, etc.) of the nucleosides are independently modified sugars. In some embodiments, the first one or several sugars are independently modified sugars. In some embodiments, the last one or several sugars are independently modified sugars. In some embodiments, the first and last one or several sugars are all independently modified sugars. In some embodiments, the modified sugar is independently a non-2'-F modified sugar, e.g., a bicyclic sugar, a 2'-OR modified sugar (R is as described herein and not -H (e.g., optionally substituted). is C _1-6 aliphatic)). In some embodiments, they are independently selected from bicyclic sugars and 2'-OR modified sugars, where R is an optionally substituted C _1-6 aliphatic. In some embodiments, they are independently 2'-OR modified sugars (R is an optionally substituted C _1-6 aliphatic). In some embodiments, they are independently a 2'-OMe modified sugar and a 2'-MOE modified sugar. In some embodiments, the first few sugars are one or more 2'-OR modified sugars (R is an optionally substituted C _1-6 aliphatic) or bicyclic sugar (eg, LNA, cEt, etc.) as described herein. include In some embodiments, the first few sugars include one or more 2'-OR modified sugars, where R is an optionally substituted C _1-6 aliphatic. In some embodiments, the first few sugars include one or more 2'-OMe modified sugars. In some embodiments, the first few sugars include one or more 2'-MOE modified sugars. In some embodiments, the first few sugars include one or more 2'-OMe modified sugars and one or more 2'-MOE modified sugars. In some embodiments, the last few sugars are one or more 2'-OR modified sugars (R is an optionally substituted C _1-6 aliphatic) or bicyclic sugar (eg, LNA, cEt, etc.) as described herein. include In some embodiments, the last few sugars include one or more 2'-OR modified sugars, where R is an optionally substituted C _1-6 aliphatic. In some embodiments, the last few sugars include one or more 2'-OMe modified sugars. In some embodiments, the last few sugars include one or more 2'-MOE modified sugars. In some embodiments, the last few sugars include one or more 2'-OMe modified sugars and one or more 2'-MOE modified sugars. In some embodiments, the last several sugars are independently 2'-OMe modified sugars. In some embodiments, the first few sugars are two or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10, etc.) contiguous bicyclic sugars or 2'-OR variant sugars ( R is an optionally substituted C _1-6 aliphatic; In some embodiments, the first few sugars are 2 or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10, etc.) consecutive 2'-OR variant sugars (R is optionally substituted C _1-6 aliphatic). In some embodiments, the first few sugars include two or more (eg, 2, 3, 4, 5, 6, 7, 8, 9, or 10, etc.) consecutive modified sugars, wherein each modified sugar The sugars are independently 2'-OMe modified sugars or 2'-MOE modified sugars. In some embodiments, the first few sugars contain two or more (eg, 2, 3, 4, 5, 6, 7, 8, 9, or 10, etc.) contiguous 2'-OMe modified sugars. In some embodiments, the first few sugars contain two or more (eg, 2, 3, 4, 5, 6, 7, 8, 9, or 10, etc.) contiguous 2'-MOE modified sugars. In some embodiments, the last few sugars are 2 or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10, etc.) consecutive 2'-OR variants (R is optionally substituted C _1-6 aliphatic). In some embodiments, the last few sugars include two or more (eg, 2, 3, 4, 5, 6, 7, 8, 9, or 10, etc.) consecutive modified sugars, wherein each modified sugar The sugars are independently 2'-OMe modified sugars or 2'-MOE modified sugars. In some embodiments, the last few sugars comprise two or more (eg, 2, 3, 4, 5, 6, 7, 8, 9, or 10, etc.) contiguous 2'-OMe modified sugars. In some embodiments, the last few sugars include 3 or more contiguous 2'-OMe modified sugars. In some embodiments, the last few sugars include 4 or more contiguous 2'-OMe modified sugars. In some embodiments, the last few sugars include 5 or more contiguous 2'-OMe modified sugars. In some embodiments, the last few sugars include 6 or more contiguous 2'-OMe modified sugars. In some embodiments, the last few sugars comprise two or more (eg, 2, 3, 4, 5, 6, 7, 8, 9, or 10, etc.) contiguous 2'-MOE modified sugars.

In some embodiments, one or more (1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) is per strain. In some embodiments, one or more of the first few (1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) sugars (1, 2, 3, 4, 5, 6, 7 , 8, 9, or 10) are each independently a 2'-OR modified sugar (R is an optionally substituted C _1-6 aliphatic) and a bicyclic sugar (eg, 2'-O-CH ₂ -4' (-CH ₂ - is optionally substituted) (eg, LNA sugar, cET sugar (eg, (S)-cEt))). In some embodiments, two or more of the first few sugars are each independently a modified sugar selected from a 2'-OR modified sugar (R is an optionally substituted C _1-6 aliphatic) and a bicyclic sugar. In some embodiments, 3 or more of the first few sugars are each independently a modified sugar selected from a 2′-OR modified sugar (R is an optionally substituted C _1-6 aliphatic) and a bicyclic sugar. In some embodiments, at least 4 of the first few sugars are each independently a modified sugar selected from a 2′-OR modified sugar (R is an optionally substituted C _1-6 aliphatic) and a bicyclic sugar. In some embodiments, one or more sugars are consecutive. In some embodiments, the first 1, 2, 3 or 4 sugars are modified sugars. In some embodiments, each of the first two sugars is independently a modified sugar selected from a 2′-OR modified sugar (R is an optionally substituted C _1-6 aliphatic) and a bicyclic sugar. In some embodiments, each of the first three sugars is independently a modified sugar selected from a 2'-OR modified sugar (R is an optionally substituted C _1-6 aliphatic) and a bicyclic sugar. In some embodiments, each of the first four sugars is independently a modified sugar selected from a 2'-OR modified sugar (R is an optionally substituted C _1-6 aliphatic) and a bicyclic sugar. In some embodiments, each 2'-OR modified sugar is independently a 2'-OMe or 2'-MOE modified sugar. In some embodiments, each bicyclic sugar is independently an LNA sugar or a cEt sugar. In some embodiments, each of one or more (e.g., 1, 2, 3, 4, or 5) sugars of the first few sugars, or each of the first few (e.g., 1, 2, 3, 4, or 5) sugars are independently 2'-OR modified sugars (R is an optionally substituted C _1-6 aliphatic). In some embodiments, each of one or more (e.g., 1, 2, 3, 4, or 5) sugars of the first few sugars, or each of the first few (e.g., 1, 2, 3, 4, or 5) sugars are independently 2'-OMe or 2'-MOE modified sugars. In some embodiments, each of one or more (e.g., 1, 2, 3, 4, or 5) sugars of the first few sugars, or each of the first few (e.g., 1, 2, 3, 4, or 5) sugars are independently 2'-OMe modified sugars. In some embodiments, each of one or more (e.g., 1, 2, 3, 4, or 5) sugars of the first few sugars, or each of the first few (e.g., 1, 2, 3, 4, or 5) sugars are independently 2'-MOE modified sugars. In some embodiments, the first 1, 2, 3, 4 or more sugars are independently 2'-OMe modified sugars. In some embodiments, the first sugar is a 2'-OMe modified sugar. In some embodiments, the first two sugars are independently 2'-OMe modified sugars. In some embodiments, the first three sugars are independently 2'-OMe modified sugars. In some embodiments, the first 4 sugars are independently 2'-OMe modified sugars. In some embodiments, the first 1, 2, 3, 4 or more sugars are independently 2'-MOE modified sugars. In some embodiments, the first sugar is a 2'-MOE modified sugar. In some embodiments, the first two sugars are independently 2'-MOE modified sugars. In some embodiments, the first three sugars are independently 2'-MOE modified sugars. In some embodiments, the first 4 sugars are independently 2'-MOE modified sugars. In some embodiments, each such modification is independently A, T, C, G, or U, wherein its nucleobase is optionally substituted or protected, or an optionally substituted or protected tautomer of A, T, C, G, or U. It is the sugar of a chain nucleoside. In some embodiments, one or more such sugars are independently linked to non-negatively charged internucleotidic linkages. In some embodiments, one or more such sugars are independently linked to neutral internucleotidic linkages, such as n001. In some embodiments, the non-negatively charged internucleotide linkages or neutral internucleotide linkages, eg n001, are chirally controlled. In some embodiments, it is R p. In some embodiments, one or more of these sugars are independently linked to phosphorothioate internucleotidic linkages. In some embodiments, the phosphorothioate internucleotidic linkages are chirally controlled. In some embodiments, it is Sp . In some embodiments, as described herein, the internucleoside linkage between the first nucleoside and the second nucleoside is a non-negatively charged internucleoside linkage. In some embodiments, it is a neutral internucleotide linkage. In some embodiments, it is a phosphoryl guanidine internucleotide linkage. In some embodiments, this is n001. In some embodiments, it is chirally controlled. In some embodiments, it is R p. In some embodiments, each internucleotide linkage linked to a nucleoside comprising one or more of the first few or the first several modified sugars is independently It is a phosphorothioate internucleotidic linkage. In some embodiments, each is chirally controlled. In some embodiments, each is Sp . In some embodiments, the first nucleoside is linked to an additional moiety, eg Mod001, optionally through a linker, eg L001, through its 5'-terminal carbon (in some embodiments via a phosphate group).

In some embodiments, one or more (1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) is per strain. In some embodiments, one or more of the last few (1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) sugars (1, 2, 3, 4, 5, 6, 7 , 8, 9, or 10) are each independently a 2'-OR modified sugar (R is an optionally substituted C _1-6 aliphatic) and a bicyclic sugar (eg, 2'-O-CH ₂ -4' (-CH ₂ - is optionally substituted) (eg, LNA sugar, cET sugar (eg, (S)-cEt))). In some embodiments, two or more of the last few sugars are each independently a modified sugar selected from a 2'-OR modified sugar (R is an optionally substituted C _1-6 aliphatic) and a bicyclic sugar. In some embodiments, 3 or more of the last few sugars are each independently a modified sugar selected from a 2′-OR modified sugar (R is an optionally substituted C _1-6 aliphatic) and a bicyclic sugar. In some embodiments, at least 4 of the last few sugars are each independently a modified sugar selected from a 2′-OR modified sugar (R is an optionally substituted C _1-6 aliphatic) and a bicyclic sugar. In some embodiments, one or more sugars are consecutive. In some embodiments, the last 1, 2, 3 or 4 sugars are modified sugars. In some embodiments, the last two sugars are each independently a modified sugar selected from a 2'-OR modified sugar (R is an optionally substituted C _1-6 aliphatic) and a bicyclic sugar. In some embodiments, each of the last three sugars is independently a modified sugar selected from a 2′-OR modified sugar (R is an optionally substituted C _1-6 aliphatic) and a bicyclic sugar. In some embodiments, each of the last four sugars is independently a modified sugar selected from a 2'-OR modified sugar (R is an optionally substituted C _1-6 aliphatic) and a bicyclic sugar. In some embodiments, each 2'-OR modified sugar is independently a 2'-OMe or 2'-MOE modified sugar. In some embodiments, each bicyclic sugar is independently an LNA sugar or a cEt sugar. In some embodiments, each of one or more (e.g., 1, 2, 3, 4, or 5) sugars of the last few sugars, or each of the last few (e.g., 1, 2, 3, 4, or 5) sugars are independently 2'-OR modified sugars (R is an optionally substituted C _1-6 aliphatic). In some embodiments, each of one or more (e.g., 1, 2, 3, 4, or 5) sugars of the last few sugars, or each of the last few (e.g., 1, 2, 3, 4, or 5) sugars are independently 2'-OMe or 2'-MOE modified sugars. In some embodiments, each of one or more (e.g., 1, 2, 3, 4, or 5) sugars of the last few sugars, or each of the last few (e.g., 1, 2, 3, 4, or 5) sugars are independently 2'-OMe modified sugars. In some embodiments, each of one or more (e.g., 1, 2, 3, 4, or 5) sugars of the last few sugars, or each of the last few (e.g., 1, 2, 3, 4, or 5) sugars are independently 2'-MOE modified sugars. In some embodiments, the last 1, 2, 3, 4 or more sugars are independently 2'-OMe modified sugars. In some embodiments, the last sugar is a 2'-OMe modified sugar. In some embodiments, the last two sugars are independently 2'-OMe modified sugars. In some embodiments, the last three sugars are independently 2'-OMe modified sugars. In some embodiments, the last 4 sugars are independently 2'-OMe modified sugars. In some embodiments, the last 1, 2, 3, 4 or more sugars are independently 2'-MOE modified sugars. In some embodiments, the last sugar is a 2'-MOE modified sugar. In some embodiments, the last two sugars are independently 2'-MOE modified sugars. In some embodiments, the last three sugars are independently 2'-MOE modified sugars. In some embodiments, the last four sugars are independently 2'-MOE modified sugars. In some embodiments, each such modification is independently A, T, C, G, or U, wherein its nucleobase is optionally substituted or protected, or an optionally substituted or protected tautomer of A, T, C, G, or U. It is the sugar of a chain nucleoside. In some embodiments, one or more such sugars are independently linked to non-negatively charged internucleotidic linkages. In some embodiments, one or more such sugars are independently linked to neutral internucleotidic linkages, such as n001. In some embodiments, the non-negatively charged internucleotide linkages or neutral internucleotide linkages, eg n001, are chirally controlled. In some embodiments, it is R p. In some embodiments, one or more of these sugars are independently linked to phosphorothioate internucleotidic linkages. In some embodiments, the phosphorothioate internucleotidic linkages are chirally controlled. In some embodiments, it is Sp . In some embodiments, as described herein, the internucleoside linkage between the last nucleoside and the penultimate nucleoside is a non-negatively charged internucleoside linkage. In some embodiments, it is a neutral internucleotide linkage. In some embodiments, it is a phosphoryl guanidine internucleotide linkage. In some embodiments, this is n001. In some embodiments, it is chirally controlled. In some embodiments, it is R p. In some embodiments, each internucleotide linkage linked to a nucleoside comprising one or more of the last several or the last several modified sugars, except for internucleotide linkages between the last and penultimate nucleosides, is are independently phosphorothioate internucleotidic linkages. In some embodiments, each is chirally controlled. In some embodiments, each is Sp .

In some embodiments, the sugar at position +1 is a 2'-F modified sugar. In some embodiments, the sugar at position +1 is a natural DNA sugar. In some embodiments, the sugar at position 0 is a natural DNA sugar (the nucleoside at position 0 is opposite the target adenosine when aligned). In some embodiments, the sugar at position -1 is a DNA sugar. In some embodiments, the sugar at position -2 is a 2'-OR modified sugar (R is an optionally substituted C _1-6 aliphatic) or a bicyclic sugar (eg, 2'-O-CH ₂ -4'). sugars (-CH ₂ - is optionally substituted) (eg, LNA sugar, cET sugar (eg, (S)-cEt))). In some embodiments, it is a 2′-OR modified sugar (R is an optionally substituted C _1-6 aliphatic). In some embodiments, it is a 2'-OMe modified sugar. In some embodiments, it is a 2'-MOE modified sugar. In some embodiments, it is a bicyclic sugar. In some embodiments, it is an LNA sugar. In some embodiments, it is per cEt. In some embodiments, the sugar at position -3 is a 2'-F modified sugar. In some embodiments, each sugar following position -3 (eg, positions -4, -5, -6, etc.) is independently a 2'-OR modified sugar (R is an optionally substituted C _1-6 are aliphatic) or bicyclic sugars (eg, sugars containing 2'-O-CH ₂ -4' (-CH ₂ - is optionally substituted) (eg, LNA sugars, cET sugars (eg, (S)-cEt))). In some embodiments, each is independently a 2′-OR modified sugar (R is an optionally substituted C _1-6 aliphatic) or a bicyclic sugar. In some embodiments, each is independently a 2'-OMe or 2'-MOE modified sugar. In some embodiments, each is a 2'-OMe modified sugar. In some embodiments, each is a 2'-MOE modified sugar. In some embodiments, one or more are independently 2'-OMe modified sugars and one or more are independently 2'-MOE modified sugars. In some embodiments, as described herein, the internucleotide linkage between the nucleoside at position -1 and the nucleoside at position -2 is a non-negatively charged internucleotide linkage. In some embodiments, it is a neutral internucleotide linkage. In some embodiments, it is a phosphoryl guanidine internucleotide linkage. In some embodiments, this is n001. In some embodiments, it is chirally controlled. In some embodiments, it is Sp . In some embodiments, it is R p. In some embodiments, the internucleotide linkage between the nucleoside at position -2 and the nucleoside at position -3 is a natural phosphate linkage. In some embodiments, as described herein, the internucleoside linkage between the last nucleoside and the penultimate nucleoside is a non-negatively charged internucleoside linkage. In some embodiments, it is a neutral internucleotide linkage. In some embodiments, it is a phosphoryl guanidine internucleotide linkage. In some embodiments, this is n001. In some embodiments, it is chirally controlled. In some embodiments, it is R p. In some embodiments, between the nucleoside at position -1 and the nucleoside at position -2, between the nucleoside at position -2 and the nucleoside at position -3, and between the last and penultimate nucleoside Each internucleoside linkage between nucleosides to the 3' side of the nucleoside opposite the target adenosine, except between the cleosides, is independently a phosphorothioate internucleotidic linkage. In some embodiments, each phosphorothioate internucleotidic linkage is chirally controlled. In some embodiments, each is Sp .

In some embodiments, the first and/or last one or several sugars are a modified sugar, eg a bicyclic sugar and/or a 2'-OR modified sugar (R is an optionally substituted C _1-6 aliphatic) (eg , per 2'-OMe strain, per 2'-MOE strain, etc.). In some embodiments, such sugars may increase the stability, affinity and/or activity of the oligonucleotide. In some embodiments, when conjugated to one or more additional chemical moieties, the sugar at the 5'- and/or 3'-end of the oligonucleotide is a bicyclic sugar or a 2'-OR modified sugar (R is an optionally substituted C _{1- 6} is aliphatic). In some embodiments, the 5'-terminal sugar is a bicyclic sugar or a 2'-OR modified sugar, where R is an optionally substituted C _1-6 aliphatic. In some embodiments, these 5'-terminal sugars are not linked to additional chemical moieties. In some embodiments, the 5'-end sugar is a 2'-F modified sugar. In some embodiments, the 5'-terminal sugar is a 2'-F modified sugar conjugated to an additional chemical moiety. In some embodiments, the 3'-terminal sugar is a bicyclic sugar or a 2'-OR modified sugar, where R is an optionally substituted C _1-6 aliphatic. In some embodiments, these 3'-terminal sugars are not linked to additional chemical moieties. In some embodiments, the 3'-terminal sugar is a 2'-F modified sugar. In some embodiments, the 3'-terminal sugar is a 2'-F modified sugar conjugated to an additional chemical moiety. In some embodiments, the last few sugars are the sugars on the 3' side of the opposite nucleoside of the target adenosine (eg, the sugars on the 3' side of the nucleoside, such as N _-1 , N _{-2 ,} etc.). In some embodiments, the last few sugars or 3' side sugars have one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more) 2'-F modifications. contains sugar In some embodiments, the last few sugars or sugars on the 3' side are two or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more) contiguous 2'-F modifications. contains sugar In some embodiments, the last few sugars or 3' side sugars comprise one or more or two or more contiguous 2'-F modified sugars, and the sugar of the last nucleoside of the oligonucleotide is a bicyclic sugar or 2'-OR modified sugar. sugar (R is an optionally substituted C _1-6 aliphatic). In some embodiments, as described herein, a 2'-OR modified sugar is a 2'-OMe modified sugar or a 2'-MOE modified sugar; In some embodiments it is a 2'-OMe modified sugar; In some embodiments it is a 2'-MOE modified sugar. In some embodiments, the last few sugars or 3' side sugars comprise one or more or two or more contiguous 2'-F modified sugars, and the sugar of the last nucleoside of the oligonucleotide is a 2'-OR modified sugar (R is optionally substituted C _1-6 aliphatic). In some embodiments, the last few sugars or 3' side sugars comprise one or more or two or more contiguous 2'-F modified sugars, and the sugar of the last nucleoside of the oligonucleotide is a 2'-OMe modified sugar or 2 '- is per MOE strain. In some embodiments, the last few sugars or 3' side sugars include one or more or two or more contiguous 2'-F modified sugars, and the sugar of the last nucleoside of the oligonucleotide is a 2'-OMe modified sugar. In some embodiments, the last few sugars or 3' side sugars include one or more or two or more contiguous 2'-F modified sugars, and the sugar of the last nucleoside of the oligonucleotide is a 2'-MOE modified sugar. In some embodiments, no more than two nucleosides on the 3' side of the nucleoside opposite to adenosine independently have a 2'-F modified sugar. In some embodiments, they are at positions -4 and -5. In some embodiments, these are the penultimate and third last nucleosides of an oligonucleotide. In some embodiments, no more than one nucleoside on the 3' side of the nucleoside opposite to adenosine has a 2'-F modified sugar. In some embodiments, it is at position -3. In some embodiments, it is the fourth to last nucleoside of an oligonucleotide.

In some embodiments, a bicyclic sugar or a 2'-OR modified sugar (R is an optionally substituted C _1-6 aliphatic) is one or more (eg, 1-30, 1-25, 1-20, 1-15 , 1-10, 2-30, 2-25, 2-20, 2-25, 2-10, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 , 13, 14, 15, 16, 17, 18, 19, 20) sugars are present in the region containing the 2'-F modified sugar. In some embodiments, most of the sugars as described herein in these regions are 2'-F modified sugars. In some embodiments, two or more 2'-F modified sugars are contiguous. In some embodiments, the region is a first domain. In some embodiments, bicyclic sugars are present in these regions. In some embodiments, a 2'-OR modified sugar (R is an optionally substituted C _1-6 aliphatic) is present in this region. In some embodiments, 2'-OMe modified sugars are present in these regions. In some embodiments, 2'-MOE modified sugars are present in these regions.

In some embodiments, one or more sugars at positions -5, -4, -3, +1, +2, +4, +5, +6, +7, and +8 (position 0 is opposite the target adenosine). is the position of the nucleoside; "+" goes to the nucleoside opposite the target adenosine towards the 5'-end of the oligonucleotide, and "-" goes to the nucleoside opposite the target adenosine towards the 3'-end of the oligonucleotide. from the seed; for example, in 5'-N ₁ N ₀ N _-1 -3', where N ₀ is the nucleoside opposite to the target adenosine, it is at position 0 and N ₁ is at position +1 position, and N _-1 is at position -1) are independently per 2'-F modification. In some embodiments, the sugar at position +1 and the one or more sugars at positions -5, -4, -3, +2, +4, +5, +6, +7 and +8 are independently 2' -F is per strain. In some embodiments, the sugar at position +1 and one sugar at positions -5, -4, -3, +2, +4, +5, +6, +7 and +8 are independently 2' -F is per strain.

In some embodiments, the oligonucleotide is one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more, 2-10, 3-10, 2-5, 2 ~4, 2-3, 3-5, 3-4, etc.) of natural DNA sugars. In some embodiments, the one or more natural DNA sugars are in the editing region, eg at positions +1, 0 and/or -1. In some embodiments, the natural DNA sugar is within the first few nucleosides (e.g., the first 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleosides) of the oligonucleotide. there is. In some embodiments, the first, second and/or third nucleoside of an oligonucleotide independently has a natural DNA sugar. In some embodiments, the natural DNA sugar is a non-negatively charged internucleotide linkage, a neutral internucleotide linkage, a phosphorylguanidine internucleotide linkage, n001, or a phosphorothioate internucleotide linkage (in various embodiments, Sp ) and are bound to the same modified internucleotide linkages.

Oligonucleotides may contain various types of internucleotidic linkages. In some embodiments, an oligonucleotide comprises one or more modified internucleotidic linkages. In some embodiments, the modified internucleotidic linkage is a chiral internucleotidic linkage. In some embodiments, the modified internucleotidic linkage is a phosphorothioate internucleotidic linkage. In some embodiments, the modified internucleotide linkage is a non-negatively charged internucleotide linkage. In some embodiments, a modified internucleotide linkage is a neutral internucleotide linkage. In some embodiments, the modified internucleotide linkage is a phosphoryl guanidine internucleotide linkage. In some embodiments, the modified internucleotide linkage is n001. In some embodiments, an oligonucleotide comprises one or more native phosphate linkages. In some embodiments, a natural phosphate linkage binds to a nucleoside comprising a modified sugar that can improve stability (eg, resistance to nucleases). In some embodiments, a natural phosphate linkage binds to a bicyclic sugar. In some embodiments, a native phosphate linkage binds to a 2'-modified sugar. In some embodiments, a native phosphate linkage joins a 2'-OR modified sugar, where R is an optionally substituted C _1-6 aliphatic. In some embodiments, a native phosphate linkage binds to a 2'-OMe modified sugar. In some embodiments, a native phosphate linkage binds to a 2'-MOE modified sugar. In some embodiments, the oligonucleotide comprises phosphorothioate internucleotidic linkages, non-negatively charged internucleotidic linkages, and natural phosphate linkages. In some embodiments, the oligonucleotide comprises phosphorothioate internucleotidic linkages, neutral internucleotidic linkages and native phosphate linkages. In some embodiments, the oligonucleotide comprises phosphorothioate internucleotidic linkages, phosphorylguanidine internucleotidic linkages, and native phosphate linkages. In some embodiments, the oligonucleotide comprises phosphorothioate internucleotidic linkages, n001 and natural phosphate linkages. In some embodiments, each chiral internucleotidic linkage is independently chirally controlled. In some embodiments, one or more chiral internucleotidic linkages are not chirally controlled. In some embodiments, each phosphorothioate internucleotidic linkage is independently chirally controlled. In some embodiments, each chiral internucleotidic linkage is independently chirally controlled. In some embodiments, most or each phosphorothioate internucleotidic linkage is Sp as described herein. In some embodiments, most or each non-negatively charged internucleotidic linkage, eg n001, is R p. In some embodiments, most or each non-negatively charged internucleotidic linkage, eg n001, is Sp .

In some embodiments, the oligonucleotide comprises phosphorothioate internucleotidic linkages and non-negatively charged internucleotidic linkages. In some embodiments, the oligonucleotide comprises phosphorothioate internucleotidic linkages and neutral internucleotidic linkages. In some embodiments, the oligonucleotide comprises phosphorothioate internucleotide linkages and phosphoryl guanidine internucleotide linkages. In some embodiments, the oligonucleotide comprises phosphorothioate internucleotidic linkages and n001. In some embodiments, each chiral internucleotidic linkage is independently chirally controlled. In some embodiments, one or more chiral internucleotidic linkages are not chirally controlled. In some embodiments, each phosphorothioate internucleotidic linkage is independently chirally controlled. In some embodiments, each chiral internucleotidic linkage is independently chirally controlled. In some embodiments, most or each phosphorothioate internucleotidic linkage is Sp as described herein. In some embodiments, the one or more (eg, 1, 2, 3, 4 or 5) phosphorothioate internucleotidic linkages are R p . In some embodiments, most or each non-negatively charged internucleotidic linkage, eg n001, is R p. In some embodiments, most or each non-negatively charged internucleotidic linkage, eg n001, is Sp . In some embodiments, oligonucleotides do not contain natural phosphate linkages. In some embodiments, each internucleotidic linkage is independently a phosphorothioate or non-negatively charged internucleotidic linkage. In some embodiments, each internucleotidic linkage is independently a phosphorothioate or neutrally charged internucleotidic linkage. In some embodiments, each internucleotide linkage is independently a phosphorothioate or phosphoryl guanidine internucleotide linkage. In some embodiments, each internucleotide linkage is independently a phosphorothioate or n001 internucleotide linkage. In some embodiments, the last internucleotide linkage of an oligonucleotide is a non-negatively charged internucleotide linkage, a neutral internucleotide linkage, a phosphoryl guanidine internucleotide linkage, or n001.

In some embodiments, an oligonucleotide of the invention comprises one or more modified nucleobases. Various modifications can be introduced to sugars and/or nucleobases according to the present invention. For example, in some embodiments, the modification is a modification described in US 9006198. In some embodiments, modifications are described in US 9394333, US 9744183, US 9605019, US 9982257, US 20170037399, US 20180216108, US 20180216107, US 9598458, WO 2017/062862, WO 2018/06 7973, WO 2017/160741, WO 2017/192679 , WO 2017/210647, WO 2018/098264, WO 2018/022473, WO 2018/223056, WO 2018/223073, WO 2018/223081, WO 2018/237194, WO 2019/032607, WO 2019/0 32612, WO 2019/055951 , WO 2019/075357, WO 2019/200185, WO 2019/217784, WO 2019/032612, WO 2020/191252, and/or WO 2021/071858, wherein each sugar, base, and internucleotide linkage is independently is incorporated herein by reference.

In some embodiments, the nucleobase of the nucleoside is BA-I, BA-I-a, BA-I-b, BA-II, BA-II-a, BA-II-b, BA-III, BA-III-a, Ring BA or tautomers of Ring BA having structures BA-III-b, BA-IV, BA-IV-a, BA-IV-b, BA-V, BA-V-a, BA-V-b, or BA-VI is or comprises, wherein the nucleobase is optionally substituted or protected.

In some embodiments, the sugar is a modified sugar including a 2'-modification, e.g., 2'-F, 2'-OR (R is optionally substituted aliphatic), or a bicyclic sugar (eg, LNA sugar), or It is an acyclic sugar (e.g. UNA sugar).

In some embodiments, as described herein, provided oligonucleotides include one or more domains, each domain independently having a particular length, strain, linkage phosphorus stereochemistry, etc., as described herein. In some embodiments, the present invention is an oligonucleotide comprising one or more modified sugars and/or one or more modified internucleotidic linkages, comprising a first domain and a second domain, each independently comprising one or more nucleobases. It provides oligonucleotides that do. In some embodiments, the present invention provides oligonucleotides comprising one or more domains and/or subdomains as described herein. In some embodiments, the present invention provides an oligonucleotide comprising a first domain as described herein. In some embodiments, the present invention provides an oligonucleotide comprising a second domain as described herein. In some embodiments, the present invention provides an oligonucleotide comprising a first subdomain as described herein. In some embodiments, the present invention provides an oligonucleotide comprising a second subdomain as described herein. In some embodiments, the present invention provides an oligonucleotide comprising a third subdomain as described herein. In some embodiments, the invention provides one or more regions each independently selected from a first domain, a second domain, a first subdomain, a second subdomain, and a third subdomain, each of which is independently as described herein. Provides an oligonucleotide comprising a. In some embodiments, the present invention

first domain; and

Provides an oligonucleotide comprising a second domain,

At this time,

the first domain comprises one or more 2'-F modifications;

The second domain contains one or more sugars without 2'-F modifications.

In some embodiments, an oligonucleotide or portion thereof (eg, a first domain, a second domain, a first subdomain, a second subdomain, a third subdomain, etc.) comprises a certain level of a modified sugar. In some embodiments, a modified sugar comprises a 2'-modification. In some embodiments, a modified sugar is a bicyclic sugar. In some embodiments, the modified sugar is acyclic (eg, by breaking the C2-C3 bonds of the corresponding cyclic sugar). In some embodiments, a modified sugar comprises a 5'-modification. In general, oligonucleotides of the present invention have a free 5'-OH at the 5'-end and a free 3'-OH at the 3'-end, eg, unless the context dictates otherwise. In some embodiments, the 5'-terminal sugar of an oligonucleotide can include a modified 5'-OH.

In some embodiments, the level is, for example, about 5% to 100%, about 10% to 100%, 20 to 100%, 30% to 100%, 40% to 100% of all sugars in each oligonucleotide or portion thereof. , 50%~80%, 50%~85%, 50%~90%, 50%~95%, 60%~80%, 60%~85%, 60%~90%, 60%~95%, 60 %~100%, 65%~80%, 65%~85%, 65%~90%, 65%~95%, 65%~100%, 70%~80%, 70%~85%, 70%~ 90%, 70% to 95%, 70% to 100%, 75% to 80%, 75% to 85%, 75% to 90%, 75% to 95%, 75% to 100%, 80% to 85% , 80%~90%, 80%~95%, 80%~100%, 85%~90%, 85%~95%, 85%~100%, 90%~95%, 90%~100%, 10 %, 20%, 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%. In some embodiments, the percentage is at least about 50%. In some embodiments, the percentage is at least about 55%. In some embodiments, the percentage is at least about 60%. In some embodiments, the percentage is at least about 65%. In some embodiments, the percentage is at least about 70%. In some embodiments, the percentage is at least about 75%. In some embodiments, the percentage is at least about 80%. In some embodiments, the percentage is at least about 85%. In some embodiments, the percentage is at least about 90%. In some embodiments, the percentage is at least about 95%. In some embodiments, the percentage is about 100%.

In some embodiments, the majority is at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more. In some embodiments, the majority is between about 50%-100%, 50%-80%, 50%-85%, 50%-90%, 50%-95%, 60%-80%, 60%-85%, 60%~90%, 60%~95%, 60%~100%, 65%~80%, 65%~85%, 65%~90%, 65%~95%, 65%~100%, 70% ~80%, 70%~85%, 70%~90%, 70%~95%, 70%~100%, 75%~80%, 75%~85%, 75%~90%, 75%~95 %, 75%~100%, 80%~85%, 80%~90%, 80%~95%, 80%~100%, 85%~90%, 85%~95%, 85%~100%, 90%~95%, 90%~100%, 10%, 20%, 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% , or 100%. In some embodiments, the majority is about or at least about 50%. In some embodiments, the majority is about or at least about 55%. In some embodiments, the majority is about or at least about 60%. In some embodiments, the majority is about or at least about 65%. In some embodiments, the majority is about or at least about 70%. In some embodiments, the majority is about or at least about 75%. In some embodiments, the majority is about or at least about 80%. In some embodiments, the majority is about or at least about 85%. In some embodiments, the majority is about or at least about 90%. In some embodiments, the majority is about or at least about 95%.

In some embodiments, an oligonucleotide or portion thereof (e.g., a first domain, a second domain, a first subdomain, a second subdomain, a third subdomain, etc.) comprises a certain level of modified internucleotidic linkages. . In some embodiments, an oligonucleotide or portion thereof (e.g., a first domain, a second domain, a first subdomain, a second subdomain, a third subdomain, etc.) comprises a certain level of chiral internucleotidic linkages. . In some embodiments, the level is, for example, about 5% to 100%, about 10% to 100%, 20 to 100%, 30% to 100%, 40% of all internucleotide linkages in each oligonucleotide or portion thereof. ~100%, 50%~80%, 50%~85%, 50%~90%, 50%~95%, 60%~80%, 60%~85%, 60%~90%, 60%~95 %, 60%~100%, 65%~80%, 65%~85%, 65%~90%, 65%~95%, 65%~100%, 70%~80%, 70%~85%, 70% to 90%, 70% to 95%, 70% to 100%, 75% to 80%, 75% to 85%, 75% to 90%, 75% to 95%, 75% to 100%, 80% ~85%, 80%~90%, 80%~95%, 80%~100%, 85%~90%, 85%~95%, 85%~100%, 90%~95%, 90%~100 %, 10%, 20%, 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%. In some embodiments, the percentage is at least about 50%. In some embodiments, the percentage is at least about 55%. In some embodiments, the percentage is at least about 60%. In some embodiments, the percentage is at least about 65%. In some embodiments, the percentage is at least about 70%. In some embodiments, the percentage is at least about 75%. In some embodiments, the percentage is at least about 80%. In some embodiments, the percentage is at least about 85%. In some embodiments, the percentage is at least about 90%. In some embodiments, the percentage is at least about 95%. In some embodiments, the percentage is about 100%.

In some embodiments, an oligonucleotide or portion thereof (e.g., a first domain, a second domain, a first subdomain, a second subdomain, a third subdomain, etc.) comprises a degree of chirally controlled internucleotidic linkage do. In some embodiments, an oligonucleotide or portion thereof (e.g., a first domain, a second domain, a first subdomain, a second subdomain, a third subdomain, etc.) comprises a certain level of Sp internucleotidic linkages. do. In some embodiments, the level is, for example, about 5% to 100%, about 10% to 100%, 20 to 100%, 30% to 100%, 40% of all internucleotide linkages in each oligonucleotide or portion thereof. ~100%, 50%~80%, 50%~85%, 50%~90%, 50%~95%, 60%~80%, 60%~85%, 60%~90%, 60%~95 %, 60%~100%, 65%~80%, 65%~85%, 65%~90%, 65%~95%, 65%~100%, 70%~80%, 70%~85%, 70% to 90%, 70% to 95%, 70% to 100%, 75% to 80%, 75% to 85%, 75% to 90%, 75% to 95%, 75% to 100%, 80% ~85%, 80%~90%, 80%~95%, 80%~100%, 85%~90%, 85%~95%, 85%~100%, 90%~95%, 90%~100 %, 10%, 20%, 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%. In some embodiments, the level is, for example, between about 5% and 100%, about 10% and 100%, 20 and 100%, 30% and 100%, 40% of all chiral internucleotide linkages in each oligonucleotide or portion thereof. %~100%, 50%~80%, 50%~85%, 50%~90%, 50%~95%, 60%~80%, 60%~85%, 60%~90%, 60%~ 95%, 60% to 100%, 65% to 80%, 65% to 85%, 65% to 90%, 65% to 95%, 65% to 100%, 70% to 80%, 70% to 85% , 70%~90%, 70%~95%, 70%~100%, 75%~80%, 75%~85%, 75%~90%, 75%~95%, 75%~100%, 80 %~85%, 80%~90%, 80%~95%, 80%~100%, 85%~90%, 85%~95%, 85%~100%, 90%~95%, 90%~ 100%, 10%, 20%, 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%. In some embodiments, the percentage is at least about 50%. In some embodiments, the percentage is at least about 55%. In some embodiments, the percentage is at least about 60%. In some embodiments, the percentage is at least about 65%. In some embodiments, the percentage is at least about 70%. In some embodiments, the percentage is at least about 75%. In some embodiments, the percentage is at least about 80%. In some embodiments, the percentage is at least about 85%. In some embodiments, the percentage is at least about 90%. In some embodiments, the percentage is at least about 95%. In some embodiments, the percentage is about 100%.

In some embodiments, an oligonucleotide or portion thereof (e.g., a first domain, a second domain, a first subdomain, a second subdomain, a third subdomain, etc.) comprises a certain level of Sp internucleotidic linkages. do. In some embodiments, the level is, for example, about 5% to 100%, about 10% to 100%, 20 to 100%, 30% to 100%, 40% of all internucleotide linkages in each oligonucleotide or portion thereof. ~100%, 50%~80%, 50%~85%, 50%~90%, 50%~95%, 60%~80%, 60%~85%, 60%~90%, 60%~95 %, 60%~100%, 65%~80%, 65%~85%, 65%~90%, 65%~95%, 65%~100%, 70%~80%, 70%~85%, 70% to 90%, 70% to 95%, 70% to 100%, 75% to 80%, 75% to 85%, 75% to 90%, 75% to 95%, 75% to 100%, 80% ~85%, 80%~90%, 80%~95%, 80%~100%, 85%~90%, 85%~95%, 85%~100%, 90%~95%, 90%~100 %, 10%, 20%, 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%. In some embodiments, the level is, for example, between about 5% and 100%, about 10% and 100%, 20 and 100%, 30% and 100%, 40% of all chiral internucleotide linkages in each oligonucleotide or portion thereof. %~100%, 50%~80%, 50%~85%, 50%~90%, 50%~95%, 60%~80%, 60%~85%, 60%~90%, 60%~ 95%, 60% to 100%, 65% to 80%, 65% to 85%, 65% to 90%, 65% to 95%, 65% to 100%, 70% to 80%, 70% to 85% , 70%~90%, 70%~95%, 70%~100%, 75%~80%, 75%~85%, 75%~90%, 75%~95%, 75%~100%, 80 %~85%, 80%~90%, 80%~95%, 80%~100%, 85%~90%, 85%~95%, 85%~100%, 90%~95%, 90%~ 100%, 10%, 20%, 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%. In some embodiments, the level is, for example, about 5% to 100%, about 10% to 100%, 20 to 100%, 30% to 100% of all chiral control internucleotide linkages in each oligonucleotide or portion thereof, 40%~100%, 50%~80%, 50%~85%, 50%~90%, 50%~95%, 60%~80%, 60%~85%, 60%~90%, 60% ~95%, 60%~100%, 65%~80%, 65%~85%, 65%~90%, 65%~95%, 65%~100%, 70%~80%, 70%~85 %, 70%~90%, 70%~95%, 70%~100%, 75%~80%, 75%~85%, 75%~90%, 75%~95%, 75%~100%, 80%~85%, 80%~90%, 80%~95%, 80%~100%, 85%~90%, 85%~95%, 85%~100%, 90%~95%, 90% ~100%, 10%, 20%, 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%. In some embodiments, the percentage is at least about 50%. In some embodiments, the percentage is at least about 55%. In some embodiments, the percentage is at least about 60%. In some embodiments, the percentage is at least about 65%. In some embodiments, the percentage is at least about 70%. In some embodiments, the percentage is at least about 75%. In some embodiments, the percentage is at least about 80%. In some embodiments, the percentage is at least about 85%. In some embodiments, the percentage is at least about 90%. In some embodiments, the percentage is at least about 95%. In some embodiments, the percentage is about 100%. In some embodiments, about 1-50, 1-40, 1-30, for example about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 , 15, 16, 17, 18, 19, or 20 internucleotide linkages are independently Sp chiral internucleotide linkages. In many embodiments, a high (e.g., higher than R p internucleotidic linkage and/or higher than native phosphate linkage) percentage of S p internucleotidic linkages in an oligonucleotide or a particular portion thereof may result in improved properties and/or activity; For example, it has been observed to provide high stability and/or high adenosine editing activity.

In some embodiments, an oligonucleotide or portion thereof (eg, a first domain, a second domain, a first subdomain, a second subdomain, a third subdomain, etc.) comprises specific R p internucleotidic linkages. In some embodiments, the level is, for example, about 5% to 100%, about 10% to 100%, 20 to 100%, 30% to 100%, 40% of all internucleotide linkages in each oligonucleotide or portion thereof. ~100%, 50%~80%, 50%~85%, 50%~90%, 50%~95%, 60%~80%, 60%~85%, 60%~90%, 60%~95 %, 60%~100%, 65%~80%, 65%~85%, 65%~90%, 65%~95%, 65%~100%, 70%~80%, 70%~85%, 70% to 90%, 70% to 95%, 70% to 100%, 75% to 80%, 75% to 85%, 75% to 90%, 75% to 95%, 75% to 100%, 80% ~85%, 80%~90%, 80%~95%, 80%~100%, 85%~90%, 85%~95%, 85%~100%, 90%~95%, 90%~100 %, 10%, 20%, 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%. In some embodiments, the level is, for example, between about 5% and 100%, about 10% and 100%, 20 and 100%, 30% and 100%, 40%, of all chiral internucleotide linkages in each oligonucleotide or portion thereof. %~100%, 50%~80%, 50%~85%, 50%~90%, 50%~95%, 60%~80%, 60%~85%, 60%~90%, 60%~ 95%, 60% to 100%, 65% to 80%, 65% to 85%, 65% to 90%, 65% to 95%, 65% to 100%, 70% to 80%, 70% to 85% , 70%~90%, 70%~95%, 70%~100%, 75%~80%, 75%~85%, 75%~90%, 75%~95%, 75%~100%, 80 %~85%, 80%~90%, 80%~95%, 80%~100%, 85%~90%, 85%~95%, 85%~100%, 90%~95%, 90%~ 100%, 10%, 20%, 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%. In some embodiments, the level is, for example, about 5% to 100%, about 10% to 100%, 20 to 100%, 30% to 100% of all chiral control internucleotide linkages in each oligonucleotide or portion thereof, 40%~100%, 50%~80%, 50%~85%, 50%~90%, 50%~95%, 60%~80%, 60%~85%, 60%~90%, 60% ~95%, 60%~100%, 65%~80%, 65%~85%, 65%~90%, 65%~95%, 65%~100%, 70%~80%, 70%~85 %, 70%~90%, 70%~95%, 70%~100%, 75%~80%, 75%~85%, 75%~90%, 75%~95%, 75%~100%, 80%~85%, 80%~90%, 80%~95%, 80%~100%, 85%~90%, 85%~95%, 85%~100%, 90%~95%, 90% ~100%, 10%, 20%, 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%. In some embodiments, the percentage is at least about 50%. In some embodiments, the percentage is at least about 55%. In some embodiments, the percentage is at least about 60%. In some embodiments, the percentage is at least about 65%. In some embodiments, the percentage is at least about 70%. In some embodiments, the percentage is at least about 75%. In some embodiments, the percentage is at least about 80%. In some embodiments, the percentage is at least about 85%. In some embodiments, the percentage is at least about 90%. In some embodiments, the percentage is at least about 95%. In some embodiments, the percentage is about 100%. In some embodiments, the percentage is about or up to about 5%. In some embodiments, the percentage is about or up to about 10%. In some embodiments, the percentage is about or up to about 15%. In some embodiments, the percentage is about or up to about 20%. In some embodiments, the percentage is about or up to about 25%. In some embodiments, the percentage is about or up to about 30%. In some embodiments, the percentage is about or up to about 35%. In some embodiments, the percentage is about or up to about 40%. In some embodiments, the percentage is about or up to about 45%. In some embodiments, the percentage is about or up to about 50%. In some embodiments, about 1-50, 1-40, 1-30, for example about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 , 15, 16, 17, 18, 19, or 20 internucleotide linkages are independently R p chiral internucleotide linkages. In some embodiments, the number is about or up to about 1. In some embodiments, the number is about or up to about 2. In some embodiments, the number is about or up to about 3. In some embodiments, the number is about or up to about 4. In some embodiments, the number is about or up to about 5. In some embodiments, the number is about or up to about 6. In some embodiments, the number is about or up to about 7. In some embodiments, the number is about or up to about 8. In some embodiments, the number is about or up to about 9. In some embodiments, the number is about or up to about 10.

Without wishing to be bound by theory, it is believed that in some cases the R p and Sp arrangement of internucleotidic linkages may affect structural changes in the helical conformation of a double-stranded complex formed by an oligonucleotide and a target nucleic acid, such as RNA, and ADAR proteins. It is noted that this multi-domain can recognize and interact with various targets (eg, double-stranded complexes formed by oligonucleotides and target nucleic acids such as RNA). In some embodiments, provided oligonucleotides and compositions thereof promote and/or enhance the interaction profile of oligonucleotides, target nucleic acids, and/or ADAR proteins through incorporation of various modifications and/or control of stereochemistry to bind to ADAR proteins. Provides efficient adenosine modification by

In some embodiments, an oligonucleotide comprises a base sequence; internucleotide linkages, base modifications, sugar modifications, additional chemical moieties, or patterns thereof; and/or may have or include any other structural element described herein (eg, in a Table).

In some embodiments, a provided oligonucleotide or composition, when contacted with a target nucleic acid comprising a target adenosine in a system (eg, an ADAR-mediated deamination system), modifies a target adenosine (eg, deamination of target A). amination) is characterized as being improved over that observed at baseline conditions (eg, selected from the group consisting of the absence of the composition, the presence of the reference oligonucleotide or composition, and combinations thereof). In some embodiments, the modification, e.g., ADAR-mediated deamination (e.g., endogenous ADAR-mediated deamination), is 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 , 24, 25, 26, 27, 28, 29, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000 times or more .

In some embodiments, oligonucleotides are provided in salt form. In some embodiments, an oligonucleotide is provided as a salt comprising negatively charged internucleotidic linkages (eg, phosphorothioate internucleotidic linkages, natural phosphate linkages, etc.) present in its salt form. In some embodiments, oligonucleotides are provided as pharmaceutically acceptable salts. In some embodiments, oligonucleotides are provided as metal salts. In some embodiments, oligonucleotides are provided as sodium salts. In some embodiments, oligonucleotides are provided as ammonium salts. In some embodiments, the oligonucleotide is provided as a metal salt, e.g., sodium salt, wherein each negatively charged internucleotide linkage is independently a salt form (e.g., in the sodium salt, phosphorothioate internucleotidic linkages -O-P(O)(SNa)-O- for , -O-P(O)(ONa)-O- for natural phosphate linkages, etc.).

In some embodiments, the oligonucleotide comprises one or more chirally controlled internucleotide linkages and is chirally controlled. In some embodiments, provided oligonucleotides are stereochemically pure. In some embodiments, provided oligonucleotides or compositions thereof are substantially free of other stereoisomers. In some embodiments, the present invention provides chirally controlled oligonucleotide compositions.

As described herein, oligonucleotides of the present invention may be provided in high purity (eg, 50%-100%). In some embodiments, oligonucleotides of the invention have high stereochemical purity (eg, 50%-100%). In some embodiments, oligonucleotides in a provided composition have high stereochemical purity (eg, a high proportion (eg, 50%-100%) of stereoisomers relative to other stereoisomers of the same oligonucleotide). In some embodiments, the percentage is at least or about 50%. In some embodiments, the percentage is at least or about 60%. In some embodiments, the percentage is at least or about 70%. In some embodiments, the percentage is at least or about 75%. In some embodiments, the percentage is at least or about 80%. In some embodiments, the percentage is at least or about 85%. In some embodiments, the percentage is at least or about 90%. In some embodiments, the percentage is at least or about 95%.

first domain

As described herein, in some embodiments, an oligonucleotide comprises a first domain and a second domain. In some embodiments, an oligonucleotide is composed of a first domain and a second domain. Specific implementations are described below by way of example.

In some embodiments, the first domain is about 2-50 (e.g., about 5, 6, 7, 8, 9, or 10 to about 10, 11, 12, 13, 14, 15, 16, 17 , 18, 19, 20, 21, 22, 23, 24, 25, 30, 40 or 50, or about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 40 or 50, etc.) nucleobases in length. In some embodiments, the first domain is about 5-30 nucleobases in length. In some embodiments, the first domain is about 10-30 nucleobases in length. In some embodiments, the first domain is about 10-20 nucleobases in length. In some embodiments, the first domain is about 13-16 nucleobases in length. In some embodiments, the first domain is 10 nucleobases in length. In some embodiments, the first domain is 11 nucleobases in length. In some embodiments, the first domain is 12 nucleobases in length. In some embodiments, the first domain is 13 nucleobases in length. In some embodiments, the first domain is 14 nucleobases in length. In some embodiments, the first domain is 15 nucleobases in length. In some embodiments, the first domain is 16 nucleobases in length. In some embodiments, the first domain is 17 nucleobases in length. In some embodiments, the first domain is 18 nucleobases in length. In some embodiments, the first domain is 19 nucleobases in length. In some embodiments, the first domain is 20 nucleobases in length.

In some embodiments, the first domain comprises about or at least about 5-95%, 10%-90%, 20%-80%, 30%-70%, 40%-70%, 40%-60% of the oligonucleotide. , 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85 %, 90%, 95%. In some embodiments, the percentage is between about 30% and 80%. In some embodiments, the percentage is between about 30% and 70%. In some embodiments, the percentage is between about 40% and 60%. In some embodiments, the percentage is about 20%. In some embodiments, the percentage is about 25%. In some embodiments, the percentage is about 30%. In some embodiments, the percentage is about 35%. In some embodiments, the percentage is about 40%. In some embodiments, the percentage is about 45%. In some embodiments, the percentage is about 50%. In some embodiments, the percentage is about 55%. In some embodiments, the percentage is about 60%. In some embodiments, the percentage is about 65%. In some embodiments, the percentage is about 70%. In some embodiments, the percentage is about 75%. In some embodiments, the percentage is about 80%. In some embodiments, the percentage is about 85%. In some embodiments, the percentage is about 90%.

In some embodiments, the first domain contains one or more (e.g., 1-20, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, etc.) In some implementations, there is 1 mismatch. In some implementations, there are two mismatches. In some implementations, there are three mismatches. In some implementations, there are 4 mismatches. In some implementations, there are 5 mismatches. In some implementations, there are 6 mismatches. In some implementations, there are 7 mismatches. In some implementations, there are 8 mismatches. In some implementations, there are 9 mismatches. In some implementations, there are 10 mismatches.

In some embodiments, the first domain contains one or more (e.g., 1-20, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, etc.) wobbles exist. In some implementations, there is 1 wobble. In some implementations, there are two wobbles. In some implementations, there are three wobbles. In some implementations, there are 4 wobbles. In some implementations, there are 5 wobbles. In some implementations, there are 6 wobbles. In some implementations, there are 7 wobbles. In some implementations, there are 8 wobbles. In some implementations, there are 9 wobbles. In some implementations, there are 10 wobbles.

In some embodiments, the duplex of the oligonucleotide in the first domain region and the target nucleic acid comprises one or more bulges, each independently comprising one or more mismatches that are not wobbles. In some embodiments, 0-10 (e.g., 0-1, 0-2, 0-3, 0-4, 0-5, 0-6, 0-7, 0-8, 0-9, 0~10, 1~2, 1~3, 1~4, 1~5, 1~6, 1~7, 1~8, 1~9, 1~10, 2~3, 2~4, 2~ 5, 2~6, 2~7, 2~8, 2~9, 2~10, 3~4, 3~5, 3~6, 3~7, 3~8, 3~9, 3~10, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 bulges). In some embodiments, the number is zero. In some embodiments, the number is one. In some embodiments, the number is two. In some embodiments, the number is three. In some embodiments, the number is four. In some embodiments, the number is five.

In some embodiments, the first domain is fully complementary to the target nucleic acid.

In some embodiments, the first domain comprises one or more modified nucleobases.

In some embodiments, the second domain comprises one or more sugars (eg, natural DNA sugars) comprising two 2'-Hs. In some embodiments, the second domain comprises one or more sugars (eg, native RNA sugars) comprising 2'-OH. In some embodiments, the first domain comprises one or more modified sugars. In some embodiments, a modified sugar comprises a 2'-modification. In some embodiments, the modified sugar is a bicyclic sugar, such as an LNA sugar. In some embodiments, the modified sugar is acyclic (eg, by breaking the C2-C3 bonds of the corresponding cyclic sugar).

In some embodiments, the first domain is about 1-50 (e.g., about 5, 6, 7, 8, 9, or 10 to about 10, 11, 12, 13, 14, 15, 16, 17 , 18, 19, 20, 21, 22, 23, 24, 25, 30, 40 or 50, or about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 40 or 50 etc, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 , 13, 14, 15, 16, 17, 18, 19, or 20, etc.) modified sugars. In some embodiments, the first domain contains about 1-50 (e.g., about 5, 6, 7, 8, 9, or 10 to about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 40 or 50, or about 5, 6, 7, 8, 9, 10, 11, 12, 13 , 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 40 or 50, etc., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, etc.) modified sugars. In some embodiments, the first domain has between about 2 and 50 (e.g., about 2, 3, 4, 5, 6, 7, 8, 9, or 10 to about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 40 or 50, or about 5, 6, 7, 8, 9, 10 , 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 40 or 50, etc., 2 to 40, 2 to 30, 2 to 25 , 2~20, 2~15, 2~10, 3~40, 3~30, 3~25, 3~20, 3~15, 3~10, 4~40, 4~30, 4~25, 4 ~20, 4~15, 4~10, 5~40, 5~30, 5~25, 5~20, 5~15, 5~10, 6~40, 6~30, 6~25, 6~20 , 6~15, 6~10, 7~40, 7~30, 7~25, 7~20, 7~15, 7~10, 8~40, 8~30, 8~25, 8~20, 8 ~15, 8~10, 9~40, 9~30, 9~25, 9~20, 9~15, 9~10, 10~40, 10~30, 10~25, 10~20, 10~15 about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 consecutive strains, etc. do. In some embodiments, the first domain comprises two consecutive 2'-F modified sugars. In some embodiments, the first domain comprises three consecutive 2'-F modified sugars. In some embodiments, the first domain comprises four consecutive 2'-F modified sugars. In some embodiments, the first domain comprises 5 contiguous 2'-F modified sugars. In some embodiments, the first domain comprises 6 contiguous 2'-F modified sugars. In some embodiments, the first domain comprises 7 contiguous 2'-F modified sugars. In some embodiments, the first domain comprises 8 contiguous 2'-F modified sugars. In some embodiments, the first domain comprises 9 contiguous 2'-F modified sugars. In some embodiments, the first domain comprises 10 contiguous 2'-F modified sugars. In some embodiments, the first domain comprises two or more blocks per 2'-F modification, wherein each sugar in the block per 2'-F modification is independently a 2'-F modification sugar. In some embodiments, each block per 2'-F variant independently comprises 2, 3, 4, 5, 6, 7, 8, 9 or 10 contiguous 2'-F variant sugars as described herein; consists of this In some embodiments, two contiguous 2'-F variant sugar blocks are independently separated by a separating block comprising one or more sugars that are not 2'-F variant sugars. In some embodiments, each sugar in the separation block is independently not 2'-F modified. In some embodiments, two or more (eg, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more) or all sugars in the separation block are independently 2'-F modified. In some embodiments, the separating block comprises one or more bicyclic sugars (eg, LNA sugars, cEt sugars, etc.) and/or one or more 2'-OR modified sugars (R is optionally substituted C _1-6 aliphatic) (eg For example, 2'-OMe, 2'-MOE, etc.). In some embodiments, the separating block comprises one or more 2'-OR modified sugars, where R is an optionally substituted C _1-6 aliphatic (eg, 2'-OMe, 2'-MOE, etc.). In some embodiments, two or more non-2'-F modified sugars are contiguous. In some embodiments, two or more 2'-OR modified sugars (R is an optionally substituted C _1-6 aliphatic (eg, 2'-OMe, 2'-MOE, etc.)) are contiguous. In some embodiments, the separating block comprises two or more (eg, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more) 2'-OR modified sugars, wherein R is optionally substituted C _1-6 aliphatic (eg, 2'-OMe, 2'-MOE, etc.). In some embodiments, the split block comprises two or more (eg, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more) consecutive 2'-OR modified sugars, wherein R is an optionally substituted C _1-6 aliphatic (eg, 2'-OMe, 2'-MOE, etc.). In some embodiments, each 2'-OR modified sugar is independently a 2'-OMe or 2'-MOE sugar. In some embodiments, each 2'-OR modified sugar is independently a 2'-OMe sugar. In some embodiments, each 2'-OR modified sugar is independently a 2'-MOE sugar. In some embodiments, the separating block comprises one or more 2'-F modified sugars. In some embodiments, none of the 2'-F strain sugars in the separating block are next to each other. In some embodiments, the separating block does not contain 2'-F modified sugars. In some embodiments, each sugar in the separating block is independently a 2′-OR modified sugar (R is an optionally substituted C _1-6 aliphatic) or a bicyclic sugar. In some embodiments, each sugar in each separating block is independently a 2′-OR modified sugar (R is an optionally substituted C _1-6 aliphatic) or a bicyclic sugar. In some embodiments, each sugar in the separating block is independently a 2'-OR modified sugar, where R is an optionally substituted C _1-6 aliphatic. In some embodiments, each sugar in each separating block is independently a 2'-OR modified sugar, where R is an optionally substituted C _1-6 aliphatic. In some embodiments, each sugar in the separation block is independently a 2'-OMe or 2'-MOE modified sugar. In some embodiments, each sugar in each separation block is independently a 2'-OMe or 2'-MOE modified sugar. In some embodiments, each sugar in the separation block is independently a 2'-OMe modified sugar. In some embodiments, each sugar in the separation block is independently a 2'-MOE modified sugar. In some embodiments, the separating block comprises a 2'-OMe sugar and a 2'-MOE modified sugar. In some embodiments, each 2'-F block and each separating block independently contains 1, 2, 3, 4 or 5 nucleosides. In some embodiments, each 2'-F block and each separating block independently contains 1, 2 or 3 nucleosides.

In some embodiments, about 5%-100% (e.g., about 10%-100%, 20-100%, 30%-100%, 40%-100%, 50%-100%) of all sugars in the first domain. 80%, 50%~85%, 50%~90%, 50%~95%, 60%~80%, 60%~85%, 60%~90%, 60%~95%, 60%~100% , 65%~80%, 65%~85%, 65%~90%, 65%~95%, 65%~100%, 70%~80%, 70%~85%, 70%~90%, 70 %~95%, 70%~100%, 75%~80%, 75%~85%, 75%~90%, 75%~95%, 75%~100%, 80%~85%, 80%~ 90%, 80% to 95%, 80% to 100%, 85% to 90%, 85% to 95%, 85% to 100%, 90% to 95%, 90% to 100%, 10%, 20% , 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%, etc.) are independently modified sugars. In some embodiments, about 5%-100% (e.g., about 10%-100%, 20-100%, 30%-100%, 40%-100%, 50%-100%) of all sugars in the first domain. 80%, 50%~85%, 50%~90%, 50%~95%, 60%~80%, 60%~85%, 60%~90%, 60%~95%, 60%~100% , 65%~80%, 65%~85%, 65%~90%, 65%~95%, 65%~100%, 70%~80%, 70%~85%, 70%~90%, 70 %~95%, 70%~100%, 75%~80%, 75%~85%, 75%~90%, 75%~95%, 75%~100%, 80%~85%, 80%~ 90%, 80% to 95%, 80% to 100%, 85% to 90%, 85% to 95%, 85% to 100%, 90% to 95%, 90% to 100%, 10%, 20% , 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%, etc.) are independently per 2'-F modification. In some embodiments, the percentage is at least about 40%. In some embodiments, the percentage is at least about 50%. In some embodiments, the percentage is at least about 55%. In some embodiments, the percentage is at least about 60%. In some embodiments, the percentage is at least about 65%. In some embodiments, the percentage is at least about 70%. In some embodiments, the percentage is at least about 75%. In some embodiments, the percentage is at least about 80%. In some embodiments, the percentage is at least about 85%. In some embodiments, the percentage is at least about 90%. In some embodiments, the percentage is at least about 95%. In some embodiments, the percentage is about 100%. In some embodiments, the percentage is about or up to about 60%. In some embodiments, the percentage is about or up to about 70%. In some embodiments, the percentage is about or up to about 80%. In some embodiments, the percentage is about or up to about 90%.

In some embodiments, the first domain does not include a heterocyclic sugar or a 2'-OR modified sugar (R is not -H). In some embodiments, the first domain is per one or more (eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) bicyclics and/or 2'-OR variants. (R is not -H). In some embodiments, the first domain is per one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) 2'-OR variants (R is -H not). In some embodiments, the first domain is per one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) 2'-OR variants (R is optionally substituted). is C _1-10 aliphatic). In some embodiments, the level of bicyclic sugars and/or 2'-OR modified sugars (R is not -H), individually or in combination, is relatively low compared to 2'-F modified sugars. In some embodiments, the level of bicyclic sugars and/or 2'-OR modified sugars (R is not -H), individually or in combination, is between about 10% and 80% (e.g., between about 10% and 75%). , 10% to 70%, 10% to 65%, 10% to 60%, 10% to 50%, about 20% to 60%, about 30% to 60%, about 20% to 50%, about 30% to 50 %, about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, or 60%, etc.). In some embodiments, the level of 2'-OR variant sugars (R is not -H) is between about 10 and 70 in combination (e.g., the combination of 2'-OMe and 2'-MOE variant sugars, if present). % (e.g., about 10% to 60%, 10% to 50%, about 20% to 60%, about 30% to 60%, about 20% to 50%, about 30 to 50%, about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, or 60%, etc.). In some embodiments, up to about 1% to 95% (e.g., up to about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%) of the sugars in the first domain. %, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, etc.) contains 2'-OMe. In some embodiments, up to about 50% of the sugars in the first domain comprise 2'-OMe. In some embodiments, up to about 1% to 95% (e.g., up to about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%) of the sugars in the first domain. %, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, etc.) contains a 2'-OR (R is optionally substituted C _1-6 is aliphatic). In some embodiments, up to about 50% of the sugars in the first domain comprise a 2'-OR (R is an optionally substituted C _1-6 aliphatic). In some embodiments, up to about 40% of the sugars in the first domain comprise a 2'-OR (R is an optionally substituted C _1-6 aliphatic). In some embodiments, up to about 30% of the sugars in the first domain comprise a 2'-OR (R is an optionally substituted C _1-6 aliphatic). In some embodiments, up to about 25% of the sugars in the first domain comprise a 2'-OR (R is an optionally substituted C _1-6 aliphatic). In some embodiments, up to about 20% of the sugars in the first domain comprise a 2'-OR (R is an optionally substituted C _1-6 aliphatic). In some embodiments, up to about 10% of the sugars in the first domain comprise a 2'-OR (R is an optionally substituted C _1-6 aliphatic). In some embodiments, as described herein, a 2'-OR is a 2'-MOE. In some embodiments, as described herein, a 2'-OR is 2'-MOE or 2'-OMe. In some embodiments, the first domain comprises _one or more (eg, about 1-20, 1, 2, 3, 4, 5, 6, 7, 8, 9 , 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, etc.) modified sugars. In some embodiments, the _first domain comprises one or more (eg, about 1-20, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10; 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, etc.) modified sugars. In some embodiments, the first domain is one or more (e.g., about 1-20, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 , 16, 17, 18, 19, 20, etc.) of heterocyclic sugars (eg, LNA sugars). In some embodiments, the first domain is one or more (e.g., about 1-20, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 , 16, 17, 18, 19, 20, etc.) of acyclic sugars (eg, UNA sugars). In some embodiments, multiple 5'-terminal sugars in the first domain are independently 2'-OR modified sugars (R is not -H). In some embodiments, multiple (eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) 5'-end sugars in the first domain are independently 2'- OR is a modified sugar (R is independently an optionally substituted C _1-6 aliphatic). In some embodiments, the first about 1-10 sugars from the 5′-end of the first domain, for example 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 sugars, are independently 2 '-OR modified sugar (R is independently optionally substituted C _1-6 aliphatic). In some embodiments, the first one is 2'-OR modified. In some embodiments, the first two are independently 2'-OR modified. In some embodiments, the first three are independently 2'-OR modified. In some embodiments, the first four are independently 2'-OR modified. In some embodiments, the first 5 are independently 2'-OR modified. In some embodiments, all 2'-OR modifications in a domain (eg, a first domain), subdomain (eg, a first subdomain), or oligonucleotide are the same. In some embodiments, a 2'-OR is a 2'-MOE. In some embodiments, 2'-OR is 2'-OMe.

In some embodiments, the sugar in the first domain does not include a 2'-OR. In some embodiments, the sugar in the first domain does not include 2'-OMe. In some embodiments, the sugar in the first domain does not include a 2'-MOE. In some embodiments, the sugar in the first domain does not include 2'-MOE or 2'-OMe. In some embodiments, the sugar in the first domain does not include a 2'-OR (R is an optionally substituted C _1-6 aliphatic). In some embodiments, each sugar in the first domain comprises a 2'-F.

In some embodiments, about 40-70% of the sugars in the first domain (e.g., about 40%-70%, 40%-60%, 50%-70%, 50%-60%, etc., or about 40%) %, 45%, 50%, 55%, 60%, 65%, 70%, etc.) are 2'-F modified, and about 10% to 60% of the sugars in the first domain (e.g., about 10% to about 10% to 50%, 20% to 60%, 30% to 60%, 30% to 50%, 40% to 50%, etc., or about 10%, 15%, 20%, 25%, 30%, 35%, 40% , 45%, 50%, 55%, or 60%) are independently 2'-OR variants (R is not -H) or bicyclic sugars (eg, LNA sugars, cEt sugars, etc.). In some embodiments, between about 20% and 60% of the sugars in the first domain are 2'-F modified. In some embodiments, about 25%-60% of the sugars in the first domain are 2'-F modified. In some embodiments, about 30%-60% of the sugars in the first domain are 2'-F modified. In some embodiments, about 35%-60% of the sugars in the first domain are 2'-F modified. In some embodiments, about 40%-60% of the sugars in the first domain are 2'-F modified. In some embodiments, about 50%-60% of the sugars in the first domain are 2'-F modified. In some embodiments, about 50%-70% of the sugars in the first domain are 2'-F modified. In some embodiments, about 20%-60% of the sugars in the first domain are independently 2'-OR variants (R is not -H) or bicyclic sugars. In some embodiments, about 30%-60% of the sugars in the first domain are independently 2'-OR variants (R is not -H) or bicyclic sugars. In some embodiments, about 40%-60% of the sugars in the first domain are independently 2'-OR variants (R is not -H) or bicyclic sugars. In some embodiments, about 30%-50% of the sugars in the first domain are independently 2'-OR variants (R is not -H) or bicyclic sugars. In some embodiments, about 40%-50% of the sugars in the first domain are independently 2'-OR variants (R is not -H) or bicyclic sugars. In some embodiments, each sugar in the first domain that is independently a 2'-OR variant (R is not -H) or a heterocyclic sugar is independently a 2'-OR variant sugar (R is not -H). In some embodiments, each of these is independently a 2'-OR modified sugar (R is C _1-6 aliphatic). In some embodiments, each of these is independently a 2'-OR modified sugar (R is C _1-6 alkyl). In some embodiments, each of these is independently a 2'-OMe or 2'-MOE modified sugar.

In some embodiments, the first domain is about 1-50 (e.g., about 5, 6, 7, 8, 9, or 10 to about 10, 11, 12, 13, 14, 15, 16, 17 , 18, 19, 20, 21, 22, 23, 24, 25, 30, 40 or 50, or about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 40 or 50 etc, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 , 13, 14, 15, 16, 17, 18, 19, or 20, etc.) modified internucleotide linkages. In some embodiments, about 5%-100% (e.g., about 10%-100%, 20-100%, 30%-100%, 40%-100%, 50%-100%) of the internucleotide linkages in the first domain %~80%, 50%~85%, 50%~90%, 50%~95%, 60%~80%, 60%~85%, 60%~90%, 60%~95%, 60%~ 100%, 65%~80%, 65%~85%, 65%~90%, 65%~95%, 65%~100%, 70%~80%, 70%~85%, 70%~90% , 70%~95%, 70%~100%, 75%~80%, 75%~85%, 75%~90%, 75%~95%, 75%~100%, 80%~85%, 80 %~90%, 80%~95%, 80%~100%, 85%~90%, 85%~95%, 85%~100%, 90%~95%, 90%~100%, 10%, 20%, 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%, etc.) are modified internucleotide linkages. In some embodiments, each internucleotide linkage in the first domain is independently a modified internucleotide linkage. In some embodiments, each modified internucleotide linkage is independently a chiral internucleotide linkage. In some embodiments, the modified or chiral internucleotidic linkage is a phosphorothioate internucleotidic linkage. In some embodiments, a modified or chiral internucleotidic linkage is a non-negatively charged internucleotidic linkage. In some embodiments, the modified or chiral internucleotide linkage is a neutral internucleotide linkage (eg n001). In some embodiments, each modified internucleotide linkage is independently a phosphorothioate internucleotide linkage or a non-negatively charged internucleotide linkage. In some embodiments, each modified internucleotide linkage is independently a phosphorothioate internucleotide linkage or a neutral internucleotide linkage. In some embodiments, each modified internucleotide linkage is independently a phosphorothioate internucleotide linkage. In some embodiments, at least about 1-50 (e.g., about 5, 6, 7, 8, 9, or 10 to about 10, 11, 12, 13, 14, 15, 16 in the first domain) , 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 40 or 50, or about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 40 or 50 etc, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, or 20, etc.) chiral internucleotide linkages are chirally controlled. In some embodiments, at least 5%-100% (e.g., about 10%-100%, 20-100%, 30%-100%, 40%-100%, 50%~80%, 50%~85%, 50%~90%, 50%~95%, 60%~80%, 60%~85%, 60%~90%, 60%~95%, 60% ~100%, 65%~80%, 65%~85%, 65%~90%, 65%~95%, 65%~100%, 70%~80%, 70%~85%, 70%~90 %, 70%~95%, 70%~100%, 75%~80%, 75%~85%, 75%~90%, 75%~95%, 75%~100%, 80%~85%, 80%~90%, 80%~95%, 80%~100%, 85%~90%, 85%~95%, 85%~100%, 90%~95%, 90%~100%, 10% , 20%, 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%, etc.) are chirally controlled. In some embodiments, at least 5%-100% (e.g., about 10%-100%, 20-100%, 30%-100%, 40%-100%) of the phosphorothioate internucleotide linkages in the first domain 100%, 50%~80%, 50%~85%, 50%~90%, 50%~95%, 60%~80%, 60%~85%, 60%~90%, 60%~95% , 60%~100%, 65%~80%, 65%~85%, 65%~90%, 65%~95%, 65%~100%, 70%~80%, 70%~85%, 70 %~90%, 70%~95%, 70%~100%, 75%~80%, 75%~85%, 75%~90%, 75%~95%, 75%~100%, 80%~ 85%, 80% to 90%, 80% to 95%, 80% to 100%, 85% to 90%, 85% to 95%, 85% to 100%, 90% to 95%, 90% to 100% , 10%, 20%, 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%, etc.) are chirally controlled. In some embodiments, each is independently chirally controlled. In some embodiments, at least about 1-50 (e.g., about 5, 6, 7, 8, 9, or 10 to about 10, 11, 12, 13, 14, 15, 16 in the first domain) , 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 40 or 50, or about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 40 or 50 etc, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, or 20, etc.) chiral internucleotide linkages are Sp . In some embodiments, at least about 1-50 (e.g., about 5, 6, 7, 8, 9, or 10 to about 10, 11, 12, 13, 14, 15, 16 in the first domain) , 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 40 or 50, or about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 40 or 50 etc, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, or 20, etc.) phosphorothioate internucleotide linkages are Sp . In some embodiments, at least 5%-100% (e.g., about 10%-100%, 20-100%, 30%-100%, 40%-100%, 50%~80%, 50%~85%, 50%~90%, 50%~95%, 60%~80%, 60%~85%, 60%~90%, 60%~95%, 60% ~100%, 65%~80%, 65%~85%, 65%~90%, 65%~95%, 65%~100%, 70%~80%, 70%~85%, 70%~90 %, 70%~95%, 70%~100%, 75%~80%, 75%~85%, 75%~90%, 75%~95%, 75%~100%, 80%~85%, 80%~90%, 80%~95%, 80%~100%, 85%~90%, 85%~95%, 85%~100%, 90%~95%, 90%~100%, 10% , 20%, 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%, etc.) is Sp . In some embodiments, at least 5%-100% (e.g., about 10%-100%, 20-100%, 30%-100%, 40%-100%) of the phosphorothioate internucleotide linkages in the first domain 100%, 50%~80%, 50%~85%, 50%~90%, 50%~95%, 60%~80%, 60%~85%, 60%~90%, 60%~95% , 60%~100%, 65%~80%, 65%~85%, 65%~90%, 65%~95%, 65%~100%, 70%~80%, 70%~85%, 70 %~90%, 70%~95%, 70%~100%, 75%~80%, 75%~85%, 75%~90%, 75%~95%, 75%~100%, 80%~ 85%, 80% to 90%, 80% to 95%, 80% to 100%, 85% to 90%, 85% to 95%, 85% to 100%, 90% to 95%, 90% to 100% , 10%, 20%, 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%, etc.) is Sp . In some embodiments, the number is one or more. In some embodiments, the number is two or more. In some embodiments, the number is three or more. In some embodiments, the number is 4 or more. In some embodiments, the number is 5 or more. In some embodiments, the number is 6 or more. In some embodiments, the number is 7 or more. In some embodiments, the number is 8 or more. In some embodiments, the number is 9 or more. In some embodiments, the number is 10 or more. In some embodiments, the number is 11 or more. In some embodiments, the number is 12 or more. In some embodiments, the number is 13 or more. In some embodiments, the number is 14 or more. In some embodiments, the number is 15 or more. In some embodiments, the percentage is at least about 50%. In some embodiments, the percentage is at least about 55%. In some embodiments, the percentage is at least about 60%. In some embodiments, the percentage is at least about 65%. In some embodiments, the percentage is at least about 70%. In some embodiments, the percentage is at least about 75%. In some embodiments, the percentage is at least about 80%. In some embodiments, the percentage is at least about 85%. In some embodiments, the percentage is at least about 90%. In some embodiments, the percentage is at least about 95%. In some embodiments, the percentage is about 100%. In some embodiments, each internucleotide linkage linking two first domain nucleosides is independently a modified internucleotide linkage. In some embodiments, each modified internucleotide linkage is independently a chiral internucleotide linkage. In some embodiments, each modified internucleotide linkage is independently a phosphorothioate internucleotide linkage. In some embodiments, each chiral internucleotidic linkage is independently a phosphorothioate internucleotidic linkage. In some embodiments, each modified internucleotide linkage is independently an Sp chiral internucleotide linkage. In some embodiments, each modified internucleotide linkage is independently an S p phosphorothioate internucleotide linkage. In some embodiments, each chiral internucleotidic linkage is independently an S p phosphorothioate internucleotidic linkage. In some embodiments, the internucleotide linkages of the first domain are linked to two nucleosides of the first domain. In some embodiments, an internucleoside linkage between a nucleoside of a first domain and a nucleoside of a second domain may properly be considered an internucleoside linkage of the first domain. In some embodiments, the internucleotide linkage linked to the nucleoside of the first domain and the nucleoside of the second domain is a modified internucleotide linkage, in some embodiments, it is a chiral internucleotide linkage, and in some embodiments, It is chirally controlled, and in some embodiments it is R p , and in some embodiments it is Sp . In many embodiments, a high percentage of S p internucleotidic linkages (e.g., higher than R p internucleotidic linkages and/or natural phosphate linkages) results in improved properties and/or activity, e.g., higher stability and/or It has been observed to provide high adenosine editing activity.

In some embodiments, the first domain comprises some level of R p internucleotidic linkages. In some embodiments, the level is, for example, between about 5% and 100%, about 10% and 100%, 20 and 100%, 30% and 100%, 40% and 100% of all internucleotide linkages in the first domain. , 50%~80%, 50%~85%, 50%~90%, 50%~95%, 60%~80%, 60%~85%, 60%~90%, 60%~95%, 60 %~100%, 65%~80%, 65%~85%, 65%~90%, 65%~95%, 65%~100%, 70%~80%, 70%~85%, 70%~ 90%, 70% to 95%, 70% to 100%, 75% to 80%, 75% to 85%, 75% to 90%, 75% to 95%, 75% to 100%, 80% to 85% , 80%~90%, 80%~95%, 80%~100%, 85%~90%, 85%~95%, 85%~100%, 90%~95%, 90%~100%, 10 %, 20%, 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%. In some embodiments, the level is, for example, between about 5% and 100%, about 10% and 100%, 20 and 100%, 30% and 100%, 40% and 100% of all chiral internucleotide linkages in the first domain. %, 50%~80%, 50%~85%, 50%~90%, 50%~95%, 60%~80%, 60%~85%, 60%~90%, 60%~95%, 60%~100%, 65%~80%, 65%~85%, 65%~90%, 65%~95%, 65%~100%, 70%~80%, 70%~85%, 70% ~90%, 70%~95%, 70%~100%, 75%~80%, 75%~85%, 75%~90%, 75%~95%, 75%~100%, 80%~85 %, 80%~90%, 80%~95%, 80%~100%, 85%~90%, 85%~95%, 85%~100%, 90%~95%, 90%~100%, 10%, 20%, 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%. In some embodiments, the level is, for example, between about 5% and 100%, about 10% and 100%, 20 and 100%, 30% and 100%, 40% and 40% of all chiral control internucleotide linkages in the first domain. 100%, 50%~80%, 50%~85%, 50%~90%, 50%~95%, 60%~80%, 60%~85%, 60%~90%, 60%~95% , 60%~100%, 65%~80%, 65%~85%, 65%~90%, 65%~95%, 65%~100%, 70%~80%, 70%~85%, 70 %~90%, 70%~95%, 70%~100%, 75%~80%, 75%~85%, 75%~90%, 75%~95%, 75%~100%, 80%~ 85%, 80% to 90%, 80% to 95%, 80% to 100%, 85% to 90%, 85% to 95%, 85% to 100%, 90% to 95%, 90% to 100% , 10%, 20%, 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%. In some embodiments, the percentage is about or up to about 50%. In some embodiments, the percentage is at least about 55%. In some embodiments, the percentage is at least about 60%. In some embodiments, the percentage is at least about 65%. In some embodiments, the percentage is at least about 70%. In some embodiments, the percentage is at least about 75%. In some embodiments, the percentage is at least about 80%. In some embodiments, the percentage is at least about 85%. In some embodiments, the percentage is at least about 90%. In some embodiments, the percentage is at least about 95%. In some embodiments, the percentage is about 100%. In some embodiments, the percentage is about or up to about 5%. In some embodiments, the percentage is about or up to about 10%. In some embodiments, the percentage is about or up to about 15%. In some embodiments, the percentage is about or up to about 20%. In some embodiments, the percentage is about or up to about 25%. In some embodiments, the percentage is about or up to about 30%. In some embodiments, the percentage is about or up to about 35%. In some embodiments, the percentage is about or up to about 40%. In some embodiments, the percentage is about or up to about 45%. In some embodiments, the percentage is about or up to about 50%. In some embodiments, about 1-50, 1-40, 1-30, 1-25, 1-20, 1-15, 1-10, 1-5, for example about 1, 2, 3, 4 , 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 internucleotide linkages are independently R p chiral internucleotide linkages. In some embodiments, the number is about or up to about 1. In some embodiments, the number is about or up to about 2. In some embodiments, the number is about or up to about 3. In some embodiments, the number is about or up to about 4. In some embodiments, the number is about or up to about 5. In some embodiments, the number is about or up to about 6. In some embodiments, the number is about or up to about 7. In some embodiments, the number is about or up to about 8. In some embodiments, the number is about or up to about 9. In some embodiments, the number is about or up to about 10.

In some embodiments, each phosphorothioate internucleotidic linkage in the first domain is independently chirally controlled. In some embodiments, each is independently Sp or R p. In some embodiments, the high level is Sp as described herein. In some embodiments, each phosphorothioate internucleotidic linkage in the first domain is chirally controlled and is Sp .

In some embodiments, as exemplified in certain examples, the first domain comprises one or more non-negatively charged internucleotide linkages, each optionally and independently chirally controlled. In some embodiments, each non-negatively charged internucleotide linkage is independently n001. In some embodiments, non-negatively charged chiral internucleotidic linkages are not chirally controlled. In some embodiments, each chiral internucleotidic linkage that is not negatively charged is not chirally controlled. In some embodiments, non-negatively charged chiral internucleotidic linkages are chirally controlled. In some embodiments, non-negatively charged chiral internucleotidic linkages are chirally controlled and R p . In some embodiments, the non-negatively charged chiral internucleotidic linkages are chirally controlled and are Sp . In some embodiments, each non-negatively charged chiral internucleotidic linkage is chirally controlled. In some embodiments, the number of non-negatively charged internucleotide linkages in the first domain is about 1-10, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 . In some embodiments, the number is about 1. In some embodiments, the number is about two. In some embodiments, the number is about 3. In some embodiments, the number is about 4. In some embodiments, the number is about 5. In some embodiments, linkages between two or more nucleotides that are not negatively charged are contiguous. In some embodiments, linkages between two non-negatively charged nucleotides are not contiguous. In some embodiments, all non-negatively charged internucleotide linkages in the first domain are contiguous (eg, three consecutive non-negatively charged internucleotidic linkages). In some embodiments, the non-negatively charged internucleotide linkage, or two or more consecutive non-negatively charged internucleotide linkages, is at the 5′-end of the first domain. In some embodiments, the internucleoside linkage connecting the last two nucleosides of the first domain is a non-negatively charged internucleoside linkage. In some embodiments, the internucleoside linkage connecting the last two nucleosides of the first domain is a non-negatively charged S p internucleoside linkage. In some embodiments, the internucleoside linkage connecting the last two nucleosides of the first domain is a non-negatively charged R p internucleoside linkage. In some embodiments, the internucleoside linkage connecting the last two nucleosides of the first domain is a phosphorothioate internucleoside linkage. In some embodiments, the internucleoside linkage connecting the last two nucleosides of the first domain is an S p phosphorothioate internucleoside linkage. In some embodiments, the internucleoside linkage connecting the first two nucleosides of the first domain is a non-negatively charged internucleoside linkage. In some embodiments, the internucleoside linkage connecting the first two nucleosides of the first domain is a non-negatively charged S p internucleoside linkage. In some embodiments, the internucleoside linkage connecting the first two nucleosides of the first domain is a non-negatively charged R p internucleoside linkage. In some embodiments, the internucleoside linkage connecting the first two nucleosides of the first domain is a phosphorothioate internucleoside linkage. In some embodiments, the internucleoside linkage connecting the first two nucleosides of the first domain is an S p phosphorothioate internucleoside linkage. In some embodiments, the non-negatively charged internucleotidic linkage is a neutral internucleotidic linkage, such as n001. In some embodiments, the first two nucleosides of the first domain are the first two nucleosides of an oligonucleotide.

In some embodiments, the first domain comprises one or more native phosphate linkages. In some embodiments, the first domain does not contain native phosphate linkages. In some embodiments, one or more 2'-OR modified sugars (R is not -H) are independently linked to a natural phosphate linkage. In some embodiments, one or more 2'-OR modified sugars (R is an optionally substituted C _1-6 aliphatic) are independently linked to a natural phosphate linkage. In some embodiments, one or more 2'-OMe modified sugars are independently linked to a natural phosphate linkage. In some embodiments, one or more 2'-MOE modified sugars are independently linked to a natural phosphate linkage. In some embodiments, each 2'-MOE modified sugar is independently linked to a natural phosphate linkage. In some embodiments, at least 50% (e.g., 50%-100%, 50%-90%, 50-80%, or about 50%, 60%, 66%, 70%, 75%, 80%, 90% or more) of the 2'-OR modified sugars (R is not -H) are independently linked to a natural phosphate linkage. In some embodiments, at least 50% (e.g., 50%-100%, 50%-90%, 50-80%, or about 50%, 60%, 66%, 70%, 75%, 80%, 90% or more) of the 2'-OMe modified sugars are independently linked to natural phosphate linkages. In some embodiments, at least 50% (e.g., 50%-100%, 50%-90%, 50-80%, or about 50%, 60%, 66%, 70%, 75%, 80%, 90% or more) of the 2'-MOE modified sugars are independently linked to natural phosphate linkages. In some embodiments, 50% or more (e.g., 50%-100%, 50%-90%, 50-80%, or about 50%, 60%, 66%, 70%, 75%, 80%, 90% or more) of the internucleotide linkages are independently natural phosphate linkages. In some embodiments, at least 50% (e.g., 50%-100%, 50%-90%, 50-80%, or about 50%) bound to two 2'-OMe or 2'-MOE modified sugars. %, 60%, 66%, 70%, 75%, 80%, 90% or more) of the internucleotide linkages are independently natural phosphate linkages.

In some embodiments, in an oligonucleotide of the invention or portion thereof, e.g., a first domain, a second domain, a first subdomain, a second subdomain, a third subdomain, etc., per two 2'-F modifications Each internucleotide linkage linked to is independently a modified internucleotide linkage. In some embodiments, it is independently a non-negatively charged internucleotide linkage, such as a phosphorothioate internucleotide linkage or a phosphorylguanidine internucleotide linkage such as n001. In some embodiments, it is independently a non-negatively charged internucleotidic linkage, such as an S p phosphorothioate internucleotidic linkage or a phosphorylguanidine internucleotidic linkage such as n001. In some embodiments, it is independently an S p phosphorothioate internucleotide linkage or an R p phosphoryl guanidine internucleotide linkage, such as R p n001. In some embodiments, each phosphorothioate internucleotidic linkage linked to two 2'-F modified sugars is independently Sp .

In some embodiments, the first domain recruits, facilitates, or contributes to recruitment of a protein, such as an ADAR protein (eg, ADAR1, ADAR2, etc.). In some embodiments, the first domain supplements, facilitates, or contributes to an interaction with a protein, such as an ADAR protein. In some embodiments, the first domain contacts the RNA binding domain (RBD) of an ADAR. In some embodiments, the first domain does not substantially contact the second RBD domain of the ADAR. In some embodiments, the first domain does not substantially contact the catalytic domain of an ADAR having deaminase activity. In some embodiments, various nucleobases, sugars, and/or internucleotidic linkages may interact with one or more residues of a protein (eg, an ADAR protein).

2nd domain

As described herein, in some embodiments, an oligonucleotide comprises a first domain and a second domain in a 5' to 3' direction. In some embodiments, an oligonucleotide is composed of a first domain and a second domain. Specific implementations of the second domain are described below by way of example. In some embodiments, the second domain comprises the nucleoside opposite the target adenosine to be modified (eg, converted to I).

In some embodiments, the second domain is about 2-50 (e.g., about 5, 6, 7, 8, 9, or 10 to about 10, 11, 12, 13, 14, 15, 16, 17 , 18, 19, 20, 21, 22, 23, 24, 25, 30, 40 or 50, or about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 40 or 50, etc.) nucleobases in length. In some embodiments, the second domain is about 5-30 nucleobases in length. In some embodiments, the second domain is about 10-30 nucleobases in length. In some embodiments, the second domain is about 10-20 nucleobases in length. In some embodiments, the second domain is about 5-15 nucleobases in length. In some embodiments, the second domain is about 13-16 nucleobases in length. In some embodiments, the second domain is about 1-7 nucleobases in length. In some embodiments, the second domain is 10 nucleobases in length. In some embodiments, the second domain is 11 nucleobases in length. In some embodiments, the second domain is 12 nucleobases in length. In some embodiments, the second domain is 13 nucleobases in length. In some embodiments, the second domain is 14 nucleobases in length. In some embodiments, the second domain is 15 nucleobases in length. In some embodiments, the second domain is 16 nucleobases in length. In some embodiments, the second domain is 17 nucleobases in length. In some embodiments, the second domain is 18 nucleobases in length. In some embodiments, the second domain is 19 nucleobases in length. In some embodiments, the second domain is 20 nucleobases in length.

In some embodiments, the second domain comprises about or at least about 5-95%, 10%-90%, 20%-80%, 30%-70%, 40%-70%, 40%-60% of the oligonucleotide. , 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85 %, 90%, 95%. In some embodiments, the percentage is between about 30% and 80%. In some embodiments, the percentage is between about 30% and 70%. In some embodiments, the percentage is between about 40% and 60%. In some embodiments, the percentage is about 20%. In some embodiments, the percentage is about 25%. In some embodiments, the percentage is about 30%. In some embodiments, the percentage is about 35%. In some embodiments, the percentage is about 40%. In some embodiments, the percentage is about 45%. In some embodiments, the percentage is about 50%. In some embodiments, the percentage is about 55%. In some embodiments, the percentage is about 60%. In some embodiments, the percentage is about 65%. In some embodiments, the percentage is about 70%. In some embodiments, the percentage is about 75%. In some embodiments, the percentage is about 80%. In some embodiments, the percentage is about 85%. In some embodiments, the percentage is about 90%.

In some embodiments, the second domain contains one or more (e.g., 1-20, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, etc.) In some implementations, there is 1 mismatch. In some implementations, there are two mismatches. In some implementations, there are three mismatches. In some implementations, there are 4 mismatches. In some implementations, there are 5 mismatches. In some implementations, there are 6 mismatches. In some implementations, there are 7 mismatches. In some implementations, there are 8 mismatches. In some implementations, there are 9 mismatches. In some implementations, there are 10 mismatches.

In some embodiments, the second domain contains one or more (e.g., 1-20, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, etc.) wobbles exist. In some implementations, there is 1 wobble. In some implementations, there are two wobbles. In some implementations, there are three wobbles. In some implementations, there are 4 wobbles. In some implementations, there are 5 wobbles. In some implementations, there are 6 wobbles. In some implementations, there are 7 wobbles. In some implementations, there are 8 wobbles. In some implementations, there are 9 wobbles. In some implementations, there are 10 wobbles.

In some embodiments, the duplex of the oligonucleotide in the second domain region and the target nucleic acid comprises one or more bulges, each independently comprising one or more mismatches that are not wobbles. In some embodiments, 0-10 (e.g., 0-1, 0-2, 0-3, 0-4, 0-5, 0-6, 0-7, 0-8, 0-9, 0~10, 1~2, 1~3, 1~4, 1~5, 1~6, 1~7, 1~8, 1~9, 1~10, 2~3, 2~4, 2~ 5, 2~6, 2~7, 2~8, 2~9, 2~10, 3~4, 3~5, 3~6, 3~7, 3~8, 3~9, 3~10, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 bulges). In some embodiments, the number is zero. In some embodiments, the number is one. In some embodiments, the number is two. In some embodiments, the number is three. In some embodiments, the number is four. In some embodiments, the number is five.

In some embodiments, the second domain is fully complementary to the target nucleic acid.

In some embodiments, the second domain comprises one or more modified nucleobases.

In some embodiments, the second domain comprises a nucleoside opposite the target adenosine, for example when the oligonucleotide forms a duplex with the target nucleic acid. In some embodiments, the opposite nucleobase is an optionally substituted or protected U or an optionally substituted or protected tautomer of U. In some embodiments, the opposite nucleobase is U.

In some embodiments, the opposite nucleobases have weaker hydrogen bonds with the target adenine of the target adenosine than U. In some embodiments, the opposing nucleobases form fewer hydrogen bonds with the target adenine of the target adenosine than U. In some embodiments, opposite nucleobases form one or more hydrogen bonds with one or more amino acid residues of a protein (eg, ADAR) and residues form one or more hydrogen bonds with opposite U of a target adenosine. In some embodiments, the opposite nucleobases form one or more hydrogen bonds with each amino acid residue of the ADAR that forms one or more hydrogen bonds with the opposite U of the target adenosine. In some embodiments, by weakening hydrogen bonds with target A and/or maintaining or enhancing interactions with proteins such as ADAR1, ADAR2, etc., certain opposing nucleobases are adenosine by, for example, ADAR proteins such as ADAR1 and ADAR2. Facilitates and/or promotes transformation.

In some embodiments, the opposite nucleobase is an optionally substituted or protected C, or an optionally substituted or protected tautomer of C. In some embodiments, the opposite nucleobase is C. In some embodiments, the opposite nucleobase is an optionally substituted or protected A, or an optionally substituted or protected tautomer of A. In some embodiments, the opposite nucleobase is A. In some embodiments, the opposite nucleobase is an optionally substituted or protected nucleobase of a pseudoisocytosine, or an optionally substituted or protected tautomer of a nucleobase of pseudoisocytosine. In some embodiments, the opposite nucleobase is a pseudoisocytosine nucleobase.

In some embodiments, a nucleoside, e.g., a nucleoside opposite a target adenosine (which may also be referred to as an “opposite nucleoside”) is as described herein (e.g., L010, L012, L028, etc. having the structure of) is non-basic.

Many useful embodiments of modified nucleobases, for example relative to opposite nucleobases, are also described below. In some embodiments, as described herein (e.g., in various oligonucleotides), the present invention provides, for example, A, T, C, G, U, hypoxanthine, b001U, b002U, b003U, b004U, b005U, b006U, b007U, b008U, b009U, b011U, b012U, b013U, b001A, b002A, b003A, b001G, b002G, b001C, b002C, b003C, b004C, b005C, b006C, b007C, b008C, b0 09C, b002I, b003I, b004I, and zdnp An oligonucleotide comprising the nucleobase of the nucleoside opposite to the target nucleoside, such as A, is or comprises the same. In some embodiments, as described herein (e.g., in various oligonucleotides), the present invention provides, for example, b001U, b002U, b003U, b004U, b005U, b006U, b007U, b008U, b009U, b011U, b012U, b013U A target nucleoside, such as A, which is or comprises b001A, b002A, b003A, b001G, b002G, b001C, b002C, b003C, b004C, b005C, b006C, b007C, b008C, b009C, b002I, b003I, b004I and zdnp the other side of An oligonucleotide comprising a nucleobase of a nucleoside is provided. In some embodiments, as described herein (e.g., in various oligonucleotides), the present invention provides, for example, C, A, b007U, b001U, b001A, b002U, b001C, b003U, b002C, b004U, b003C, b005U . In some embodiments, the nucleobase is C. In some embodiments, the nucleobase is A. In some embodiments, the nucleobase is hypoxanthine. In some embodiments, the nucleobase is b002I. In some embodiments, the nucleobase is b003I. In some embodiments, the nucleobase is b004I. In some embodiments, the nucleobase is b014I. In some embodiments, the nucleobase is b001C. In some embodiments, the nucleobase is b002C. In some embodiments, the nucleobase is b003C. In some embodiments, the nucleobase is b004C. In some embodiments, the nucleobase is b005C. In some embodiments, the nucleobase is b006C. In some embodiments, the nucleobase is b007C. In some embodiments, the nucleobase is b008C. In some embodiments, the nucleobase is b009C. In some embodiments, the nucleobase is b001U. In some embodiments, the nucleobase is b002U. In some embodiments, the nucleobase is b003U. In some embodiments, the nucleobase is b004U. In some embodiments, the nucleobase is b005U. In some embodiments, the nucleobase is b006U. In some embodiments, the nucleobase is b007U. In some embodiments, the nucleobase is b008U. In some embodiments, the nucleobase is b009U. In some embodiments, the nucleobase is b011U. In some embodiments, the nucleobase is b012U. In some embodiments, the nucleobase is b013U. In some embodiments, the nucleobase is b001A. In some embodiments, the nucleobase is b002A. In some embodiments, the nucleobase is b003A. In some embodiments, the nucleobase is b001G. In some embodiments, the nucleobase is b002G. In some embodiments, the nucleobase is zdnp. In some embodiments, as will be appreciated by those skilled in the art, nucleobases are protected, for example for oligonucleotide synthesis. For example, in some embodiments, a nucleobase is is a protected b001A having the structure of (R' is as described herein). In some embodiments, R' is -C(0)R. In some embodiments, R' is -C(O)Ph.

In some embodiments, the various modified nucleobases, e.g., b001A, b008U, etc., are compared to the reference nucleobase (e.g., as under comparable conditions, including the same oligonucleotide, evaluated in the same or comparable assays, etc.) It has been observed that, when compared, it can provide improved adenosine editing efficiency. In some embodiments, the reference nucleobase is U. In some embodiments, the reference nucleobase is T. In some embodiments, the reference nucleobase is C.

specific modified nucleobases

In some embodiments, BA is or comprises Ring BA or a tautomer thereof, and Ring BA is an optionally substituted 5-20 membered monocyclic, bicyclic or polycyclic ring having 0-10 heteroatoms. In some embodiments, ring BA is or comprises an optionally substituted 5-20 membered monocyclic, bicyclic or polycyclic having 1-10 heteroatoms, at least one heteroatom being nitrogen. In some embodiments, ring BA is saturated. In some embodiments, ring BA contains one or more unsaturations. In some embodiments, ring BA is partially unsaturated. In some embodiments, ring BA is aromatic.

In some embodiments, BA is or contains ring BA, and ring BA is an optionally substituted 5-20 membered monocyclic, bicyclic or polycyclic ring having 0-10 heteroatoms. In some embodiments, ring BA is or comprises an optionally substituted 5-20 membered monocyclic, bicyclic or polycyclic having 1-10 heteroatoms, at least one heteroatom being nitrogen. In some embodiments, ring BA is saturated. In some embodiments, ring BA contains one or more unsaturations. In some embodiments, ring BA is partially unsaturated. In some embodiments, ring BA is aromatic.

In some embodiments, BA is or comprises a ring BA. In some embodiments, BA is a ring BA. In some embodiments, BA is or comprises a tautomer of ring BA. In some embodiments, BA is a tautomer of ring BA.

In some embodiments, structures of the invention include one or more optionally substituted rings (eg, ring BA, -Cy-, ring BA ^A , R, etc., formed with an R group). In some embodiments, the rings are 1-10 (eg, 1-10, 1-5, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, etc.) hetero optionally substituted with atoms C _3-30 , C _3-20 , C _3-15 , C _3-10 , C _3-9 , C _3-8 , C _3-7 , C _3-6 , C _5-50 , C _5-20 , C _5-15 , C _5-10 , C _5-9 , C _5-8 , C _5-7 , C _5-6 , or 3-30 circles (e.g. 3-30, 3 ~20, 3~15, 3~10, 3~9, 3~8, 3~7, 3~6, 5~50, 5~20, 5~15, 5~10, 5~9, 5~8 , 5-7, 5-6, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30 membered, etc.) monocyclic, bicyclic or polycyclic ring. In some embodiments, the ring is an optionally substituted 3-10 membered monocyclic or bicyclic, saturated, partially saturated or aromatic ring having 0-3 heteroatoms. In some embodiments, rings are substituted. In some embodiments, a ring is unsubstituted. In some embodiments, a ring is 3, 4, 5, 6, 7, 8, 9, or 10 membered. In some embodiments, a ring is 5, 6, or 7 membered. In some embodiments, a ring is 5 membered. In some embodiments, a ring is 6 membered. In some embodiments, the ring is 7 membered. In some embodiments, a ring is monocyclic. In some embodiments, a ring is bicyclic. In some embodiments, a ring is polycyclic. In some embodiments, a ring is saturated. In some embodiments, a ring contains at least one unsaturation. In some embodiments, the ring is partially unsaturated. In some embodiments, a ring is aromatic. In some embodiments, the ring has 0-5 heteroatoms. In some embodiments, the ring has 1-5 heteroatoms. In some embodiments, the ring has one or more heteroatoms. In some embodiments, the ring has 1 heteroatom. In some embodiments, the ring has 2 heteroatoms. In some embodiments, the ring has 3 heteroatoms. In some embodiments, the ring has 4 heteroatoms. In some embodiments, the ring has 5 heteroatoms. In some embodiments, the heteroatom is nitrogen. In some embodiments, the heteroatom is oxygen. In some embodiments, a ring is substituted (eg, substituted with one or more alkyl groups and optionally one or more other substituents as described herein). In some embodiments, the substituent is methyl.

In some embodiments, each monocyclic ring unit of a monocyclic, bicyclic, or polycyclic ring (e.g., ring BA, -Cy-, ring BA ^A , R, etc. formed with a group R) is independently 0 to 5 It is an optionally substituted 5-7 membered saturated, partially unsaturated or aromatic ring having two heteroatoms. In some embodiments, one or more monocyclic units independently include one or more unsaturations. In some embodiments, one or more monocyclic units are saturated. In some embodiments, one or more monocyclic units are partially saturated. In some embodiments, one or more monocyclic units are aromatic. In some embodiments, one or more monocyclic units independently have 1-5 heteroatoms. In some embodiments, one or more monocyclic units independently have at least one nitrogen atom. In some embodiments, each monocyclic unit is independently 5 or 6 membered. In some embodiments, a monocyclic unit is 5 membered. In some embodiments, monocyclic units are 5-membered and have 1-2 nitrogen atoms. In some embodiments, a monocyclic unit is 6 membered. In some embodiments, monocyclic units are 6-membered and have 1-2 nitrogen atoms. Unless otherwise specified, rings and monocyclic units thereof are optionally substituted.

Without intending to be limited by any particular theory, the present invention, in some embodiments, the structure of a nucleobase (e.g., BA) interacts with a protein (e.g., an ADAR protein such as ADAR1, ADAR2, etc.) Recognize that it can affect In some embodiments, a provided oligonucleotide comprises a nucleobase capable of facilitating interaction of the oligonucleotide with an enzyme (eg, ADAR1). In some embodiments, provided oligonucleotides include nucleobases that can reduce the strength of base pairing (eg, relative to A-T/U or C-G). In some embodiments, the invention maintains and/or enhances interactions (eg, hydrogen bonding) of the first nucleobase with a protein (eg, an enzyme such as ADAR1) and/or the first nucleobase with the other strand of the duplex. It is recognized that by reducing the interaction (eg hydrogen bonding) of the corresponding nucleobase (eg A) on the phase, modification of the corresponding nucleobase by a protein (eg an enzyme such as ADAR1) can be significantly improved. In some embodiments, the invention provides an oligonucleotide comprising such a first nucleobase (eg, various embodiments of BA described herein). Exemplary embodiments of such a first nucleobase are as described herein. In some embodiments, when an oligonucleotide comprising such a first nucleobase is aligned with another nucleic acid for maximum complementarity, the first nucleobase is opposite A. In some embodiments, such an A opposite the first nucleobase, as exemplified in many embodiments of the present invention, can be efficiently modified using the techniques of the present invention.

In some embodiments, ring BA is a moiety X ² X ³ and each variable is independently as described herein. In some embodiments, ring BA is a moiety X ² X ³ X ⁴ and each variable is independently as described herein. In some embodiments, ring BA is a moiety -X ¹ ( ) X ² X ³ and each variable is independently as described herein. In some embodiments, ring BA is a moiety -X ¹ ( ) X ² X ³ X ⁴ and each variable is independently as described herein. In some embodiments, X ¹ is linked to a sugar. In some embodiments, X ¹ is -N(-)-. In some embodiments, X ¹ is -C(=)-. In some embodiments, X ² is -C(O)-. In some embodiments, X ³ is -NH-. In some embodiments, X ⁴ is not -C(O)-. In some embodiments, X ⁴ is -C(O)-, eg together with a moiety of the same nucleotide unit within the same BA unit (eg, a hydrogen bond donor of X ⁵ eg -OH, SH, etc. ) together) to form intramolecular hydrogen bonds. In some embodiments, X ⁴ is -C(=NH)-. In some embodiments, ring BA is a moiety X ^4' X ^5' and each variable is independently as described herein. In some embodiments, X ^4' is -C(O)-. In some embodiments, X ^5' is -NH-.

In some embodiments, BA is optionally substituted or protected C or a tautomer thereof. In some embodiments, BA is optionally substituted or optionally protected C. In some embodiments, BA is an optionally substituted or optionally protected tautomer of C. In some embodiments, BA is C. In some embodiments, BA is a substituted C. In some embodiments, BA is protected C. In some embodiments, BA is a substituted tautomer of C. In some embodiments, BA is a protected tautomer of C.

In some embodiments, ring BA has the structure of Formula BA-I,

[Formula BA-I]

In the above formula,

ring BA is an optionally substituted 5-20 membered monocyclic, bicyclic or polycyclic, saturated, partially saturated or aromatic ring having 1-10 heteroatoms;

Each are independently single or double bonds;

X ¹ is -N(-)- or -C(-)=;

X ² is -C(0)-, -C(R ^B2 )=, or -C(OR ^B2 )=, and R ^B2 is -L ^B2 -R';

X ³ is -N(R ^B3 )- or -N=, R ^B3 is -L ^B3 -R';

X ⁴ is -C(R ^B4 )=, -C(-N(R ^B4 ) ₂ )=, -C(R ^B4 ) ₂ -, -C(O)-, or -C(=NR ^B4 )-; , each R ^B4 is independently -L ^B4 -R ^B41 , or two R ^B4 on the same atom together =O, =C(-L ^B4 -R ^B41 ) ₂ , =NL ^B4 -R ^B41 , or optionally substituted ═CH ₂ or ═NH, and each R ^B41 is independently R′;

each of L ^B2 , L ^B3 , and L ^B4 is independently L ^B ;

each ^LB is independently a covalent bond or an optionally substituted divalent C _1-10 saturated or partially unsaturated chain having 0-6 heteroatoms, and one or more methylene units are optionally and independently -Cy-, -O -, -S-, -N(R')-, -C(O)-, -C(S)-, -C(NR')-, -C(O)N(R')-, -N (R')C(O)N(R')-, -N(R')C(O)O-, -S(O)-, -S(O) ₂ -, -S(O) ₂ N (R')-, -C(O)S-, or -C(O)O-;

each -Cy- is independently an optionally substituted 3-20 membered monocyclic, bicyclic or polycyclic ring having 0-10 heteroatoms;

each R′ is independently -R, -C(O)R, -C(O)OR, -C(O)N(R) ₂ , or -SO ₂ R;

Each R is independently -H, C _1-20 aliphatic, C _1-20 heteroaliphatic having 1-10 heteroatoms, C _6-20 aryl, C _6-20 arylaliphatic, 1-10 heteroatoms is an optionally substituted group selected from C _6-20 arylheteroaliphatic having , 5-20 membered heteroaryl having 1-10 heteroatoms, and 3-20 membered heterocyclyl having 1-10 heteroatoms;

two R groups optionally and independently together form a covalent bond;

two or more R groups on the same atom optionally and independently form an optionally substituted 3-20 membered monocyclic, bicyclic or polycyclic ring having, in addition to the corresponding atoms, 0-10 heteroatoms together with the corresponding atoms;

Two or more R groups on two or more atoms are optionally and independently substituted 3 to 30 membered monocyclic, bicyclic or polycyclic rings having 0 to 10 heteroatoms in addition to the intervening atoms together with the atoms intervening therebetween. form

In some embodiments, ring BA (eg, of formula BA-I) has the structure of formula BA-I-a.

[Formula BA-I-a]

In some embodiments, ring BA (eg, of formula BA-I, BA-I-a, etc.) has the structure of formula BA-I-b.

[Formula BA-I-b]

In some embodiments, ring BA (eg, of formula BA-I) has the structure of formula BA-II,

[Formula BA-II]

In the above formula,

X ⁵ is -C(R ^B5 ) ₂ -, -N(R ^B5 )-, -C(R ^B5 )=, -C(O)-, or -N=, and each R ^B5 is independently halogen; or -L ^B5 -R ^B51 , wherein R ^B51 is -R', -N(R') ₂ , -OR', or -SR';

L ^B5 is L ^B ;

Each other variable is independently as described herein.

In some embodiments, ring BA (eg, of formula BA-I, BA-I-a, BA-II, etc.) has the structure of formula BA-II-a.

[Formula BA-II-a]

In some embodiments, ring BA (eg, of formula BA-I, BA-I-a, BA-I-b, BA-II, BA-II-a, etc.) has a structure of formula BA-II-b.

[Formula BA-II-b]

In some embodiments, ring BA (eg, of formula BA-I, BA-II, etc.) has the structure of formula BA-III.

[Formula BA-III]

In the above formula,

X ⁶ is -C(R ^B6 )=, -C(OR ^B6 )=, -C(R ^B6 ) ₂ -, -C(O)-, or -N=, and each R ^B6 is independently -L ^B6 -R ^B61 or two R ^B6 on the same atom together form =O, =C(-L ^B6 -R ^B61 ) ₂ , =NL ^B6 -R ^B61 , or optionally substituted =CH ₂ or =NH; each R ^B61 is independently R';

L ^B6 is L ^B ;

Each other variable is independently as described herein.

In some embodiments, ring BA (eg, of formula BA-I, BA-I-a, BA-II, BA-II-a, BA-III, etc.) has the structure of formula BA-III-a.

[Formula BA-III-a]

In some embodiments, a ring BA (e.g., Formula BA-I, BA-I-a, BA-I-b, BA-II, BA-II-a, BA-II-b, BA-III, BA-III-a etc.) has a structure of the formula BA-III-b.

[Formula BA-III-b]

In some embodiments, ring BA (eg, of formula BA-I, BA-II, etc.) has the structure of formula BA-IV.

[Formula BA-IV]

In the above formula,

ring BA ^A is an optionally substituted 5-14 membered monocyclic, bicyclic or polycyclic ring having 0-5 heteroatoms;

Each other variable is independently as described herein.

In some embodiments, ring BA (eg, one of formula BA-I, BA-I-a, BA-II, BA-II-a, etc.) has the structure of formula BA-IV-a.

[Formula BA-IV-a]

In some embodiments, ring BA (eg, Formula BA-I, BA-I-a, BA-II, BA-II-a, etc.) has the structure BA-IV-b.

[Formula BA-IV-b]

In some embodiments, ring BA (eg, of formula BA-I, BA-II, BA-III, BA-IV, etc.) has the structure of formula BA-V.

[Formula BA-V]

In some embodiments, a ring BA (e.g., Formula BA-I, BA-I-a, BA-II, BA-II-a, BA-III, BA-III-a, BA-IV, BA-IV-a , BA-V, etc.) has a structure of the formula BA-V-a.

[Formula BA-V-a]

In some embodiments, a ring BA (e.g., Formula BA-I, BA-I-a, BA-I-b, BA-II, BA-II-a, BA-II-b, BA-III, BA-III-a , BA-III-b, BA-IV, BA-IV-a, BA-IV-b, BA-V, BA-V-a, etc.) has a structure of formula BA-V-a.

[Formula BA-V-b]

In some embodiments, ring BA has the structure of Formula BA-VI.

[Formula BA-VI]

In the above formula,

X ^1' is -N(-)- or -C(-)=;

X ^2' is -C(0)- or -C(R ^B2' )=, R ^B2' is -L ^B2' -R';

Each are independently single or double bonds;

X ^3' is -N(R ^B3' )- or -N=, R ^B3' is -L ^B3' -R';

X ^4' is -C(R ^B4' )=, -C(OR ^B4' )=, -C(-N(R ^B4' ) ₂ )=, -C(R ^B4' ) ₂ -, -C(O )-, or -C(=NR ^B4' )-, and each R ^B4' is independently -L ^B4' -R ^B41' , or two R ^B4' on the same atom together =O, =C(- L ^B4' -R ^B41' ) ₂ , =NL ^B4' -R ^B41' , or optionally substituted =CH ₂ or =NH, wherein each R ^B41' is independently -R';

X ⁵ 'is -N(R ^B5- )- or -N=, R ^B5' is -L ^B5' -R';

X ^6' is -C(R ^B6' )=, -C(OR ^B6' )=, -C(R ^B6' ) ₂ -, -C(O)- or -N=, and each R ^B6' is independently -L ^B6' -R ^B61' , or two R ^B6' on the same atom together =O, =C(-L ^B6' -R ^B61' ) ₂ , =NL ^B6' -R ^B61' , or optionally substituted =CH ₂ or =NH, and each R ^B61' is independently R';

X ^7' is -C(R ^B7' )=, -C(OR ^B6' )=, -C(R ^B7' ) ₂ -, -C(O)-, -N(R ^B7' )-, or - N=, each R ^B7' is independently -L ^7' -R ^B71' , or two R ^B7' on the same atom together =O, =C(-L ^7' -R ^B71' ) ₂ , =NL ^7' -R ^B71' , or optionally substituted =CH ₂ or =NH, each R ^B71' is independently R';

each of L ^B2' , L ^B3' , L ^B4' , L ^B5' , and L ^B6' is independently L ^B ;

Each other variable is independently as described herein.

In some embodiments, is a single bond. In some embodiments, is a double bond.

In some embodiments, X ¹ is -(N-)-. In some embodiments, X ¹ is -C(-)=.

In some embodiments, X ² is -C(O)-. In some embodiments, X ² is -C(R ^B2 )=. In some embodiments, X ² is -C(OR ^B2 )=. In some embodiments, X ² is -CH=.

In some embodiments, L ^B2 is a covalent bond.

In some embodiments, R ^B2 is a suitable protecting group for oligonucleotide synthesis, eg a hydroxyl protecting group. In some embodiments, R ^B2 is R'. In some embodiments, R ^B2 is -H.

In some embodiments, X ³ is -N(R ^B3 )-. In some embodiments, X ³ is -NH-. In some embodiments, X ³ is -N=.

In some embodiments, L ^B3 is a covalent bond.

In some embodiments, R ^B3 is a suitable protecting group for oligonucleotide synthesis, eg an amino protecting group (eg Bz). In some embodiments, R ^B3 is R'. In some embodiments, R ^B3 is -C(O)R. In some embodiments, R ^B3 is R. In some embodiments, R ^B3 is -H.

In some embodiments, X ⁴ is -C(R ^B4 )=. In some embodiments, X ⁴ is -C(R)=. In some embodiments, X ⁴ is -CH=. In some embodiments, X ⁴ is -C(OR ^B4 )=. In some embodiments, X ⁴ is -C(-N(R ^B4 ) ₂ )=. In some embodiments, X ⁴ is -C(-NHR ^B4 )=. In some embodiments, X ⁴ is -C(-NHR')=. In some embodiments, X ⁴ is -C(-NHR')=. In some embodiments, X ⁴ is -C(-NH ₂ )=. In some embodiments, X ⁴ is -C(-NHC(O)R)=. In some embodiments, X ⁴ is -C(R ^B4 ) ₂ -. In some embodiments, X ⁴ is -CH ₂ -. In some embodiments, X ⁴ is -C(O)-. In some embodiments, X ⁴ is -C(O)-, and O forms an intramolecular hydrogen bond. In some embodiments, O forms a hydrogen bond with a hydrogen bond donor of X ⁵ of the same BA. In some embodiments, X ⁴ is -C(=NR ^B4 )-. In some embodiments, X ⁴ is -C(=NR ^B4 )- and N forms an intramolecular hydrogen bond. In some embodiments, N forms a hydrogen bond with a hydrogen bond donor of X ⁵ of the same BA.

In some embodiments, R ^B4 is -L ^B4 -R ^B41 . In some embodiments, two R ^B4 on the same atom together form =O, =C(-L ^B4 -R ^B41 ) ₂ , =NL ^B4 -R ^B41 , or optionally substituted =CH ₂ or =NH.

In some embodiments, two R ^B4 on the same atom together form ═O. In some embodiments, two R ^B4 on the same atom together form =C(-L ^B4 -R ^B41 ) ₂ . In some embodiments, =C(-L ^B4 -R ^B41 ) ₂ is =CH-L ^B4 -R ^B41 . In some embodiments, =C(-L ^B4 -R ^B41 ) ₂ is =CHR′. In some embodiments, =C(-L ^B4 -R ^B41 ) ₂ is =CHR. In some embodiments, two R ^B4 on the same atom together form =NL ^B4 -R ^B41 . In some embodiments, =NL ^B4 -R ^B41 is =NR. In some embodiments, two R ^B4 on the same atom together form =CH ₂ . In some embodiments, two R ^B4 on the same atom together form =NH. In some embodiments, the group formed is a protecting group suitable for oligonucleotide synthesis, such as an amino protecting group.

In some embodiments, X ⁴ is -C(-N=C(-L ^B4 -R ^B41 ) ₂ )=. In some embodiments, X ⁴ is -C(-N=CH-L ^B4 -R ^B41 )=. In some embodiments, X ⁴ is -C(-N=CH-N(CH ₃ ) ₂ )=.

In some embodiments, R of X ⁴ (eg, -C(=NR)-, =C(R)-, etc.) is optionally together with another R, eg, R of X ⁵ is a ring as described herein form

In some embodiments, R ^B4 is R'. In some embodiments, R ^B4 is R. In some embodiments, R ^B4 is -H.

In some embodiments, R ^B4 is a suitable protecting group for oligonucleotide synthesis, eg an amino or hydroxyl protecting group. In some embodiments, R ^B4 is R'. In some embodiments, R ^B4 is -CH ₂ CH ₂ -(4-nitrophenyl).

In some embodiments, L ^B4 is a covalent bond. In some embodiments, L ^B4 is not a covalent bond. In some embodiments, at least one methylene unit is replaced with -C(O)-. In some embodiments, at least one methylene unit is replaced with -C(O)N(R')-. In some embodiments, at least one methylene unit is replaced with -N(R')-. In some embodiments, at least one methylene unit is replaced with -NH-. In some embodiments, L ^B4 is or comprises optionally substituted -N=CH-.

In some embodiments, R ^B41 is R'. In some embodiments, R ^B41 is -H. In some embodiments, R ^B41 is R. In some embodiments, R is an optionally substituted phenyl. In some embodiments, R is phenyl.

In some embodiments, X ⁵ is -C(R ^B5 ) ₂ -. In some embodiments, X ⁵ is -ChR ^B5 -. In some embodiments, X ⁵ is -CH ₂ -. In some embodiments, X ⁵ is -N(R ^B5 )-. In some embodiments, X ⁵ is -NH-. In some embodiments, X ⁵ is -C(R ^B5 )=. In some embodiments, X ⁵ is -C(R)=. In some embodiments, X ⁵ is -CH=. In some embodiments, X ⁵ is -N=. In some embodiments, X ⁵ is -C(O)-.

In some embodiments, R ^B5 is halogen. In some embodiments, R ^B5 is -L ^B5 -R ^B51 . In some embodiments, R ^B5 is -L ^B5 -R ^B51 , and R ^B51 is R', -NHR', -OH, or -SH. In some embodiments, R ^B5 is -L ^B5 -R ^B51 and R ^B51 is -NHR, -OH, or -SH. In some embodiments, R ^B5 is -L ^B5 -R ^B51 and R ^B51 is -NH ₂ , -OH, or -SH. In some embodiments, R ^B5 is -C(O)-R ^B51 . In some embodiments, R ^B5 is R'. In some embodiments, R ^B5 is R. In some embodiments, R ^B5 is -H. In some embodiments, R ^B5 is -OH. In some embodiments, R ^B5 is -CH ₂ OH.

In some embodiments, when X ⁴ is -C(O)-, X ⁵ is -C(R ^B5 ) ₂ -, -C(R ^B5 )=, or -N(R ^B5 )-, and R ^B5 is -L ^B5 -R ^B51 , and R ^B51 is -NHR', -OH, or -SH. In some embodiments, X ⁴ is -C(O)-, and R ^B51 is or comprises a hydrogen bond donor that forms a hydrogen bond with O of X ⁴ .

In some embodiments, L ^B5 is a covalent bond. In some embodiments, L ^B5 is or comprises -C(O)-. In some embodiments, L ^B5 is or comprises -O-. In some embodiments, L ^B5 is or comprises -OC(O)-. In some embodiments, L ^B5 is or comprises -CH ₂ OC(O)-.

In some embodiments, R ⁵¹ is -R'. In some embodiments, R ⁵¹ is -R. In some embodiments, R ⁵¹ is -H. In some embodiments, R ⁵¹ is -N(R') ₂ . In some embodiments, R ⁵¹ is -NHR'. In some embodiments, R ⁵¹ is -NHR. In some embodiments, R ⁵¹ is -NH ₂ . In some embodiments, R ⁵¹ is -OR'. In some embodiments, R ⁵¹ is -OR. In some embodiments, R ⁵¹ is —OH. In some embodiments, R ⁵¹ is -SR'. In some embodiments, R ⁵¹ is -SR. In some embodiments, R ⁵¹ is -SH. In some embodiments, R is benzyl. In some embodiments, R is an optionally substituted phenyl. In some embodiments, R is phenyl. In some embodiments, R is methyl.

In some embodiments, R ^B5 is -C(O)-R ^B51 . In some embodiments, R ^B5 is -C(O)NHCH ₂ Ph. In some embodiments, R ^B5 is -C(O)NHPh. In some embodiments, R ^B5 is -C(O)NHCH ₃ . In some embodiments, R ^B5 is -OC(O)-R ^B51 . In some embodiments, R ^B5 is -OC(O)-R. In some embodiments, R ^B5 is -OC(O)CH ₃ .

In some embodiments, X ⁵ is directly bonded to X ¹ and ring BA is 5-membered.

In some embodiments, X ⁶ is -C(R ^B6 )=. In some embodiments, X ⁶ is -CH=. In some embodiments, X ⁶ is -C(OR ^B6 )=. In some embodiments, X ⁶ is -C(R ^B6 ) ₂ -. In some embodiments, X ⁶ is -CH ₂ -. In some embodiments, X ⁶ is -C(O)-. In some embodiments, X ⁶ is -N=.

In some embodiments, R ^B6 is -L ^B6 -R ^B61 . In some embodiments, two R ^B6 on the same atom together form =O, =C(-L ^B6 -R ^B61 ) ₂ , =NL ^B6 -R ^B61 , or optionally substituted =CH ₂ or =NH. In some embodiments, two R ^B6 on the same atom together form ═O. In some embodiments, L ^B6 is a covalent bond. In some embodiments, R ^B6 is R. In some embodiments, R ^B6 is -H.

In some embodiments, R ^B6 is a suitable protecting group for oligonucleotide synthesis, eg an amino or hydroxyl protecting group. In some embodiments, R ^B6 is R. In some embodiments,

In some embodiments, L ^B6 is a covalent bond. In some embodiments, L ^B6 is an optionally substituted C _1-10 alkylene. In some embodiments, L ^B6 is -CH ₂ CH ₂ -. In some embodiments, R ^B6 is -CH ₂ CH ₂ -(4-nitrophenyl).

In some embodiments, R ^B61 is R'. In some embodiments, R ^B61 is R. In some embodiments, R ^B61 is -H.

In some embodiments, ring BA ^A is 5 membered. In some embodiments, ring BA ^A is 5 membered. In some embodiments, ring BA ^A has 1 heteroatom. In some embodiments, ring BA ^A has 2 heteroatoms. In some embodiments, the heteroatom is nitrogen. In some embodiments, the heteroatom is oxygen.

In some embodiments, X ^1′ is -(N-)-. In some embodiments, X ^1' is -C(-)=.

In some embodiments, X ^2' is -C(O)-. In some embodiments, X ^2' is -C(R ^B2' )=. In some embodiments, X ^2' is -CH=.

In some embodiments, L ^B2' is a covalent bond.

In some embodiments, R ^B2' is R'. In some embodiments, R ^B2′ is R. In some embodiments, R ^B2' is -H. In some embodiments, X ^2' is -CH=.

In some embodiments, X ^3' is -N(R ^B3' )-. In some embodiments, X ^3' is -N(R')-. In some embodiments, X ^3' is -NH-. In some embodiments, X ^3' is -N=.

In some embodiments, L ^B3' is a covalent bond.

In some embodiments, R ^B3' is R'. In some embodiments, R ^B3′ is R. In some embodiments, R ^B3' is -H.

In some embodiments, X ^4' is -C(R ^B4' )=. In some embodiments, X ^4' is -C(OR ^B4' )=. In some embodiments, X ^4′ is -C(-N(R ^B4′ ) ₂ )=. In some embodiments, X ^4' is -C(-NHR ^B4' )=. In some embodiments, X ^4′ is -C(-NH ₂ )=. In some embodiments, X ^4' is -C(-NHR')=. In some embodiments, X ^4′ is -C(-NHC(O)R)=. In some embodiments, X ^4' is -C(R ^B4' ) ₂ -. In some embodiments, X ^4' is -C(O)-. In some embodiments, X ^4' is -C(=NR ^B4' )-.

In some embodiments, R ^B4' is -L ^B4' -R ^B41' . In some embodiments, two R ^B4' on the same atom together =O, =C(-L ^B4' -R ^B41' ) ₂ , =NL ^B4' -R ^B41' , or optionally substituted =CH ₂ or = form NH. In some embodiments, two R ^B4' on the same atom together form ═O. In some embodiments, two R ^B4' on the same atom together form =C(-L ^B4' -R ^B41' ) ₂ . In some embodiments, two R ^B4' on the same atom together form =NL ^B4' -R ^B41' . In some embodiments, two R ^B4' on the same atom together form =CH ₂ . In some embodiments, two R ^B4' on the same atom together form =NH. In some embodiments, the group formed is a protecting group suitable for oligonucleotide synthesis, such as an amino protecting group.

In some embodiments, X ^4′ is -C(-N=C(-L ^B4′ -R ^B41′ ) ₂ )=. In some embodiments, X ^4' is -C(-N=CH-L ^B4' -R ^B41' )=. In some embodiments, X ^4′ is -C(-N=CH-N(CH ₃ ) ₂ )=.

In some embodiments, R ^B4' is R'. In some embodiments, R ^B4′ is R. In some embodiments, R ^B4' is -H.

In some embodiments, R ^B4' is a suitable protecting group for oligonucleotide synthesis, such as an amino or hydroxyl protecting group. In some embodiments, R ^B4' is R'. In some embodiments, R ^B4' is -CH ₂ CH ₂ -(4-nitrophenyl).

In some embodiments, L ^B4' is a covalent bond. In some embodiments, L ^B4' is an optionally substituted C _1-10 alkylene. In some embodiments, L ^B4' is -CH ₂ CH ₂ -. In some embodiments, at least one methylene unit is replaced with -N(R')-. In some embodiments, R' is R. In some embodiments, R is an optionally substituted phenyl. In some embodiments, R is phenyl. In some embodiments, R is methyl. In some embodiments, R is -H.

In some embodiments, R ^B41' is R'. In some embodiments, R ^B41' is R. In some embodiments, R ^B41' is -H.

In some embodiments, X ^5' is -N(R ^B5' )-. In some embodiments, X ^5' is -NH-. In some embodiments, X ^5′ is -N=.

In some embodiments, L ^B5' is a covalent bond.

In some embodiments, R ^B5' is R'. In some embodiments, R ^B5′ is R. In some embodiments, R ^B5' is -H.

In some embodiments, X ^6' is -C(R ^B6' )=. In some embodiments, X ^6' is -CH=. In some embodiments, X ^6' is -C(OR ^B6' )=. In some embodiments, X ^6' is -C(R ^B6' ) ₂ -. In some embodiments, X ^6' is -C(O)-. In some embodiments, X ^6' is -N=.

In some embodiments, R ^B6' is -L ^B6' -R ^B61' . In some embodiments, two R ^B6' on the same atom together =O, =C(-L ^B6' -R ^B61' ) ₂ , =NL ^B6' -R ^B61' , or optionally substituted =CH ₂ or = form NH. In some embodiments, two R ^B6' on the same atom together form ═O.

In some embodiments, L ^B6' is a covalent bond. In some embodiments, L ^B6' is an optionally substituted C _1-10 alkylene. In some embodiments, L ^B6' is -CH ₂ CH ₂ -.

In some embodiments, R ^B6' is R'. In some embodiments, R ^B6' is R. In some embodiments, R ^B6' is -H. In some embodiments, R ^B6' is a suitable protecting group for oligonucleotide synthesis, such as an amino or hydroxyl protecting group. In some embodiments, R ^B6' is R'. In some embodiments, R ^B6' is -CH ₂ CH ₂ -(4-nitrophenyl).

In some embodiments, R ^B61' is R'. In some embodiments, R ^B61' is R. In some embodiments, R ^B61' is -H.

In some embodiments, X ^7' is -C(R ^B7' )=. In some embodiments, X ^7' is -CH=. In some embodiments, X ^7' is -C(OR ^B7' )=. In some embodiments, X ^7' is -C(R ^B7' ) ₂ -. In some embodiments, X ^7' is -C(O)-. In some embodiments, X ^7' is -N(R ^B7' )-. In some embodiments, X ^7' is -NH-. In some embodiments, X ^7' is -N=.

In some embodiments, R ^B7' is -L ^7' -R ^B71' . In some embodiments, two R ^B7' on the same atom together =O, =C(-L ^7' -R ^B71' ) ₂ , =NL ^7' -R ^B71' , or optionally substituted =CH ₂ or = form NH. In some embodiments, two R ^B7' on the same atom together form ═O. In some embodiments, L ^7' is a covalent bond. In some embodiments, R ^B7′ is R. In some embodiments, R ^B7' is -H.

In some embodiments, R ^B71' is R'. In some embodiments, R ^B71' is R. In some embodiments, R ^B71' is -H.

In some embodiments, L ^B is a covalent bond. In some embodiments, ^LB is an optionally substituted divalent C _1-10 saturated or partially unsaturated aliphatic chain, and one or more methylene units are optionally and independently -Cy-, -O-, -S-, -N(R ')-, -C(O)-, -C(S)-, -C(NR')-, -C(O)N(R')-, -N(R')C(O)N( R')-, -N(R')C(O)O-, -S(O)-, -S(O) ₂ -, -S(O) ₂ N(R')-, -C(O) )S-, or -C(O)O-. In some embodiments, ^LB is an optionally substituted divalent C _1-10 saturated or partially unsaturated heteroaliphatic chain having 1-6 heteroatoms, wherein one or more methylene units are optionally and independently -Cy-, -O- , -S-, -N(R')-, -C(O)-, -C(S)-, -C(NR')-, -C(O)N(R')-, -N( R')C(O)N(R')-, -N(R')C(O)O-, -S(O)-, -S(O) ₂ -, -S(O) ₂ N( R′)-, -C(O)S-, or -C(O)O-. In some embodiments, at least methylene units are replaced. In some embodiments, ^LB is an optionally substituted C _1-10 alkylene. In some embodiments, L ^B is -CH ₂ CH ₂ -. In some embodiments, at least one methylene unit is replaced with -C(O)-. In some embodiments, at least one methylene unit is replaced with -C(O)N(R')-. In some embodiments, at least one methylene unit is replaced with -N(R')-. In some embodiments, at least one methylene unit is replaced with -NH-. In some embodiments, at least one methylene unit is replaced with -Cy-. In some embodiments, L ^B is or comprises optionally substituted -N=CH-. In some embodiments, L ^B is or comprises -C(O)-. In some embodiments, L ^B is or comprises -O-. In some embodiments, L ^B is or comprises -OC(O)-. In some embodiments, L ^B is or comprises -CH ₂ OC(O)-.

In some embodiments, each -Cy- is independently an optionally substituted 3-20 membered monocyclic, bicyclic or polycyclic, saturated, partially saturated or aromatic ring having 0-10 heteroatoms. Suitable monocyclic unit(s) of -Cy- are described herein. In some embodiments, -Cy- is monocyclic. In some embodiments, -Cy- is bicyclic. In some embodiments, -Cy- is polycyclic. In some embodiments, -Cy- is an optionally substituted divalent 3-10 membered monocyclic, saturated or partially unsaturated ring having 0-5 heteroatoms. In some embodiments, -Cy- is an optionally substituted divalent 5-10 membered aromatic ring having 0-5 heteroatoms. In some embodiments, -Cy- is an optionally substituted phenylene. In some embodiments, -Cy- is phenylene.

In some embodiments, R' is R. In some embodiments, R' is -C(0)R. In some embodiments, R' is -C(O)OR. In some embodiments, R' is -C(O)N(R) ₂ . In some embodiments, R' is -SO ₂ R.

In some embodiments, R' of various structures is a protecting group (eg, for amino, hydroxyl, etc.), such as those suitable for oligonucleotide synthesis. In some embodiments, R is an optionally substituted phenyl. In some embodiments, R is phenyl. In some embodiments, R is 4-nitrophenyl. In some embodiments, R is -CH ₂ CH ₂ -(4-nitrophenyl). In some embodiments, R' is -C(O)NPh ₂ .

In some embodiments, each R is independently —H, C _1-20 aliphatic, C _1-20 heteroaliphatic having 1-10 heteroatoms, C _6-30 aryl, C _6-30 arylaliphatic, 1 optionally selected from C _6-30 arylheteroaliphatic having ~10 heteroatoms, 5-20 membered heteroaryl having 1-10 heteroatoms, and 3-30 membered heterocyclyl having 1-10 heteroatoms; is a substituted group. In some embodiments, two R groups optionally and independently form a covalent bond together. In some embodiments, two or more R groups on the same atom optionally and independently form an optionally substituted 3-20 membered monocyclic, bicyclic or polycyclic ring having 0-10 heteroatoms in addition to the corresponding atom together with the corresponding atom. In some embodiments, two R groups on the same atom optionally and independently form an optionally substituted 3-20 membered monocyclic, bicyclic or polycyclic ring having 0-10 heteroatoms in addition to the corresponding atom together with the corresponding atom. In some embodiments, two or more R groups on two or more atoms, optionally and independently with atoms intervening therebetween, are optionally substituted 3-30 membered monocycles having 0-10 heteroatoms in addition to those intervening atoms. , form bicyclic or polycyclic rings. In some embodiments, two R groups on two or more atoms, optionally and independently with atoms intervening therebetween, have 0-10 heteroatoms in addition to those intervening atoms, optionally substituted 3-30 membered monocycles; form bicyclic or polycyclic rings. In some embodiments, the formed ring is monocyclic. In some embodiments, the rings formed are bicyclic. In some embodiments, the rings formed are polycyclic. In some embodiments, each monocyclic ring unit independently has 3-10 members (e.g., 3-8, 3-7, 3-6, 5-10, 5-8, 5-7, 5-6, 3, 4, 5, 6, 7, 8, 9, or 10 members, etc.), independently saturated, partially saturated, or aromatic, and independently having 0 to 5 heteroatoms. In some embodiments, a ring is saturated. In some embodiments, the ring is partially saturated. In some embodiments, a ring is aromatic. In some embodiments, the formed ring has 1-5 heteroatoms. In some embodiments, the formed ring has 1 heteroatom. In some embodiments, the formed ring has 2 heteroatoms. In some embodiments, the heteroatom is nitrogen. In some embodiments, the heteroatom is oxygen.

In some embodiments, R is -H.

In some embodiments, R is an optionally substituted C _1-20 , C _1-15 , C _1-10 , C _1-8 , C _1-6 , C _1-5 , C _1-4 , C _1-3 , or C _1-2 aliphatic. In some embodiments, R is an optionally substituted alkyl. In some embodiments, R is an optionally substituted C _1-6 alkyl. In some embodiments, R is optionally substituted methyl. In some embodiments, R is an optionally substituted cycloaliphatic. In some embodiments, R is an optionally substituted cycloalkyl.

In some embodiments, R is an optionally substituted C _1-20 heteroaliphatic having 1-10 heteroatoms.

In some embodiments, R is an optionally substituted C _6-20 aryl. In some embodiments, R is an optionally substituted phenyl. In some embodiments, R is phenyl.

In some embodiments, R is an optionally substituted C _6-20 arylaliphatic. In some embodiments, R is an optionally substituted C _6-20 arylalkyl. In some embodiments, R is benzyl. In some embodiments, R is an optionally substituted C _6-20 arylheteroaliphatic having 1-10 heteroatoms.

In some embodiments, R is an optionally substituted 5-20 membered heteroaryl having 1-10 heteroatoms. In some embodiments, R is an optionally substituted 5-membered heteroaryl having 1-4 heteroatoms. In some embodiments, R is an optionally substituted 6-membered heteroaryl having 1-4 heteroatoms. In some embodiments, R is an optionally substituted 3-20 membered heterocyclyl having 1-10 heteroatoms. In some embodiments, R is an optionally substituted 3-10 membered heterocyclyl having 1-5 heteroatoms. In some embodiments, R is an optionally substituted 5-6 membered heterocyclyl having 1-5 heteroatoms. In some embodiments, a heterocyclyl is saturated. In some embodiments, a heterocyclyl is partially saturated.

In some embodiments, the heteroatom is selected from boron, nitrogen, oxygen, sulfur, silicon, and phosphorus. In some embodiments, the heteroatom is selected from nitrogen, oxygen, sulfur, and silicon. In some embodiments, the heteroatom is selected from nitrogen, oxygen, and silicon. In some embodiments, the heteroatom is nitrogen. In some embodiments, the heteroatom is oxygen. In some embodiments, the heteroatom is sulfur.

As will be appreciated by those skilled in the art, implementations described for variables can be readily combined to provide a variety of structures. One skilled in the art also understands that implementations described for a variable can be readily used for other variables that can be that variable (e.g., R', R ^B2 , R ^B3 , R ^B4 , R ^B5 , R ^B6 , R ^B2' , R ^{B3 ' ,} R ^{B4 ' ,} R ^{B5 ' ,} R ^{B6 ',} etc. embodiment of R ^; an embodiment of L ^B for L ^B5' , L ^B6', etc.). Exemplary embodiments and combinations thereof include, but are not limited to, the structures illustrated herein. Specific examples are described below.

For example, in some embodiments, ring BA is optionally substituted or protected. am. In some embodiments, ring BA is am. In some embodiments, ring BA is am.

In some embodiments, X ⁴ is -C(O)-, and O in -C(O)- of X ⁴ is -H of R ⁵ , for example -NHR', -OH, or -H of R ^{5' .} Can form a hydrogen bond with -H in -SH. In some embodiments, X ⁴ is -C(O)- and X ⁵ is -C(R ⁵ )=. In some embodiments, R ^5' is -NHR'. In some embodiments, R ^5' is -L ^B5 -NHR'. In some embodiments, L ^B5 is optionally substituted -CH ₂ -. In some embodiments, methylene units are replaced with -C(O)-. In some embodiments, L ^B5 is -C(O)-. In some embodiments, R' is optionally substituted methyl. In some embodiments, R' is -CH ₂ Ph. In some embodiments, R' is an optionally substituted phenyl. In some embodiments, R' is phenyl. In some embodiments, R' is an optionally substituted C _1-6 aliphatic. In some embodiments, R' is an optionally substituted C _1-6 alkyl. In some embodiments, R' is optionally substituted methyl. In some embodiments, R' is methyl. In some embodiments, ring BA is optionally protected am. In some embodiments, ring BA is am. In some embodiments, ring BA is optionally protected am. In some embodiments, ring BA is am. In some embodiments, ring BA is optionally protected am. In some embodiments, ring BA is am. In some embodiments, ring BA is optionally protected am. In some embodiments, ring BA is am. In some embodiments, ring BA is optionally protected am. In some embodiments, ring BA is am. In some embodiments, ring BA is optionally protected am. In some embodiments, ring BA is am. In some embodiments, ring BA is optionally protected am. In some embodiments, ring BA is am.

In some embodiments, X ¹ is -C(-)= and X ⁴ is =C(-N(R ^B4 ) ₂ )-. In some embodiments, two R groups on the same atom (eg, nitrogen atom) together form an optionally substituted =CH ₂ or =NH. In some embodiments, two R groups on the same atom (eg, nitrogen atom) together form an optionally substituted =C(-L ^B4 -R) ₂ , =NL ^B4 -R. In some embodiments, the group formed is =CHN(R) ₂ . In some embodiments, the group formed is =CHN(CH ₃ ) ₂ . In some embodiments, X ⁴ is =C(-N=CHN(CH ₃ ) ₂ )-. In some embodiments, -N(R ^B4 ) ₂ is -NR ^B4 . In some embodiments, R ^B4 is -NHC(O)R. In some embodiments, ring BA is optionally substituted or protected am. In some embodiments, ring BA is am. In some embodiments, ring BA is am.

In some embodiments, X ¹ is -N(-)-, X ² is -C(O)-, and X ³ is -N(R ^B3 )-. In some embodiments, X ¹ is -N(-)-, X ² is -C(O)-, X ³ is -N(R ^B3 )-, and X ⁴ is -C(R ^B4 )= . In some embodiments, X ¹ is -N(-)-, X ² is -C(O)-, X ³ is -N(R ^B3 )-, and X ⁴ is -C(R ^B4 )= , X ⁵ is -C(R ^B5 )=. In some embodiments, ring BA is optionally substituted or protected am. In some embodiments, ring BA is am.

In some embodiments, X ³ is -N(R')-. In some embodiments, R' is -C(0)R. In some embodiments, X ⁴ is -C(R ^B4 ) ₂ -. In some embodiments, R ^B4 is -R. In some embodiments, R ^B4 is -H. In some embodiments, X ⁴ is -CH ₂ -. In some embodiments, X ⁵ is -C(R ^B5 ) ₂ -. In some embodiments, R ^B5 is -R. In some embodiments, R ^B5 is -H. In some embodiments, X ⁵ is -CH ₂ -. In some embodiments, ring BA is optionally substituted or protected am. In some embodiments, ring BA is am. In some embodiments, ring BA is am.

In some embodiments, X ⁴ is -C(R ^B4 )=. In some embodiments, X ⁴ is -CH=. In some embodiments, X ⁵ is -C(R ^B5 )=. In some embodiments, X ⁵ is -CH=. In some embodiments, ring BA is optionally substituted or protected am. In some embodiments, ring BA is am. In some embodiments, ring BA is optionally substituted or protected am. In some embodiments, ring BA is am.

In some embodiments, X ⁴ is -C(R ^B4 ) ₂ -. In some embodiments, X ⁴ is -CH ₂ -. In some embodiments, X ⁵ is -C(R ^B5 )=. In some embodiments, X ⁵ is -CH=. In some embodiments, ring BA is optionally substituted or protected am. In some embodiments, ring BA is am. In some embodiments, ring BA is am.

In some embodiments, X ¹ is -N(-)-, X ² is -C(O)-, X ³ is -N(R ^B3 )-, and X ⁴ is -C(R ^B4 )= , X ⁵ is -C(R ^B5 )=, and X ⁶ is -C(O)-. In some embodiments, each of R ^B3 , R ^B4 , and R ^B5 is independently R. In some embodiments, R ^B3 is -H. In some embodiments, R ^B4 is -H. In some embodiments, R ^B5 is -H. In some embodiments, BA is optionally substituted or protected is or includes In some embodiments, BA is am.

In some embodiments, X ¹ is -N(-)-, X ² is -C(O)-, and X ³ is -N(R ^B3 )-. In some embodiments, X ⁴ is -C(R ^B4 ) ₂ -, and two R ^B4 together form =O, or =C(-L ^B4 -R ^B41 ) ₂ , =NL ^B4 -R ^B41 . In some embodiments, X ⁴ is -C(=NR ^B4 )-. In some embodiments, X ⁵ is -C(R ^B5 )=. In some embodiments, R ^B41 or R ^B4 and R ^B5 are R and together with the intervening atoms form an optionally substituted ring as described herein. In some embodiments, ring BA is optionally substituted or protected am. In some embodiments, ring BA is am. In some embodiments, ring BA is optionally substituted or protected am. In some embodiments, ring BA is am. In some embodiments, X ¹ is -N(-)-, X ² is -C(O)-, and X ³ is -N=. In some embodiments, X ⁴ is -C(-N(R ^B4 ) ₂ )=. In some embodiments, X ⁴ is -C(-NHR ^B4 )=. In some embodiments, X ⁵ is -C(R ^B5 )=. In some embodiments, one R ^B4 and R ^B5 together form an optionally substituted ring as described herein. In some embodiments, the ring formed is an optionally substituted 5-membered ring having a nitrogen atom. In some embodiments, ring BA is optionally substituted or protected am. In some embodiments, ring BA is am. In some embodiments, ring BA is optionally substituted or protected am. In some embodiments, ring BA is am. In some embodiments, ring BA is optionally substituted or protected am. In some embodiments, ring BA is am. In some embodiments, ring BA is optionally substituted or protected am. In some embodiments, ring BA is am.

In some embodiments, ring BA has the structure of Formula BA-IV or BA-V. In some embodiments, X ¹ is -N(-)-, X ² is -C(O)-, and X ³ is -N=. In some embodiments, X ¹ is -N(-)-, X ² is -C(O)-, X ³ is -N=, and X ⁶ is -C(R ^B6 )=. In some embodiments, ring BA ^A is 5-6 members. In some embodiments, ring BA ^A is monocyclic. In some embodiments, ring BA ^A is partially unsaturated. In some embodiments, ring BA ^A is aromatic. In some embodiments, ring BA ^A has 0-2 heteroatoms. In some embodiments, ring BA ^A has 1-2 heteroatoms. In some embodiments, ring BA ^A has 1 heteroatom. In some embodiments, ring BA ^A has 2 heteroatoms. In some embodiments, the heteroatom is nitrogen. In some embodiments, the heteroatom is oxygen. In some embodiments, ring BA is optionally substituted or protected , , or am. In some embodiments, ring BA is , , or am.

In some embodiments, ring BA is an optionally substituted 5-membered ring. In some embodiments, X ¹ is bonded to X ⁵ . In some embodiments, each of X ⁴ and X ⁵ is independently -CH=. In some embodiments, X ¹ is -N(-)-, X ² is -C(O)-, X ³ is -NH-, X ⁴ is -CH=, and X ⁵ is -CH= . In some embodiments, ring BA is optionally substituted or protected am. In some embodiments, ring BA is am.

In some embodiments, ring BA has the structure of Formula BA-VI. In some embodiments, X ^1' is -N(-)-, X ^2' is -C(O)-, and X ^3' is -N(R ^B3 )-. In some embodiments, X ^1' is -N(-)-, X ^2' is -C(O)-, X ^3' is -N(R ^B3 )-, and X ^4' is -C(R ^B4' )=, X ^5' is -N=, X ^6' is -C(R ^B6' )=, and X ^7' is -N=. In some embodiments, X ^1' is -N(-)-, X ^2' is -C(O)-, X ^3' is -N(R ^B3 )-, and X ^4' is -C(R ^B4' )=, X ^5' is -C(R ^B5' )=, X ^6' is -C(R ^B6' )=, and X ^7' is -C(R ^B7' )=. In some embodiments, ring BA is optionally substituted or protected am. In some embodiments, ring BA is am. In some embodiments, ring BA is optionally substituted or protected am. In some embodiments, ring BA is am. In some embodiments, ring BA is am. In some embodiments, ring BA is optionally substituted or protected am. In some embodiments, ring BA is am. In some embodiments, ring BA is am. In some embodiments, ring BA is optionally substituted or protected am. In some embodiments, ring BA is am. In some embodiments, ring BA is am. In some embodiments, ring BA is optionally substituted or protected am. In some embodiments, ring BA is am. In some embodiments, ring BA is optionally substituted or protected am. In some embodiments, ring BA is am. In some embodiments, X ^1' is -N(-)-, X ^2' is -C(R ^B2' )=, and X ^3' is -N=. In some embodiments, X ^1' is -N(-)-, X ^2' is -C(R ^B2' )=, X ^3' is -N=, and X ^4' is -C(-N( R ^B4' ) ₂ )=, X ^5' is -N=, X ^6' is -C(O)-, and X ^7' is -N(R ^B7' )-. In some embodiments, ring BA is optionally substituted or protected am. In some embodiments, ring BA is am.

In some embodiments, X ¹ is -C(-)=, X ² is -C(O)-, and X ³ is -N(R ^B3 )-. In some embodiments, X ¹ is -C(-)=, X ² is -C(O)-, and X ³ is -N(R ^B3 )-, -C(-N(R ^B4 ) ₂ )= , and X ⁴ is -C(R ^B4 )=. In some embodiments, X ¹ is -C(-)=, X ² is -C(O)-, X ³ is -N(R ^B3 )-, -C(-N(R ^B4 ) ₂ )= , X ⁴ is -C(R ^B4 )=, and X ⁶ is -C(R ^B6 )=. In some embodiments, each of R ^B3 , R ^B4 , and R ^B6 is independently -H. In some embodiments, ring BA is optionally substituted or protected am. In some embodiments, ring BA is am. In some embodiments, ring BA is optionally substituted or protected am. In some embodiments, ring BA is am.

In some embodiments, ring BA is has the structure of In some embodiments, R ^B4 is optionally substituted aryl. In some embodiments, R ^B4 is optionally substituted am. In some embodiments, R ^B4 is am. In some embodiments, R ^B5 is -H. In some embodiments, R ^B5 is -N(R′) ₂ . In some embodiments, R ^B5 is -NH ₂ . In some embodiments, ring BA is am. In some embodiments, ring BA is am.

As described herein, ring BA may be optionally substituted. In some embodiments, X ² , X ³ , X ⁴ , X ⁵ , X ⁶ , X ^2′ , X ^3′ , X ^4′ , X ^5′ , X ^6′ , and X ^7′ each represent -CH=, independently and optionally substituted when -C(OH)=, -C(-NH ₂ )=, -CH ₂ -, -C(=NH)-, or -NH-. In some embodiments, X ² , X ³ , X ⁴ , X ⁵ , X ⁶ , X ^2′ , X ^3′ , X ^4′ , X ^5′ , X ^6′ , and X ^7′ each represent -CH=, -CH ₂ -, or -NH-, independently and optionally substituted. In some embodiments, each of X ² , X ³ , X ⁴ , X ⁵ , X ⁶ , X ^2' , X ^3' , X ^4' , X ^5' , X ^6' , and X ^7' is -CH= are independently and optionally substituted. In some embodiments, each of X ² , X ³ , X ⁴ , X ⁵ , X ⁶ , X ^2' , X ^3' , X ^4' , X ^5' , X ^6' , and X ^7' is -CH ₂ - are independently and optionally substituted. In some embodiments, each of X ² , X ³ , X ⁴ , X ⁵ , X ⁶ , X ^2′ , X ^3′ , X ^4′ , X ^5′ , X ^6′ , and X ^7′ is -NH- are independently and optionally substituted. In some embodiments, X ² is optionally substituted -CH=, -C(OH)=, -C(-NH ₂ )=, -CH ₂ -, -C(=NH)-, or -NH-. In some embodiments, X ³ is optionally substituted -CH=, -C(OH)=, -C(-NH ₂ )=, -CH ₂ -, -C(=NH)-, or -NH-. In some embodiments, X ⁴ is optionally substituted -CH=, -C(OH)=, -C(-NH ₂ )=, -CH ₂ -, -C(=NH)-, or -NH-. In some embodiments, X ⁵ is optionally substituted -CH=, -C(OH)=, -C(-NH ₂ )=, -CH ₂ -, -C(=NH)-, or -NH-. In some embodiments, X ⁶ is optionally substituted -CH=, -C(OH)=, -C(-NH ₂ )=, -CH ₂ -, -C(=NH)-, or -NH-. In some embodiments, X ^2′ is optionally substituted -CH=, -C(OH)=, -C(-NH ₂ )=, -CH ₂ -, -C(=NH)-, or -NH- . In some embodiments, X ^3′ is optionally substituted -CH=, -C(OH)=, -C(-NH ₂ )=, -CH ₂ -, -C(=NH)-, or -NH- . In some embodiments, X ^4′ is optionally substituted -CH=, -C(OH)=, -C(-NH ₂ )=, -CH ₂ -, -C(=NH)-, or -NH- . In some embodiments, X ^5′ is optionally substituted -CH=, -C(OH)=, -C(-NH ₂ )=, -CH ₂ -, -C(=NH)-, or -NH- . In some embodiments, X ^6′ is optionally substituted -CH=, -C(OH)=, -C(-NH ₂ )=, -CH ₂ -, -C(=NH)-, or -NH- . In some embodiments, X ^7′ is optionally substituted -CH=, -C(OH)=, -C(-NH ₂ )=, -CH ₂ -, -C(=NH)-, or -NH- .

As shown herein, in some embodiments, a provided oligonucleotide comprising a specific nucleobase (eg, b001A, b002A, b008U, C, A, etc.) opposite a target adenosine is specifically selected (eg, a reference nucleus such as U). base) can provide improved editing efficiency. In some embodiments, the opposite nucleoside is linked to I on the 3' side.

In some embodiments, the nucleobase is ring BA as described herein. In some embodiments, an oligonucleotide comprises one or more ring BAs as described herein.

In some embodiments, the opposing nucleoside is, for example, L010 ( ), L012( ), or L028( ) is a base with a structure of As will be appreciated by those skilled in the art and as shown in various oligonucleotides, free base nucleosides may be used in other portions of oligonucleotides, and oligonucleotides may contain one or more (e.g., 1, 2, 3, 4, 5 one or more), optionally contiguous, abasic nucleosides. In some embodiments, the first domain comprises one or more optionally contiguous base free nucleosides. In some embodiments, an oligonucleotide comprises no more than one abasic nucleoside. In some embodiments, each free base nucleoside is independently in the first domain or the first subdomain of the second domain. In some embodiments, each free base nucleoside is independently in the first domain. In some embodiments, each free base nucleoside is independently in the first subdomain of the second domain. In some embodiments, the abasic nucleoside is opposite the target adenosine. As shown herein, a single free base nucleoside can replace one or more nucleosides, each independently comprising a nucleobase, in a reference oligonucleotide. For example, L010 can be used to replace 1 nucleoside containing a nucleobase, and L012 can be used to replace 1, 2, or 3 nucleosides (each independently containing a nucleobase). , and L028 can be used to replace 1, 2, or 3 nucleosides, each independently containing a nucleobase. In some embodiments, a basic nucleoside is linked to an adjacent nucleoside (optionally abasic) in the 3′ direction via a sterically random linkage (eg, a sterically random phosphorothioate internucleoside linkage). In some embodiments, each basic nucleoside is independently linked to an adjacent nucleoside (optionally abasic) in the 3' direction via a sterically random linkage (e.g., a sterically random phosphorothioate internucleoside linkage). .

In some embodiments, a modified nucleobase opposite the target adenine can greatly improve the properties and/or activity of the oligonucleotide. In some embodiments, a deformed nucleus in an opposite position, even if flanked by a G (e.g., on the 3' side) and/or another nucleobase (e.g., C) provides much lower activity or exhibits little activity. Bases can provide high activity.

In some embodiments, the second domain comprises one or more sugars (eg, natural DNA sugars) comprising two 2'-Hs. In some embodiments, the second domain comprises one or more sugars (eg, native RNA sugars) comprising 2'-OH. In some embodiments, the second domain comprises one or more modified sugars. In some embodiments, a modified sugar comprises a 2'-modification. In some embodiments, the modified sugar is a bicyclic sugar, such as an LNA sugar. In some embodiments, the modified sugar is acyclic (eg, by breaking the C2-C3 bonds of the corresponding cyclic sugar).

In some embodiments, the second domain is about 1-50 (e.g., about 5, 6, 7, 8, 9, or 10 to about 10, 11, 12, 13, 14, 15, 16, 17 , 18, 19, 20, 21, 22, 23, 24, 25, 30, 40 or 50, or about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 40 or 50 etc, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 , 13, 14, 15, 16, 17, 18, 19, or 20, etc.) modified sugars. In some embodiments, the second domain is about 1-50 (e.g., about 5, 6, 7, 8, 9, or 10 to about 10, 11, 12, 13, 14, 15, 16, 17 , 18, 19, 20, 21, 22, 23, 24, 25, 30, 40 or 50, or about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 40 or 50 etc, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 , 13, 14, 15, 16, 17, 18, 19, or 20, etc.) independently of bicyclic sugars (eg, LNA sugars) or 2'-OR modified sugars (R are independently optionally substituted C _1-6 and modified sugars that are aliphatic). In some embodiments, the second domain is about 1-50 (e.g., about 5, 6, 7, 8, 9, or 10 to about 10, 11, 12, 13, 14, 15, 16, 17 , 18, 19, 20, 21, 22, 23, 24, 25, 30, 40 or 50, or about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 40 or 50 etc, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 , 13, 14, 15, 16, 17, 18, 19, or 20, etc.) independently 2'-OR modified sugars (R is independently optionally substituted C _1-6 aliphatic). . In some embodiments, the number is one. In some embodiments, the number is two. In some embodiments, the number is three. In some embodiments, the number is four. In some embodiments, the number is five. In some embodiments, the number is six. In some embodiments, the number is 7. In some embodiments, the number is eight. In some embodiments, the number is 9. In some embodiments, the number is 10. In some embodiments, the number is 11. In some embodiments, the number is 12. In some embodiments, the number is 13. In some embodiments, the number is 14. In some embodiments, the number is 15. In some embodiments, the number is 16. In some embodiments, the number is 17. In some embodiments, the number is 18. In some embodiments, the number is 19. In some embodiments, the number is 20. In some embodiments, R is methyl.

In some embodiments, about 5%-100% (e.g., about 10%-100%, 20-100%, 30%-100%, 40%-100%, 50%-100%) of all sugars in the second domain. 80%, 50%~85%, 50%~90%, 50%~95%, 60%~80%, 60%~85%, 60%~90%, 60%~95%, 60%~100% , 65%~80%, 65%~85%, 65%~90%, 65%~95%, 65%~100%, 70%~80%, 70%~85%, 70%~90%, 70 %~95%, 70%~100%, 75%~80%, 75%~85%, 75%~90%, 75%~95%, 75%~100%, 80%~85%, 80%~ 90%, 80% to 95%, 80% to 100%, 85% to 90%, 85% to 95%, 85% to 100%, 90% to 95%, 90% to 100%, 10%, 20% , 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%, etc.) are independently modified sugars. In some embodiments, about 5%-100% (e.g., about 10%-100%, 20-100%, 30%-100%, 40%-100%, 50%-100%) of all sugars in the second domain. 80%, 50%~85%, 50%~90%, 50%~95%, 60%~80%, 60%~85%, 60%~90%, 60%~95%, 60%~100% , 65%~80%, 65%~85%, 65%~90%, 65%~95%, 65%~100%, 70%~80%, 70%~85%, 70%~90%, 70 %~95%, 70%~100%, 75%~80%, 75%~85%, 75%~90%, 75%~95%, 75%~100%, 80%~85%, 80%~ 90%, 80% to 95%, 80% to 100%, 85% to 90%, 85% to 95%, 85% to 100%, 90% to 95%, 90% to 100%, 10%, 20% , 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%, etc.) independently of the affected party (e.g., LNA) or a 2′-OR modified sugar, wherein R is independently optionally substituted C _1-6 aliphatic. In some embodiments, about 5%-100% (e.g., about 10%-100%, 20-100%, 30%-100%, 40%-100%, 50%-100%) of all sugars in the second domain. 80%, 50%~85%, 50%~90%, 50%~95%, 60%~80%, 60%~85%, 60%~90%, 60%~95%, 60%~100% , 65%~80%, 65%~85%, 65%~90%, 65%~95%, 65%~100%, 70%~80%, 70%~85%, 70%~90%, 70 %~95%, 70%~100%, 75%~80%, 75%~85%, 75%~90%, 75%~95%, 75%~100%, 80%~85%, 80%~ 90%, 80% to 95%, 80% to 100%, 85% to 90%, 85% to 95%, 85% to 100%, 90% to 95%, 90% to 100%, 10%, 20% , 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%, etc.) independently per 2'-OR variant (R are independently optionally substituted C _1-6 aliphatic). In some embodiments, the percentage is at least about 50%. In some embodiments, the percentage is at least about 55%. In some embodiments, the percentage is at least about 60%. In some embodiments, the percentage is at least about 65%. In some embodiments, the percentage is at least about 70%. In some embodiments, the percentage is at least about 75%. In some embodiments, the percentage is at least about 80%. In some embodiments, the percentage is at least about 85%. In some embodiments, the percentage is at least about 90%. In some embodiments, the percentage is at least about 95%. In some embodiments, the percentage is about 100%. In some embodiments, R is methyl.

In some embodiments, the second domain has about 1-50 (e.g., about 5, 6, 7, 8, 9, or 10 to about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 40 or 50, or about 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 40 or 50, etc., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, etc.) modified sugars. In some embodiments, about 5%-100% (e.g., about 10%-100%, 20-100%, 30%-100%, 40%-100%, 50%-80%) of the sugars in the second domain. %, 50%~85%, 50%~90%, 50%~95%, 60%~80%, 60%~85%, 60%~90%, 60%~95%, 60%~100%, 65%~80%, 65%~85%, 65%~90%, 65%~95%, 65%~100%, 70%~80%, 70%~85%, 70%~90%, 70% ~95%, 70%~100%, 75%~80%, 75%~85%, 75%~90%, 75%~95%, 75%~100%, 80%~85%, 80%~90 %, 80%~95%, 80%~100%, 85%~90%, 85%~95%, 85%~100%, 90%~95%, 90%~100%, 10%, 20%, 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%, etc.) independently, a non-2'-F variant It is a transforming party in In some embodiments, between about 50% and 100% of the sugars in the second domain (e.g., between about 50% and 80%, 50% and 85%, 50% and 90%, 50% and 95%, 60% and 80%, 60%-85%, 60%-90%, 60%-95%, 60%-100%, 65%-80%, 65%-85%, 65%-90%, 65%-95% , 65%~100%, 70%~80%, 70%~85%, 70%~90%, 70%~95%, 70%~100%, 75%~80%, 75%~85%, 75 %~90%, 75%~95%, 75%~100%, 80%~85%, 80%~90%, 80%~95%, 80%~100%, 85%~90%, 85%~ 95%, 85% to 100%, 90% to 95%, 90% to 100%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100 %, etc.) are, independently, modified sugars with non-2'-F modifications. In some embodiments, each modified sugar in the second domain is independently a bicyclic sugar (eg, LNA sugar), an acyclic sugar (eg, UNA sugar), a sugar with a 2'-OR variant, or a 2'-N (R ) is selected from sugars with ₂ modifications (each R is independently an optionally substituted C _1-6 aliphatic).

In some embodiments, the second domain comprises one or more 2'-F modified sugars. In some embodiments, the second domain does not include a 2'-F modified sugar. In some embodiments, the second domain comprises one or more heterocyclic sugars and/or 2'-OR modified sugars (R is not -H). In some embodiments, the level of bicyclic sugars and/or 2'-OR modified sugars (R is not -H), individually or in combination, is relatively high compared to 2'-F modified sugars. In some embodiments, up to about 1% to 95% (e.g., up to about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%) of the sugars in the second domain. %, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, etc.) includes 2'-F. In some embodiments, up to about 50% of the sugars in the second domain include a 2'-F. In some embodiments, the second domain comprises _one or more (eg, about 1-20, 1, 2, 3, 4, 5, 6, 7, 8, 9 , 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, etc.) modified sugars. In some embodiments, the _second domain comprises one or more (eg, about 1-20, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10; 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, etc.) modified sugars. In some embodiments, the second domain is one or more (e.g., about 1-20, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 , 16, 17, 18, 19, 20, etc.) of heterocyclic sugars (eg, LNA sugars). In some embodiments, the second domain is one or more (e.g., about 1-20, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 , 16, 17, 18, 19, 20, etc.) of acyclic sugars (eg, UNA sugars).

In some embodiments, up to about 1% to 95% (e.g., up to about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%) of the sugars in the second domain. %, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, etc.) contain the 2'-MOE. In some embodiments, up to about 50% of the sugars in the second domain comprise a 2'-MOE. In some embodiments, the sugar in the second domain does not include a 2'-MOE.

In some embodiments, the second domain is about 1-50 (e.g., about 5, 6, 7, 8, 9, or 10 to about 10, 11, 12, 13, 14, 15, 16, 17 , 18, 19, 20, 21, 22, 23, 24, 25, 30, 40 or 50, or about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 40 or 50 etc, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 , 13, 14, 15, 16, 17, 18, 19, or 20, etc.) modified internucleotide linkages. In some embodiments, about 5%-100% (e.g., about 10%-100%, 20-100%, 30%-100%, 40%-100%, 50%) of the internucleotide linkages in the second domain %~80%, 50%~85%, 50%~90%, 50%~95%, 60%~80%, 60%~85%, 60%~90%, 60%~95%, 60%~ 100%, 65%~80%, 65%~85%, 65%~90%, 65%~95%, 65%~100%, 70%~80%, 70%~85%, 70%~90% , 70%~95%, 70%~100%, 75%~80%, 75%~85%, 75%~90%, 75%~95%, 75%~100%, 80%~85%, 80 %~90%, 80%~95%, 80%~100%, 85%~90%, 85%~95%, 85%~100%, 90%~95%, 90%~100%, 10%, 20%, 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%, etc.) are modified internucleotide linkages. In some embodiments, each internucleotide linkage in the second domain is independently a modified internucleotide linkage. In some embodiments, each modified internucleotide linkage is independently a chiral internucleotide linkage. In some embodiments, the modified or chiral internucleotidic linkage is a phosphorothioate internucleotidic linkage. In some embodiments, a modified or chiral internucleotidic linkage is a non-negatively charged internucleotidic linkage. In some embodiments, the modified or chiral internucleotide linkage is a neutral internucleotide linkage (eg n001). In some embodiments, each modified internucleotide linkage is independently a phosphorothioate internucleotide linkage or a non-negatively charged internucleotide linkage. In some embodiments, each modified internucleotide linkage is independently a phosphorothioate internucleotide linkage or a neutral internucleotide linkage. In some embodiments, each modified internucleotide linkage is independently a phosphorothioate internucleotide linkage. In some embodiments, at least about 1-50 (e.g., about 5, 6, 7, 8, 9, or 10 to about 10, 11, 12, 13, 14, 15, 16 in the second domain) , 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 40 or 50, or about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 40 or 50 etc, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, or 20, etc.) chiral internucleotide linkages are chirally controlled. In some embodiments, at least 5%-100% (e.g., about 10%-100%, 20-100%, 30%-100%, 40%-100%, 50%~80%, 50%~85%, 50%~90%, 50%~95%, 60%~80%, 60%~85%, 60%~90%, 60%~95%, 60% ~100%, 65%~80%, 65%~85%, 65%~90%, 65%~95%, 65%~100%, 70%~80%, 70%~85%, 70%~90 %, 70%~95%, 70%~100%, 75%~80%, 75%~85%, 75%~90%, 75%~95%, 75%~100%, 80%~85%, 80%~90%, 80%~95%, 80%~100%, 85%~90%, 85%~95%, 85%~100%, 90%~95%, 90%~100%, 10% , 20%, 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%, etc.) are chirally controlled. In some embodiments, at least 5%-100% (e.g., about 10%-100%, 20-100%, 30%-100%, 40%-100%) of the phosphorothioate internucleotide linkages in the second domain 100%, 50%~80%, 50%~85%, 50%~90%, 50%~95%, 60%~80%, 60%~85%, 60%~90%, 60%~95% , 60%~100%, 65%~80%, 65%~85%, 65%~90%, 65%~95%, 65%~100%, 70%~80%, 70%~85%, 70 %~90%, 70%~95%, 70%~100%, 75%~80%, 75%~85%, 75%~90%, 75%~95%, 75%~100%, 80%~ 85%, 80% to 90%, 80% to 95%, 80% to 100%, 85% to 90%, 85% to 95%, 85% to 100%, 90% to 95%, 90% to 100% , 10%, 20%, 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%, etc.) are chirally controlled. In some embodiments, each is independently chirally controlled. In some embodiments, at least about 1-50 (e.g., about 5, 6, 7, 8, 9, or 10 to about 10, 11, 12, 13, 14, 15, 16 in the second domain) , 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 40 or 50, or about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 40 or 50 etc, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, or 20, etc.) chiral internucleotide linkages are Sp . In some embodiments, each is independently chirally controlled. In some embodiments, at least about 1-50 (e.g., about 5, 6, 7, 8, 9, or 10 to about 10, 11, 12, 13, 14, 15, 16 in the second domain) , 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 40 or 50, or about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 40 or 50 etc, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, or 20, etc.) phosphorothioate internucleotide linkages are Sp . In some embodiments, at least 5%-100% (e.g., about 10%-100%, 20-100%, 30%-100%, 40%-100%, 50%~80%, 50%~85%, 50%~90%, 50%~95%, 60%~80%, 60%~85%, 60%~90%, 60%~95%, 60% ~100%, 65%~80%, 65%~85%, 65%~90%, 65%~95%, 65%~100%, 70%~80%, 70%~85%, 70%~90 %, 70%~95%, 70%~100%, 75%~80%, 75%~85%, 75%~90%, 75%~95%, 75%~100%, 80%~85%, 80%~90%, 80%~95%, 80%~100%, 85%~90%, 85%~95%, 85%~100%, 90%~95%, 90%~100%, 10% , 20%, 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%, etc.) is Sp . In some embodiments, at least 5%-100% (e.g., about 10%-100%, 20-100%, 30%-100%, 40%-100%) of the phosphorothioate internucleotide linkages in the second domain 100%, 50%~80%, 50%~85%, 50%~90%, 50%~95%, 60%~80%, 60%~85%, 60%~90%, 60%~95% , 60%~100%, 65%~80%, 65%~85%, 65%~90%, 65%~95%, 65%~100%, 70%~80%, 70%~85%, 70 %~90%, 70%~95%, 70%~100%, 75%~80%, 75%~85%, 75%~90%, 75%~95%, 75%~100%, 80%~ 85%, 80% to 90%, 80% to 95%, 80% to 100%, 85% to 90%, 85% to 95%, 85% to 100%, 90% to 95%, 90% to 100% , 10%, 20%, 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%, etc.) is Sp . In some embodiments, the number is one or more. In some embodiments, the number is two or more. In some embodiments, the number is three or more. In some embodiments, the number is 4 or more. In some embodiments, the number is 5 or more. In some embodiments, the number is 6 or more. In some embodiments, the number is 7 or more. In some embodiments, the number is 8 or more. In some embodiments, the number is 9 or more. In some embodiments, the number is 10 or more. In some embodiments, the number is 11 or more. In some embodiments, the number is 12 or more. In some embodiments, the number is 13 or more. In some embodiments, the number is 14 or more. In some embodiments, the number is 15 or more. In some embodiments, the percentage is at least about 50%. In some embodiments, the percentage is at least about 55%. In some embodiments, the percentage is at least about 60%. In some embodiments, the percentage is at least about 65%. In some embodiments, the percentage is at least about 70%. In some embodiments, the percentage is at least about 75%. In some embodiments, the percentage is at least about 80%. In some embodiments, the percentage is at least about 85%. In some embodiments, the percentage is at least about 90%. In some embodiments, the percentage is at least about 95%. In some embodiments, the percentage is about 100%. In some embodiments, each internucleotide linkage linking two second domain nucleosides is independently a modified internucleotide linkage. In some embodiments, each modified internucleotide linkage is independently a chiral internucleotide linkage. In some embodiments, each modified internucleotide linkage is independently a phosphorothioate internucleotide linkage. In some embodiments, each chiral internucleotidic linkage is independently a phosphorothioate internucleotidic linkage. In some embodiments, each modified internucleotide linkage is independently an Sp chiral internucleotide linkage. In some embodiments, each modified internucleotide linkage is independently an S p phosphorothioate internucleotide linkage. In some embodiments, each chiral internucleotidic linkage is independently an S p phosphorothioate internucleotidic linkage. In some embodiments, the internucleotide linkages of the second domain are linked to two nucleosides of the second domain. In some embodiments, an internucleoside linkage between a nucleoside of a first domain and a nucleoside of a second domain may properly be considered an internucleoside linkage of a second domain. In some embodiments, a high (e.g., high relative to R p internucleotidic linkages and/or natural phosphate linkages) percentage of S p internucleotidic linkages results in improved properties and/or activity, e.g., higher stability and/or It has been observed to provide high adenosine editing activity.

In some embodiments, the second domain comprises some level of R p internucleotidic linkages. In some embodiments, the level is, for example, between about 5% and 100%, about 10% and 100%, 20 and 100%, 30% and 100%, 40% and 100% of all internucleotide linkages in the second domain. , 50%~80%, 50%~85%, 50%~90%, 50%~95%, 60%~80%, 60%~85%, 60%~90%, 60%~95%, 60 %~100%, 65%~80%, 65%~85%, 65%~90%, 65%~95%, 65%~100%, 70%~80%, 70%~85%, 70%~ 90%, 70% to 95%, 70% to 100%, 75% to 80%, 75% to 85%, 75% to 90%, 75% to 95%, 75% to 100%, 80% to 85% , 80%~90%, 80%~95%, 80%~100%, 85%~90%, 85%~95%, 85%~100%, 90%~95%, 90%~100%, 10 %, 20%, 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%. In some embodiments, the level is, for example, between about 5% and 100%, about 10% and 100%, 20 and 100%, 30% and 100%, 40% and 100% of all chiral internucleotide linkages in the second domain. %, 50%~80%, 50%~85%, 50%~90%, 50%~95%, 60%~80%, 60%~85%, 60%~90%, 60%~95%, 60%~100%, 65%~80%, 65%~85%, 65%~90%, 65%~95%, 65%~100%, 70%~80%, 70%~85%, 70% ~90%, 70%~95%, 70%~100%, 75%~80%, 75%~85%, 75%~90%, 75%~95%, 75%~100%, 80%~85 %, 80%~90%, 80%~95%, 80%~100%, 85%~90%, 85%~95%, 85%~100%, 90%~95%, 90%~100%, 10%, 20%, 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%. In some embodiments, the level is between about 5% and 100%, about 10% and 100%, 20 and 100%, 30% and 100%, 40% and 40% of all chiral control internucleotide linkages in the second domain, for example. 100%, 50%~80%, 50%~85%, 50%~90%, 50%~95%, 60%~80%, 60%~85%, 60%~90%, 60%~95% , 60%~100%, 65%~80%, 65%~85%, 65%~90%, 65%~95%, 65%~100%, 70%~80%, 70%~85%, 70 %~90%, 70%~95%, 70%~100%, 75%~80%, 75%~85%, 75%~90%, 75%~95%, 75%~100%, 80%~ 85%, 80% to 90%, 80% to 95%, 80% to 100%, 85% to 90%, 85% to 95%, 85% to 100%, 90% to 95%, 90% to 100% , 10%, 20%, 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%. In some embodiments, the percentage is about or up to about 50%. In some embodiments, the percentage is at least about 55%. In some embodiments, the percentage is at least about 60%. In some embodiments, the percentage is at least about 65%. In some embodiments, the percentage is at least about 70%. In some embodiments, the percentage is at least about 75%. In some embodiments, the percentage is at least about 80%. In some embodiments, the percentage is at least about 85%. In some embodiments, the percentage is at least about 90%. In some embodiments, the percentage is at least about 95%. In some embodiments, the percentage is about 100%. In some embodiments, the percentage is about or up to about 5%. In some embodiments, the percentage is about or up to about 10%. In some embodiments, the percentage is about or up to about 15%. In some embodiments, the percentage is about or up to about 20%. In some embodiments, the percentage is about or up to about 25%. In some embodiments, the percentage is about or up to about 30%. In some embodiments, the percentage is about or up to about 35%. In some embodiments, the percentage is about or up to about 40%. In some embodiments, the percentage is about or up to about 45%. In some embodiments, the percentage is about or up to about 50%. In some embodiments, about 1-50, 1-40, 1-30, 1-25, 1-20, 1-15, 1-10, 1-5, for example about 1, 2, 3, 4 , 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 internucleotide linkages are independently R p chiral internucleotide linkages. In some embodiments, the number is about or up to about 1. In some embodiments, the number is about or up to about 2. In some embodiments, the number is about or up to about 3. In some embodiments, the number is about or up to about 4. In some embodiments, the number is about or up to about 5. In some embodiments, the number is about or up to about 6. In some embodiments, the number is about or up to about 7. In some embodiments, the number is about or up to about 8. In some embodiments, the number is about or up to about 9. In some embodiments, the number is about or up to about 10.

In some embodiments, each phosphorothioate internucleotidic linkage in the second domain is independently chirally controlled. In some embodiments, each is independently Sp or R p. In some embodiments, the high level is Sp as described herein. In some embodiments, each phosphorothioate internucleotidic linkage in the second domain is chirally controlled and is Sp . In some embodiments, one or more, for example about 1-5 (eg, about 1, 2, 3, 4, or 5) is R p .

In some embodiments, each phosphorothioate internucleotidic linkage in the second domain is independently chirally controlled. In some embodiments, each is independently Sp or R p. In some embodiments, the high level is Sp as described herein. In some embodiments, each phosphorothioate internucleotidic linkage in the second domain is chirally controlled and is Sp . In some embodiments, one or more, for example about 1-5 (eg, about 1, 2, 3, 4, or 5) is R p .

In some embodiments, as exemplified in certain examples, the second domain comprises one or more non-negatively charged internucleotide linkages, each optionally and independently chirally controlled. In some embodiments, each non-negatively charged internucleotide linkage is independently n001. In some embodiments, non-negatively charged chiral internucleotidic linkages are not chirally controlled. In some embodiments, each chiral internucleotidic linkage that is not negatively charged is not chirally controlled. In some embodiments, non-negatively charged chiral internucleotidic linkages are chirally controlled. In some embodiments, non-negatively charged chiral internucleotidic linkages are chirally controlled and R p . In some embodiments, the non-negatively charged chiral internucleotidic linkages are chirally controlled and are Sp . In some embodiments, each non-negatively charged chiral internucleotidic linkage is chirally controlled. In some embodiments, the number of non-negatively charged internucleotide linkages in the second domain is about 1-10, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 . In some embodiments, the number is about 1. In some embodiments, the number is about two. In some embodiments, the number is about 3. In some embodiments, the number is about 4. In some embodiments, the number is about 5. In some embodiments, linkages between two or more nucleotides that are not negatively charged are contiguous. In some embodiments, linkages between two non-negatively charged nucleotides are not contiguous. In some embodiments, all non-negatively charged internucleotide linkages in the second domain are contiguous (eg, three consecutive non-negatively charged internucleotidic linkages). In some embodiments, the non-negatively charged internucleotidic linkages, or two or more (e.g., about 2, about 3, about 4, etc.) consecutive non-negatively charged internucleotidic linkages are formed in a second at the 3'-end of the domain. In some embodiments, the last two or three or four internucleotide linkages of the second domain comprise at least one internucleotide linkage that is not a negatively charged internucleotide linkage. In some embodiments, the last 2 or 3 or 4 internucleotide linkages of the second domain comprise at least one internucleotide linkage other than n001.

In some embodiments, the internucleoside linkage connecting the last two nucleosides of the second domain is a non-negatively charged internucleoside linkage. In some embodiments, the internucleoside linkage connecting the last two nucleosides of the second domain is a non-negatively charged S p internucleoside linkage. In some embodiments, the internucleoside linkage connecting the last two nucleosides of the second domain is a non-negatively charged R p internucleoside linkage. In some embodiments, the internucleoside linkage connecting the last two nucleosides of the second domain is a phosphorothioate internucleoside linkage. In some embodiments, the internucleotide linkage connecting the last two nucleosides of the second domain is an S p phosphorothioate internucleoside linkage. In some embodiments, the last two nucleosides of the second domain are the last two nucleosides of the oligonucleotide. In some embodiments, the internucleoside linkage connecting the first two nucleosides of the second domain is a non-negatively charged internucleoside linkage. In some embodiments, the internucleoside linkage connecting the first two nucleosides of the second domain is a non-negatively charged S p internucleoside linkage. In some embodiments, the internucleoside linkage connecting the first two nucleosides of the second domain is a non-negatively charged R p internucleoside linkage. In some embodiments, the internucleoside linkage connecting the first two nucleosides of the second domain is a phosphorothioate internucleoside linkage. In some embodiments, the internucleoside linkage connecting the first two nucleosides of the second domain is an S p phosphorothioate internucleoside linkage. In some embodiments, the non-negatively charged internucleotidic linkage is a neutral internucleotidic linkage, such as n001.

In some embodiments, the second domain comprises one or more native phosphate linkages. In some embodiments, the second domain does not contain native phosphate linkages.

In some embodiments, the second domain recruits, promotes recruiting, or contributes to recruiting of a protein, such as an ADAR protein. In some embodiments, the second domain supplements, facilitates, or contributes to an interaction with a protein, such as an ADAR protein. In some embodiments, the second domain contacts the RNA binding domain (RBD) of an ADAR. In some embodiments, the second domain contacts the catalytic domain of an ADAR having deaminase activity. In some embodiments, various nucleobases, sugars, and/or internucleotidic linkages may interact with one or more residues of a protein (eg, an ADAR protein).

In some embodiments, the second domain comprises or consists of a first subdomain as described herein. In some embodiments, the second domain comprises or consists of a second subdomain as described herein. In some embodiments, the second domain comprises or consists of a third subdomain as described herein. In some embodiments, the second domain comprises or consists of a first subdomain, a second subdomain, and a third subdomain in a 5' to 3' direction. Specific implementations of these subdomains are described below.

1st subdomain

As described herein, in some embodiments, an oligonucleotide comprises a first domain and a second domain in a 5' to 3' direction. In some embodiments, the second domain comprises or consists of a first subdomain, a second subdomain, and a third subdomain in a 5' to 3' direction. Specific implementations of the first subdomain are described below by way of example. In some embodiments, the first subdomain comprises the nucleoside opposite the target adenosine to be modified (eg, converted to I).

In some embodiments, the first subdomain is about 1-50, 1-40, 1-30, 1-20 (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9 , or 10 to about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 40 or 50, or about 1, 2 , 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 40 or 50, etc.) nucleobases in length. In some embodiments, the first subdomain is about 5-30 nucleobases in length. In some embodiments, the first subdomain is about 10-30 nucleobases in length. In some embodiments, the tenth subdomain is about 1-20 nucleobases in length. In some embodiments, the first subdomain is about 5-15 nucleobases in length. In some embodiments, the first subdomain is about 13-16 nucleobases in length. In some embodiments, the first subdomain is about 6-12 nucleobases in length. In some embodiments, the first subdomain is about 6-9 nucleobases in length. In some embodiments, the first subdomain is about 1-10 nucleobases in length. In some embodiments, the first subdomain is about 1-7 nucleobases in length. In some embodiments, the first subdomain is about 1-5 nucleobases in length. In some embodiments, the first subdomain is about 1-3 nucleobases in length. In some embodiments, the first subdomain is 1 nucleobase in length. In some embodiments, the first subdomain is two nucleobases in length. In some embodiments, the first subdomain is 3 nucleobases in length. In some embodiments, the first subdomain is 4 nucleobases in length. In some embodiments, the first subdomain is 5 nucleobases in length. In some embodiments, the first subdomain is 6 nucleobases in length. In some embodiments, the first subdomain is 7 nucleobases in length. In some embodiments, the first subdomain is 8 nucleobases in length. In some embodiments, the first subdomain is 9 nucleobases in length. In some embodiments, the first subdomain is 10 nucleobases in length. In some embodiments, the first subdomain is 11 nucleobases in length. In some embodiments, the first subdomain is 12 nucleobases in length. In some embodiments, the first subdomain is 13 nucleobases in length. In some embodiments, the first subdomain is 14 nucleobases in length. In some embodiments, the first subdomain is 15 nucleobases in length.

In some embodiments, the first subdomain is about or at least about 5-95%, 10%-90%, 20%-80%, 30%-70%, 40%-70%, 40%-of the second domain. 60%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80% , 85%, 90%, 95%. In some embodiments, the percentage is between about 30% and 80%. In some embodiments, the percentage is between about 30% and 70%. In some embodiments, the percentage is between about 40% and 60%. In some embodiments, the percentage is about 20%. In some embodiments, the percentage is about 25%. In some embodiments, the percentage is about 30%. In some embodiments, the percentage is about 35%. In some embodiments, the percentage is about 40%. In some embodiments, the percentage is about 45%. In some embodiments, the percentage is about 50%. In some embodiments, the percentage is about 55%. In some embodiments, the percentage is about 60%. In some embodiments, the percentage is about 65%. In some embodiments, the percentage is about 70%. In some embodiments, the percentage is about 75%. In some embodiments, the percentage is about 80%. In some embodiments, the percentage is about 85%. In some embodiments, the percentage is about 90%.

In some embodiments, the first subdomain contains one or more (e.g., 1-20, 1, 2, 3, 4, 5, 6, 7, 8, 9 , or 10, etc.) mismatches exist. In some implementations, there is 1 mismatch. In some implementations, there are two mismatches. In some implementations, there are three mismatches. In some implementations, there are 4 mismatches. In some implementations, there are 5 mismatches. In some implementations, there are 6 mismatches. In some implementations, there are 7 mismatches. In some implementations, there are 8 mismatches. In some implementations, there are 9 mismatches. In some implementations, there are 10 mismatches.

In some embodiments, the first subdomain contains one or more (e.g., 1-20, 1, 2, 3, 4, 5, 6, 7, 8, 9 , or 10, etc.) wobbles exist. In some implementations, there is 1 wobble. In some implementations, there are two wobbles. In some implementations, there are three wobbles. In some implementations, there are 4 wobbles. In some implementations, there are 5 wobbles. In some implementations, there are 6 wobbles. In some implementations, there are 7 wobbles. In some implementations, there are 8 wobbles. In some implementations, there are 9 wobbles. In some implementations, there are 10 wobbles.

In some embodiments, the duplex of the oligonucleotide and the target nucleic acid in the first subdomain region comprises one or more bulges, each independently comprising one or more mismatches that are not wobbles. In some embodiments, 0-10 (e.g., 0-1, 0-2, 0-3, 0-4, 0-5, 0-6, 0-7, 0-8, 0-9, 0~10, 1~2, 1~3, 1~4, 1~5, 1~6, 1~7, 1~8, 1~9, 1~10, 2~3, 2~4, 2~ 5, 2~6, 2~7, 2~8, 2~9, 2~10, 3~4, 3~5, 3~6, 3~7, 3~8, 3~9, 3~10, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 bulges). In some embodiments, the number is zero. In some embodiments, the number is one. In some embodiments, the number is two. In some embodiments, the number is three. In some embodiments, the number is four. In some embodiments, the number is five.

In some embodiments, the first subdomain is fully complementary to the target nucleic acid.

In some embodiments, the first subdomain comprises one or more modified nucleobases.

In some embodiments, the first subdomain comprises a nucleoside opposite the target adenosine, for example when the oligonucleotide forms a duplex with the target nucleic acid. Suitable nucleobases comprising modified nucleobases on opposing nucleosides are described herein. For example, in some embodiments, opposite nucleobases are C, a tautomer of C, U, a tautomer of U, A, a tautomer of A, and BA-I, BA-I-a, BA-I-b, BA-II, BA-II-a, BA-II-b, BA-III, BA-III-a, BA-III-b, BA-IV, BA-IV-a, BA-IV-b, BA-V, BA- an optionally substituted or protected nucleobase selected from ring BA having the structure V-a, BA-V-b, or BA-VI or a nucleobase comprising or a tautomer of ring BA.

In some embodiments, the first subdomain comprises one or more sugars (eg, natural DNA sugars) comprising two 2'-Hs. In some embodiments, the first subdomain includes one or more sugars including 2'-OH (eg, native RNA sugars). In some embodiments, the first subdomain comprises one or more modified sugars. In some embodiments, a modified sugar comprises a 2'-modification. In some embodiments, the modified sugar is a bicyclic sugar, such as an LNA sugar. In some embodiments, the modified sugar is acyclic (eg, by breaking the C2-C3 bonds of the corresponding cyclic sugar).

In some embodiments, the first subdomain is about 1-50, 1-40, 1-30, 1-25, 1-20, 1-15, 1-10 (e.g., about 1, 2, 3 , 4, 5, 6, 7, 8, 9, or 10 to about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 40 or 50, or about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 , 30, 40 or 50, etc., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 dog, etc.) In some embodiments, the first subdomain is about 1-50, 1-40, 1-30, 1-25, 1-20, 1-15, 1-10 (e.g., about 1, 2, 3 , 4, 5, 6, 7, 8, 9, or 10 to about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 40 or 50, or about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 , 30, 40 or 50, etc., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 modified sugars, which are independently bicyclic sugars (eg, LNA sugars) or 2'-OR modified sugars (R is independently optionally substituted C _1-6 aliphatic). In some embodiments, the first subdomain is about 1-50, 1-40, 1-30, 1-25, 1-20, 1-15, 1-10 (e.g., about 1, 2, 3 , 4, 5, 6, 7, 8, 9, or 10 to about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 40 or 50, or about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 , 30, 40 or 50, etc., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 modified sugars, which are independently 2'-OR modified sugars (R is independently optionally substituted C _1-6 aliphatic). In some embodiments, the number is one. In some embodiments, the number is two. In some embodiments, the number is three. In some embodiments, the number is four. In some embodiments, the number is five. In some embodiments, the number is six. In some embodiments, the number is 7. In some embodiments, the number is eight. In some embodiments, the number is 9. In some embodiments, the number is 10. In some embodiments, the number is 11. In some embodiments, the number is 12. In some embodiments, the number is 13. In some embodiments, the number is 14. In some embodiments, the number is 15. In some embodiments, the number is 16. In some embodiments, the number is 17. In some embodiments, the number is 18. In some embodiments, the number is 19. In some embodiments, the number is 20. In some embodiments, R is methyl.

In some embodiments, about 5%-100% (e.g., about 10%-100%, 20-100%, 30%-100%, 40%-100%, 50%) of all sugars in the first subdomain ~80%, 50%~85%, 50%~90%, 50%~95%, 60%~80%, 60%~85%, 60%~90%, 60%~95%, 60%~100 %, 65%~80%, 65%~85%, 65%~90%, 65%~95%, 65%~100%, 70%~80%, 70%~85%, 70%~90%, 70%~95%, 70%~100%, 75%~80%, 75%~85%, 75%~90%, 75%~95%, 75%~100%, 80%~85%, 80% ~90%, 80%~95%, 80%~100%, 85%~90%, 85%~95%, 85%~100%, 90%~95%, 90%~100%, 10%, 20 %, 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%, etc.) are independently modified sugars. In some embodiments, about 5%-100% (e.g., about 10%-100%, 20-100%, 30%-100%, 40%-100%, 50%) of all sugars in the first subdomain ~80%, 50%~85%, 50%~90%, 50%~95%, 60%~80%, 60%~85%, 60%~90%, 60%~95%, 60%~100 %, 65%~80%, 65%~85%, 65%~90%, 65%~95%, 65%~100%, 70%~80%, 70%~85%, 70%~90%, 70%~95%, 70%~100%, 75%~80%, 75%~85%, 75%~90%, 75%~95%, 75%~100%, 80%~85%, 80% ~90%, 80%~95%, 80%~100%, 85%~90%, 85%~95%, 85%~100%, 90%~95%, 90%~100%, 10%, 20 %, 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%, etc.) independently of the affected party (e.g., LNA). ) or a 2'-OR modified sugar (R is independently an optionally substituted C _1-6 aliphatic). In some embodiments, about 5%-100% (e.g., about 10%-100%, 20-100%, 30%-100%, 40%-100%, 50%) of all sugars in the first subdomain ~80%, 50%~85%, 50%~90%, 50%~95%, 60%~80%, 60%~85%, 60%~90%, 60%~95%, 60%~100 %, 65%~80%, 65%~85%, 65%~90%, 65%~95%, 65%~100%, 70%~80%, 70%~85%, 70%~90%, 70%~95%, 70%~100%, 75%~80%, 75%~85%, 75%~90%, 75%~95%, 75%~100%, 80%~85%, 80% ~90%, 80%~95%, 80%~100%, 85%~90%, 85%~95%, 85%~100%, 90%~95%, 90%~100%, 10%, 20 %, 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%, etc.) independently per 2'-OR variant ( R is independently optionally substituted C _1-6 aliphatic). In some embodiments, the percentage is at least about 50%. In some embodiments, the percentage is at least about 55%. In some embodiments, the percentage is at least about 60%. In some embodiments, the percentage is at least about 65%. In some embodiments, the percentage is at least about 70%. In some embodiments, the percentage is at least about 75%. In some embodiments, the percentage is at least about 80%. In some embodiments, the percentage is at least about 85%. In some embodiments, the percentage is at least about 90%. In some embodiments, the percentage is at least about 95%. In some embodiments, the percentage is about 100%. In some embodiments, R is methyl.

In some embodiments, the first subdomain comprises about 1-50 (e.g., about 5, 6, 7, 8, 9, or 10 to about 10, 11) independently having non-2'-F modifications. , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 40 or 50, or about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 40 or 50, etc., about 1, 2, 3, 4, 5, 6 , 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, etc.) modified sugars. In some embodiments, from about 5% to 100% (e.g., from about 10% to 100%, 20 to 100%, 30% to 100%, 40% to 100%, 50% to 100%) of the sugars in the first subdomain. 80%, 50%~85%, 50%~90%, 50%~95%, 60%~80%, 60%~85%, 60%~90%, 60%~95%, 60%~100% , 65%~80%, 65%~85%, 65%~90%, 65%~95%, 65%~100%, 70%~80%, 70%~85%, 70%~90%, 70 %~95%, 70%~100%, 75%~80%, 75%~85%, 75%~90%, 75%~95%, 75%~100%, 80%~85%, 80%~ 90%, 80% to 95%, 80% to 100%, 85% to 90%, 85% to 95%, 85% to 100%, 90% to 95%, 90% to 100%, 10%, 20% , 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%) independently, non-2′-F variants This is a transforming party. In some embodiments, about 50%-100% (e.g., about 50%-80%, 50%-85%, 50%-90%, 50%-95%, 60%) of the sugars in the first subdomain ~80%, 60%~85%, 60%~90%, 60%~95%, 60%~100%, 65%~80%, 65%~85%, 65%~90%, 65%~95 %, 65%~100%, 70%~80%, 70%~85%, 70%~90%, 70%~95%, 70%~100%, 75%~80%, 75%~85%, 75%~90%, 75%~95%, 75%~100%, 80%~85%, 80%~90%, 80%~95%, 80%~100%, 85%~90%, 85% ~95%, 85%~100%, 90%~95%, 90%~100%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%, etc.) are independently modified sugars with modifications other than 2'-F. In some embodiments, each modified sugar in the first subdomain is independently a bicyclic sugar (eg, LNA sugar), an acyclic sugar (eg, UNA sugar), a sugar with a 2'-OR variant, or a 2'-N ( R) is selected from sugars with ₂ modifications (each R is independently an optionally substituted C _1-6 aliphatic). In some embodiments, each sugar in the first domain is a 2'-F modified sugar.

In some embodiments, the first subdomain is a bicyclic sugar (eg, LNA sugar), an acyclic sugar (eg, UNA sugar), a sugar with a 2'-OR strain, or a 2'-N(R) ₂ strain. from about 1 to 50 (eg, about 5, 6, 7, 8, 9, or 10 to about 10) independently selected from sugars (each R is independently optionally substituted C _1-6 aliphatic) , 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 40 or 50, or about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 40 or 50, etc., about 1, 2, 3, 4, 5 , 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, etc.) modified sugars. In some embodiments, from about 5% to 100% (e.g., from about 10% to 100%, 20 to 100%, 30% to 100%, 40% to 100%, 50% to 100%) of the sugars in the first subdomain. 80%, 50%~85%, 50%~90%, 50%~95%, 60%~80%, 60%~85%, 60%~90%, 60%~95%, 60%~100% , 65%~80%, 65%~85%, 65%~90%, 65%~95%, 65%~100%, 70%~80%, 70%~85%, 70%~90%, 70 %~95%, 70%~100%, 75%~80%, 75%~85%, 75%~90%, 75%~95%, 75%~100%, 80%~85%, 80%~ 90%, 80% to 95%, 80% to 100%, 85% to 90%, 85% to 95%, 85% to 100%, 90% to 95%, 90% to 100%, 10%, 20% , 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%, etc.) independently of the affected party (e.g., LNA) , an acyclic sugar (eg UNA sugar), a sugar with a 2'-OR strain, or a sugar with a 2'-N(R) ₂ strain, where each R is independently an optionally substituted C _1-6 aliphatic It is a modified sugar selected from In some embodiments, about 50%-100% (e.g., about 50%-80%, 50%-85%, 50%-90%, 50%-95%, 60%) of the sugars in the first subdomain ~80%, 60%~85%, 60%~90%, 60%~95%, 60%~100%, 65%~80%, 65%~85%, 65%~90%, 65%~95 %, 65%~100%, 70%~80%, 70%~85%, 70%~90%, 70%~95%, 70%~100%, 75%~80%, 75%~85%, 75%~90%, 75%~95%, 75%~100%, 80%~85%, 80%~90%, 80%~95%, 80%~100%, 85%~90%, 85% ~95%, 85%~100%, 90%~95%, 90%~100%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%, etc.) are independently bicyclic (e.g., LNA sugars), acyclic sugars (e.g., UNA sugars), sugars with 2'-OR modifications, or sugars with 2'-N(R) ₂ modifications (each R of is independently a modified sugar selected from optionally substituted C _1-6 aliphatic.

In some embodiments, each sugar in the first subdomain independently comprises a 2'-OR variant (R is an optionally substituted C _1-6 aliphatic) or a 2'-OL ^B -4' variant. In some embodiments, each sugar in the first subdomain is independently a 2'-OR variant (R is an optionally substituted C _1-6 aliphatic) or a 2'-OL ^B -4' variant (L ^B is optionally substituted C 1-6 aliphatic). substituted -CH ₂ -). In some embodiments, each sugar in the first subdomain independently comprises 2'-OMe.

In some embodiments, the first subdomain includes one or more 2'-F modified sugars. In some embodiments, the first subdomain does not include a 2'-F modified sugar. In some embodiments, the first subdomain comprises one or more bicyclic sugars and/or 2'-OR modified sugars (R is not -H). In some embodiments, the first subdomain is one or more (e.g., about 1-20, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, etc.) of 2'-OMe modified sugars. In some embodiments, the first subdomain is one or more (e.g., about 1-20, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, etc.) of consecutive 2'-OMe modified sugars. In some embodiments, the level of bicyclic sugars and/or 2'-OR modified sugars (R is not -H), individually or in combination, is relatively high compared to 2'-F modified sugars. In some embodiments, up to about 1% to 95% (e.g., up to about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, etc.) include 2'-F. In some embodiments, up to about 50% of the sugars in the first subdomain comprise 2'-F. In some embodiments, the first subdomain comprises _one or more (eg, about 1-20, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, etc.) modified sugars. In some embodiments, the first subdomain comprises one or more (eg, about 1-20, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10) containing 2′-NH ₂ modifications. , 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, etc.) modified sugars. In some embodiments, the first subdomain is one or more (e.g., about 1-20, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, etc.) of heterocyclic sugars (eg, LNA sugars). In some embodiments, the first subdomain is one or more (e.g., about 1-20, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, etc.) of acyclic sugars (eg, UNA sugars).

In some embodiments, up to about 1% to 95% (e.g., up to about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, etc.) contain a 2'-MOE. In some embodiments, up to about 50% of the sugars in the first subdomain comprise a 2'-MOE. In some embodiments, the sugar in the first subdomain does not include a 2'-MOE.

In some embodiments, the first subdomain contains more 2'-OR modified sugars than 2'-F modified sugars. In some embodiments, each sugar in the first subdomain is independently a 2'-OR modified sugar or a 2'-F modified sugar. In some embodiments, the first subdomain contains only three nucleosides, two of which are independently 2'-OR modified sugars and one is a 2'-F modified sugar. In some embodiments, the 2'-F modified nucleoside is at the 3'-end of the first subdomain and linked to the second subdomain. In some embodiments, each 2'-OR modified sugar is independently a 2'-OR modified sugar (R is an optionally substituted C _1-6 aliphatic) or a 2'-OL ^B -4' strain. In some embodiments, each 2'-OR modified sugar is independently a 2'-OR modified sugar, wherein R is an optionally substituted C _1-6 aliphatic. In some embodiments, each 2'-OR modified sugar is independently a 2'-OMe or 2'-MOE modified sugar. In some embodiments, each 2'-OR modified sugar is independently a 2'-OMe modified sugar. In some embodiments, each 2'-OR modified sugar is independently a 2'-MOE modified sugar. In some embodiments, the sugar is 2'-OMe modified and the sugar is 2'-MOE. In some embodiments, the first subdomain contains only three nucleosides, which are N ₂ , N ₃ and N ₄ .

In some embodiments, the first subdomain is about 1-50, 1-40, 1-30, 1-25, 1-20, 1-15, 1-10 (e.g., about 5, 6, 7 , 8, 9, or 10 to about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 40, or 50, or About 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 40 or 50, etc. , about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, etc.) modified internucleotide linkages includes In some embodiments, about 5%-100% (e.g., about 10%-100%, 20-100%, 30%-100%, 40%-100%, 50%~80%, 50%~85%, 50%~90%, 50%~95%, 60%~80%, 60%~85%, 60%~90%, 60%~95%, 60% ~100%, 65%~80%, 65%~85%, 65%~90%, 65%~95%, 65%~100%, 70%~80%, 70%~85%, 70%~90 %, 70%~95%, 70%~100%, 75%~80%, 75%~85%, 75%~90%, 75%~95%, 75%~100%, 80%~85%, 80%~90%, 80%~95%, 80%~100%, 85%~90%, 85%~95%, 85%~100%, 90%~95%, 90%~100%, 10% , 20%, 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%, etc.) are modified internucleotide linkages. In some embodiments, each internucleotide linkage in the first subdomain is independently a modified internucleotide linkage. In some embodiments, each modified internucleotide linkage is independently a chiral internucleotide linkage. In some embodiments, the modified or chiral internucleotidic linkage is a phosphorothioate internucleotidic linkage. In some embodiments, a modified or chiral internucleotidic linkage is a non-negatively charged internucleotidic linkage. In some embodiments, the modified or chiral internucleotide linkage is a neutral internucleotide linkage (eg n001). In some embodiments, each modified internucleotide linkage is independently a phosphorothioate internucleotide linkage or a non-negatively charged internucleotide linkage. In some embodiments, each modified internucleotide linkage is independently a phosphorothioate internucleotide linkage or a neutral internucleotide linkage. In some embodiments, each modified internucleotide linkage is independently a phosphorothioate internucleotide linkage. In some embodiments, at least about 1-50, 1-40, 1-30, 1-25, 1-20, 1-15, 1-10 (e.g., about 5, 6 , 7, 8, 9, or 10 to about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 40, or 50 , or about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 40 or 50 about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 chiral nucleotides, etc. Interconnections are chirally controlled. In some embodiments, at least 5%-100% (e.g., about 10%-100%, 20-100%, 30%-100%, 40%-100%) of the chiral internucleotide linkages in the first subdomain , 50%~80%, 50%~85%, 50%~90%, 50%~95%, 60%~80%, 60%~85%, 60%~90%, 60%~95%, 60 %~100%, 65%~80%, 65%~85%, 65%~90%, 65%~95%, 65%~100%, 70%~80%, 70%~85%, 70%~ 90%, 70% to 95%, 70% to 100%, 75% to 80%, 75% to 85%, 75% to 90%, 75% to 95%, 75% to 100%, 80% to 85% , 80%~90%, 80%~95%, 80%~100%, 85%~90%, 85%~95%, 85%~100%, 90%~95%, 90%~100%, 10 %, 20%, 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%, etc.) are chirally controlled. In some embodiments, at least 5%-100% (e.g., about 10%-100%, 20-100%, 30%-100%, 40%) of the phosphorothioate internucleotide linkages in the first subdomain ~100%, 50%~80%, 50%~85%, 50%~90%, 50%~95%, 60%~80%, 60%~85%, 60%~90%, 60%~95 %, 60%~100%, 65%~80%, 65%~85%, 65%~90%, 65%~95%, 65%~100%, 70%~80%, 70%~85%, 70% to 90%, 70% to 95%, 70% to 100%, 75% to 80%, 75% to 85%, 75% to 90%, 75% to 95%, 75% to 100%, 80% ~85%, 80%~90%, 80%~95%, 80%~100%, 85%~90%, 85%~95%, 85%~100%, 90%~95%, 90%~100 %, 10%, 20%, 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%, etc.) are chiral controlled. . In some embodiments, each is independently chirally controlled. In some embodiments, at least about 1-50, 1-40, 1-30, 1-25, 1-20, 1-15, 1-10 (e.g., about 5, 6 , 7, 8, 9, or 10 to about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 40, or 50 , or about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 40 or 50 about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 chiral nucleotides, etc. The connection between them is S p. In some embodiments, at least about 1-50, 1-40, 1-30, 1-25, 1-20, 1-15, 1-10 (e.g., about 5, 6 , 7, 8, 9, or 10 to about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 40, or 50 , or about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 40 or 50 about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, etc.) The thioate internucleotidic linkage is Sp . In some embodiments, at least 5%-100% (e.g., about 10%-100%, 20-100%, 30%-100%, 40%-100%) of the chiral internucleotide linkages in the first subdomain , 50%~80%, 50%~85%, 50%~90%, 50%~95%, 60%~80%, 60%~85%, 60%~90%, 60%~95%, 60 %~100%, 65%~80%, 65%~85%, 65%~90%, 65%~95%, 65%~100%, 70%~80%, 70%~85%, 70%~ 90%, 70% to 95%, 70% to 100%, 75% to 80%, 75% to 85%, 75% to 90%, 75% to 95%, 75% to 100%, 80% to 85% , 80%~90%, 80%~95%, 80%~100%, 85%~90%, 85%~95%, 85%~100%, 90%~95%, 90%~100%, 10 %, 20%, 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%, etc.) is Sp . In some embodiments, at least 5%-100% (e.g., about 10%-100%, 20-100%, 30%-100%, 40%) of the phosphorothioate internucleotide linkages in the first subdomain ~100%, 50%~80%, 50%~85%, 50%~90%, 50%~95%, 60%~80%, 60%~85%, 60%~90%, 60%~95 %, 60%~100%, 65%~80%, 65%~85%, 65%~90%, 65%~95%, 65%~100%, 70%~80%, 70%~85%, 70% to 90%, 70% to 95%, 70% to 100%, 75% to 80%, 75% to 85%, 75% to 90%, 75% to 95%, 75% to 100%, 80% ~85%, 80%~90%, 80%~95%, 80%~100%, 85%~90%, 85%~95%, 85%~100%, 90%~95%, 90%~100 %, 10%, 20%, 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%, etc.) is Sp . In some embodiments, the number is one or more. In some embodiments, the number is two or more. In some embodiments, the number is three or more. In some embodiments, the number is 4 or more. In some embodiments, the number is 5 or more. In some embodiments, the number is 6 or more. In some embodiments, the number is 7 or more. In some embodiments, the number is 8 or more. In some embodiments, the number is 9 or more. In some embodiments, the number is 10 or more. In some embodiments, the number is 11 or more. In some embodiments, the number is 12 or more. In some embodiments, the number is 13 or more. In some embodiments, the number is 14 or more. In some embodiments, the number is 15 or more. In some embodiments, the percentage is at least about 50%. In some embodiments, the percentage is at least about 55%. In some embodiments, the percentage is at least about 60%. In some embodiments, the percentage is at least about 65%. In some embodiments, the percentage is at least about 70%. In some embodiments, the percentage is at least about 75%. In some embodiments, the percentage is at least about 80%. In some embodiments, the percentage is at least about 85%. In some embodiments, the percentage is at least about 90%. In some embodiments, the percentage is at least about 95%. In some embodiments, the percentage is about 100%. In some embodiments, each internucleotide linkage linking the two first subdomain nucleosides is independently a modified internucleotide linkage. In some embodiments, each modified internucleotide linkage is independently a chiral internucleotide linkage. In some embodiments, each modified internucleotide linkage is independently a phosphorothioate internucleotide linkage. In some embodiments, each chiral internucleotidic linkage is independently a phosphorothioate internucleotidic linkage. In some embodiments, each modified internucleotidic linkage is independently an Sp chiral internucleotidic linkage. In some embodiments, each modified internucleotide linkage is independently an S p phosphorothioate internucleotide linkage. In some embodiments, each chiral internucleotidic linkage is independently an S p phosphorothioate internucleotidic linkage. In some embodiments, the internucleotide linkages of the first subdomain are linked to two nucleosides of the first subdomain. In some embodiments, an internucleotide linkage between a nucleoside of a first subdomain and a nucleoside of a second subdomain may properly be considered an internucleotide linkage of the first subdomain. In some embodiments, the internucleoside linkage linked to the nucleoside of the first subdomain and the nucleoside of the second subdomain is a modified internucleotide linkage, in some embodiments, it is a chiral internucleoside linkage, and in some embodiments , it is chirally controlled, in some embodiments it is R p , and in some embodiments it is Sp .

In some embodiments, the first subdomain comprises a certain level of R p internucleotidic linkages. In some embodiments, the level is, for example, between about 5% and 100%, about 10% and 100%, 20 and 100%, 30% and 100%, 40% and 100% of all internucleotide linkages in the first subdomain. %, 50%~80%, 50%~85%, 50%~90%, 50%~95%, 60%~80%, 60%~85%, 60%~90%, 60%~95%, 60%~100%, 65%~80%, 65%~85%, 65%~90%, 65%~95%, 65%~100%, 70%~80%, 70%~85%, 70% ~90%, 70%~95%, 70%~100%, 75%~80%, 75%~85%, 75%~90%, 75%~95%, 75%~100%, 80%~85 %, 80%~90%, 80%~95%, 80%~100%, 85%~90%, 85%~95%, 85%~100%, 90%~95%, 90%~100%, 10%, 20%, 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%. In some embodiments, the level is between about 5% and 100%, about 10% and 100%, 20 and 100%, 30% and 100%, 40% and 40% of all chiral internucleotide linkages in the first subdomain, for example. 100%, 50%~80%, 50%~85%, 50%~90%, 50%~95%, 60%~80%, 60%~85%, 60%~90%, 60%~95% , 60%~100%, 65%~80%, 65%~85%, 65%~90%, 65%~95%, 65%~100%, 70%~80%, 70%~85%, 70 %~90%, 70%~95%, 70%~100%, 75%~80%, 75%~85%, 75%~90%, 75%~95%, 75%~100%, 80%~ 85%, 80% to 90%, 80% to 95%, 80% to 100%, 85% to 90%, 85% to 95%, 85% to 100%, 90% to 95%, 90% to 100% , 10%, 20%, 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%. In some embodiments, the level is, for example, between about 5% and 100%, about 10% and 100%, 20 and 100%, 30% and 100%, 40% of all chiral controlled internucleotide linkages in the first subdomain. ~100%, 50%~80%, 50%~85%, 50%~90%, 50%~95%, 60%~80%, 60%~85%, 60%~90%, 60%~95 %, 60%~100%, 65%~80%, 65%~85%, 65%~90%, 65%~95%, 65%~100%, 70%~80%, 70%~85%, 70% to 90%, 70% to 95%, 70% to 100%, 75% to 80%, 75% to 85%, 75% to 90%, 75% to 95%, 75% to 100%, 80% ~85%, 80%~90%, 80%~95%, 80%~100%, 85%~90%, 85%~95%, 85%~100%, 90%~95%, 90%~100 %, 10%, 20%, 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%. In some embodiments, the percentage is about or up to about 50%. In some embodiments, the percentage is at least about 55%. In some embodiments, the percentage is at least about 60%. In some embodiments, the percentage is at least about 65%. In some embodiments, the percentage is at least about 70%. In some embodiments, the percentage is at least about 75%. In some embodiments, the percentage is at least about 80%. In some embodiments, the percentage is at least about 85%. In some embodiments, the percentage is at least about 90%. In some embodiments, the percentage is at least about 95%. In some embodiments, the percentage is about 100%. In some embodiments, the percentage is about or up to about 5%. In some embodiments, the percentage is about or up to about 10%. In some embodiments, the percentage is about or up to about 15%. In some embodiments, the percentage is about or up to about 20%. In some embodiments, the percentage is about or up to about 25%. In some embodiments, the percentage is about or up to about 30%. In some embodiments, the percentage is about or up to about 35%. In some embodiments, the percentage is about or up to about 40%. In some embodiments, the percentage is about or up to about 45%. In some embodiments, the percentage is about or up to about 50%. In some embodiments, about 1-50, 1-40, 1-30, 1-25, 1-20, 1-15, 1-10, 1-5, for example about 1, 2, 3, 4 , 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 internucleotide linkages are independently R p chiral internucleotide linkages. In some embodiments, the number is about or up to about 1. In some embodiments, the number is about or up to about 2. In some embodiments, the number is about or up to about 3. In some embodiments, the number is about or up to about 4. In some embodiments, the number is about or up to about 5. In some embodiments, the number is about or up to about 6. In some embodiments, the number is about or up to about 7. In some embodiments, the number is about or up to about 8. In some embodiments, the number is about or up to about 9. In some embodiments, the number is about or up to about 10.

In some embodiments, each phosphorothioate internucleotidic linkage in the first subdomain is independently chirally controlled. In some embodiments, each is independently Sp or R p. In some embodiments, the high level is Sp as described herein. In some embodiments, each phosphorothioate internucleotidic linkage in the first subdomain is chirally controlled and is Sp . In some embodiments, one or more, for example about 1-5 (eg, about 1, 2, 3, 4, or 5) is R p .

In some embodiments, as exemplified in certain examples, the first subdomain comprises one or more non-negatively charged internucleotide linkages, each optionally and independently chirally controlled. In some embodiments, each non-negatively charged internucleotide linkage is independently n001. In some embodiments, non-negatively charged chiral internucleotidic linkages are not chirally controlled. In some embodiments, each chiral internucleotidic linkage that is not negatively charged is not chirally controlled. In some embodiments, non-negatively charged chiral internucleotidic linkages are chirally controlled. In some embodiments, non-negatively charged chiral internucleotidic linkages are chirally controlled and R p . In some embodiments, the non-negatively charged chiral internucleotidic linkages are chirally controlled and are Sp . In some embodiments, each non-negatively charged chiral internucleotidic linkage is chirally controlled. In some embodiments, the number of non-negatively charged internucleotide linkages in the first subdomain is about 1-10, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 It is a dog. In some embodiments, the number is about 1. In some embodiments, the number is about two. In some embodiments, the number is about 3. In some embodiments, the number is about 4. In some embodiments, the number is about 5. In some embodiments, linkages between two or more nucleotides that are not negatively charged are contiguous. In some embodiments, linkages between two non-negatively charged nucleotides are not contiguous. In some embodiments, all non-negatively charged internucleotide linkages in the first subdomain are contiguous (eg, three consecutive non-negatively charged internucleotidic linkages). In some embodiments, the non-negatively charged internucleotidic linkages, or two or more (e.g., about 2, about 3, about 4, etc.) consecutive non-negatively charged internucleotidic linkages are at the 3'-end of the subdomain. In some embodiments, the last two or three or four internucleotide linkages of the first subdomain comprise at least one internucleotide linkage that is not a negatively charged internucleotide linkage. In some embodiments, the last 2 or 3 or 4 internucleotide linkages of the first subdomain comprise at least one internucleotide linkage other than n001. In some embodiments, the internucleoside linkage connecting the last two nucleosides of the first subdomain is a non-negatively charged internucleoside linkage. In some embodiments, the internucleoside linkage connecting the last two nucleosides of the first subdomain is a non-negatively charged S p internucleoside linkage. In some embodiments, the internucleoside linkage connecting the last two nucleosides of the first subdomain is a non-negatively charged R p internucleoside linkage. In some embodiments, the internucleoside linkage connecting the last two nucleosides of the first subdomain is a phosphorothioate internucleoside linkage. In some embodiments, the internucleoside linkage connecting the last two nucleosides of the first subdomain is an S p phosphorothioate internucleoside linkage. In some embodiments, the internucleoside linkage connecting the first two nucleosides of the first subdomain is a non-negatively charged internucleoside linkage. In some embodiments, the internucleotide linkage linking the first two nucleosides of the first subdomain is a non-negatively charged S p internucleoside linkage. In some embodiments, the internucleoside linkage connecting the first two nucleosides of the first subdomain is a non-negatively charged R p internucleotide linkage. In some embodiments, the internucleoside linkage connecting the first two nucleosides of the first subdomain is a phosphorothioate internucleoside linkage. In some embodiments, the internucleoside linkage connecting the first two nucleosides of the first subdomain is an S p phosphorothioate internucleoside linkage. In some embodiments, the non-negatively charged internucleotidic linkage is a neutral internucleotidic linkage, such as n001.

In some embodiments, the first subdomain comprises one or more native phosphate linkages. In some embodiments, the first subdomain does not contain native phosphate linkages. In some embodiments, one or more 2'-OR modified sugars (R is not -H) are independently linked to a natural phosphate linkage. In some embodiments, one or more 2'-OR modified sugars (R is an optionally substituted C _1-6 aliphatic) are independently linked to a natural phosphate linkage. In some embodiments, one or more 2'-OMe modified sugars are independently linked to a natural phosphate linkage. In some embodiments, one or more 2'-MOE modified sugars are independently linked to a natural phosphate linkage. In some embodiments, each 2'-MOE modified sugar is independently linked to a natural phosphate linkage. In some embodiments, at least 50% (e.g., 50%-100%, 50%-90%, 50-80%, or about 50%, 60%, 66%, 70%, 75%, 80%, 90% or more) of the 2'-OR modified sugars (R is not -H) are independently linked to a natural phosphate linkage. In some embodiments, at least 50% (e.g., 50%-100%, 50%-90%, 50-80%, or about 50%, 60%, 66%, 70%, 75%, 80%, 90% or more) of the 2'-OMe modified sugars are independently linked to natural phosphate linkages. In some embodiments, at least 50% (e.g., 50%-100%, 50%-90%, 50-80%, or about 50%, 60%, 66%, 70%, 75%, 80%, 90% or more) of the 2'-MOE modified sugars are independently linked to natural phosphate linkages. In some embodiments, 50% or more (e.g., 50%-100%, 50%-90%, 50-80%, or about 50%, 60%, 66%, 70%, 75%, 80%, 90% or more) of the internucleotide linkages are independently natural phosphate linkages. In some embodiments, at least 50% (e.g., 50%-100%, 50%-90%, 50-80%, or about 50%) bound to two 2'-OMe or 2'-MOE modified sugars. %, 60%, 66%, 70%, 75%, 80%, 90% or more) of the internucleotide linkages are independently natural phosphate linkages.

In some embodiments, the first subdomain is 5'-end, for example about 1-20, 1-15, 1-10, 3-8, or about 1, 2, 3, 4, 5, 6, including those having a length of 7, 8, 9, or 10 nucleobases. In some embodiments, the 5'-end portion is about 3-6 nucleobases in length. In some embodiments, the length is 1 nucleobase. In some embodiments, the length is 2 nucleobases. In some embodiments, the length is 3 nucleobases. In some embodiments, the length is 4 nucleobases. In some embodiments, the length is 5 nucleobases. In some embodiments, the length is 6 nucleobases. In some embodiments, the length is 7 nucleobases. In some embodiments, the length is 8 nucleobases. In some embodiments, the length is 9 nucleobases. In some embodiments, the length is 10 nucleobases. In some embodiments, the 5'-terminal portion comprises the 5'-terminal nucleobase of the first subdomain.

In some embodiments, the 5'-end portion includes one or more sugars with two 2'-Hs (eg, natural DNA sugars). In some embodiments, the 5'-end portion includes one or more sugars with 2'-OH (eg, native RNA sugars). In some embodiments, one or more (e.g., about 1-20, 1-15, 1-10, 3-8, or about 1, 2, 3, 4, 5, 6) sugars at the 5'-end. , 7, 8, 9, or 10) are independently modified sugars. In some embodiments, between about 5% and 100% (e.g., between about 10% and 100%, 20 and 100%, 30% and 100%, 40% and 100%, 50% and 80%, 50%~85%, 50%~90%, 50%~95%, 60%~80%, 60%~85%, 60%~90%, 60%~95%, 60%~100% , 65%~80%, 65%~85%, 65%~90%, 65%~95%, 65%~100%, 70%~80%, 70%~85%, 70%~90%, 70 %~95%, 70%~100%, 75%~80%, 75%~85%, 75%~90%, 75%~95%, 75%~100%, 80%~85%, 80%~ 90%, 80% to 95%, 80% to 100%, 85% to 90%, 85% to 95%, 85% to 100%, 90% to 95%, 90% to 100%, 10%, 20% , 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%, etc.) are independently modified sugars. In some embodiments, each sugar is independently a modified sugar. In some embodiments, the modified sugar is independently a bicyclic sugar (eg, LNA sugar), an acyclic sugar (eg, UNA sugar), a sugar with a 2'-OR strain, or a 2'-N(R) ₂ strain. sugar (each R is independently optionally substituted C _1-6 aliphatic).

In some embodiments, one or more of the modified sugars independently comprises 2'-F or 2'-OR (R is independently optionally substituted C _1-6 aliphatic). In some embodiments, one or more of the modified sugars are independently 2'-F or 2'-OMe. In some embodiments, each modified sugar at the 5'-end is independently a bicyclic sugar (eg, an LNA sugar) or a sugar with a 2'-OR variant (R is an optionally substituted C _1-6 aliphatic). In some embodiments, each modified sugar at the 5'-end is independently a bicyclic sugar (eg, an LNA sugar) or a sugar with a 2'-OR variant (R is an optionally substituted C _1-6 aliphatic). In some embodiments, each modified sugar at the 5'-end is independently a sugar with a 2'-OR variant (R is an optionally substituted C _1-6 aliphatic). In some embodiments, R is methyl.

In some embodiments, between one or more (e.g., about 1-10, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) nucleotides of the 5'-end. Linkages are independently modified internucleotide linkages. In some embodiments, between one or more (e.g., about 1-10, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) nucleotides of the 5'-end. Linkages are independently chiral internucleotidic linkages. In some embodiments, between one or more (e.g., about 1-10, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) nucleotides of the 5'-end. Linkages are independently chirally controlled internucleotidic linkages. In some embodiments, between one or more (e.g., about 1-10, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) nucleotides of the 5'-end. The connection is R p. In some embodiments, between one or more (e.g., about 1-10, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) nucleotides of the 5'-end. The connection is Sp . In some embodiments, each internucleotide linkage at the 5'-end is Sp .

In some embodiments, the 5'-end is one or more (e.g., about 1-10, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) as described herein. ), including inconsistency. In some embodiments, the 5'-end is one or more (e.g., about 1-10, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) as described herein. ) of wobbles. In some embodiments, the 5'-end is about 60-100% (eg, 66%, 70%, 75%, 80%, 85%, 90%, 95% or more) complementary to the target nucleic acid. In some embodiments, complementarity is greater than 60%. In some embodiments, complementarity is greater than 70%. In some embodiments, complementarity is greater than 75%. In some embodiments, complementarity is greater than 80%. In some embodiments, complementarity is greater than 90%.

In some embodiments, the first subdomain is 3'-terminal, e.g., about 1-20, 1-15, 1-10, 1-5, 1-3, 3-8, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleobases in length. In some embodiments, the 3'-end portion is about 1-3 nucleobases in length. In some embodiments, the length is 1 nucleobase. In some embodiments, the length is 2 nucleobases. In some embodiments, the length is 3 nucleobases. In some embodiments, the length is 4 nucleobases. In some embodiments, the length is 5 nucleobases. In some embodiments, the length is 6 nucleobases. In some embodiments, the length is 7 nucleobases. In some embodiments, the length is 8 nucleobases. In some embodiments, the length is 9 nucleobases. In some embodiments, the length is 10 nucleobases. In some embodiments, the 3'-terminal portion comprises the 3'-terminal nucleobase of the first subdomain. In some embodiments, the first subdomain comprises or consists of a 5'-end and a 3'-end.

In some embodiments, the 5'-end portion includes one or more sugars with two 2'-Hs (eg, natural DNA sugars). In some embodiments, the 5'-end portion includes one or more sugars with 2'-OH (eg, native RNA sugars). In some embodiments, one or more (e.g., about 1-20, 1-15, 1-10, 3-8, or about 1, 2, 3, 4, 5, 6) sugars at the 3'-terminus. , 7, 8, 9, or 10) are independently modified sugars. In some embodiments, about 5%-100% (e.g., about 10%-100%, 20-100%, 30%-100%, 40%-100%, 50%-100%) of the sugar at the 3'-end. 80%, 50%~85%, 50%~90%, 50%~95%, 60%~80%, 60%~85%, 60%~90%, 60%~95%, 60%~100% , 65%~80%, 65%~85%, 65%~90%, 65%~95%, 65%~100%, 70%~80%, 70%~85%, 70%~90%, 70 %~95%, 70%~100%, 75%~80%, 75%~85%, 75%~90%, 75%~95%, 75%~100%, 80%~85%, 80%~ 90%, 80% to 95%, 80% to 100%, 85% to 90%, 85% to 95%, 85% to 100%, 90% to 95%, 90% to 100%, 10%, 20% , 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%, etc.) are independently modified sugars. In some embodiments, each sugar is independently a modified sugar. In some embodiments, the modified sugar is independently a bicyclic sugar (eg, LNA sugar), an acyclic sugar (eg, UNA sugar), a sugar with a 2'-OR strain, or a 2'-N(R) ₂ strain. sugar (each R is independently optionally substituted C _1-6 aliphatic).

In some embodiments, one or more of the modified sugars independently comprises 2'-F or 2'-OR (R is independently optionally substituted C _1-6 aliphatic). In some embodiments, one or more of the modified sugars are independently 2'-F or 2'-OMe. In some embodiments, each modified sugar at the 5'-end is independently a bicyclic sugar (eg, an LNA sugar) or a sugar with a 2'-OR variant (R is an optionally substituted C _1-6 aliphatic). In some embodiments, each modified sugar at the 5'-end is independently a bicyclic sugar (eg, an LNA sugar) or a sugar with a 2'-OR variant (R is an optionally substituted C _1-6 aliphatic). In some embodiments, each modified sugar at the 5'-end is independently a sugar with a 2'-OR variant (R is an optionally substituted C _1-6 aliphatic). In some embodiments, R is methyl.

In some embodiments, compared to the 5'-end portion, the 3'-end portion has a higher level (number and/or percentage) of a 2'-F modification sugar and/or a sugar comprising two 2'-Hs (e.g., per native DNA), and/or to a lower level (number and/or percentage) of other types of modifications, such as bicyclic sugars and/or sugars with 2'-OR modifications (R is independently optionally substituted C _1-6 are aliphatic). In some embodiments, relative to the 5'-end portion, the 3'-end portion per higher level of 2'-F modification and/or lower level of 2'-OR modification (R is optionally substituted C _1-6 are aliphatic). In some embodiments, compared to the 5'-end portion, the 3'-end portion comprises a higher level of a 2'-F modified sugar and/or a lower level of a 2'-OMe modified sugar. In some embodiments, relative to the 5'-end portion, the 3'-end portion per lower level of 2'-F modification and/or higher level of 2'-OR modification (R is optionally substituted C _1-6 are aliphatic). In some embodiments, compared to the 5'-end portion, the 3'-end portion comprises a lower level of a 2'-F modified sugar and/or a higher level of a 2'-OMe modified sugar. In some embodiments, relative to the 5'-end, the 3'-end has higher levels of natural DNA sugars and/or lower levels of 2'-OR modified sugars, where R is optionally substituted C _1-6 aliphatic. includes In some embodiments, compared to the 5'-end, the 3'-end comprises higher levels of natural DNA sugars and/or lower levels of 2'-OMe modified sugars. In some embodiments, the 3'-terminus is a bicyclic sugar or a sugar comprising a 2'-OR, where R is an optionally substituted C _1-6 aliphatic (eg, methyl), to a low level (eg, up to 50%). , 40%, 30%, 25%, 20%, or 10%, or up to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) modified sugars. In some embodiments, the 3′-end portion does not include a modified sugar that is a bicyclic sugar or a sugar comprising a 2′-OR, where R is an optionally substituted C _1-6 aliphatic (eg, methyl).

In some embodiments, one or more modified sugars independently include 2'-F. In some embodiments, the modified sugar does not include 2'-OMe or other 2'-OR modifications (R is an optionally substituted C _1-6 aliphatic). In some embodiments, each sugar at the 3'-end independently comprises two 2'-H or 2'-F modifications. In some embodiments, the 3'-end portion comprises 1, 2, 3, 4, or 5 2'-F modified sugars. In some embodiments, the 3'-end portion contains 1-3 2'-F modified sugars. In some embodiments, the 3'-end comprises 1, 2, 3, 4, or 5 natural DNA sugars. In some embodiments, the 3'-end comprises 1-3 natural DNA sugars.

In some embodiments, between one or more (e.g., about 1-10, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) nucleotides of the 3'-end. Linkages are independently modified internucleotide linkages. In some embodiments, between one or more (e.g., about 1-10, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) nucleotides of the 3'-end. Linkages are independently chiral internucleotidic linkages. In some embodiments, between one or more (e.g., about 1-10, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) nucleotides of the 3'-end. Linkages are independently chirally controlled internucleotidic linkages. In some embodiments, between one or more (e.g., about 1-10, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) nucleotides of the 3'-end. The connection is R p. In some embodiments, between one or more (e.g., about 1-10, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) nucleotides of the 3'-end. The connection is Sp . In some embodiments, each internucleotide linkage at the 3'-end is Sp . In some embodiments, the 3'-end portion comprises a higher level (number and/or percentage) of R p internucleotidic linkages and/or native phosphate linkages relative to the 5'-end portion.

In some embodiments, the 3'-end is one or more (e.g., about 1-10, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) as described herein. ), including inconsistency. In some embodiments, the 3'-end is one or more (e.g., about 1-10, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) as described herein. ) of wobbles. In some embodiments, the 3'-end is about 60-100% (eg, 66%, 70%, 75%, 80%, 85%, 90%, 95% or more) complementary to the target nucleic acid. In some embodiments, complementarity is greater than 60%. In some embodiments, complementarity is greater than 70%. In some embodiments, complementarity is greater than 75%. In some embodiments, complementarity is greater than 80%. In some embodiments, complementarity is greater than 90%.

In some embodiments, the first subdomain enables, promotes, or contributes to recruitment of a protein, such as an ADAR protein, eg, ADAR1, ADAR2, etc.). In some embodiments, the first subdomain supplements, promotes, or contributes to an interaction with a protein, such as an ADAR protein. In some embodiments, the first subdomain contacts the RNA binding domain (RBD) of an ADAR. In some embodiments, the first subdomain contacts a catalytic domain of an ADAR having deaminase activity. In some embodiments, the first subdomain contacts a domain having deaminase activity of ADAR1. In some embodiments, the first subdomain contacts a domain having deaminase activity of ADAR2. In some embodiments, the various nucleobases, sugars and/or internucleotide linkages of the first subdomain can interact with one or more residues of a protein (eg, an ADAR protein).

2nd subdomain

As described herein, in some embodiments, an oligonucleotide comprises a first domain and a second domain in a 5' to 3' direction. In some embodiments, the second domain comprises or consists of a first subdomain, a second subdomain, and a third subdomain in a 5' to 3' direction. Specific implementations of the second subdomain are described below by way of example. In some embodiments, the second subdomain comprises the nucleoside opposite the target adenosine to be modified (eg, converted to I). In some embodiments, the second subdomain comprises no more than one nucleoside opposite the target adenosine. In some embodiments, each nucleoside opposite the target adenosine of the oligonucleotide is in a second subdomain.

In some embodiments, the second subdomain is about 1-10, 1-5, 1-3, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleobases have a length In some embodiments, the second subdomain is about 1-10 nucleobases in length. In some embodiments, the second subdomain is about 1-5 nucleobases in length. In some embodiments, the second subdomain is about 1-3 nucleobases in length. In some embodiments, the second subdomain is 1 nucleobase in length. In some embodiments, the second subdomain is two nucleobases in length. In some embodiments, the second subdomain is 3 nucleobases in length. In some embodiments, all nucleosides in the second subdomain are 5'-N ₁ N ₀ N _-1 -3'.

In some embodiments, one or more (e.g., 1-20, 1, 2, 3, 4, 5, 6, 7, 8, 9 , or 10, etc.) mismatches exist. In some implementations, there is 1 mismatch. In some implementations, there are two mismatches. In some implementations, there are three mismatches. In some implementations, there are 4 mismatches. In some implementations, there are 5 mismatches. In some implementations, there are 6 mismatches. In some implementations, there are 7 mismatches. In some implementations, there are 8 mismatches. In some implementations, there are 9 mismatches. In some implementations, there are 10 mismatches.

In some implementations, the second subdomain contains no more than one mismatch. In some embodiments, the second subdomain contains no more than two mismatches. In some embodiments, the second subdomain comprises no more than two mismatches, one mismatch between a target adenosine and its opposite nucleoside, and/or one mismatch between a nucleoside next to a target adenosine and an oligo between the corresponding nucleosides of a nucleotide. In some embodiments, mismatches between the nucleoside next to the target adenosine and the corresponding nucleoside of the oligonucleotide are wobbles. In some implementations, the wobble is I-C. In some embodiments, C is immediately adjacent to the target adenosine, eg, on the 3' side.

In some embodiments, one or more (e.g., 1-20, 1, 2, 3, 4, 5, 6, 7, 8, 9 , or 10, etc.) wobbles exist. In some implementations, there is 1 wobble. In some implementations, there are two wobbles. In some implementations, there are three wobbles. In some implementations, there are 4 wobbles. In some implementations, there are 5 wobbles. In some implementations, there are 6 wobbles. In some implementations, there are 7 wobbles. In some implementations, there are 8 wobbles. In some implementations, there are 9 wobbles. In some implementations, there are 10 wobbles.

In some embodiments, the duplex of the oligonucleotide and the target nucleic acid in the second subdomain region comprises one or more bulges, each independently comprising one or more mismatches that are not wobbles. In some embodiments, 0-10 (e.g., 0-1, 0-2, 0-3, 0-4, 0-5, 0-6, 0-7, 0-8, 0-9, 0~10, 1~2, 1~3, 1~4, 1~5, 1~6, 1~7, 1~8, 1~9, 1~10, 2~3, 2~4, 2~ 5, 2~6, 2~7, 2~8, 2~9, 2~10, 3~4, 3~5, 3~6, 3~7, 3~8, 3~9, 3~10, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 bulges). In some embodiments, the number is zero. In some embodiments, the number is one. In some embodiments, the number is two. In some embodiments, the number is three. In some embodiments, the number is four. In some embodiments, the number is five.

In some embodiments, the second subdomain is fully complementary to the target nucleic acid.

In some embodiments, the second subdomain comprises one or more modified nucleobases.

In some embodiments, the second subdomain comprises a nucleoside opposite the target adenosine, for example when the oligonucleotide forms a duplex with the target nucleic acid. Suitable nucleobases comprising modified nucleobases on opposing nucleosides are described herein. For example, in some embodiments, opposite nucleobases are C, a tautomer of C, U, a tautomer of U, A, a tautomer of A, and BA-I, BA-Ia, BA-Ib, BA-II, BA-II-a, BA-II-b, BA-III, BA-III-a, BA-III-b, BA-IV, BA-IV-a, BA-IV-b, BA-V, BA- an optionally substituted or protected nucleobase selected from nucleobases that are or contain ring BA or a tautomer of ring BA having the structure Va, BA-Vb, or BA-VI. For example, in some embodiments, opposite nucleobases are , , , , , , , , , , , or is selected from In some embodiments, opposite nucleobases are am. In some embodiments, opposite nucleobases are am. In some embodiments, opposite nucleobases are am. In some embodiments, opposite nucleobases are am. In some embodiments, opposite nucleobases are am. In some embodiments, opposite nucleobases are am. In some embodiments, opposite nucleobases are am. In some embodiments, opposite nucleobases are am. In some embodiments, opposite nucleobases are am. In some embodiments, opposite nucleobases are am. In some embodiments, opposite nucleobases are am. In some embodiments, opposite nucleobases are am.

In some embodiments, the second subdomain comprises a modified nucleobase flanking the opposing nucleobase. In some embodiments, it is on the 5' side. In some embodiments, it is on the 3' side. In some embodiments, each side independently has a modified nucleobase. In particular, the present invention provides that nucleobases adjacent to (eg, flanking) opposite nucleobases interfere with recognition, binding, interaction, and/or modification of target nucleic acids, oligonucleotides, and/or duplexes thereof ( eg, steric hindrance). In some embodiments, the interference is related to adjacent G. In some embodiments, the invention provides nucleobases that can replace G and provide improved stability and/or activity relative to G. For example, in some embodiments, adjacent nucleobases (e.g., adjacent nucleosides 3' of opposite nucleosides) replace hypoxanthines (G to reduce interference (e.g., steric hindrance) and/or to C and C). form wobble base pairs). In some embodiments, adjacent nucleobases are derivatives of hypoxanthine. In some embodiments, the 3' adjacent nucleoside comprises a nucleobase that is or comprises ring BA having the structure of Formula BA-VI. In some embodiments, adjacent nucleobases are am. In some embodiments, adjacent nucleobases are am.

In some embodiments, the second subdomain comprises one or more sugars (eg, natural DNA sugars) comprising two 2'-Hs. In some embodiments, the second subdomain includes one or more sugars including 2'-OH (eg, native RNA sugars). In some embodiments, the second subdomain includes one or more modified sugars. In some embodiments, a modified sugar comprises a 2'-modification. In some embodiments, the modified sugar is a bicyclic sugar, such as an LNA sugar. In some embodiments, the modified sugar is acyclic (eg, by breaking the C2-C3 bonds of the corresponding cyclic sugar). In some embodiments, the opposite nucleoside comprises an acyclic sugar such as a UNA sugar. In some embodiments, these acyclic sugars provide flexibility for the protein to undergo modifications at the target adenosine.

In some embodiments, the second subdomain is a bicyclic sugar (eg, LNA sugar), an acyclic sugar (eg, UNA sugar), a sugar with a 2'-OR strain, or a 2'-N(R) ₂ strain. from about 1 to about 10 (eg about 1, 2, 3, 4, 5, 6, 7 _, 8; 9, or 10) modified sugars. In some embodiments, between about 5% and 100% (e.g., between about 10%-100%, 20-100%, 30%-100%, 40%-100%, 50%-100%) of the sugars in the second subdomain. 80%, 50%~85%, 50%~90%, 50%~95%, 60%~80%, 60%~85%, 60%~90%, 60%~95%, 60%~100% , 65%~80%, 65%~85%, 65%~90%, 65%~95%, 65%~100%, 70%~80%, 70%~85%, 70%~90%, 70 %~95%, 70%~100%, 75%~80%, 75%~85%, 75%~90%, 75%~95%, 75%~100%, 80%~85%, 80%~ 90%, 80% to 95%, 80% to 100%, 85% to 90%, 85% to 95%, 85% to 100%, 90% to 95%, 90% to 100%, 10%, 20% , 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%, etc.) independently of the affected party (e.g., LNA) , an acyclic sugar (eg UNA sugar), a sugar with a 2'-OR strain, or a sugar with a 2'-N(R) ₂ strain, where each R is independently an optionally substituted C _1-6 aliphatic It is a modified sugar selected from

In some embodiments, a lower level in the second subdomain (e.g., up to 50%, 40%, 30%, 25%, 20%, or 10%, or up to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) sugars independently comprise a 2'-OR variant (R is an optionally substituted C _1-6 aliphatic), or a 2'-OL ^B -4' variant. In some embodiments, each sugar in the second subdomain is independently a 2'-OR variant (R is an optionally substituted C _1-6 aliphatic) or a 2'-OL ^B -4' variant (L ^B is optionally substituted C 1-6 aliphatic). substituted -CH ₂ -). In some embodiments, each sugar in the second subdomain independently does not contain 2'-OMe.

In some embodiments, a high level in the second subdomain (e.g., greater than 50%, 60%, 70%, 80%, 90%, or 95%, 99%, or 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, more than 20) sugars are independently a 2'-OR variant (R is an optionally substituted C _1-6 aliphatic), or a 2'-OL ^B -4' variant includes In some embodiments, each sugar in the second subdomain is independently a 2'-OR variant (R is an optionally substituted C _1-6 aliphatic) or a 2'-OL ^B -4' variant (L ^B is optionally substituted C 1-6 aliphatic). substituted -CH ₂ -). In some embodiments, each sugar in the second subdomain independently comprises 2'-OMe.

In some embodiments, the second subdomain includes one or more 2'-F modified sugars.

In some embodiments, a high level (e.g., about 60-100%, or about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more) in the second subdomain , or 100%) of or all sugars are independently 2'-F modified sugars, sugars containing two 2'-H (e.g., natural DNA sugars), or sugars containing 2'-OH (e.g., natural DNA sugars). per RNA). In some embodiments, a high level (e.g., about 60-100%, or about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more) in the second subdomain , or 100%) of or all sugars are independently 2'-F modified sugars, native DNA sugars, or native RNA sugars. In some embodiments, a high level (e.g., about 60-100%, or about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more) in the second subdomain , or 100%) or all sugars are independently 2'-F modified sugars and natural DNA sugars. In some embodiments, the level is 100%. In some embodiments, the second subdomain comprises 1, 2, 3, 4 or 5 2′-F modified sugars. In some embodiments, the second subdomain comprises 1, 2, 3, 4 or 5 sugars including two 2′-Hs. In some embodiments, the second subdomain comprises 1, 2, 3, 4 or 5 natural DNA sugars. In some embodiments, the second subdomain comprises 1, 2, 3, 4 or 5 sugars including 2'-OH. In some embodiments, the second subdomain comprises 1, 2, 3, 4 or 5 native RNA sugars. In some embodiments, the number is one. In some embodiments, the number is two. In some embodiments, the number is three. In some embodiments, the number is four. In some embodiments, the number is five.

In some embodiments, a lower level in the second subdomain (e.g., up to 50%, 40%, 30%, 25%, 20%, or 10%, or up to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) sugars independently contain 2'-F modifications. In some embodiments, each sugar in the second subdomain independently does not contain a 2'-F modification. In some embodiments, each sugar in the second subdomain independently does not include a 2'-F.

In some embodiments, the sugar of the opposite nucleoside to the target adenosine ("opposite sugar"), the sugar of the 5' adjacent nucleoside to the opposite nucleoside ("5' adjacent sugar"), and/or Sugars of adjacent nucleosides in the 3' direction to opposite nucleosides ("3' adjacent sugars") are independently and optionally sugars containing two 2'-H per 2'-F variant (e.g., natural DNA sugars), or sugars containing 2'-OH (e.g., native RNA sugars). In some embodiments, the opposite sugar is a 2'-F modified sugar. In some embodiments, the opposite sugar is a sugar comprising two 2'-Hs. In some embodiments, the opposite sugar is a natural DNA sugar. In some embodiments, the opposite sugar is a sugar comprising 2'-OH. In some embodiments, the opposite sugar is a native RNA sugar. For example, in some embodiments, each of the 5' adjacent sugar, opposite sugar, and 3' adjacent sugar of an oligonucleotide is independently a natural DNA sugar. In some embodiments, the 5' adjacent sugar is a 2'-F modified sugar, and each opposite sugar and 3' adjacent sugar are independently natural DNA sugars.

In some embodiments, the 5' adjacent sugar is a 2'-F modified sugar. In some embodiments, a 5' adjacent sugar is a sugar comprising two 2'-Hs. In some embodiments, the 5' adjacent sugar is a natural DNA sugar. In some embodiments, a 5' adjacent sugar is a sugar comprising a 2'-OH. In some embodiments, the 5' adjacent sugar is a native RNA sugar.

In some embodiments, the 3' adjacent sugar is a 2'-F modified sugar. In some embodiments, a 3' adjacent sugar is a sugar comprising two 2'-Hs. In some embodiments, the 3' adjacent sugar is a natural DNA sugar. In some embodiments, the 3' adjacent sugar is a sugar comprising a 2'-OH. In some embodiments, the 3' adjacent sugar is a native RNA sugar.

In some embodiments, up to about 1% to 95% (e.g., up to about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, etc.) contain a 2'-MOE. In some embodiments, up to about 50% of the sugars in the second subdomain comprise a 2'-MOE. In some embodiments, the sugar in the second subdomain does not include a 2'-MOE.

In some embodiments, the second subdomain is about 1-10 (e.g., about 1-5, 1-4, 1-3, about 1, 2, 3, 4, 5, 6, 7, 8 , 9, or 10) modified internucleotide linkages. In some embodiments, about 5%-100% (e.g., about 10%-100%, 20-100%, 30%-100%, 40%-100%, 50%~80%, 50%~85%, 50%~90%, 50%~95%, 60%~80%, 60%~85%, 60%~90%, 60%~95%, 60% ~100%, 65%~80%, 65%~85%, 65%~90%, 65%~95%, 65%~100%, 70%~80%, 70%~85%, 70%~90 %, 70%~95%, 70%~100%, 75%~80%, 75%~85%, 75%~90%, 75%~95%, 75%~100%, 80%~85%, 80%~90%, 80%~95%, 80%~100%, 85%~90%, 85%~95%, 85%~100%, 90%~95%, 90%~100%, 10% , 20%, 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%, etc.) are modified internucleotide linkages. In some embodiments, each internucleotide linkage in the second subdomain is independently a modified internucleotide linkage. In some embodiments, each modified internucleotide linkage is independently a chiral internucleotide linkage. In some embodiments, the modified or chiral internucleotidic linkage is a phosphorothioate internucleotidic linkage. In some embodiments, a modified or chiral internucleotidic linkage is a non-negatively charged internucleotidic linkage. In some embodiments, the modified or chiral internucleotide linkage is a neutral internucleotide linkage (eg n001). In some embodiments, each modified internucleotide linkage is independently a phosphorothioate internucleotide linkage or a non-negatively charged internucleotide linkage. In some embodiments, each modified internucleotide linkage is independently a phosphorothioate internucleotide linkage or a neutral internucleotide linkage. In some embodiments, each modified internucleotide linkage is independently a phosphorothioate internucleotide linkage. In some embodiments, at least about 1-10 (e.g., about 1-5, 1-4, 1-3, about 1, 2, 3, 4, 5, 6, 7) in the second subdomain. , 8, 9, or 10) chiral internucleotide linkages are chirally controlled. In some embodiments, at least 5%-100% (e.g., about 10%-100%, 20-100%, 30%-100%, 40%-100%) of the chiral internucleotide linkages in the second subdomain , 50%~80%, 50%~85%, 50%~90%, 50%~95%, 60%~80%, 60%~85%, 60%~90%, 60%~95%, 60 %~100%, 65%~80%, 65%~85%, 65%~90%, 65%~95%, 65%~100%, 70%~80%, 70%~85%, 70%~ 90%, 70% to 95%, 70% to 100%, 75% to 80%, 75% to 85%, 75% to 90%, 75% to 95%, 75% to 100%, 80% to 85% , 80%~90%, 80%~95%, 80%~100%, 85%~90%, 85%~95%, 85%~100%, 90%~95%, 90%~100%, 10 %, 20%, 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%, etc.) are chirally controlled. In some embodiments, at least 5%-100% (e.g., about 10%-100%, 20-100%, 30%-100%, 40%) of the phosphorothioate internucleotide linkages in the second subdomain ~100%, 50%~80%, 50%~85%, 50%~90%, 50%~95%, 60%~80%, 60%~85%, 60%~90%, 60%~95 %, 60%~100%, 65%~80%, 65%~85%, 65%~90%, 65%~95%, 65%~100%, 70%~80%, 70%~85%, 70% to 90%, 70% to 95%, 70% to 100%, 75% to 80%, 75% to 85%, 75% to 90%, 75% to 95%, 75% to 100%, 80% ~85%, 80%~90%, 80%~95%, 80%~100%, 85%~90%, 85%~95%, 85%~100%, 90%~95%, 90%~100 %, 10%, 20%, 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%, etc.) are chiral controlled. . In some embodiments, each is independently chirally controlled. In some embodiments, at least about 1-10 (e.g., about 1-5, 1-4, 1-3, about 1, 2, 3, 4, 5, 6, 7) in the second subdomain. , 8, 9, or 10) chiral internucleotide linkages are Sp . In some embodiments, at least about 1-10 (e.g., about 1-5, 1-4, 1-3, about 1, 2, 3, 4, 5, 6, 7) in the second subdomain. , 8, 9, or 10) phosphorothioate internucleotide linkages are Sp . In some embodiments, at least 5%-100% (e.g., about 10%-100%, 20-100%, 30%-100%, 40%-100%) of the chiral internucleotide linkages in the second subdomain , 50%~80%, 50%~85%, 50%~90%, 50%~95%, 60%~80%, 60%~85%, 60%~90%, 60%~95%, 60 %~100%, 65%~80%, 65%~85%, 65%~90%, 65%~95%, 65%~100%, 70%~80%, 70%~85%, 70%~ 90%, 70% to 95%, 70% to 100%, 75% to 80%, 75% to 85%, 75% to 90%, 75% to 95%, 75% to 100%, 80% to 85% , 80%~90%, 80%~95%, 80%~100%, 85%~90%, 85%~95%, 85%~100%, 90%~95%, 90%~100%, 10 %, 20%, 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%, etc.) is Sp . In some embodiments, at least 5%-100% (e.g., about 10%-100%, 20-100%, 30%-100%, 40%) of the phosphorothioate internucleotide linkages in the second subdomain ~100%, 50%~80%, 50%~85%, 50%~90%, 50%~95%, 60%~80%, 60%~85%, 60%~90%, 60%~95 %, 60%~100%, 65%~80%, 65%~85%, 65%~90%, 65%~95%, 65%~100%, 70%~80%, 70%~85%, 70% to 90%, 70% to 95%, 70% to 100%, 75% to 80%, 75% to 85%, 75% to 90%, 75% to 95%, 75% to 100%, 80% ~85%, 80%~90%, 80%~95%, 80%~100%, 85%~90%, 85%~95%, 85%~100%, 90%~95%, 90%~100 %, 10%, 20%, 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%, etc.) is Sp . In some embodiments, the number is one or more. In some embodiments, the number is two or more. In some embodiments, the number is three or more. In some embodiments, the number is 4 or more. In some embodiments, the percentage is at least about 50%. In some embodiments, the percentage is at least about 55%. In some embodiments, the percentage is at least about 60%. In some embodiments, the percentage is at least about 65%. In some embodiments, the percentage is at least about 70%. In some embodiments, the percentage is at least about 75%. In some embodiments, the percentage is at least about 80%. In some embodiments, the percentage is at least about 85%. In some embodiments, the percentage is at least about 90%. In some embodiments, the percentage is at least about 95%. In some embodiments, the percentage is about 100%. In some embodiments, each internucleotide linkage linking two second subdomain nucleosides is independently a modified internucleotide linkage. In some embodiments, each modified internucleotide linkage is independently a chiral internucleotide linkage. In some embodiments, each modified internucleotide linkage is independently a phosphorothioate internucleotide linkage. In some embodiments, each chiral internucleotidic linkage is independently a phosphorothioate internucleotidic linkage. In some embodiments, each modified internucleotidic linkage is independently an Sp chiral internucleotidic linkage. In some embodiments, each modified internucleotide linkage is independently an S p phosphorothioate internucleotide linkage. In some embodiments, each chiral internucleotidic linkage is independently an S p phosphorothioate internucleotidic linkage. In some embodiments, the internucleotide linkages of the second subdomain are linked to two nucleosides of the second subdomain. In some embodiments, an internucleotide linkage between a nucleoside of the second subdomain and a nucleoside of the first or third subdomain may properly be considered an internucleotide linkage of the second subdomain. In some embodiments, the internucleotide linkage linked to the nucleoside of the second subdomain and the nucleoside of the first or third subdomain is a modified internucleotide linkage, in some embodiments, it is a chiral internucleotide linkage, In some embodiments, it is chirally controlled, in some embodiments it is R p, and in some embodiments it is Sp .

In some embodiments, the second subdomain comprises a certain level of R p internucleotidic linkages. In some embodiments, the level is, for example, between about 5% and 100%, about 10% and 100%, 20 and 100%, 30% and 100%, 40% and 100% of all internucleotide linkages in the second subdomain. %, 50%~80%, 50%~85%, 50%~90%, 50%~95%, 60%~80%, 60%~85%, 60%~90%, 60%~95%, 60%~100%, 65%~80%, 65%~85%, 65%~90%, 65%~95%, 65%~100%, 70%~80%, 70%~85%, 70% ~90%, 70%~95%, 70%~100%, 75%~80%, 75%~85%, 75%~90%, 75%~95%, 75%~100%, 80%~85 %, 80%~90%, 80%~95%, 80%~100%, 85%~90%, 85%~95%, 85%~100%, 90%~95%, 90%~100%, 10%, 20%, 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%. In some embodiments, the level is between about 5% and 100%, about 10% and 100%, 20 and 100%, 30% and 100%, 40% and 40% of all chiral internucleotide linkages in the second subdomain, for example. 100%, 50%~80%, 50%~85%, 50%~90%, 50%~95%, 60%~80%, 60%~85%, 60%~90%, 60%~95% , 60%~100%, 65%~80%, 65%~85%, 65%~90%, 65%~95%, 65%~100%, 70%~80%, 70%~85%, 70 %~90%, 70%~95%, 70%~100%, 75%~80%, 75%~85%, 75%~90%, 75%~95%, 75%~100%, 80%~ 85%, 80% to 90%, 80% to 95%, 80% to 100%, 85% to 90%, 85% to 95%, 85% to 100%, 90% to 95%, 90% to 100% , 10%, 20%, 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%. In some embodiments, the level is, for example, between about 5% and 100%, about 10% and 100%, 20 and 100%, 30% and 100%, 40% of all chiral controlled internucleotide linkages in the second subdomain. ~100%, 50%~80%, 50%~85%, 50%~90%, 50%~95%, 60%~80%, 60%~85%, 60%~90%, 60%~95 %, 60%~100%, 65%~80%, 65%~85%, 65%~90%, 65%~95%, 65%~100%, 70%~80%, 70%~85%, 70% to 90%, 70% to 95%, 70% to 100%, 75% to 80%, 75% to 85%, 75% to 90%, 75% to 95%, 75% to 100%, 80% ~85%, 80%~90%, 80%~95%, 80%~100%, 85%~90%, 85%~95%, 85%~100%, 90%~95%, 90%~100 %, 10%, 20%, 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%. In some embodiments, the percentage is about or up to about 50%. In some embodiments, the percentage is at least about 55%. In some embodiments, the percentage is at least about 60%. In some embodiments, the percentage is at least about 65%. In some embodiments, the percentage is at least about 70%. In some embodiments, the percentage is at least about 75%. In some embodiments, the percentage is at least about 80%. In some embodiments, the percentage is at least about 85%. In some embodiments, the percentage is at least about 90%. In some embodiments, the percentage is at least about 95%. In some embodiments, the percentage is about 100%. In some embodiments, the percentage is about or up to about 5%. In some embodiments, the percentage is about or up to about 10%. In some embodiments, the percentage is about or up to about 15%. In some embodiments, the percentage is about or up to about 20%. In some embodiments, the percentage is about or up to about 25%. In some embodiments, the percentage is about or up to about 30%. In some embodiments, the percentage is about or up to about 35%. In some embodiments, the percentage is about or up to about 40%. In some embodiments, the percentage is about or up to about 45%. In some embodiments, the percentage is about or up to about 50%. In some embodiments, 1-10 (e.g., about 1-5, 1-4, 1-3, about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) ) are independently R p chiral internucleotidic linkages. In some embodiments, the number is about or up to about 1. In some embodiments, the number is about or up to about 2. In some embodiments, the number is about or up to about 3. In some embodiments, the number is about or up to about 4. In some embodiments, the number is about or up to about 5. In some embodiments, the number is about or up to about 6. In some embodiments, the number is about or up to about 7. In some embodiments, the number is about or up to about 8. In some embodiments, the number is about or up to about 9. In some embodiments, the number is about or up to about 10. In some implementations, the second subdomain is at a higher level (in number and/or percentage) of R p internucleotidic linkages. In some embodiments, the second subdomain comprises a higher level (number and/or percentage) of R p internucleotide linkages than S p internucleotide linkages.

In some embodiments, each phosphorothioate internucleotidic linkage in the second subdomain is independently chirally controlled. In some embodiments, each is independently Sp or R p. In some embodiments, the high level is Sp as described herein. In some embodiments, each phosphorothioate internucleotidic linkage in the second subdomain is chirally controlled and is Sp . In some embodiments, one or more, for example about 1-5 (eg, about 1, 2, 3, 4, or 5) is R p .

In some embodiments, each internucleotide linkage linked to the native DNA or RNA or 2′-F modified sugar in the second subdomain is independently a modified internucleotide linkage as described herein. In some embodiments, each such modified internucleotide linkage is independently a non-negatively charged internucleotide linkage, such as a phosphorothioate or phosphorylguanidine internucleotide linkage such as n001. In some embodiments, each such modified internucleotide linkage is independently a phosphorothioate or n001 internucleotide linkage. In some embodiments, each internucleotide linkage linked to two second subdomain nucleosides is independently a phosphorothioate internucleotide linkage. In some embodiments, each phosphorothioate internucleoside linkage linked to two second subdomain nucleosides is chirally controlled and is Sp . In some embodiments, the one or more internucleoside linkages linked to the second subdomain nucleoside are independently non-negatively charged internucleotide linkages, such as phosphoryl guanidine internucleotide linkages such as n001. In some embodiments, the internucleotidic linkages linked to N _-1 and N _-2 are non-negatively charged internucleotidic linkages. In some embodiments, it is a phosphoryl guanidine internucleotide linkage. In some embodiments, this is n001. In some embodiments, it is chirally controlled and R p . In some embodiments, it is chirally controlled and is Sp . In some embodiments, N _-1 includes hypoxanthine and in some embodiments is deoxyinosine. In some embodiments, the phosphoryl guanidine internucleotide linkage, such as n001 linked to the 3' position of a nucleoside comprising hypoxanthine, is chirally controlled and is Sp . In some embodiments, an oligonucleotide comprising a sp phosphorylguanidine internucleotide linkage, such as Sp n001 linked to the 3' position of a nucleoside comprising hypoxanthine (eg, deoxyinosine), is Provides advantages such as higher activity, better properties, lower manufacturing cost and/or more readily available manufacturing materials.

In some embodiments, as exemplified in certain examples, the second subdomain comprises one or more non-negatively charged internucleotide linkages, each optionally and independently chirally controlled. In some embodiments, each non-negatively charged internucleotide linkage is independently n001. In some embodiments, non-negatively charged chiral internucleotidic linkages are not chirally controlled. In some embodiments, each chiral internucleotidic linkage that is not negatively charged is not chirally controlled. In some embodiments, non-negatively charged chiral internucleotidic linkages are chirally controlled. In some embodiments, non-negatively charged chiral internucleotidic linkages are chirally controlled and R p . In some embodiments, the non-negatively charged chiral internucleotidic linkages are chirally controlled and are Sp . In some embodiments, each non-negatively charged chiral internucleotidic linkage is chirally controlled. In some embodiments, the number of non-negatively charged internucleotide linkages in the second subdomain is about 1-5, or about 1, 2, 3, 4, or 5. In some embodiments, the number is about 1. In some embodiments, the number is about two. In some embodiments, the number is about 3. In some embodiments, the number is about 4. In some embodiments, the number is about 5. In some embodiments, linkages between two or more nucleotides that are not negatively charged are contiguous. In some embodiments, linkages between two non-negatively charged nucleotides are not contiguous. In some embodiments, all non-negatively charged internucleotide linkages in the second subdomain are contiguous (eg, three consecutive non-negatively charged internucleotidic linkages). In some embodiments, the non-negatively charged internucleotidic linkages, or two or more (e.g., about 2, about 3, about 4, etc.) consecutive non-negatively charged internucleotidic linkages are formed in a second at the 3'-end of the subdomain. In some embodiments, the last two or three or four internucleotide linkages of the second subdomain comprise at least one internucleotide linkage that is not a negatively charged internucleotide linkage. In some embodiments, the last 2 or 3 or 4 internucleotide linkages of the second subdomain comprise at least one internucleotide linkage other than n001. In some embodiments, the internucleoside linkage connecting the last two nucleosides of the second subdomain is a non-negatively charged internucleoside linkage. In some embodiments, the internucleoside linkage connecting the last two nucleosides of the second subdomain is a non-negatively charged S p internucleoside linkage. In some embodiments, the internucleoside linkage connecting the last two nucleosides of the second subdomain is a non-negatively charged R p internucleoside linkage. In some embodiments, the internucleoside linkage connecting the last two nucleosides of the second subdomain is a phosphorothioate internucleoside linkage. In some embodiments, the internucleoside linkage connecting the last two nucleosides of the second subdomain is an S p phosphorothioate internucleoside linkage. In some embodiments, the internucleoside linkage connecting the first two nucleosides of the second subdomain is a non-negatively charged internucleotidic linkage. In some embodiments, the internucleoside linkage connecting the first two nucleosides of the second subdomain is a non-negatively charged S p internucleoside linkage. In some embodiments, the internucleoside linkage connecting the first two nucleosides of the second subdomain is a non-negatively charged R p internucleoside linkage. In some embodiments, the internucleoside linkage connecting the first two nucleosides of the second subdomain is a phosphorothioate internucleoside linkage. In some embodiments, the internucleoside linkage connecting the first two nucleosides of the second subdomain is an S p phosphorothioate internucleoside linkage. In some embodiments, the internucleoside connecting the last nucleoside of the second subdomain and the first nucleoside of the third subdomain is a non-negatively charged internucleoside linkage. In some embodiments, the internucleotide linkage connecting the last nucleoside of the second subdomain and the first nucleoside of the third subdomain is a non-negatively charged S p internucleoside linkage. In some embodiments, the internucleoside linking the last nucleoside of the second subdomain and the first nucleoside of the third subdomain is a non-negatively charged R p internucleoside linkage. In some embodiments, the internucleoside connecting the last nucleoside of the second subdomain to the first nucleoside of the third subdomain is a phosphorothioate internucleoside linkage. In some embodiments, the internucleotide linkage connecting the last nucleoside of the second subdomain and the first nucleoside of the third subdomain is an S p phosphorothioate internucleoside linkage. In some embodiments, the non-negatively charged internucleotidic linkage is a neutral internucleotidic linkage, such as n001.

In some embodiments, the second subdomain comprises one or more native phosphate linkages. In some embodiments, the second subdomain does not contain native phosphate linkages. In some embodiments, the second subdomain comprises at least one native phosphate linkage. In some embodiments, the second subdomain comprises at least two native phosphate linkages. In some embodiments, the second subdomain comprises at least 3 native phosphate linkages. In some embodiments, the second subdomain comprises at least 4 natural phosphate linkages. In some embodiments, the second subdomain comprises at least 5 natural phosphate linkages.

In some embodiments, the opposite nucleoside is linked to the adjacent nucleoside in the 5' direction via a natural phosphate linkage. In some embodiments, the opposite nucleoside is linked to the adjacent nucleoside in the 5' direction via a natural phosphate linkage. In some embodiments, the opposing nucleoside is linked to the adjacent nucleoside in the 5' direction via a modified internucleotide linkage. In some embodiments, the modified internucleotidic linkage is a chiral internucleotidic linkage. In some embodiments, the modified internucleotidic linkage is a phosphorothioate internucleotidic linkage. In some embodiments, the modified internucleotide linkage is a non-negatively charged internucleotide linkage. In some embodiments, the modified internucleotide linkage is a neutrally charged internucleotide linkage. In some embodiments, chiral internucleotidic linkages are chirally controlled. In some embodiments, the chiral internucleotidic linkage is R p. In some embodiments, the chiral internucleotidic linkage is Sp .

In some embodiments, the opposing nucleoside is linked to the 3′ adjacent nucleoside (at position -1 relative to the opposing nucleoside) via a natural phosphate linkage. In some embodiments, the opposing nucleoside is linked to the adjacent nucleoside in the 3' direction via a modified internucleotide linkage. In some embodiments, the modified internucleotidic linkage is a chiral internucleotidic linkage. In some embodiments, the modified internucleotidic linkage is a phosphorothioate internucleotidic linkage. In some embodiments, the modified internucleotide linkage is a non-negatively charged internucleotide linkage. In some embodiments, the modified internucleotide linkage is a neutrally charged internucleotide linkage. In some embodiments, chiral internucleotidic linkages are chirally controlled. In some embodiments, the chiral internucleotidic linkage is R p. In some embodiments, the chiral internucleotidic linkage is Sp . In some embodiments, chiral internucleotidic linkages are phosphorothioate internucleotidic linkages and are chirally controlled. In some embodiments, the chiral internucleotidic linkage is a phosphorothioate internucleotidic linkage and is Sp . In some embodiments, the chiral internucleotidic linkage is a phosphorothioate internucleotidic linkage and is R p . In some embodiments, a chiral internucleotide linkage is a non-negatively charged internucleotide linkage (eg, n001) and is chirally controlled. In some embodiments, the chiral internucleotide linkage is a non-negatively charged internucleotide linkage (eg n001) and is chirally controlled and R p . In some embodiments, a chiral internucleotidic linkage is a non-negatively charged internucleotidic linkage (eg n001) and is chirally controlled and is Sp . In some embodiments, a chiral internucleotide linkage is a non-negatively charged internucleotide linkage (eg, n001) and is not chirally controlled.

In some embodiments, the nucleoside at position -1 to the opposite nucleoside and the nucleoside at position -2 to the opposite nucleoside are (e.g., 5'-...N ₀ N _-1 N _{- 2} …3', where N ₀ is the opposite nucleoside, N _-1 is position -1 and N _-2 is position -2) linked via natural phosphate linkages. In some embodiments, they are linked via modified internucleotide linkages. In some embodiments, the modified internucleotidic linkage is a chiral internucleotidic linkage. In some embodiments, the modified internucleotidic linkage is a phosphorothioate internucleotidic linkage. In some embodiments, the modified internucleotide linkage is a non-negatively charged internucleotide linkage. In some embodiments, the modified internucleotide linkage is a neutrally charged internucleotide linkage. In some embodiments, chiral internucleotidic linkages are chirally controlled. In some embodiments, the chiral internucleotidic linkage is R p. In some embodiments, the chiral internucleotidic linkage is Sp . In some embodiments, chiral internucleotidic linkages are phosphorothioate internucleotidic linkages and are chirally controlled. In some embodiments, the chiral internucleotidic linkage is a phosphorothioate internucleotidic linkage and is Sp . In some embodiments, the chiral internucleotidic linkage is a phosphorothioate internucleotidic linkage and is R p . In some embodiments, a chiral internucleotide linkage is a non-negatively charged internucleotide linkage (eg, n001) and is chirally controlled. In some embodiments, the chiral internucleotide linkage is a non-negatively charged internucleotide linkage (eg n001) and is chirally controlled and R p . In some embodiments, the chiral internucleotide linkage is a non-negatively charged internucleotide linkage (eg n001) and is chirally controlled and is Sp . In some embodiments, a chiral internucleotide linkage is a non-negatively charged internucleotide linkage (eg, n001) and is not chirally controlled.

In some embodiments, the nucleosides of the second subdomain and the nucleosides of the third subdomain are linked via natural phosphate linkages. In some embodiments, they are linked via modified internucleotide linkages. In some embodiments, the modified internucleotidic linkage is a chiral internucleotidic linkage. In some embodiments, the modified internucleotidic linkage is a phosphorothioate internucleotidic linkage. In some embodiments, the modified internucleotide linkage is a non-negatively charged internucleotide linkage. In some embodiments, the modified internucleotide linkage is a neutrally charged internucleotide linkage. In some embodiments, chiral internucleotidic linkages are chirally controlled. In some embodiments, the chiral internucleotidic linkage is R p. In some embodiments, the chiral internucleotidic linkage is Sp . In some embodiments, chiral internucleotidic linkages are phosphorothioate internucleotidic linkages and are chirally controlled. In some embodiments, the chiral internucleotidic linkage is a phosphorothioate internucleotidic linkage and is Sp . In some embodiments, the chiral internucleotidic linkage is a phosphorothioate internucleotidic linkage and is R p . In some embodiments, a chiral internucleotide linkage is a non-negatively charged internucleotide linkage (eg, n001) and is chirally controlled. In some embodiments, the chiral internucleotide linkage is a non-negatively charged internucleotide linkage (eg n001) and is chirally controlled and R p . In some embodiments, the chiral internucleotide linkage is a non-negatively charged internucleotide linkage (eg n001) and is chirally controlled and is Sp . In some embodiments, a chiral internucleotide linkage is a non-negatively charged internucleotide linkage (eg, n001) and is not chirally controlled.

In some embodiments, the oligonucleotide comprises 5'-N ₁ N ₀ N _-1 -3', each of N ₁ , N ₀ , and N _-1 is independently a nucleoside, and N ₁ and N ₀ are binds to an internucleotide linkage as described herein, N _-1 and N ₀ bind to an internucleotide linkage as described herein, and N ₀ is opposite the target adenosine. In some embodiments, each sugar of N ₁ , N ₀ , and N ₋₁ is independently a natural DNA sugar or a 2′-F modified sugar. In some embodiments, each sugar of N ₁ , N ₀ , and N ₋₁ is independently a natural DNA sugar. In some embodiments, the sugar of N ₁ is a 2'-modified sugar, and each sugar of N ₀ and N ₋₁ is independently a natural DNA sugar. In some embodiments, such oligonucleotides provide high levels of editing. In some embodiments, each of the two internucleotide linkages linked to N _-1 is independently R p. In some embodiments, each of the two internucleotide linkages linked to N _-1 is independently an R p phosphorothioate internucleotide linkage. In some embodiments, each of the two internucleotide linkages linked to N _-1 is an R p phosphorothioate internucleotide linkage, and each other phosphorothioate internucleotide linkage in the oligonucleotide, if present, is independently S is p. In some embodiments, the 5' internucleotide linkage linked to N ₁ is R p. In some embodiments, the internucleotide linkage linked to N ₁ and N ₀ (ie, the 3′ internucleotide linkage linked to N ₁ ) is R p. In some embodiments, the internucleotide linkage linked to N _-1 and N ₀ is R p. In some embodiments, the 3' internucleotide linkage linked to N _-1 is R p. In some embodiments, each internucleotide linkage linked to N ₀ is independently R p . In some embodiments, each internucleotide linkage linked to N ₀ and N ₁ is independently R p . In some embodiments, each internucleotide linkage linked to N ₀ and N ₋₁ is independently R p . In some embodiments, each internucleotide linkage linked to N ₁ is independently R p . In some embodiments, each R p internucleotidic linkage is independently an R p phosphorothioate internucleotidic linkage. In some embodiments, each other chirally controlled phosphorothioate internucleotidic linkage in the oligonucleotide is independently Sp .

In some embodiments, the sugars of the 5' adjacent nucleosides (eg, N ₁ ) are independently per native DNA, per native RNA, and per 2'-F modification (eg, R ^2s is -F) is selected from In some embodiments, the sugar of the opposing nucleoside (eg, N ₀ ) is independently selected from a natural DNA sugar, a native RNA sugar, and a 2′-F modified sugar. In some embodiments, the sugar of the 3' adjacent nucleoside (eg, N _-1 ) is independently selected from natural DNA sugars, native RNA sugars, and 2'-F modified sugars. In some embodiments, the sugar of the 5' contiguous nucleoside, the opposite nucleoside, and the 3' contiguous nucleoside are each independently a natural DNA sugar. In some embodiments, the sugars of the 5' contiguous nucleoside, the opposite nucleoside, and the 3' contiguous nucleoside are a native DNA sugar, a native RNA sugar, and a native DNA sugar, respectively. In some embodiments, the sugars of the 5' contiguous nucleoside, the opposite nucleoside, and the 3' contiguous nucleoside are a 2'-F modified sugar, a native RNA sugar, and a native DNA sugar, respectively.

In some embodiments, the sugar of the opposing nucleoside is a native RNA sugar. In some embodiments, such opposite nucleosides are used with a 3' adjacent I nucleoside (optionally complementary to the C of the target nucleic acid when aligned). In some embodiments, an internucleotide linkage between a 3' adjacent nucleoside (eg, N _-1 ) and its 3' adjacent nucleoside (eg, N _-2 ) is a non-negatively charged internucleotide linkage ( Example: n001). In some embodiments, it is stereorandom. In some embodiments, it is chirally controlled and R p . In some embodiments, it is chirally controlled and is Sp .

In some embodiments, a 3' adjacent nucleoside (eg, N _-1 ) and its 3' adjacent nucleoside (eg, 5'-N ₁ N ₀ N _-1 N _-2 -3 The internucleotide linkage linked to N _-2 in ' is a modified internucleotide linkage. In some embodiments, it is a chiral internucleotidic linkage. In some embodiments, it is stereorandom. In some embodiments, it is a sterically random phosphorothioate internucleotidic linkage. In some embodiments, it is a non-negatively charged stereorandom internucleotidic linkage. In some embodiments, it is stereorandom n001. In some embodiments, it is chirally controlled. In some embodiments, it is an R p phosphorothioate internucleotidic linkage. In some embodiments, it is an S p phosphorothioate internucleotidic linkage. In some embodiments, it is chirally controlled. In some embodiments, it is a non-negatively charged R p internucleotidic linkage. In some embodiments, it is a non-negatively charged S p internucleotidic linkage. In some embodiments, the non-negatively charged internucleotidic linkages are neutral internucleotidic linkages. In some embodiments, the non-negatively charged internucleotide linkage is n001.

In some embodiments, N ₋₁ is I. In some embodiments, I is used to replace G, for example when the target nucleic acid contains 5'-CA-3' (A is the target adenosine). In some embodiments, 5'-N ₁ N ₀ N _-1 -3' is 5'-N ₁ N ₀ I-3'. In some embodiments, N ₀ is b001A, b002A, b003A, b008U, b001C, C, A, or U. In some embodiments, N ₀ is b001A, b002A, b008U, b001C, C, or A. In some embodiments, N ₀ is b001A, b002A, b008U, or b001C. In some embodiments, N ₀ is b001A. In some embodiments, N ₀ is b002A. In some embodiments, N ₀ is b003A. In some embodiments, N ₀ is b008U. In some embodiments, N ₀ is b001C. In some embodiments, N ₀ is A. In some embodiments, N ₀ is U.

As shown herein, in some embodiments, a provided oligonucleotide comprising a specific nucleobase (eg, b001A, b002A, b008U, C, A, etc.) opposite a target adenosine is specifically selected (eg, a reference nucleus such as U). base) can provide improved editing efficiency. In some embodiments, the opposite nucleoside is linked to I on the 3' side.

In some embodiments, the second subdomain comprises an editing region as described herein.

In some embodiments, the second subdomain comprises a 5'-terminal portion, eg, having a length of about 1-5, 1-3, or 1, 2, 3, 4, or 5 nucleobases. In some embodiments, the length is 1 nucleobase. In some embodiments, the length is 2 nucleobases. In some embodiments, the length is 3 nucleobases. In some embodiments, the length is 4 nucleobases. In some embodiments, the length is 5 nucleobases.

In some embodiments, the 5'-end portion includes one or more sugars with two 2'-Hs (eg, natural DNA sugars). In some embodiments, the 5'-end portion includes one or more sugars with 2'-OH (eg, native RNA sugars). In some embodiments, one or more (eg, about 1-5, 1-3, or 1, 2, 3, 4, or 5) of the sugars at the 5'-end are independently modified sugars. In some embodiments, between about 5% and 100% (e.g., between about 10% and 100%, 20 and 100%, 30% and 100%, 40% and 100%, 50% and 80%, 50%~85%, 50%~90%, 50%~95%, 60%~80%, 60%~85%, 60%~90%, 60%~95%, 60%~100% , 65%~80%, 65%~85%, 65%~90%, 65%~95%, 65%~100%, 70%~80%, 70%~85%, 70%~90%, 70 %~95%, 70%~100%, 75%~80%, 75%~85%, 75%~90%, 75%~95%, 75%~100%, 80%~85%, 80%~ 90%, 80% to 95%, 80% to 100%, 85% to 90%, 85% to 95%, 85% to 100%, 90% to 95%, 90% to 100%, 10%, 20% , 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%, etc.) are independently modified sugars. In some embodiments, each sugar is independently a modified sugar. In some embodiments, the modified sugar is independently a bicyclic sugar (eg, LNA sugar), an acyclic sugar (eg, UNA sugar), a sugar with a 2'-OR strain, or a 2'-N(R) ₂ strain. sugar (each R is independently optionally substituted C _1-6 aliphatic).

In some embodiments, at a lower level at the 5'-end (e.g., up to 50%, 40%, 30%, 25%, 20%, or 10%, or up to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) sugars independently include a 2'-OR variant (R is an optionally substituted C _1-6 aliphatic), or a 2'-OL ^B -4' variant. In some embodiments, each sugar at the 5'-end is independently a 2'-OR variant (R is an optionally substituted C _1-6 aliphatic) or a 2'-OL ^B -4' variant (L ^B is optionally substituted C 1-6 aliphatic). substituted -CH ₂ -). In some embodiments, each sugar at the 5'-end independently does not contain 2'-OMe.

In some embodiments, the 5'-end portion includes one or more 2'-F modified sugars.

In some embodiments, a high level (e.g., about 60-100%, or about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more) at the 5'-end. , or 100%) of or all sugars are independently 2'-F modified sugars, sugars containing two 2'-H (e.g., natural DNA sugars), or sugars containing 2'-OH (e.g., natural DNA sugars). per RNA). In some embodiments, a high level (e.g., about 60-100%, or about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more) at the 5'-end. , or 100%) of or all sugars are independently 2'-F modified sugars, native DNA sugars, or native RNA sugars. In some embodiments, a high level (e.g., about 60-100%, or about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more) at the 5'-end. , or 100%) or all sugars are independently 2'-F modified sugars and natural DNA sugars. In some embodiments, the level is 100%. In some embodiments, the sugar at the 5'-end is selected from sugars having two 2'-Hs (eg, native DNA sugars) and 2'-F modified sugars. In some embodiments, the 5'-end portion comprises 1, 2, 3, 4 or 5 2'-F modified sugars. In some embodiments, the 5'-end portion comprises 1, 2, 3, 4 or 5 sugars including two 2'-Hs. In some embodiments, the 5'-end comprises 1, 2, 3, 4 or 5 natural DNA sugars. In some embodiments, the 5'-end comprises 1, 2, 3, 4 or 5 sugars including 2'-OH. In some embodiments, the 5'-end comprises 1, 2, 3, 4 or 5 native RNA sugars. In some embodiments, the number is one. In some embodiments, the number is two. In some embodiments, the number is three. In some embodiments, the number is four. In some embodiments, the number is five.

In some embodiments, one or more (eg, about 1, 2, 3, 4, or 5) internucleotide linkages at the 5'-end are independently modified internucleotide linkages. In some embodiments, one or more (eg, about 1, 2, 3, 4, or 5) internucleotide linkages at the 5'-end are independently chiral internucleotide linkages. In some embodiments, one or more (eg, about 1, 2, 3, 4, or 5) internucleotide linkages at the 5'-end are independently chirally controlled internucleotide linkages. In some embodiments, the internucleotidic linkage of one or more (eg, about 1, 2, 3, 4, or 5) of the 5'-end is R p. In some embodiments, the internucleotidic linkage of one or more (eg, about 1, 2, 3, 4, or 5) of the 5'-end is Sp . In some embodiments, each internucleotide linkage at the 5'-end is Sp .

In some embodiments, one or more (eg, about 1, 2, 3, 4, or 5) internucleotide linkages at the 5'-end are independently modified internucleotide linkages. In some embodiments, one or more (eg, about 1, 2, 3, 4, or 5) internucleotide linkages at the 5'-end are independently chiral internucleotide linkages. In some embodiments, one or more (eg, about 1, 2, 3, 4, or 5) internucleotide linkages at the 5'-end are independently chirally controlled internucleotide linkages. In some embodiments, the internucleotidic linkage of one or more (eg, about 1, 2, 3, 4, or 5) of the 5'-end is R p. In some embodiments, the internucleotidic linkage of one or more (eg, about 1, 2, 3, 4, or 5) of the 5'-end is R p. In some embodiments, each internucleotide linkage at the 5'-end is R p.

In some embodiments, the 5'-end portion comprises one or more (eg, about 1, 2, 3, 4, or 5) mismatches as described herein. In some embodiments, the 5'-end portion comprises one or more (eg, about 1, 2, 3, 4, or 5) wobbles as described herein. In some embodiments, the 5'-end is about 60-100% (eg, 66%, 70%, 75%, 80%, 85%, 90%, 95% or more) complementary to the target nucleic acid. In some embodiments, complementarity is greater than 60%. In some embodiments, complementarity is greater than 70%. In some embodiments, complementarity is greater than 75%. In some embodiments, complementarity is greater than 80%. In some embodiments, complementarity is greater than 90%.

In some embodiments, the 5'-end portion comprises an adjacent nucleoside in the 5' direction to the opposite nucleoside. In some embodiments, the adjacent nucleoside 5' to the opposing nucleoside comprises a nucleobase as described herein.

In some embodiments, the second subdomain comprises a 3′-end, eg, having a length of about 1-5, 1-3, or 1, 2, 3, 4, or 5 nucleobases. In some embodiments, the length is 1 nucleobase. In some embodiments, the length is 2 nucleobases. In some embodiments, the length is 3 nucleobases. In some embodiments, the length is 4 nucleobases. In some embodiments, the length is 5 nucleobases. In some embodiments, the second subdomain consists of a 5'-end portion and a 3'-end portion.

In some embodiments, the 3'-end portion includes one or more sugars with two 2'-Hs (eg, natural DNA sugars). In some embodiments, the 3'-end portion includes one or more sugars with 2'-OH (eg, native RNA sugars). In some embodiments, one or more (eg, about 1-5, 1-3, or 1, 2, 3, 4, or 5) sugars at the 3'-end are independently modified sugars. In some embodiments, about 5%-100% (e.g., about 10%-100%, 20-100%, 30%-100%, 40%-100%, 50%-100%) of the sugar at the 3'-end. 80%, 50%~85%, 50%~90%, 50%~95%, 60%~80%, 60%~85%, 60%~90%, 60%~95%, 60%~100% , 65%~80%, 65%~85%, 65%~90%, 65%~95%, 65%~100%, 70%~80%, 70%~85%, 70%~90%, 70 %~95%, 70%~100%, 75%~80%, 75%~85%, 75%~90%, 75%~95%, 75%~100%, 80%~85%, 80%~ 90%, 80% to 95%, 80% to 100%, 85% to 90%, 85% to 95%, 85% to 100%, 90% to 95%, 90% to 100%, 10%, 20% , 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%, etc.) are independently modified sugars. In some embodiments, each sugar is independently a modified sugar. In some embodiments, the modified sugar is independently a bicyclic sugar (eg, LNA sugar), an acyclic sugar (eg, UNA sugar), a sugar with a 2'-OR strain, or a 2'-N(R) ₂ strain. sugar (each R is independently optionally substituted C _1-6 aliphatic).

In some embodiments, at a lower level at the 3'-end (e.g., up to 50%, 40%, 30%, 25%, 20%, or 10%, or up to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) sugars independently include a 2'-OR variant (R is an optionally substituted C _1-6 aliphatic), or a 2'-OL ^B -4' variant. In some embodiments, each sugar at the 3'-terminus is independently a 2'-OR variant (R is an optionally substituted C _1-6 aliphatic) or a 2'-OL ^B -4' variant (L ^B is optionally substituted C 1-6 aliphatic). substituted -CH ₂ -). In some embodiments, each sugar at the 3'-end independently does not contain 2'-OMe.

In some embodiments, the 3'-end portion includes one or more 2'-F modified sugars.

In some embodiments, a high level (e.g., about 60-100%, or about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more) at the 3'-end. , or 100%) of or all sugars are independently 2'-F modified sugars, sugars containing two 2'-H (e.g., natural DNA sugars), or sugars containing 2'-OH (e.g., natural DNA sugars). per RNA). In some embodiments, a high level (e.g., about 60-100%, or about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more) at the 3'-end. , or 100%) of or all sugars are independently 2'-F modified sugars, native DNA sugars, or native RNA sugars. In some embodiments, a high level (e.g., about 60-100%, or about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more) at the 3'-end. , or 100%) or all sugars are independently 2'-F modified sugars and natural DNA sugars. In some embodiments, the level is 100%. In some embodiments, the sugar at the 3'-end is selected from sugars having two 2'-Hs (eg, native DNA sugars) and 2'-F modified sugars. In some embodiments, the 3'-end portion comprises 1, 2, 3, 4 or 5 2'-F modified sugars. In some embodiments, the 3'-end portion contains 1, 2, 3, 4 or 5 sugars including two 2'-Hs. In some embodiments, the 3'-end comprises 1, 2, 3, 4 or 5 natural DNA sugars. In some embodiments, the 3'-end comprises 1, 2, 3, 4 or 5 sugars including 2'-OH. In some embodiments, the 3'-end comprises 1, 2, 3, 4 or 5 native RNA sugars. In some embodiments, the number is one. In some embodiments, the number is two. In some embodiments, the number is three. In some embodiments, the number is four. In some embodiments, the number is five.

In some embodiments, one or more (eg, about 1, 2, 3, 4, or 5) internucleotide linkages at the 3'-end are independently modified internucleotide linkages. In some embodiments, one or more (eg, about 1, 2, 3, 4, or 5) internucleotidic linkages of the 3'-terminus are independently chiral internucleotidic linkages. In some embodiments, one or more (eg, about 1, 2, 3, 4, or 5) internucleotide linkages at the 3'-end are independently chirally controlled internucleotide linkages. In some embodiments, the internucleotide linkage of one or more (eg, about 1, 2, 3, 4, or 5) of the 3'-terminus is R p. In some embodiments, the internucleotidic linkage of one or more (eg, about 1, 2, 3, 4, or 5) of the 3'-end is Sp . In some embodiments, each internucleotide linkage at the 3'-end is Sp .

In some embodiments, one or more (eg, about 1, 2, 3, 4, or 5) internucleotide linkages at the 3'-end are independently modified internucleotide linkages. In some embodiments, one or more (eg, about 1, 2, 3, 4, or 5) internucleotidic linkages of the 3'-terminus are independently chiral internucleotidic linkages. In some embodiments, one or more (eg, about 1, 2, 3, 4, or 5) internucleotide linkages at the 3'-end are independently chirally controlled internucleotide linkages. In some embodiments, the internucleotide linkage of one or more (eg, about 1, 2, 3, 4, or 5) of the 3'-terminus is R p. In some embodiments, the internucleotide linkage of one or more (eg, about 1, 2, 3, 4, or 5) of the 3'-terminus is R p. In some embodiments, each internucleotide linkage at the 3'-end is R p.

In some embodiments, the 3'-end portion comprises one or more (eg, about 1, 2, 3, 4, or 5) mismatches as described herein. In some embodiments, the 3'-end portion comprises one or more (eg, about 1, 2, 3, 4, or 5) wobbles as described herein. In some embodiments, the 3'-end is about 60-100% (eg, 66%, 70%, 75%, 80%, 85%, 90%, 95% or more) complementary to the target nucleic acid. In some embodiments, complementarity is greater than 60%. In some embodiments, complementarity is greater than 70%. In some embodiments, complementarity is greater than 75%. In some embodiments, complementarity is greater than 80%. In some embodiments, complementarity is greater than 90%.

In some embodiments, the 3'-end portion comprises an adjacent nucleoside in the 3' direction to the opposite nucleoside. In some embodiments, the adjacent nucleoside in the 3' direction to the opposing nucleoside comprises a nucleobase as described herein. In some embodiments, adjacent nucleosides 3' to opposite nucleosides form wobble pairs with the corresponding nucleosides of the target nucleic acid. In some embodiments, the nucleobase of the 3' adjacent nucleoside to the opposite nucleoside is hypoxanthine, and in some embodiments it is a derivative of hypoxanthine.

In some embodiments, the second subdomain enables, promotes, or contributes to recruitment of a protein, such as an ADAR protein, eg, ADAR1, ADAR2, etc.). In some embodiments, the second subdomain supplements, promotes, or contributes to an interaction with a protein, such as an ADAR protein. In some embodiments, the second subdomain contacts the RNA binding domain (RBD) of an ADAR. In some embodiments, the second subdomain contacts the catalytic domain of an ADAR having deaminase activity. In some embodiments, the second subdomain contacts the domain having deaminase activity of ADAR1. In some embodiments, the second subdomain contacts the domain having deaminase activity of ADAR2. In some embodiments, the various nucleobases, sugars and/or internucleotide linkages of the second subdomain can interact with one or more residues of a protein (eg, an ADAR protein).

3rd subdomain

As described herein, in some embodiments, an oligonucleotide comprises a first domain and a second domain in a 5' to 3' direction. In some embodiments, the second domain comprises or consists of a first subdomain, a second subdomain, and a third subdomain in a 5' to 3' direction. Specific implementations of the third subdomain are described below by way of example.

In some embodiments, the third subdomain is about 1-50, 1-40, 1-30, 1-20 (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9 , or 10 to about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 40 or 50, or about 1, 2 , 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 40 or 50, etc.) nucleobases in length. In some embodiments, the third subdomain is about 5-30 nucleobases in length. In some embodiments, the third subdomain is about 10-30 nucleobases in length. In some embodiments, the third subdomain is about 10-20 nucleobases in length. In some embodiments, the third subdomain is about 5-15 nucleobases in length. In some embodiments, the third subdomain is about 13-16 nucleobases in length. In some embodiments, the third subdomain is about 6-12 nucleobases in length. In some embodiments, the third subdomain is about 6-9 nucleobases in length. In some embodiments, the third subdomain is about 1-10 nucleobases in length. In some embodiments, the third subdomain is about 1-7 nucleobases in length. In some embodiments, the third subdomain is 1 nucleobase in length. In some embodiments, the third subdomain is two nucleobases in length. In some embodiments, the third subdomain is 3 nucleobases in length. In some embodiments, the third subdomain is 4 nucleobases in length. In some embodiments, the third subdomain is 5 nucleobases in length. In some embodiments, the sixth subdomain is 3 nucleobases in length. In some embodiments, the third subdomain is 7 nucleobases in length. In some embodiments, the third subdomain is 8 nucleobases in length. In some embodiments, the third subdomain is 9 nucleobases in length. In some embodiments, the third subdomain is 10 nucleobases in length. In some embodiments, the third subdomain is 11 nucleobases in length. In some embodiments, the third subdomain is 12 nucleobases in length. In some embodiments, the third subdomain is 13 nucleobases in length. In some embodiments, the third subdomain is 14 nucleobases in length. In some embodiments, the third subdomain is 15 nucleobases in length. In some implementations, the third subdomain is shorter than the first subdomain. In some implementations, the third subdomain is shorter than the first domain. In some embodiments, the third subdomain comprises the 3′-terminal nucleobase of the second domain.

In some embodiments, the third subdomain comprises about or at least about 5-95%, 10%-90%, 20%-80%, 30%-70%, 40%-70%, 40%-of the second domain. 60%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80% , 85%, 90%, 95%. In some embodiments, the percentage is between about 30% and 80%. In some embodiments, the percentage is between about 30% and 70%. In some embodiments, the percentage is between about 40% and 60%. In some embodiments, the percentage is about 20%. In some embodiments, the percentage is about 25%. In some embodiments, the percentage is about 30%. In some embodiments, the percentage is about 35%. In some embodiments, the percentage is about 40%. In some embodiments, the percentage is about 45%. In some embodiments, the percentage is about 50%. In some embodiments, the percentage is about 55%. In some embodiments, the percentage is about 60%. In some embodiments, the percentage is about 65%. In some embodiments, the percentage is about 70%. In some embodiments, the percentage is about 75%. In some embodiments, the percentage is about 80%. In some embodiments, the percentage is about 85%. In some embodiments, the percentage is about 90%. In some embodiments, the 5'-terminal nucleoside of the third subdomain is N _-2 . In some embodiments, all nucleosides from N _-2 to the 3'-end are in the third subdomain.

In some embodiments, one or more (e.g., 1-20, 1, 2, 3, 4, 5, 6, 7, 8, 9 , or 10, etc.) mismatches exist. In some implementations, there is 1 mismatch. In some implementations, there are two mismatches. In some implementations, there are three mismatches. In some implementations, there are 4 mismatches. In some implementations, there are 5 mismatches. In some implementations, there are 6 mismatches. In some implementations, there are 7 mismatches. In some implementations, there are 8 mismatches. In some implementations, there are 9 mismatches. In some implementations, there are 10 mismatches.

In some embodiments, one or more (e.g., 1-20, 1, 2, 3, 4, 5, 6, 7, 8, 9 , or 10, etc.) wobbles exist. In some implementations, there is 1 wobble. In some implementations, there are two wobbles. In some implementations, there are three wobbles. In some implementations, there are 4 wobbles. In some implementations, there are 5 wobbles. In some implementations, there are 6 wobbles. In some implementations, there are 7 wobbles. In some implementations, there are 8 wobbles. In some implementations, there are 9 wobbles. In some implementations, there are 10 wobbles.

In some embodiments, the duplex of the oligonucleotide and the target nucleic acid in the third subdomain region comprises one or more bulges, each independently comprising one or more mismatches that are not wobbles. In some embodiments, 0-10 (e.g., 0-1, 0-2, 0-3, 0-4, 0-5, 0-6, 0-7, 0-8, 0-9, 0~10, 1~2, 1~3, 1~4, 1~5, 1~6, 1~7, 1~8, 1~9, 1~10, 2~3, 2~4, 2~ 5, 2~6, 2~7, 2~8, 2~9, 2~10, 3~4, 3~5, 3~6, 3~7, 3~8, 3~9, 3~10, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 bulges). In some embodiments, the number is zero. In some embodiments, the number is one. In some embodiments, the number is two. In some embodiments, the number is three. In some embodiments, the number is four. In some embodiments, the number is five.

In some embodiments, the third subdomain is fully complementary to the target nucleic acid.

In some embodiments, the third subdomain comprises one or more modified nucleobases.

In some embodiments, the third subdomain comprises the opposite nucleoside of the target adenosine (opposite nucleoside). In some embodiments, the third subdomain comprises an adjacent nucleoside in the 3' direction to the opposite nucleoside. In some embodiments, the third subdomain comprises an adjacent nucleoside 5' to the opposite nucleoside. A variety of suitable opposite nucleosides, including sugars and their nucleobases, have been described herein.

In some embodiments, the third subdomain comprises the opposite nucleoside of the target adenosine, for example when the oligonucleotide forms a duplex with the target nucleic acid. Suitable nucleobases comprising modified nucleobases on opposing nucleosides are described herein. For example, in some embodiments, opposite nucleobases are C, a tautomer of C, U, a tautomer of U, A, a tautomer of A, and BA-I, BA-I-a, BA-I-b, BA-II, BA-II-a, BA-II-b, BA-III, BA-III-a, BA-III-b, BA-IV, BA-IV-a, BA-IV-b, BA-V, BA- an optionally substituted or protected nucleobase selected from ring BA having the structure V-a, BA-V-b, or BA-VI or a nucleobase comprising or a tautomer of ring BA.

In some embodiments, the third subdomain comprises one or more sugars (eg, natural DNA sugars) comprising two 2'-Hs. In some embodiments, the third subdomain includes one or more sugars including 2'-OH (eg, native RNA sugars). In some embodiments, the third subdomain includes one or more modified sugars. In some embodiments, a modified sugar comprises a 2'-modification. In some embodiments, the modified sugar is a bicyclic sugar, such as an LNA sugar. In some embodiments, the modified sugar is acyclic (eg, by breaking the C2-C3 bonds of the corresponding cyclic sugar).

In some embodiments, the third subdomain is about 1-50, 1-40, 1-30, 1-25, 1-20, 1-15, 1-10 (e.g., about 1, 2, 3 , 4, 5, 6, 7, 8, 9, or 10 to about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 40 or 50, or about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 , 30, 40 or 50, etc., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 dog, etc.) In some embodiments, the third subdomain is about 1-50, 1-40, 1-30, 1-25, 1-20, 1-15, 1-10 (e.g., about 1, 2, 3 , 4, 5, 6, 7, 8, 9, or 10 to about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 40 or 50, or about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 , 30, 40 or 50, etc., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 modified sugars, which are independently bicyclic sugars (eg, LNA sugars) or 2'-OR modified sugars (R is independently optionally substituted C _1-6 aliphatic). In some embodiments, the third subdomain is about 1-50, 1-40, 1-30, 1-25, 1-20, 1-15, 1-10 (e.g., about 1, 2, 3 , 4, 5, 6, 7, 8, 9, or 10 to about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 40 or 50, or about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 , 30, 40 or 50, etc., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 modified sugars, which are independently 2'-OR modified sugars (R is independently optionally substituted C _1-6 aliphatic). In some embodiments, the number is one. In some embodiments, the number is two. In some embodiments, the number is three. In some embodiments, the number is four. In some embodiments, the number is five. In some embodiments, the number is six. In some embodiments, the number is 7. In some embodiments, the number is eight. In some embodiments, the number is 9. In some embodiments, the number is 10. In some embodiments, the number is 11. In some embodiments, the number is 12. In some embodiments, the number is 13. In some embodiments, the number is 14. In some embodiments, the number is 15. In some embodiments, the number is 16. In some embodiments, the number is 17. In some embodiments, the number is 18. In some embodiments, the number is 19. In some embodiments, the number is 20. In some embodiments, R is methyl.

In some embodiments, the third subdomain is one or more (e.g., about 1-20, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11) comprising 2'-OH. , 12, 13, 14, 15, 16, 17, 18, 19, 20, etc.) sugars. In some embodiments, the third subdomain is one or more (e.g., about 1-20, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10) comprising two 2'-Hs. , 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, etc.) sugars. In some embodiments, the third subdomain is one or more (e.g., about 1-20, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, etc.) RNA sugars. In some embodiments, the third subdomain is one or more (e.g., about 1-20, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, etc.) DNA sugars.

In some embodiments, about 5%-100% (e.g., about 10%-100%, 20-100%, 30%-100%, 40%-100%, 50%) of all sugars in the third subdomain ~80%, 50%~85%, 50%~90%, 50%~95%, 60%~80%, 60%~85%, 60%~90%, 60%~95%, 60%~100 %, 65%~80%, 65%~85%, 65%~90%, 65%~95%, 65%~100%, 70%~80%, 70%~85%, 70%~90%, 70%~95%, 70%~100%, 75%~80%, 75%~85%, 75%~90%, 75%~95%, 75%~100%, 80%~85%, 80% ~90%, 80%~95%, 80%~100%, 85%~90%, 85%~95%, 85%~100%, 90%~95%, 90%~100%, 10%, 20 %, 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%, etc.) are independently modified sugars. In some embodiments, about 5%-100% (e.g., about 10%-100%, 20-100%, 30%-100%, 40%-100%, 50%) of all sugars in the third subdomain ~80%, 50%~85%, 50%~90%, 50%~95%, 60%~80%, 60%~85%, 60%~90%, 60%~95%, 60%~100 %, 65%~80%, 65%~85%, 65%~90%, 65%~95%, 65%~100%, 70%~80%, 70%~85%, 70%~90%, 70%~95%, 70%~100%, 75%~80%, 75%~85%, 75%~90%, 75%~95%, 75%~100%, 80%~85%, 80% ~90%, 80%~95%, 80%~100%, 85%~90%, 85%~95%, 85%~100%, 90%~95%, 90%~100%, 10%, 20 %, 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%, etc.) independently of the affected party (e.g., LNA). ) or a 2'-OR modified sugar (R is independently an optionally substituted C _1-6 aliphatic). In some embodiments, about 5%-100% (e.g., about 10%-100%, 20-100%, 30%-100%, 40%-100%, 50%) of all sugars in the third subdomain ~80%, 50%~85%, 50%~90%, 50%~95%, 60%~80%, 60%~85%, 60%~90%, 60%~95%, 60%~100 %, 65%~80%, 65%~85%, 65%~90%, 65%~95%, 65%~100%, 70%~80%, 70%~85%, 70%~90%, 70%~95%, 70%~100%, 75%~80%, 75%~85%, 75%~90%, 75%~95%, 75%~100%, 80%~85%, 80% ~90%, 80%~95%, 80%~100%, 85%~90%, 85%~95%, 85%~100%, 90%~95%, 90%~100%, 10%, 20 %, 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%, etc.) independently per 2'-OR variant ( R is independently optionally substituted C _1-6 aliphatic). In some embodiments, the percentage is at least about 50%. In some embodiments, the percentage is at least about 55%. In some embodiments, the percentage is at least about 60%. In some embodiments, the percentage is at least about 65%. In some embodiments, the percentage is at least about 70%. In some embodiments, the percentage is at least about 75%. In some embodiments, the percentage is at least about 80%. In some embodiments, the percentage is at least about 85%. In some embodiments, the percentage is at least about 90%. In some embodiments, the percentage is at least about 95%. In some embodiments, the percentage is about 100%. In some embodiments, R is methyl. In some embodiments, N _-2 comprises a 2'-OR modified sugar (R is not -H). In some embodiments, N _-3 comprises a 2'-F modified sugar. In some embodiments, each nucleoside following N _-3 independently comprises a 2'-OR modified sugar (R is not -H). In some embodiments, N _-3 comprises a 2'-F modified sugar and each nucleoside in the third subdomain independently comprises a 2'-OR modified sugar (R is not -H). In some embodiments, the 2'-OR modified sugars are independently 2'-OMe or 2'-MOE modified sugars. In some embodiments, a 2'-OR modified sugar is independently a 2'-OMe modified sugar. In some embodiments, each 2'-OR modified sugar is independently a 2'-OR modified sugar (R is an optionally substituted C _1-6 aliphatic) or a bicyclic sugar. In some embodiments, each 2'-OR modified sugar is independently a 2'-OR modified sugar, wherein R is an optionally substituted C _1-6 aliphatic. In some embodiments, each 2'-OR modified sugar is independently a 2'-OMe or 2'-MOE modified sugar. In some embodiments, each 2'-OR modified sugar is independently a 2'-OMe modified sugar.

In some embodiments, the third subdomain comprises about 1-50 (e.g., about 5, 6, 7, 8, 9, or 10 to about 10, 11) independently having non-2'-F modifications. , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 40 or 50, or about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 40 or 50, etc., about 1, 2, 3, 4, 5, 6 , 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, etc.) modified sugars. In some embodiments, between about 5% and 100% (e.g., between about 10%-100%, 20-100%, 30%-100%, 40%-100%, 50%-100%) of the sugars in the third subdomain. 80%, 50%~85%, 50%~90%, 50%~95%, 60%~80%, 60%~85%, 60%~90%, 60%~95%, 60%~100% , 65%~80%, 65%~85%, 65%~90%, 65%~95%, 65%~100%, 70%~80%, 70%~85%, 70%~90%, 70 %~95%, 70%~100%, 75%~80%, 75%~85%, 75%~90%, 75%~95%, 75%~100%, 80%~85%, 80%~ 90%, 80% to 95%, 80% to 100%, 85% to 90%, 85% to 95%, 85% to 100%, 90% to 95%, 90% to 100%, 10%, 20% , 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%) independently, non-2′-F variants This is a transforming party. In some embodiments, about 50%-100% (e.g., about 50%-80%, 50%-85%, 50%-90%, 50%-95%, 60%) of the sugars in the third subdomain ~80%, 60%~85%, 60%~90%, 60%~95%, 60%~100%, 65%~80%, 65%~85%, 65%~90%, 65%~95 %, 65%~100%, 70%~80%, 70%~85%, 70%~90%, 70%~95%, 70%~100%, 75%~80%, 75%~85%, 75%~90%, 75%~95%, 75%~100%, 80%~85%, 80%~90%, 80%~95%, 80%~100%, 85%~90%, 85% ~95%, 85%~100%, 90%~95%, 90%~100%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%, etc.) are independently modified sugars with modifications other than 2'-F. In some embodiments, each modified sugar in the third subdomain is independently a bicyclic sugar (eg, LNA sugar), an acyclic sugar (eg, UNA sugar), a sugar with a 2'-OR variant, or a 2'-N ( R) is selected from sugars with ₂ modifications (each R is independently an optionally substituted C _1-6 aliphatic).

In some embodiments, the third subdomain is a bicyclic sugar (eg, LNA sugar), an acyclic sugar (eg, UNA sugar), a sugar with a 2'-OR strain, or a 2'-N(R) ₂ strain. from about 1 to 50 (eg, about 5, 6, 7, 8, 9, or 10 to about 10) independently selected from sugars (each R is independently optionally substituted C _1-6 aliphatic) , 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 40 or 50, or about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 40 or 50, etc., about 1, 2, 3, 4, 5 , 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, etc.) modified sugars. In some embodiments, between about 5% and 100% (e.g., between about 10%-100%, 20-100%, 30%-100%, 40%-100%, 50%-100%) of the sugars in the third subdomain. 80%, 50%~85%, 50%~90%, 50%~95%, 60%~80%, 60%~85%, 60%~90%, 60%~95%, 60%~100% , 65%~80%, 65%~85%, 65%~90%, 65%~95%, 65%~100%, 70%~80%, 70%~85%, 70%~90%, 70 %~95%, 70%~100%, 75%~80%, 75%~85%, 75%~90%, 75%~95%, 75%~100%, 80%~85%, 80%~ 90%, 80% to 95%, 80% to 100%, 85% to 90%, 85% to 95%, 85% to 100%, 90% to 95%, 90% to 100%, 10%, 20% , 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%, etc.) independently of the affected party (e.g., LNA) , an acyclic sugar (eg UNA sugar), a sugar with a 2'-OR strain, or a sugar with a 2'-N(R) ₂ strain, where each R is independently an optionally substituted C _1-6 aliphatic It is a modified sugar selected from In some embodiments, about 50%-100% (e.g., about 50%-80%, 50%-85%, 50%-90%, 50%-95%, 60%) of the sugars in the third subdomain ~80%, 60%~85%, 60%~90%, 60%~95%, 60%~100%, 65%~80%, 65%~85%, 65%~90%, 65%~95 %, 65%~100%, 70%~80%, 70%~85%, 70%~90%, 70%~95%, 70%~100%, 75%~80%, 75%~85%, 75%~90%, 75%~95%, 75%~100%, 80%~85%, 80%~90%, 80%~95%, 80%~100%, 85%~90%, 85% ~95%, 85%~100%, 90%~95%, 90%~100%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%, etc.) are independently bicyclic (e.g., LNA sugars), acyclic sugars (e.g., UNA sugars), sugars with 2'-OR modifications, or sugars with 2'-N(R) ₂ modifications (each R of is independently a modified sugar selected from optionally substituted C _1-6 aliphatic.

In some embodiments, each sugar in the third subdomain independently comprises a 2'-OR variant (R is an optionally substituted C _1-6 aliphatic) or a 2'-OL ^B -4' variant. In some embodiments, each sugar in the third subdomain is independently a 2'-OR variant (R is an optionally substituted C _1-6 aliphatic) or a 2'-OL ^B -4' variant (L ^B is optionally substituted C 1-6 aliphatic). substituted -CH ₂ -). In some embodiments, each sugar in the third subdomain independently comprises 2'-OMe.

In some embodiments, the third subdomain is one or more (e.g., about 1-20, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, etc.) of 2'-F modified sugars. In some embodiments, the third subdomain does not contain a 2'-F modified sugar. In some embodiments, the third subdomain comprises one or more bicyclic sugars and/or 2'-OR modified sugars (R is not -H). In some embodiments, the level of bicyclic sugars and/or 2'-OR modified sugars (R is not -H), individually or in combination, is relatively high compared to 2'-F modified sugars. In some embodiments, up to about 1% to 95% (e.g., up to about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, etc.) include 2'-F. In some embodiments, up to about 50% of the sugars in the third subdomain include 2'-F. In some embodiments, the third subdomain comprises _one or more (eg, about 1-20, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, etc.) modified sugars. In some embodiments, the third subdomain comprises one or more (eg, about 1-20, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10) containing 2′-NH ₂ modifications. , 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, etc.) modified sugars. In some embodiments, the third subdomain is one or more (e.g., about 1-20, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, etc.) of heterocyclic sugars (eg, LNA sugars). In some embodiments, the third subdomain is one or more (e.g., about 1-20, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, etc.) of acyclic sugars (eg, UNA sugars).

In some embodiments, up to about 1% to 95% (e.g., up to about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, etc.) contain a 2'-MOE. In some embodiments, up to about 50% of the sugars in the third subdomain comprise a 2'-MOE. In some embodiments, the sugar in the third subdomain does not include a 2'-MOE.

In some embodiments, the third subdomain is about 1-50, 1-40, 1-30, 1-25, 1-20, 1-15, 1-10 (e.g., about 5, 6, 7 , 8, 9, or 10 to about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 40, or 50, or About 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 40 or 50, etc. , about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, etc.) modified internucleotide linkages includes In some embodiments, about 5%-100% (e.g., about 10%-100%, 20-100%, 30%-100%, 40%-100%, 50%~80%, 50%~85%, 50%~90%, 50%~95%, 60%~80%, 60%~85%, 60%~90%, 60%~95%, 60% ~100%, 65%~80%, 65%~85%, 65%~90%, 65%~95%, 65%~100%, 70%~80%, 70%~85%, 70%~90 %, 70%~95%, 70%~100%, 75%~80%, 75%~85%, 75%~90%, 75%~95%, 75%~100%, 80%~85%, 80%~90%, 80%~95%, 80%~100%, 85%~90%, 85%~95%, 85%~100%, 90%~95%, 90%~100%, 10% , 20%, 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%, etc.) are modified internucleotide linkages. In some embodiments, each internucleotide linkage in the third subdomain is independently a modified internucleotide linkage. In some embodiments, each modified internucleotide linkage is independently a chiral internucleotide linkage. In some embodiments, the modified or chiral internucleotidic linkage is a phosphorothioate internucleotidic linkage. In some embodiments, a modified or chiral internucleotidic linkage is a non-negatively charged internucleotidic linkage. In some embodiments, the modified or chiral internucleotide linkage is a neutral internucleotide linkage (eg n001). In some embodiments, each modified internucleotide linkage is independently a phosphorothioate internucleotide linkage or a non-negatively charged internucleotide linkage. In some embodiments, each modified internucleotide linkage is independently a phosphorothioate internucleotide linkage or a neutral internucleotide linkage. In some embodiments, each modified internucleotide linkage is independently a phosphorothioate internucleotide linkage. In some embodiments, at least about 1-50, 1-40, 1-30, 1-25, 1-20, 1-15, 1-10 (e.g., about 5, 6 , 7, 8, 9, or 10 to about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 40, or 50 , or about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 40 or 50 about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 chiral nucleotides, etc. Interconnections are chirally controlled. In some embodiments, at least 5%-100% (e.g., about 10%-100%, 20-100%, 30%-100%, 40%-100%) of the chiral internucleotide linkages in the third subdomain , 50%~80%, 50%~85%, 50%~90%, 50%~95%, 60%~80%, 60%~85%, 60%~90%, 60%~95%, 60 %~100%, 65%~80%, 65%~85%, 65%~90%, 65%~95%, 65%~100%, 70%~80%, 70%~85%, 70%~ 90%, 70% to 95%, 70% to 100%, 75% to 80%, 75% to 85%, 75% to 90%, 75% to 95%, 75% to 100%, 80% to 85% , 80%~90%, 80%~95%, 80%~100%, 85%~90%, 85%~95%, 85%~100%, 90%~95%, 90%~100%, 10 %, 20%, 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%, etc.) are chirally controlled. In some embodiments, at least 5%-100% (e.g., about 10%-100%, 20-100%, 30%-100%, 40%) of the phosphorothioate internucleotide linkages in the third subdomain ~100%, 50%~80%, 50%~85%, 50%~90%, 50%~95%, 60%~80%, 60%~85%, 60%~90%, 60%~95 %, 60%~100%, 65%~80%, 65%~85%, 65%~90%, 65%~95%, 65%~100%, 70%~80%, 70%~85%, 70% to 90%, 70% to 95%, 70% to 100%, 75% to 80%, 75% to 85%, 75% to 90%, 75% to 95%, 75% to 100%, 80% ~85%, 80%~90%, 80%~95%, 80%~100%, 85%~90%, 85%~95%, 85%~100%, 90%~95%, 90%~100 %, 10%, 20%, 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%, etc.) are chiral controlled. . In some embodiments, each is independently chirally controlled. In some embodiments, at least about 1-50, 1-40, 1-30, 1-25, 1-20, 1-15, 1-10 (e.g., about 5, 6 , 7, 8, 9, or 10 to about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 40, or 50 , or about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 40 or 50 about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 chiral nucleotides, etc. The connection between them is S p. In some embodiments, each is independently chirally controlled. In some embodiments, at least about 1-50, 1-40, 1-30, 1-25, 1-20, 1-15, 1-10 (e.g., about 5, 6 , 7, 8, 9, or 10 to about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 40, or 50 , or about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 40 or 50 about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, etc.) The thioate internucleotidic linkage is Sp . In some embodiments, at least 5%-100% (e.g., about 10%-100%, 20-100%, 30%-100%, 40%-100%) of the chiral internucleotide linkages in the third subdomain , 50%~80%, 50%~85%, 50%~90%, 50%~95%, 60%~80%, 60%~85%, 60%~90%, 60%~95%, 60 %~100%, 65%~80%, 65%~85%, 65%~90%, 65%~95%, 65%~100%, 70%~80%, 70%~85%, 70%~ 90%, 70% to 95%, 70% to 100%, 75% to 80%, 75% to 85%, 75% to 90%, 75% to 95%, 75% to 100%, 80% to 85% , 80%~90%, 80%~95%, 80%~100%, 85%~90%, 85%~95%, 85%~100%, 90%~95%, 90%~100%, 10 %, 20%, 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%, etc.) is Sp . In some embodiments, at least 5%-100% (e.g., about 10%-100%, 20-100%, 30%-100%, 40%) of the phosphorothioate internucleotide linkages in the third subdomain ~100%, 50%~80%, 50%~85%, 50%~90%, 50%~95%, 60%~80%, 60%~85%, 60%~90%, 60%~95 %, 60%~100%, 65%~80%, 65%~85%, 65%~90%, 65%~95%, 65%~100%, 70%~80%, 70%~85%, 70% to 90%, 70% to 95%, 70% to 100%, 75% to 80%, 75% to 85%, 75% to 90%, 75% to 95%, 75% to 100%, 80% ~85%, 80%~90%, 80%~95%, 80%~100%, 85%~90%, 85%~95%, 85%~100%, 90%~95%, 90%~100 %, 10%, 20%, 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%, etc.) is Sp . In some embodiments, the number is one or more. In some embodiments, the number is two or more. In some embodiments, the number is three or more. In some embodiments, the number is 4 or more. In some embodiments, the number is 5 or more. In some embodiments, the number is 6 or more. In some embodiments, the number is 7 or more. In some embodiments, the number is 8 or more. In some embodiments, the number is 9 or more. In some embodiments, the number is 10 or more. In some embodiments, the number is 11 or more. In some embodiments, the number is 12 or more. In some embodiments, the number is 13 or more. In some embodiments, the number is 14 or more. In some embodiments, the number is 15 or more. In some embodiments, the percentage is at least about 50%. In some embodiments, the percentage is at least about 55%. In some embodiments, the percentage is at least about 60%. In some embodiments, the percentage is at least about 65%. In some embodiments, the percentage is at least about 70%. In some embodiments, the percentage is at least about 75%. In some embodiments, the percentage is at least about 80%. In some embodiments, the percentage is at least about 85%. In some embodiments, the percentage is at least about 90%. In some embodiments, the percentage is at least about 95%. In some embodiments, the percentage is about 100%. In some embodiments, each internucleotide linkage linking two third subdomain nucleosides is independently a modified internucleotide linkage. In some embodiments, each modified internucleotide linkage is independently a chiral internucleotide linkage. In some embodiments, each modified internucleotide linkage is independently a phosphorothioate internucleotide linkage. In some embodiments, each chiral internucleotidic linkage is independently a phosphorothioate internucleotidic linkage. In some embodiments, each modified internucleotide linkage is independently an Sp chiral internucleotide linkage. In some embodiments, each modified internucleotide linkage is independently an S p phosphorothioate internucleotide linkage. In some embodiments, each chiral internucleotidic linkage is independently an S p phosphorothioate internucleotidic linkage. In some embodiments, the internucleotide linkages of the third subdomain are linked to two nucleosides of the third subdomain. In some embodiments, an internucleotide linkage between a nucleoside of the third subdomain and a nucleoside of the second subdomain may properly be considered an internucleotide linkage of the third subdomain. In some embodiments, the internucleotide linkage linked to the nucleoside of the third subdomain and the nucleoside of the second subdomain is a modified internucleotide linkage, in some embodiments, it is a chiral internucleotide linkage, and in some embodiments , it is chirally controlled, in some embodiments it is R p , and in some embodiments it is Sp .

In some embodiments, the third subdomain comprises some level of R p internucleotidic linkages. In some embodiments, the level is, for example, between about 5% and 100%, about 10% and 100%, 20 and 100%, 30% and 100%, 40% and 100% of all internucleotide linkages in the third subdomain. %, 50%~80%, 50%~85%, 50%~90%, 50%~95%, 60%~80%, 60%~85%, 60%~90%, 60%~95%, 60%~100%, 65%~80%, 65%~85%, 65%~90%, 65%~95%, 65%~100%, 70%~80%, 70%~85%, 70% ~90%, 70%~95%, 70%~100%, 75%~80%, 75%~85%, 75%~90%, 75%~95%, 75%~100%, 80%~85 %, 80%~90%, 80%~95%, 80%~100%, 85%~90%, 85%~95%, 85%~100%, 90%~95%, 90%~100%, 10%, 20%, 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%. In some embodiments, the level is between about 5% and 100%, about 10% and 100%, 20 and 100%, 30% and 100%, 40% and 40% of all chiral internucleotide linkages in the third subdomain, for example. 100%, 50%~80%, 50%~85%, 50%~90%, 50%~95%, 60%~80%, 60%~85%, 60%~90%, 60%~95% , 60%~100%, 65%~80%, 65%~85%, 65%~90%, 65%~95%, 65%~100%, 70%~80%, 70%~85%, 70 %~90%, 70%~95%, 70%~100%, 75%~80%, 75%~85%, 75%~90%, 75%~95%, 75%~100%, 80%~ 85%, 80% to 90%, 80% to 95%, 80% to 100%, 85% to 90%, 85% to 95%, 85% to 100%, 90% to 95%, 90% to 100% , 10%, 20%, 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%. In some embodiments, the level is, for example, between about 5% and 100%, about 10% and 100%, 20 and 100%, 30% and 100%, 40% of all chirally controlled internucleotide linkages in the third subdomain. ~100%, 50%~80%, 50%~85%, 50%~90%, 50%~95%, 60%~80%, 60%~85%, 60%~90%, 60%~95 %, 60%~100%, 65%~80%, 65%~85%, 65%~90%, 65%~95%, 65%~100%, 70%~80%, 70%~85%, 70% to 90%, 70% to 95%, 70% to 100%, 75% to 80%, 75% to 85%, 75% to 90%, 75% to 95%, 75% to 100%, 80% ~85%, 80%~90%, 80%~95%, 80%~100%, 85%~90%, 85%~95%, 85%~100%, 90%~95%, 90%~100 %, 10%, 20%, 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%. In some embodiments, the percentage is about or up to about 50%. In some embodiments, the percentage is at least about 55%. In some embodiments, the percentage is at least about 60%. In some embodiments, the percentage is at least about 65%. In some embodiments, the percentage is at least about 70%. In some embodiments, the percentage is at least about 75%. In some embodiments, the percentage is at least about 80%. In some embodiments, the percentage is at least about 85%. In some embodiments, the percentage is at least about 90%. In some embodiments, the percentage is at least about 95%. In some embodiments, the percentage is about 100%. In some embodiments, the percentage is about or up to about 5%. In some embodiments, the percentage is about or up to about 10%. In some embodiments, the percentage is about or up to about 15%. In some embodiments, the percentage is about or up to about 20%. In some embodiments, the percentage is about or up to about 25%. In some embodiments, the percentage is about or up to about 30%. In some embodiments, the percentage is about or up to about 35%. In some embodiments, the percentage is about or up to about 40%. In some embodiments, the percentage is about or up to about 45%. In some embodiments, the percentage is about or up to about 50%. In some embodiments, about 1-50, 1-40, 1-30, 1-25, 1-20, 1-15, 1-10, 1-5, for example about 1, 2, 3, 4 , 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 internucleotide linkages are independently R p chiral internucleotide linkages. In some embodiments, the number is about or up to about 1. In some embodiments, the number is about or up to about 2. In some embodiments, the number is about or up to about 3. In some embodiments, the number is about or up to about 4. In some embodiments, the number is about or up to about 5. In some embodiments, the number is about or up to about 6. In some embodiments, the number is about or up to about 7. In some embodiments, the number is about or up to about 8. In some embodiments, the number is about or up to about 9. In some embodiments, the number is about or up to about 10.

In some embodiments, each phosphorothioate internucleotidic linkage in the third subdomain is independently chirally controlled. In some embodiments, each is independently Sp or R p. In some embodiments, the high level is Sp as described herein. In some embodiments, each phosphorothioate internucleotidic linkage in the third subdomain is chirally controlled and is Sp . In some embodiments, one or more, for example about 1-5 (eg, about 1, 2, 3, 4, or 5) is R p .

In some embodiments, as exemplified in certain examples, the third subdomain comprises one or more non-negatively charged internucleotide linkages, each optionally and independently chirally controlled. In some embodiments, each non-negatively charged internucleotide linkage is independently n001. In some embodiments, non-negatively charged chiral internucleotidic linkages are not chirally controlled. In some embodiments, each chiral internucleotidic linkage that is not negatively charged is not chirally controlled. In some embodiments, non-negatively charged chiral internucleotidic linkages are chirally controlled. In some embodiments, non-negatively charged chiral internucleotidic linkages are chirally controlled and R p . In some embodiments, the non-negatively charged chiral internucleotidic linkages are chirally controlled and are Sp . In some embodiments, each non-negatively charged chiral internucleotidic linkage is chirally controlled. In some embodiments, the number of non-negatively charged internucleotide linkages in the third subdomain is about 1-10, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 It is a dog. In some embodiments, the number is about 1. In some embodiments, the number is about two. In some embodiments, the number is about 3. In some embodiments, the number is about 4. In some embodiments, the number is about 5. In some embodiments, linkages between two or more nucleotides that are not negatively charged are contiguous. In some embodiments, linkages between two non-negatively charged nucleotides are not contiguous. In some embodiments, all non-negatively charged internucleotide linkages in the third subdomain are contiguous (eg, three consecutive non-negatively charged internucleotidic linkages). In some embodiments, the non-negatively charged internucleotidic linkages, or two or more (e.g., about 2, about 3, about 4, etc.) consecutive non-negatively charged internucleotidic linkages are formed in a third at the 3'-end of the subdomain. In some embodiments, the last two or three or four internucleotide linkages of the third subdomain comprise at least one internucleotide linkage that is not a negatively charged internucleotide linkage. In some embodiments, the last 2 or 3 or 4 internucleotide linkages of the third subdomain comprise at least one internucleotide linkage other than n001. In some embodiments, the internucleoside linkage connecting the last two nucleosides of the third subdomain is a non-negatively charged internucleoside linkage. In some embodiments, the internucleoside linkage connecting the last two nucleosides of the third subdomain is a non-negatively charged S p internucleoside linkage. In some embodiments, the internucleoside linkage connecting the last two nucleosides of the third subdomain is a non-negatively charged R p internucleoside linkage. In some embodiments, the internucleoside linkage connecting the last two nucleosides of the third subdomain is a phosphorothioate internucleoside linkage. In some embodiments, the internucleoside linkage connecting the last two nucleosides of the third subdomain is an S p phosphorothioate internucleoside linkage. In some embodiments, the last two nucleosides of the third subdomain are the last two nucleosides of the second domain. In some embodiments, the last two nucleosides of the third subdomain are the last two nucleosides of the oligonucleotide. In some embodiments, the internucleoside linkage connecting the first two nucleosides of the third subdomain is a non-negatively charged internucleoside linkage. In some embodiments, the internucleoside linkage connecting the first two nucleosides of the third subdomain is a non-negatively charged S p internucleoside linkage. In some embodiments, the internucleoside linkage connecting the first two nucleosides of the third subdomain is a non-negatively charged R p internucleoside linkage. In some embodiments, the internucleoside linkage connecting the first two nucleosides of the third subdomain is a phosphorothioate internucleoside linkage. In some embodiments, the internucleoside linkage connecting the first two nucleosides of the third subdomain is an S p phosphorothioate internucleoside linkage. In some embodiments, the non-negatively charged internucleotidic linkage is a neutral internucleotidic linkage, such as n001. In some embodiments, it is chirally controlled and R p . In some embodiments, the last and/or penultimate internucleotide linkage of an oligonucleotide is a non-negatively charged internucleotide linkage, such as a phosphoryl guanidine internucleotide linkage, such as n001. In some embodiments, it is chirally controlled and R p .

In some embodiments, the third subdomain comprises one or more (eg, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10) natural phosphate linkages. In some embodiments, the third subdomain does not contain native phosphate linkages. In some embodiments, the internucleotide linkages linked to N _-2 and N _-3 are natural phosphate linkages. In some embodiments, the sugar of N _-3 is a 2'-F modified sugar and the sugar of N _-2 is a 2'-OR modified sugar (R is not -H) (eg, a 2'-OMe modified sugar) am. In some embodiments, one of all internucleotide linkages linked to two nucleosides of the third subdomain is a natural phosphate linkage (e.g., between N _-2 and N _-3 as described herein), and one is a negatively charged R p internucleotidic linkage (e.g., the last or penultimate internucleotide linkage of an oligonucleotide), such as a phosphorylguanidine internucleotide linkage n001, all others are Sp phosphorothioate nucleotides is a connection between

In some embodiments, the third subdomain is at the 5'-end, e.g., about 1-20, 1-15, 1-10, 1-8, 1-5, 1-3, 3-8, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleobases in length. In some embodiments, the 5'-end portion is about 1-3 nucleobases in length. In some embodiments, the length is 1 nucleobase. In some embodiments, the length is 2 nucleobases. In some embodiments, the length is 3 nucleobases. In some embodiments, the length is 4 nucleobases. In some embodiments, the length is 5 nucleobases. In some embodiments, the length is 6 nucleobases. In some embodiments, the length is 7 nucleobases. In some embodiments, the length is 8 nucleobases. In some embodiments, the length is 9 nucleobases. In some embodiments, the length is 10 nucleobases. In some embodiments, the 5'-terminal portion comprises the 5'-terminal nucleobase of the third subdomain. In some embodiments, the third subdomain comprises or consists of a 3'-end and a 5'-end. In some embodiments, the 5'-terminal portion comprises the 5'-terminal nucleobase of the third subdomain. In some embodiments, the 5′-end of the third subdomain is joined to the second subdomain.

In some embodiments, the 5'-end portion includes one or more sugars with two 2'-Hs (eg, natural DNA sugars). In some embodiments, the 5'-end portion includes one or more sugars with 2'-OH (eg, native RNA sugars). In some embodiments, one or more (e.g., about 1-20, 1-15, 1-10, 3-8, or about 1, 2, 3, 4, 5, 6) sugars at the 5'-end. , 7, 8, 9, or 10) are independently modified sugars. In some embodiments, between about 5% and 100% (e.g., between about 10% and 100%, 20 and 100%, 30% and 100%, 40% and 100%, 50% and 80%, 50%~85%, 50%~90%, 50%~95%, 60%~80%, 60%~85%, 60%~90%, 60%~95%, 60%~100% , 65%~80%, 65%~85%, 65%~90%, 65%~95%, 65%~100%, 70%~80%, 70%~85%, 70%~90%, 70 %~95%, 70%~100%, 75%~80%, 75%~85%, 75%~90%, 75%~95%, 75%~100%, 80%~85%, 80%~ 90%, 80% to 95%, 80% to 100%, 85% to 90%, 85% to 95%, 85% to 100%, 90% to 95%, 90% to 100%, 10%, 20% , 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%, etc.) are independently modified sugars. In some embodiments, each sugar is independently a modified sugar. In some embodiments, the modified sugar is independently a bicyclic sugar (eg, LNA sugar), an acyclic sugar (eg, UNA sugar), a sugar with a 2'-OR strain, or a 2'-N(R) ₂ strain. sugar (each R is independently optionally substituted C _1-6 aliphatic).

In some embodiments, one or more of the modified sugars independently comprises 2'-F or 2'-OR (R is independently optionally substituted C _1-6 aliphatic). In some embodiments, one or more of the modified sugars are independently 2'-F or 2'-OMe. In some embodiments, each modified sugar at the 5'-end is independently a bicyclic sugar (eg, an LNA sugar) or a sugar with a 2'-OR variant (R is an optionally substituted C _1-6 aliphatic). In some embodiments, each modified sugar at the 5'-end is independently a bicyclic sugar (eg, an LNA sugar) or a sugar with a 2'-OR variant (R is an optionally substituted C _1-6 aliphatic). In some embodiments, each modified sugar at the 5'-end is independently a sugar with a 2'-OR variant (R is an optionally substituted C _1-6 aliphatic). In some embodiments, R is methyl.

In some embodiments, relative to the 3'-end portion, the 5'-end portion has a higher level (number and/or percentage) of a 2'-F modification sugar and/or a sugar comprising two 2'-Hs (e.g., per native DNA), and/or to a lower level (number and/or percentage) of other types of modifications, such as bicyclic sugars and/or sugars with 2'-OR modifications (R is independently optionally substituted C _1-6 are aliphatic). In some embodiments, relative to the 3'-end portion, the 5'-end portion per higher level of 2'-F modification and/or lower level of 2'-OR modification (R is optionally substituted C _1-6 are aliphatic). In some embodiments, compared to the 3'-end portion, the 5'-end portion comprises higher levels of 2'-F modified sugars and/or lower levels of 2'-OMe modified sugars. In some embodiments, compared to the 3'-end, the 5'-end has higher levels of natural DNA sugars and/or lower levels of 2'-OR modified sugars, where R is optionally substituted C _1-6 aliphatic. includes In some embodiments, compared to the 3'-end, the 5'-end comprises higher levels of natural DNA sugars and/or lower levels of 2'-OMe modified sugars. In some embodiments, the 5'-terminus is a bicyclic sugar or a sugar comprising a 2'-OR, where R is an optionally substituted C _1-6 aliphatic (eg, methyl), to a low level (eg, up to 50%). , 40%, 30%, 25%, 20%, or 10%, or up to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) modified sugars. In some embodiments, the 5′-end portion does not include a modified sugar that is a bicyclic sugar or a sugar comprising a 2′-OR, where R is an optionally substituted C _1-6 aliphatic (eg, methyl).

In some embodiments, one or more modified sugars independently include 2'-F. In some embodiments, the modified sugar does not include 2'-OMe or other 2'-OR modifications (R is an optionally substituted C _1-6 aliphatic). In some embodiments, each sugar at the 5'-end independently comprises two 2'-H or 2'-F modifications. In some embodiments, the 5'-end portion comprises 1, 2, 3, 4, or 5 2'-F modified sugars. In some embodiments, the 5'-end portion contains 1-3 2'-F modified sugars. In some embodiments, the 5'-end portion comprises 1, 2, 3, 4, or 5 natural DNA sugars. In some embodiments, the 5'-end comprises 1-3 natural DNA sugars.

In some embodiments, between one or more (e.g., about 1-10, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) nucleotides of the 5'-end. Linkages are independently modified internucleotide linkages. In some embodiments, between one or more (e.g., about 1-10, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) nucleotides of the 5'-end. Linkages are independently chiral internucleotidic linkages. In some embodiments, between one or more (e.g., about 1-10, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) nucleotides of the 5'-end. Linkages are independently chirally controlled internucleotidic linkages. In some embodiments, between one or more (e.g., about 1-10, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) nucleotides of the 5'-end. The connection is R p. In some embodiments, between one or more (e.g., about 1-10, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) nucleotides of the 5'-end. The connection is Sp . In some embodiments, each internucleotide linkage at the 5'-end is Sp . In some embodiments, the 5'-end portion comprises a higher level (number and/or percentage) of R p internucleotidic linkages and/or native phosphate linkages relative to the 3'-end portion.

In some embodiments, the 5'-end is one or more (e.g., about 1-10, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) as described herein. ), including inconsistency. In some embodiments, the 5'-end is one or more (e.g., about 1-10, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) as described herein. ) of wobbles. In some embodiments, the 5'-end is about 60-100% (eg, 66%, 70%, 75%, 80%, 85%, 90%, 95% or more) complementary to the target nucleic acid. In some embodiments, complementarity is greater than 60%. In some embodiments, complementarity is greater than 70%. In some embodiments, complementarity is greater than 75%. In some embodiments, complementarity is greater than 80%. In some embodiments, complementarity is greater than 90%.

In some embodiments, the third subdomain is 3'-end, e.g., about 1-20, 1-15, 1-10, 1-8, 1-4, 3-8, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleobases in length. In some embodiments, the 3'-end portion is about 3-6 nucleobases in length. In some embodiments, the length is 1 nucleobase. In some embodiments, the length is 2 nucleobases. In some embodiments, the length is 3 nucleobases. In some embodiments, the length is 4 nucleobases. In some embodiments, the length is 5 nucleobases. In some embodiments, the length is 6 nucleobases. In some embodiments, the length is 7 nucleobases. In some embodiments, the length is 8 nucleobases. In some embodiments, the length is 9 nucleobases. In some embodiments, the length is 10 nucleobases. In some embodiments, the 3'-terminal portion comprises the 3'-terminal nucleobase of the third subdomain.

In some embodiments, the 3'-end portion includes one or more sugars with two 2'-Hs (eg, natural DNA sugars). In some embodiments, the 3'-end portion includes one or more sugars with 2'-OH (eg, native RNA sugars). In some embodiments, one or more (e.g., about 1-20, 1-15, 1-10, 3-8, or about 1, 2, 3, 4, 5, 6) sugars at the 3'-terminus. , 7, 8, 9, or 10) are independently modified sugars. In some embodiments, about 5%-100% (e.g., about 10%-100%, 20-100%, 30%-100%, 40%-100%, 50%-100%) of the sugar at the 3'-end. 80%, 50%~85%, 50%~90%, 50%~95%, 60%~80%, 60%~85%, 60%~90%, 60%~95%, 60%~100% , 65%~80%, 65%~85%, 65%~90%, 65%~95%, 65%~100%, 70%~80%, 70%~85%, 70%~90%, 70 %~95%, 70%~100%, 75%~80%, 75%~85%, 75%~90%, 75%~95%, 75%~100%, 80%~85%, 80%~ 90%, 80% to 95%, 80% to 100%, 85% to 90%, 85% to 95%, 85% to 100%, 90% to 95%, 90% to 100%, 10%, 20% , 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%, etc.) are independently modified sugars. In some embodiments, each sugar is independently a modified sugar. In some embodiments, the modified sugar is independently a bicyclic sugar (eg, LNA sugar), an acyclic sugar (eg, UNA sugar), a sugar with a 2'-OR strain, or a 2'-N(R) ₂ strain. sugar (each R is independently optionally substituted C _1-6 aliphatic).

In some embodiments, one or more of the modified sugars independently comprises 2'-F or 2'-OR (R is independently optionally substituted C _1-6 aliphatic). In some embodiments, one or more of the modified sugars are independently 2'-F or 2'-OMe. In some embodiments, each modified sugar at the 3'-end is independently a bicyclic sugar (eg, an LNA sugar) or a sugar with a 2'-OR variant (R is an optionally substituted C _1-6 aliphatic). In some embodiments, each modified sugar at the 3'-end is independently a bicyclic sugar (eg, an LNA sugar) or a sugar with a 2'-OR variant (R is an optionally substituted C _1-6 aliphatic). In some embodiments, each modified sugar at the 3'-end is independently a sugar with a 2'-OR variant (R is an optionally substituted C _1-6 aliphatic). In some embodiments, R is methyl.

In some embodiments, one or more sugars at the 3'-end independently comprise a 2'-OR variant (R is an optionally substituted C _1-6 aliphatic) or a 2'-OL ^B -4' variant. In some embodiments, each sugar at the 3'-end independently comprises a 2'-OR strain (R is an optionally substituted C _1-6 aliphatic) or a 2'-OL ^B -4' strain. In some embodiments, L ^B is optionally substituted -CH ₂ -. In some embodiments, L ^B is -CH ₂ -. In some embodiments, each sugar at the 3'-end independently comprises 2'-OMe.

In some embodiments, between one or more (e.g., about 1-10, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) nucleotides of the 3'-end. Linkages are independently modified internucleotide linkages. In some embodiments, between one or more (e.g., about 1-10, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) nucleotides of the 3'-end. Linkages are independently chiral internucleotidic linkages. In some embodiments, between one or more (e.g., about 1-10, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) nucleotides of the 3'-end. Linkages are independently chirally controlled internucleotidic linkages. In some embodiments, between one or more (e.g., about 1-10, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) nucleotides of the 3'-end. The connection is R p. In some embodiments, between one or more (e.g., about 1-10, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) nucleotides of the 3'-end. The connection is Sp . In some embodiments, each internucleotide linkage at the 3'-end is Sp .

In some embodiments, the 3'-end is one or more (e.g., about 1-10, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) as described herein. ), including inconsistency. In some embodiments, the 3'-end is one or more (e.g., about 1-10, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) as described herein. ) of wobbles. In some embodiments, the 3'-end is about 60-100% (eg, 66%, 70%, 75%, 80%, 85%, 90%, 95% or more) complementary to the target nucleic acid. In some embodiments, complementarity is greater than 60%. In some embodiments, complementarity is greater than 70%. In some embodiments, complementarity is greater than 75%. In some embodiments, complementarity is greater than 80%. In some embodiments, complementarity is greater than 90%.

In some embodiments, the third subdomain enables, facilitates, or contributes to recruitment of a protein, such as an ADAR protein, eg, ADAR1, ADAR2, etc.). In some embodiments, the third subdomain supplements, promotes, or contributes to an interaction with a protein, such as an ADAR protein. In some embodiments, the third subdomain contacts the RNA binding domain (RBD) of an ADAR. In some embodiments, the third subdomain contacts the catalytic domain of an ADAR having deaminase activity. In some embodiments, the third subdomain contacts the domain having deaminase activity of ADAR1. In some embodiments, the third subdomain contacts the domain having deaminase activity of ADAR2. In some embodiments, the various nucleobases, sugars and/or internucleotide linkages of the third subdomain can interact with one or more residues of a protein (eg, an ADAR protein).

As shown herein, chiral control of linkages of chiral internucleotidic linkages in oligonucleotides can be used to provide various properties and/or activities. In some embodiments, R p internucleotidic linkages (e.g., R p phosphorothioate internucleotide linkages, S p internucleotide linkages (e.g., R p phosphorothioate internucleotide linkages), or non-chiral Controlled internucleotidic linkages (e.g., non-chiral controlled phosphorothioate internucleotide linkages) are -8, -7, -6, -5, -4, -3, -2 of the nucleoside opposite the target adenosine. , -1, +1, +2, +3, +4, +5, +6, +7, and +8 positions ("+" is the 5'- of the oligonucleotide in the nucleoside) Counting towards the end, the internucleotide linkage at position +1 is an internucleotide linkage between the nucleoside opposite the target adenosine and its 5' adjacent nucleoside (e.g., at the 5'-carbon of the nucleoside opposite the target adenosine). a linked internucleotidic linkage, or between N _{1 and N 0 of} 5′-N ₁ N ₀ N _-1 -3′, where N ₀ is the nucleoside opposite the target adenosine, as described herein; "-" counts toward the 3'-end of the oligonucleotide in the nucleoside, and the internucleotide linkage at position -1 is an internucleotide linkage between the nucleoside opposite the target adenosine and its 3'-side adjacent nucleoside (e.g. For example, _{an internucleotide linkage attached to the 3'-carbon of the nucleoside opposite the target adenosine, or between N -1 and N 0 of} 5'-N ₁ N ₀ N _-1 -3' (wherein as described herein , N ₀ is the nucleoside opposite the target adenosine). In some embodiments, the R p internucleoside linkage (eg, the R p phosphorothioate internucleoside linkage) is the nucleoside opposite the target adenosine. Positions -8, -7, -6, -5, -4, -3, -2, -1, +1, +2, +3, +4, +5, +6, +7, and +8 In some embodiments, the R p internucleotide linkage (eg, the R p phosphorothioate internucleotide linkage) is -2, -1, +3, + of the nucleoside opposite the target adenosine. at one or more of positions 4, +5, +6, +7, and +8. In some embodiments, the S p internucleotidic linkage (eg, the S p phosphorothioate internucleotide linkage) is -8, -7, -6, -5, -4, - of the nucleoside opposite the target adenosine. at one or more of positions 3, -2, -1, +1, +2, +3, +4, +5, +6, +7, and +8. In some embodiments, the S p internucleotidic linkage (eg, the S p phosphorothioate internucleotide linkage) is -2, -1, +3, +4, +5, + of the nucleoside opposite the target adenosine. at one or more of positions 6, +7, and +8. In some embodiments, the non-chirally controlled internucleotidic linkage (eg, the non-chirally controlled phosphorothioate internucleotidic linkage) is -8, -7, -6, -5, -8, -7, -6, -5, at one or more of positions -4, -3, -2, -1, +1, +2, +3, +4, +5, +6, +7, and +8. In some embodiments, a non-chirally controlled internucleotide linkage (e.g., a non-chirally controlled phosphorothioate internucleotide linkage) is -2, -1, +3, +4, -2, -1, +3, +4, It is at one or more of positions +5, +6, +7, and +8.

In some embodiments, Rp is at position +8. In some embodiments, Rp is at position +7. In some embodiments, Rp is at position -6. In some embodiments, Rp is at position +5. In some embodiments, Rp is at position +4. In some embodiments, Rp is at position +3. In some embodiments, Rp is at position +2. In some embodiments, Rp is at position +1. In some embodiments, Rp is at position -1. In some embodiments, Rp is at position -2. In some embodiments, Rp is at position -3. In some embodiments, Rp is at position -4. In some embodiments, Rp is at position -5. In some embodiments, Rp is at position -6. In some embodiments, Rp is at position -7. In some embodiments, Rp is at position -8. In some embodiments, Rp is a sequence of chirally controlled phosphorothioate internucleotidic linkages. In some embodiments, Sp is at position +8. In some embodiments, Sp is at position +7. In some embodiments, Sp is at position -6. In some embodiments, Sp is at position +5. In some embodiments, Sp is at position +4. In some embodiments, Sp is at position +3. In some embodiments, Sp is at position +2. In some embodiments, Sp is at position +1. In some embodiments, Sp is at position -1. In some embodiments, Sp is at position -2. In some embodiments, Sp is at position -3. In some embodiments, Sp is at position -4. In some embodiments, Sp is at position -5. In some embodiments, Sp is at position -6. In some embodiments, Sp is at position -7. In some embodiments, Sp is at position -8. In some embodiments, Sp is a sequence of chirally controlled phosphorothioate internucleotidic linkages. In some embodiments, the non-chiral control internucleotidic linkage is at position +8. In some embodiments, the non-chiral control internucleotidic linkage is at position +7. In some embodiments, the non-chiral control internucleotidic linkage is at position -6. In some embodiments, the non-chiral control internucleotidic linkage is at position +5. In some embodiments, the non-chiral control internucleotidic linkage is at position +4. In some embodiments, the non-chiral control internucleotidic linkage is at position +3. In some embodiments, the non-chiral control internucleotidic linkage is at position +2. In some embodiments, the non-chiral control internucleotidic linkage is at position +1. In some embodiments, the non-chiral control internucleotidic linkage is at position -1. In some embodiments, the non-chiral control internucleotidic linkage is at position -2. In some embodiments, the non-chiral control internucleotidic linkage is at position -3. In some embodiments, the non-chiral control internucleotidic linkage is at position -4. In some embodiments, the non-chiral control internucleotidic linkage is at position -5. In some embodiments, the non-chiral control internucleotidic linkage is at position -6. In some embodiments, the non-chiral control internucleotidic linkage is at position -7. In some embodiments, the non-chiral control internucleotidic linkage is at position -8. In some embodiments, the non-chirally controlled internucleotidic linkage is a non-chirally controlled phosphorothioate internucleotidic linkage.

In some embodiments, the first domain comprises one or more (eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) R p internucleotide linkages (eg, R p phosphorothioate internucleotidic linkages). In some embodiments, the first domain comprises one or more (eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) S p internucleotide linkages (eg, S p phosphorothioate internucleotidic linkages). In some embodiments, the first domain comprises one or more (eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) non-chiral controlled internucleotide linkages (eg, , non-chirally controlled phosphorothioate internucleotidic linkages). In some embodiments, these internucleotidic linkages are contiguous. In some embodiments, at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%, or all of the internucleotide linkages in the first domain are It is chirally controlled and is Sp . In some embodiments, at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of the phosphorothioate internucleotide linkages in the first domain. , or all are chirally controlled and are Sp . In some embodiments, the second domain comprises one or more (eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) R p internucleotide linkages (eg, R p phosphorothioate internucleotidic linkages). In some embodiments, the second domain comprises one or more (eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) S p internucleotide linkages (eg, S p phosphorothioate internucleotidic linkages). In some embodiments, the second domain comprises one or more (eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) non-chiral controlled internucleotide linkages (eg, , non-chirally controlled phosphorothioate internucleotidic linkages). In some embodiments, these internucleotidic linkages are contiguous. In some embodiments, at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%, or all of the internucleotide linkages in the second domain are It is chirally controlled and is Sp . In some embodiments, at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of the phosphorothioate internucleotide linkages in the second domain. , or all are chirally controlled and are Sp . In some embodiments, the first subdomain comprises one or more (eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) R p internucleotide linkages (eg, R p phosphorothioate internucleotidic linkages). In some embodiments, the first subdomain comprises one or more (eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) Sp internucleotide linkages (eg, Sp phosphorothioate internucleotidic linkages). In some embodiments, the first subdomain comprises one or more (eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) non-chiral controlled internucleotide linkages (eg, eg, non-chirally controlled phosphorothioate internucleotidic linkages). In some embodiments, these internucleotidic linkages are contiguous. In some embodiments, this internucleotide linkage is at the 3'-end of the first subdomain.

In some embodiments, one or more natural phosphate linkages are used in provided oligonucleotides and compositions thereof. In some embodiments, a provided oligonucleotide or portion thereof (eg, a first domain, a second domain, a first subdomain, a second subdomain, a third subdomain, etc.) is one or more (eg, about or at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, or 50 or more) natural phosphate linkages. In some embodiments, a provided oligonucleotide or portion thereof (e.g., a first domain, a second domain, a first subdomain, a second subdomain, a third subdomain, etc.) includes two or more (e.g., about or at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 , 26, 27, 28, 29, 30, 35, 40, 45, or 50 or more) consecutive natural phosphate linkages. In some embodiments, a provided oligonucleotide or portion thereof (eg, a first domain, a second domain, a first subdomain, a second subdomain, a third subdomain, etc.) is at most about 1, 2, 3, 4 , 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 , 30, 35, 40, 45, or 50 natural phosphate linkages. In some embodiments, a provided oligonucleotide or portion thereof (e.g., a first domain, a second domain, a first subdomain, a second subdomain, a third subdomain, etc.) is at most 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, or 50 consecutive natural phosphate linkages. In some embodiments, about or at least about 5%, 10% in a provided oligonucleotide or portion thereof (e.g., a first domain, a second domain, a first subdomain, a second subdomain, a third subdomain, etc.) %, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%, or all internucleotide linkages are natural phosphate linkages. In some embodiments, about or at least about 5%, 10% in a provided oligonucleotide or portion thereof (e.g., a first domain, a second domain, a first subdomain, a second subdomain, a third subdomain, etc.) %, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%, or all, of the internucleotide linkages are not natural phosphate linkages. In some embodiments, about or at least about 5%, 10% in a provided oligonucleotide or portion thereof (e.g., a first domain, a second domain, a first subdomain, a second subdomain, a third subdomain, etc.) %, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%, or all, of the internucleotide linkages are not contiguous natural phosphate linkages.

In some embodiments, a provided oligonucleotide or portion thereof comprises one or more natural phosphate linkages and one or more modified internucleotide linkages. In some embodiments, a provided oligonucleotide or portion thereof comprises one or more natural phosphate linkages and one or more chirally controlled modified internucleotidic linkages. In some embodiments, a provided oligonucleotide or portion thereof (eg, a first domain, a second domain, a first subdomain, a second subdomain, a third subdomain, etc.) is at most about 1, 2, 3, 4 , 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 , 30, 35, 40, 45, or 50 natural phosphate linkages, each linkage being independently two sugars that do not contain a 2'-OR modification (R as described herein but not -H) join in In some embodiments, a provided oligonucleotide or portion thereof (e.g., a first domain, a second domain, a first subdomain, a second subdomain, a third subdomain, etc.) is at most 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, or 50 contiguous natural phosphate linkages, each linkage independently consisting of two sugars containing no 2'-OR modifications (R as described herein but not -H) combine In some embodiments, a provided oligonucleotide or portion thereof (eg, a first domain, a second domain, a first subdomain, a second subdomain, a third subdomain, etc.) is at most about 1, 2, 3, 4 , 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 , 30, 35, 40, 45, or 50 native phosphate linkages, each linkage independently binding to two 2'-F modified sugars. In some embodiments, a provided oligonucleotide or portion thereof (e.g., a first domain, a second domain, a first subdomain, a second subdomain, a third subdomain, etc.) is at most 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, It contains 35, 40, 45, or 50 consecutive natural phosphate linkages, each linkage independently binding to two 2'-F modified sugars. In some embodiments, a 2'-OR modification (R is herein up to about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15; 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, or 50, e.g. up to 2, up to 3 , up to 4, up to 5, etc. internucleotide linkages are natural phosphate linkages. In some embodiments, an oligonucleotide or portion thereof (eg, a first domain, a second domain, a first subdomain, a second subdomain, a third subdomain, etc.) binds to two 2'-F modified sugars. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 , 25, 26, 27, 28, 29, 30, 35, 40, 45, or 50, e.g., at most 2, at most 3, at most 4, at most 5, etc. internucleotide linkages are natural phosphate linkages am. In some embodiments, a 2'-OR modification (R is herein up to about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45% of internucleotidic linkages that bind two sugars (as described but not including -H) , 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%, such as up to 10%, up to 15%, up to 20%, up to 25% , up to about 30%, up to about 40%, up to 50%, etc. are natural phosphate linkages. In some embodiments, an oligonucleotide or portion thereof (eg, a first domain, a second domain, a first subdomain, a second subdomain, a third subdomain, etc.) binds to two 2'-F modified sugars. up to about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75 %, 80%, 85%, 90%, or 95%, such as up to 10%, up to 15%, up to 20%, up to 25%, up to about 30%, up to about 40%, up to 50%, etc. It is a phosphate linkage. In some embodiments, a 2'-OR modification (R is herein up to about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, or 50, e.g. up to 2, up to 3, up to Consecutive internucleotide linkages of 4, up to 5, etc. are natural phosphate linkages. In some embodiments, an oligonucleotide or portion thereof (eg, a first domain, a second domain, a first subdomain, a second subdomain, a third subdomain, etc.) binds to two 2'-F modified sugars. 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 , 26, 27, 28, 29, 30, 35, 40, 45, or 50 consecutive nucleotide linkages, such as at most 2, at most 3, at most 4, at most 5, etc., are natural phosphate linkages. am.

In some embodiments, the native phosphate linkage is -8, -7, -6, -5, -4, -3, -2, -1, +1, +2, +3, in one or more of the +4, +5, +6, +7, and +8 positions. In some embodiments, the native phosphate linkage is at one or more of the -1 and +1 positions. In some embodiments, the native phosphate linkages are at positions -1 and +1. In some embodiments, the native phosphate linkage is at position -1. In some embodiments, the native phosphate linkage is at position +1. In some embodiments, the natural phosphate linkage is at position +8. In some embodiments, the native phosphate linkage is at position +7. In some embodiments, the native phosphate linkage is at position -6. In some embodiments, the native phosphate linkage is at position +5. In some embodiments, the native phosphate linkage is at position +4. In some embodiments, the native phosphate linkage is at position +3. In some embodiments, the natural phosphate linkage is at position +2. In some embodiments, the native phosphate linkage is at position -2. In some embodiments, the native phosphate linkage is at position -3. In some embodiments, the native phosphate linkage is at position -4. In some embodiments, the native phosphate linkage is at position -5. In some embodiments, the native phosphate linkage is at position -6. In some embodiments, the native phosphate linkage is at position -7. In some embodiments, the native phosphate linkage is at position -8. In some embodiments, the native phosphate linkage is at position -1 and the modified internucleotide linkage is at position +1. In some embodiments, the natural phosphate linkage is at position +1 and the modified internucleotide linkage is at position -1. In some embodiments, the modified internucleotide linkages are chirally controlled. In some embodiments, the modified internucleotidic linkage is chirally controlled and is Sp. In some embodiments, the modified internucleotide linkage is a chirally controlled Sp phosphorothioate internucleotide linkage. In some embodiments, the modified internucleotidic linkage is chirally controlled and is Rp. In some embodiments, the modified internucleotide linkage is a chirally controlled Rp phosphorothioate internucleotide linkage. In some embodiments, the second domain comprises up to two natural phosphate linkages. In some embodiments, the second domain comprises at most one native phosphate linkage. In some embodiments, single natural phosphate linkages may be used at various locations of the oligonucleotide or portion thereof (eg, first domain, second domain, first subdomain, second subdomain, third subdomain, etc.) there is.

In some embodiments, a particular type of sugar is used at a particular position in an oligonucleotide or portion thereof. For example, in some embodiments, the first domain comprises multiple 2'-F modified sugars (optionally in some embodiments multiple 2'-OR modified sugars at a lower level than per 2'-F modified sugars ( R is not -H), the first subdomain contains multiple 2'-OR variants (R is not -H) (e.g., 2'-OMe variants), and/or (optionally in some embodiments comprising a plurality of 2'-F sugars at a lower level than the 2'-OR modified sugar (R is not -H)), the second domain is one or more natural DNA sugars (at the 2' position) no substitution) and/or one or more 2'-F variant sugars, and/or the third subdomain is multiple 2'-OR variant sugars (R is not -H) (e.g., 2'-OMe modified sugars) (optionally including multiple 2'-F sugars at a lower level than the 2'-OR modified sugars (R is not -H) in some embodiments). In some embodiments, a particular type of sugar is independently -8, -7, -6, -5, -4, -3, -2, -1, 0, +1, + of the nucleoside opposite the target adenosine. at one or more of positions 2, +3, +4, +5, +6, +7, and +8 ("+" counts toward the 5'-end of the oligonucleotide from the nucleoside, and "-" Count toward the 3'-end of the oligonucleotide in the nucleoside, position 0 is the position of the nucleoside opposite the target adenosine, e.g. 5'-...N ₊₂ N ₊₁ N ₀ N _-1 N _-2 …3'). In some embodiments, a particular type of sugar is independently at one or more of positions -5, -4, -3, -2, -1, 0, +1, +2, +3, +4, and +5. there is. In some embodiments, a particular type of sugar is independently at one or more of positions -3, -2, -1, 0, +1, +2, and +3. In some embodiments, a particular type of sugar is independently at one or more of positions -2, -1, 0, +1, and +2. In some embodiments, a particular type of sugar is independently at one or more of positions -1, 0, and +1. In some embodiments, the specific type of sugar is at position +8. In some embodiments, the specific type of sugar is at position +7. In some embodiments, a particular type of sugar is at position +6. In some embodiments, the specific type of sugar is at position +5. In some embodiments, the specific type of sugar is at the +4 position. In some embodiments, a particular type of sugar is at the +3 position. In some embodiments, a particular type of sugar is at position +2. In some embodiments, a particular type of sugar is at the +1 position. In some embodiments, a particular type of sugar is at position 0. In some embodiments, the specific type of sugar is at position -8. In some embodiments, the specific type of sugar is at position -7. In some embodiments, a particular type of sugar is at position -6. In some embodiments, a particular type of sugar is at position -5. In some embodiments, the specific type of sugar is at position -4. In some embodiments, a particular type of sugar is at position -3. In some embodiments, a particular type of sugar is at the -2 position. In some embodiments, a particular type of sugar is at position -1. In some embodiments, a particular type of sugar is a sugar independently selected from natural DNA sugars (two 2'-H on a 2'-carbon), 2'-OMe modified sugars, and 2'-F modified sugars. In some embodiments, a particular type of sugar is independently a sugar selected from natural DNA sugars (two 2'-H to 2'-carbon) and 2'-OMe modified sugars. In some embodiments, certain types of sugars (e.g., sugars at positions 0, -1, and/or +1) are independently natural DNA sugars (two 2'-Hs on the 2'-carbon) and 2 '-F is a sugar selected from modified sugars. In some embodiments, a particular type of sugar (eg, a sugar at positions -1, 0, or +1) is a natural DNA sugar (two 2'-Hs on the 2'-carbon). In some embodiments, a specific type of sugar (e.g., -8, -7, -6, -5, -4, -3, -2, -1, 0, +1, +2, +3, + sugars at positions 4, +5, +6, +7, and/or +8) are 2'-F modified sugars. In some embodiments, a specific type of sugar (e.g., -8, -7, -6, -5, -4, -3, -2, +2, +3, +4, +5, +6, sugars at positions +7, and/or +8) are 2'-F modified sugars. In some embodiments, the 2'-F modified sugar is at position -2. In some embodiments, the 2'-F modified sugar is at position -3. In some embodiments, the 2'-F modified sugar is at position -4. In some embodiments, the 2'-F modified sugar is at position +2. In some embodiments, the 2'-F modified sugar is at position +3. In some embodiments, the 2'-F modified sugar is at position +4. In some embodiments, the 2'-F modified sugar is at position +5. In some embodiments, the 2'-F modified sugar is at position +6. In some embodiments, the 2'-F modified sugar is at position +7. In some embodiments, the 2'-F modified sugar is at position +8. In some embodiments, a specific type of sugar (e.g., -8, -7, -6, -5, -4, -3, -2, -1, 0, +1, +2, +3, + sugars at positions 4, +5, +6, +7, and/or +8) are 2'-OMe modified sugars. In some embodiments, a specific type of sugar (e.g., -8, -7, -6, -5, -4, -3, -2, +2, +3, +4, +5, +6, sugars at positions +7, and/or +8) are 2'-OMe modified sugars. In some embodiments, the 2'-OMe modified sugar is at position -2. In some embodiments, the 2'-OMe modified sugar is at position -3. In some embodiments, the 2'-OMe modified sugar is at position -4. In some embodiments, the 2'-OMe modified sugar is at position +2. In some embodiments, the 2'-OMe modified sugar is at position +3. In some embodiments, the 2'-OMe modified sugar is at position +4. In some embodiments, the 2'-OMe modified sugar is at position +5. In some embodiments, the 2'-OMe modified sugar is at position +6. In some embodiments, the 2'-OMe modified sugar is at position +7. In some embodiments, the 2'-OMe modified sugar is at position +8. In some embodiments, the sugar at position 0 is not a 2'-MOE modified sugar. In some embodiments, the sugar at position 0 is a natural DNA sugar (two 2'-Hs to the 2'-carbon). In some embodiments, the sugar at position 0 is not a 2'-MOE modified sugar. In some embodiments, the sugar at position -1 is not a 2'-MOE modified sugar. In some embodiments, the sugar at position -2 is not a 2'-MOE modified sugar. In some embodiments, the sugar at position -3 is not a 2'-MOE modified sugar. In some embodiments, the first domain comprises one or more per 2'-F modifications, and optionally per 2'-OR modifications (in some embodiments, at a lower level than per 2'-F modifications (R is herein as described and not -H). In some embodiments, the first domain is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 per 2'-OR modification (in some embodiments, less than per 2'-F modification). level), where R is as described herein and not -H. In some embodiments, the first domain is per 1, 2, 3, or 4, or no more than 1, no more than 2, no more than 3, or no more than 4 2'-OR variants (R is C _1-6 are aliphatic). In some embodiments, the first, second, third, and/or fourth sugars of the first domain are independently 2'-OR modified sugars, where R is an optionally substituted C _1-6 aliphatic. In some embodiments, the sugar comprising the 2'-OR is contiguous. In some embodiments, the first domain comprises 2, 3, 4, 5, 6, 7, 8, 9, or 10 consecutive sugars at the 5'-end, each sugar independently 2'-OR (R is an optionally substituted C _1-6 aliphatic). In some embodiments, 2'-OR is 2'-OMe. In some embodiments, a 2'-OR is a 2'-MOE. In some embodiments, the second domain comprises one or more 2'-OR variants (in some embodiments at lower levels) (R as described herein and not -H), optionally per 2'-F variants. (in some embodiments at a lower level). In some embodiments, the first subdomain comprises one or more 2'-OR modifications (in some embodiments at lower levels) (R as described herein and not -H), optionally a 2'-F modification sugars (in some embodiments at lower levels). In some embodiments, the third subdomain comprises at least one 2'-OR modification (in some embodiments at a lower level) (R as described herein and not -H), optionally a 2'-F modification sugars (in some embodiments at lower levels; in some embodiments at higher levels). In some embodiments, the third subdomain comprises about or at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 2′-F modified sugars. In some embodiments, the third subdomain comprises about or at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 contiguous 2′-F modified sugars. In some embodiments, about or at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of the sugars in the third subdomain are independently 2 Include the '-F variant. In some embodiments, the first 2'-F modified sugar of the third subdomain (in the 5' to 3' direction) is not the first sugar of the third subdomain. In some embodiments, the first 2'-F modified sugar of the third subdomain is at position -3 relative to the nucleoside opposite the target adenosine. In some embodiments, each sugar in the third subdomain is independently a modified sugar. In some embodiments, each sugar in the third subdomain is independently a modified sugar, wherein the modification is selected from 2'-F and 2'-OR, where R is C _1-6 aliphatic. In some embodiments, the modification is selected from 2'-F and 2'-OMe. In some embodiments, each modified sugar in the third subdomain is independently a 2'-F modified sugar. In some embodiments, each modified sugar in the third subdomain is independently a 2'-OMe modified sugar. In some embodiments, the one or more modified sugars in the third subdomain are independently 2'-OMe modified sugars and the one or more modified sugars in the third subdomain are independently 2'-F modified sugars. In some embodiments, each modified sugar in the third subdomain is independently a 2'-F modified sugar, except for the first sugar in the third subdomain, which in some embodiments is a 2'-OMe modified sugar. In some embodiments, the third subdomain comprises at least one 2'-OR modification (in some embodiments at a lower level) (R as described herein and not -H), optionally a 2'-F modification sugars (in some embodiments at lower levels). In some embodiments, 2'-OR is 2'-OMe. In some embodiments, a 2'-OR is a 2'-MOE.

edit area

In some embodiments, the present invention provides an oligonucleotide comprising an editing region, eg, a region comprising or consisting of 5'-N ₁ N ₀ N _-1 -3' as described herein. In some embodiments, the editing region is the nucleoside opposite the target adenosine and its adjacent nucleosides (typically when the base sequence of the oligonucleotide aligns with the target sequence for maximum complementarity and/or the oligonucleotide hybridizes with the target nucleic acid). Is or contains a cleoside. In some embodiments, the editing region is or comprises 3 nucleobases, wherein the intermediate nucleobase is the opposite nucleoside of the target adenosine. In some embodiments, the nucleoside opposite the target adenosine is N ₀ as described herein.

In some embodiments, the nucleobase of the nucleoside opposite the target adenosine (which may be referred to as BA ₀ ) is C. In some embodiments, BA ₀ is a modified nucleobase as described herein. In some embodiments, the nucleobase, eg, BA ₀ is BA-I, BA-Ia, BA-Ib, BA-II, BA-II-a, BA-II-b, BA-III, BA-III Ring BA or Ring BA having the structure -a, BA-III-b, BA-IV, BA-IV-a, BA-IV-b, BA-V, BA-Va, BA-Vb, or BA-VI is or comprises a tautomer of, wherein the nucleobase is optionally substituted or protected. In some embodiments, the nucleobase is an optionally substituted or protected tautomer thereof, or an optionally substituted or protected tautomer thereof: C, T, U, hypoxanthine, b001U, b002U, b003U, b004U, b005U, b006U, b007U , b008U, b009U, b011U, b012U, b013U, b001A, b002A, b003A, b001G, b002G, b001C, b002C, b003C, b004C, b005C, b006C, b007C, b008C, b009C, b002I, b003I, b004I, b014I and zdnp. In some embodiments, the nucleobase is an optionally substituted or protected tautomer thereof: zdnp, b001U, b002U, b003U, b004U, b005U, b006U, b007U, b008U, b009U, b001A, b002A, b003A, b001C, b002C, b003C, b002I, b003I, or b001G. In some embodiments, the nucleobase of N ₀ is an optionally substituted or protected tautomer thereof: C, zdnp, b001U, b002U, b003U, b004U, b005U, b006U, b007U, b008U , b009U, b001A, b002A, b003A, b001C, b002C, b003C, b002I, b003I, or b001G; And the sugar of N ₀ is a natural DNA sugar. In some embodiments, the nucleobase of N ₀ is an optionally substituted or protected tautomer thereof: C, zdnp, b001U, b002U, b003U, b004U, b005U, b006U, b007U, b008U , b009U, b001A, b002A, b003A, b001C, b002C, b003C, b002I, b003I, or b001G; And the sugar of N ₀ is a native RNA sugar. In some embodiments, BA ₀ is C. In some embodiments, BA ₀ is T. In some embodiments, BA ₀ is hypoxanthine. In some embodiments, BA ₀ is U. In some embodiments, BA ₀ is b001U. In some embodiments, BA ₀ is b002U. In some embodiments, BA ₀ is b003U. In some embodiments, BA ₀ is b004U. In some embodiments, BA ₀ is b005U. In some embodiments, BA ₀ is b006U. In some embodiments, BA ₀ is b007U. In some embodiments, BA ₀ is b008U. In some embodiments, BA ₀ is b009U. In some embodiments, BA ₀ is b011U. In some embodiments, BA ₀ is b012U. In some embodiments, BA ₀ is b013U. In some embodiments, BA ₀ is b001A. In some embodiments, BA ₀ is b002A. In some embodiments, BA ₀ is b003A. In some embodiments, BA ₀ is b001C. In some embodiments, BA ₀ is b002C. In some embodiments, BA ₀ is b003C. In some embodiments, BA ₀ is b004C. In some embodiments, BA ₀ is b005C. In some embodiments, BA ₀ is b006C. In some embodiments, BA ₀ is b007C. In some embodiments, BA ₀ is b008C. In some embodiments, BA ₀ is b009C. In some embodiments, BA ₀ is b002I. In some embodiments, BA ₀ is b003I. In some embodiments, BA ₀ is b004I. In some embodiments, BA ₀ is b014I. In some embodiments, BA ₀ is b001G. In some embodiments, BA ₀ is b002G. In some embodiments, the sugar of N ₀ is a natural DNA sugar or a substituted natural DNA sugar in which one of the 2'-H is substituted with -OH or -F and the other 2'-H is unsubstituted. In some embodiments, the sugar of N ₀ is a natural DNA sugar. In some embodiments, the sugar of N ₀ is a native RNA sugar. In some embodiments, the sugar of N ₀ is an acyclic sugar. In some embodiments, the sugar of N ₀ is sm01. In some embodiments, the sugar of N ₀ is sm04. In some embodiments, the sugar of N ₀ is sm11. In some embodiments, the sugar of N ₀ is sm12. In some embodiments, the sugar of N ₀ is rsm13. In some embodiments, the sugar of N ₀ is rsm14. In some embodiments, the sugar of N ₀ is sm15. In some embodiments, the sugar of N ₀ is sm16. In some embodiments, the sugar of N ₀ is sm17. In some embodiments, the sugar of N ₀ is sm18. Among other things, the present invention has confirmed that various modified nucleobases and/or various sugars at N ₀ in oligonucleotides can be used to provide adenosine editing activity. In some embodiments, b001A as BA ₀ may provide improved adenosine editing efficiency when compared to a reference nucleobase (eg, under comparable conditions involving otherwise identical oligonucleotides, evaluated in the same or comparable assays, etc.) It was observed that there is In some embodiments, it has been observed that b008U as BA ₀ can provide improved adenosine editing efficiency. In some embodiments, the reference nucleobase is U. In some embodiments, the reference nucleobase is T. In some embodiments, the reference nucleobase is C.

In some embodiments, the nucleoside opposite the target adenosine, eg N ₀ , is dC. In some embodiments, it is rC. In some embodiments, this is fC. In some embodiments, it is dT. In some embodiments, this is rT. In some embodiments, this is fT. In some embodiments, it is dU. In some embodiments, it is rU. In some embodiments, this is fU. In some embodiments, b001A (when used for a nucleoside, unless otherwise specified, in an oligonucleotide chain or refers to). In some embodiments, Csm15 (when used for a nucleoside, unless otherwise specified, within an oligonucleotide chain or refers to). In some embodiments, it is Usm15 (when used for a nucleoside, unless otherwise specified, within an oligonucleotide chain or refers to). In some embodiments, it is rCsm13 (when used for a nucleoside, unless otherwise specified, within an oligonucleotide chain or refers to). In some embodiments, Csm04 (when used for a nucleoside, unless otherwise specified, within an oligonucleotide chain or refers to). In some embodiments, b001rA (when used for a nucleoside, unless otherwise specified, within an oligonucleotide chain or refers to). In some embodiments, the sugar is (R)-GNA sugar am. In some embodiments, the sugar is (S)-GNA sugar am. In some embodiments, Csm11 (when used herein for a nucleoside, unless otherwise specified, within an oligonucleotide chain or S-GNA C, also referred to as). In some embodiments, Csm12 (when used herein for a nucleoside, unless otherwise specified, within an oligonucleotide chain or It is R-GNA C, also referred to as). In some embodiments, b009Csm11 (when used herein for a nucleoside, unless otherwise specified, within an oligonucleotide chain or It is the S-GNA isoC, also referred to as). In some embodiments, b009Csm12 (when used herein for a nucleoside, unless otherwise specified, within an oligonucleotide chain or It is R-GNA isoC, also referred to as). In some embodiments, Gsm11 (when used herein for a nucleoside, unless otherwise specified, within an oligonucleotide chain or It is S-GNA G, also referred to as). In some embodiments, when used herein Gsm12 (nucleoside, unless otherwise specified, within an oligonucleotide chain or It is R-GNA G, also referred to as). In some embodiments, this is used herein for Tsm11 (nucleoside, unless otherwise specified, within an oligonucleotide chain or Refers to) is also referred to as S-GNA T. In some embodiments, when used herein Tsm12 (nucleoside, unless otherwise specified, within an oligonucleotide chain or It is R-GNA T, also referred to as). In some embodiments, b004C (when used for a nucleoside, unless otherwise specified, in an oligonucleotide chain or refers to). In some embodiments, b007C (when used for a nucleoside, unless otherwise specified, in an oligonucleotide chain or refers to).

In some embodiments, Csm16 (when used for a nucleoside, unless otherwise specified, within an oligonucleotide chain or refers to). In some embodiments, Csm17 (when used for a nucleoside, unless otherwise specified, within an oligonucleotide chain or refers to). In some embodiments, it is rCsm14 (when used for a nucleoside, unless otherwise specified, within an oligonucleotide chain or refers to). In some embodiments, b008U (when used for a nucleoside, unless otherwise specified, in an oligonucleotide chain or refers to). In some embodiments, b010U (when used for a nucleoside, unless otherwise specified, within an oligonucleotide chain or refers to). In some embodiments, b001C (when used for a nucleoside, unless otherwise specified, in an oligonucleotide chain or refers to). In some embodiments, b008C (when used for a nucleoside, unless otherwise specified, in an oligonucleotide chain or refers to). In some embodiments, b011U (when used for a nucleoside, unless otherwise specified, in an oligonucleotide chain or refers to). In some embodiments, b012U (when used for a nucleoside, unless otherwise specified, in an oligonucleotide chain or refers to). In some embodiments, it is abasic. In some embodiments, this is L010. In some embodiments, it is L034 (when used for a nucleoside, unless otherwise specified, within an oligonucleotide chain or refers to). In some embodiments, b002G (when used for a nucleoside, unless otherwise specified, within an oligonucleotide chain or refers to). In some embodiments, b013U (when used for a nucleoside, unless otherwise specified, in an oligonucleotide chain or refers to). In some embodiments, b002A (when used for a nucleoside, unless otherwise specified, in an oligonucleotide chain or refers to). In some embodiments, b003A (when used for a nucleoside, unless otherwise specified, in an oligonucleotide chain or refers to). In some embodiments, b004I (when used for a nucleoside, unless otherwise specified, in an oligonucleotide chain or refers to). In some embodiments, b014I (when used for a nucleoside, unless otherwise specified, in an oligonucleotide chain or refers to). In some embodiments, b009U (when used for a nucleoside, unless otherwise specified, in an oligonucleotide chain or refers to). In some embodiments, aC (when used for a nucleoside, unless otherwise specified, in an oligonucleotide chain or refers to). In some embodiments, b001U (when used for a nucleoside, unless otherwise specified, in an oligonucleotide chain or refers to). In some embodiments, b002U (when used for a nucleoside, unless otherwise specified, in an oligonucleotide chain or refers to). In some embodiments, b003U (when used for a nucleoside, unless otherwise specified, in an oligonucleotide chain or refers to). In some embodiments, b004U (when used for a nucleoside, unless otherwise specified, in an oligonucleotide chain or refers to). In some embodiments, b005U (when used for a nucleoside, unless otherwise specified, in an oligonucleotide chain or refers to). In some embodiments, b006U (when used for a nucleoside, unless otherwise specified, in an oligonucleotide chain or refers to). In some embodiments, b007U (when used for a nucleoside, unless otherwise specified, in an oligonucleotide chain or refers to). In some embodiments, b001G (when used for a nucleoside, unless otherwise specified, in an oligonucleotide chain or refers to). In some embodiments, b002C (when used for a nucleoside, unless otherwise specified, in an oligonucleotide chain or refers to). In some embodiments, b003C (when used for a nucleoside, unless otherwise specified, in an oligonucleotide chain or refers to). In some embodiments, b003mC (when used for a nucleoside, unless otherwise specified, within an oligonucleotide chain or refers to). In some embodiments, b002I (when used for a nucleoside, unless otherwise specified, in an oligonucleotide chain or refers to). In some embodiments, b003I (when used for a nucleoside, unless otherwise specified, in an oligonucleotide chain or refers to). In some embodiments, Asm01 (when used for a nucleoside, unless otherwise specified, within an oligonucleotide chain or refers to; In some embodiments, the nitrogen atom is bonded to the linking phosphorus. In some embodiments, it is Gsm01 (when used for a nucleoside, unless otherwise specified, within an oligonucleotide chain or refers to; In some embodiments, the nitrogen atom is bonded to the linking phosphorus. In some embodiments, it is Tsm01 (when used for a nucleoside, unless otherwise specified, within an oligonucleotide chain or refers to; In some embodiments, the nitrogen atom is bonded to the linking phosphorus. In some embodiments, it is 5MsfC (when used for a nucleoside, unless otherwise specified, within an oligonucleotide chain or refers to). In some embodiments, it is Usm04 (when used for a nucleoside, unless otherwise specified, within an oligonucleotide chain or refers to). In some embodiments, it is 5MRdT (when used for nucleosides, unless otherwise specified, within an oligonucleotide chain or refers to). In some embodiments, it is Tsm18 (when used for a nucleoside, unless otherwise specified, within an oligonucleotide chain or refers to; In some embodiments, the nitrogen atom is bonded to the linking phosphorus. In some embodiments, N ₀ is abasic. In some embodiments, N ₀ is L010.

In some embodiments, as demonstrated in various examples, certain modified nucleosides or nucleobases, e.g., b001A, b008U, etc., will provide improved editing, e.g., when compared to the dC at the position opposite to the target adenosine. can In some embodiments, a particular nucleoside, e.g., dC, b001A, b001rA, Csm15, b001C, etc., is identified (e.g., under comparable conditions, including the same oligonucleotide, evaluated in the same or comparable assay, etc.) It has been observed that compared to nucleosides, it can provide improved adenosine editing efficiency when used in N ₀ . In some embodiments, N ₀ is b001A. In some embodiments, N ₀ is b001rA. In some embodiments, N ₀ is b002A. In some embodiments, N ₀ is b003A. In some embodiments, N ₀ is b004I. In some embodiments, N ₀ is b014I. In some embodiments, N ₀ is b002G. In some embodiments, N ₀ is dC. In some embodiments, N ₀ is b001C. In some embodiments, N ₀ is b009U. In some implementations, N ₀ is b010U. In some implementations, N ₀ is b011U. In some implementations, N ₀ is b012U. In some implementations, N ₀ is b013U. In some embodiments, N ₀ is Csm04. In some embodiments, N ₀ is Csm11. In some embodiments, N ₀ is Csm12. In some embodiments, N ₀ is Csm15. In some embodiments, N ₀ is b009Csm11. In some embodiments, N ₀ is b009Csm12. In some embodiments, N ₀ is Gsm11. In some embodiments, N ₀ is Gsm12. In some embodiments, N ₀ is Tsm11. In some embodiments, N ₀ is Tsm12. In some embodiments, the reference nucleoside is rU. In some embodiments, the reference nucleoside is dU. In some embodiments, the reference nucleoside is dT. In some embodiments, there is no nucleobase at position N ₀ . In some embodiments, at position N ₀ it is L010. In some embodiments, the sugar of N ₀ is sm15.

In some embodiments, replacing guanine with hypoxanthine at position -1 (eg, replacing dG with dI) can provide improved editing. Specific data is provided in FIG. 17 and other data is provided as an example.

In some embodiments, the oligonucleotide comprises 5'-N ₁ N ₀ N _-1 -3', wherein each of N ₁ , N ₀ and N _-1 is independently a nucleoside as described herein. In some embodiments, the oligonucleotide comprises 5'-N ₂ N ₁ N ₀ N _-1 N _-2 -3', wherein each of N ₂ , N ₁ , N ₀ , N _-1 and N _-2 are independently is a nucleoside as described herein. In some embodiments, an oligonucleotide comprises 5'-N ₃ N ₂ N ₁ N ₀ N _-1 N _-2 N _-3 -3', wherein N ₃ , N ₂ , N ₁ , N ₀ , N _- Each of ₁ , N _-2 and N _-3 is independently a nucleoside as described herein. In some embodiments, the oligonucleotide comprises 5′-N ₄ N ₃ N ₂ N ₁ N ₀ N _-1 N _-2 N _-3 N _-4 -3′, wherein N ₄ , N ₃ , N ₂ , Each of N ₁ , N ₀ , N _-1 , N _-2 , N _-3 and N _-4 is independently a nucleoside as described herein. In some embodiments, the oligonucleotide comprises 5'-N ₅ N ₄ N ₃ N ₂ N ₁ N ₀ N _-1 N _-2 N _-3 N _-4 N _-5 -3', wherein N ₅ , N _{Each of 4} , N ₃ , N ₂ , N ₁ , N ₀ , N _-1 , N _-2 , N _-3 , N _-4 and N _-5 is independently a nucleoside as described herein. In some embodiments, the oligonucleotide comprises 5'-N ₆ N ₅ N ₄ N ₃ N ₂ N ₁ N ₀ N _-1 N -2 N _-3 N _{-4 N -5 N -6} -3'; wherein each of N ₆ , N ₅ , N ₄ , N ₃ , N ₂ , N ₁ , N ₀ , N _-1 , N _-2 , N _-3 , N _-4 , N _-5 and N _-6 is independently It is a nucleoside as described in. In some embodiments, N _n (where n is a positive number), eg, N ₁ , can also be referred to as N ₊₁ . In some embodiments, such oligonucleotides can form a duplex with a nucleic acid (eg, an RNA nucleic acid) and edit the target adenosine opposite N ₀ . In some embodiments, N _-6 is the last nucleoside (counting from the 5'-end) of the oligonucleotide.

In some embodiments, the oligonucleotide comprises 5'-N ₂ N ₁ N ₀ N _-1 N _-2 -3', wherein each of N ₂ , N ₁ , N ₀ , N _-1 and N _-2 are independently is a nucleoside. In some embodiments, the oligonucleotide comprises 5'-N ₂ N ₁ N ₀ N _-1 N _-2 -3', wherein each of N ₂ , N ₁ , N ₀ , N _-1 and N _-2 are independently is a nucleoside. In some embodiments, the oligonucleotide comprises 5'-N ₂ N ₁ N ₀ N _-1 N _-2 -3', wherein each of N ₂ , N ₁ , N ₀ , N _-1 and N _-2 are independently is a nucleoside, N ₀ is opposite to the target adenosine, and as understood by those skilled in the art, each two of N ₂ , N ₁ , N ₀ , N _-1 , and N _-2 adjacent to each other are independently linked to internucleotidic linkages as described. In some embodiments, one or more or all of N ₁ , N ₀ , and N ₋₁ independently have a native RNA sugar. In some embodiments, one or more or all of N ₁ , N ₀ , and N ₋₁ independently have a natural DNA sugar. In some embodiments, each sugar of N ₁ , N ₀ , and N ₋₁ is independently a natural DNA sugar or a 2′-F modified sugar. In some embodiments, each sugar of N ₁ , N ₀ , and N ₋₁ is independently a natural DNA sugar. In some embodiments, the sugar of N ₁ is a 2'-modified sugar, and each sugar of N ₀ and N ₋₁ is independently a natural DNA sugar. In some embodiments, the sugar of N ₁ is a 2′-F sugar, and each sugar of N ₀ and N ₋₁ is independently a natural DNA sugar. In some embodiments, the sugar of N ₁ is a modified sugar. In some embodiments, the sugar of N ₁ is a 2′-F modified sugar. In some embodiments, the sugar of N ₁ is a natural DNA sugar. In some embodiments, the sugar of N ₁ is a native RNA sugar. In some embodiments, the sugar of N ₀ is not a modified sugar. In some embodiments, the sugar of N ₀ is not a 2'-modified sugar. In some embodiments, the sugar of N ₀ is not a 2'-OR modified sugar, where R is an optionally substituted C _1-6 alkyl. In some embodiments, the sugar of N ₀ is not a 2′-F modified sugar. In some embodiments, the sugar of N ₀ is not a 2'-OMe modified sugar. In some embodiments, the sugar of N ₀ is a natural DNA or RNA sugar. In some embodiments, the sugar of N ₀ is a natural DNA sugar. In some embodiments, the sugar of N ₀ is a native RNA sugar. In some embodiments, the sugar of N _-1 is not a modified sugar. In some embodiments, the sugar of N _-1 is not a 2'-modified sugar. In some embodiments, the sugar of N _-1 is not a 2'-OR modified sugar, where R is an optionally substituted C _1-6 alkyl. In some embodiments, the sugar of N _-1 is not a 2'-F modified sugar. In some embodiments, the sugar of N _-1 is not a 2'-OMe modified sugar. In some embodiments, the sugar of N _-1 is a natural DNA or RNA sugar. In some embodiments, the sugar of N _-1 is a natural DNA sugar. In some embodiments, the sugar of N _-1 is a native RNA sugar. In some embodiments, each of N ₁ , N ₀ , and N ₋₁ independently has a native RNA sugar. In some embodiments, each of N ₁ , N ₀ , and N ₋₁ independently has a natural DNA sugar. In some embodiments, N ₁ has a 2′-F modified sugar, and each N ₀ and N ₋₁ independently has a native DNA or RNA sugar. In some embodiments, N ₁ has a 2′-F modified sugar and each N ₀ and N ₋₁ independently has a natural DNA sugar (eg, WV-22434). In some embodiments, two of N ₁ , N ₀ , and N ₋₁ independently have a natural DNA or RNA sugar. In some embodiments, two of N ₁ , N ₀ , and N ₋₁ independently have a natural DNA sugar. In some embodiments, each of N ₁ and N ₀ independently has a 2′-F modified sugar, and N ₋₁ is a natural DNA sugar.

In some embodiments, such oligonucleotides provide high levels of editing. In some embodiments, each of the two internucleotide linkages linked to N _-1 is independently R p. In some embodiments, each of the two internucleotide linkages linked to N _-1 is independently an R p phosphorothioate internucleotide linkage. In some embodiments, each of the two internucleotide linkages linked to N _-1 is an R p phosphorothioate internucleotide linkage, and each other phosphorothioate internucleotide linkage in the oligonucleotide, if present, is independently S is p. In some embodiments, the 5' internucleotide linkage linked to N ₁ is R p. In some embodiments, the internucleotide linkage linked to N ₁ and N ₀ (ie, the 3′ internucleotide linkage linked to N ₁ ) is R p. In some embodiments, the internucleotide linkage linked to N _-1 and N ₀ is R p. In some embodiments, the 3' internucleotide linkage linked to N _-1 is R p. In some embodiments, each internucleotide linkage linked to N ₀ is independently R p . In some embodiments, each internucleotide linkage linked to N ₀ and N ₁ is independently R p . In some embodiments, each internucleotide linkage linked to N ₀ and N ₋₁ is independently R p . In some embodiments, each internucleotide linkage linked to N ₁ is independently R p . In some embodiments, each R p internucleotidic linkage is independently an R p phosphorothioate internucleotidic linkage. In some embodiments, each other chirally controlled phosphorothioate internucleotidic linkage in the oligonucleotide is independently Sp . In some embodiments, the internucleotide linkage between N ₀ N _-1 is R p . In some embodiments, the internucleotide linkage between N ₀ N _-1 is an R p phosphorothioate internucleotide linkage. In some embodiments, the internucleotide linkage between N _-1 N _-2 is R p . In some embodiments, the internucleotide linkage between N _-1 N _-2 is an R p phosphorothioate internucleotide linkage. In some embodiments, all internucleotide linkages linked to N ₁ , N ₀ and N ₋₁ are independently Sp . In some embodiments, all internucleotide linkages linked to N ₁ , N ₀ and N ₋₁ are independently Sp phosphorothioate internucleotide linkages. In some embodiments, all internucleotide linkages linked to N ₂ , N ₁ , N ₀ , N _-1 and N _-2 are independently Sp . In some embodiments, all internucleotide linkages linked to N ₂ , N ₁ , N ₀ , N _-1 and N _-2 are independently Sp phosphorothioate internucleotide linkages. In some embodiments, each internucleotide linkage linked to N ₁ is both independently Sp (eg, an Sp phosphorothioate internucleotide linkage). In some embodiments, the internucleotide linkage between N ₁ and N ₀ is an S p (eg, an S p phosphorothioate internucleotide linkage). In some embodiments, the internucleotide linkage between N ₋₁ and N ₀ is an S p (eg, an S p phosphorothioate internucleotide linkage). In some embodiments, the internucleotide linkage between N _-1 and N _-2 is a neutral internucleotide linkage. In some embodiments, the internucleotide linkage between N _-1 and N _-2 is a non-negatively charged internucleotide linkage. In some embodiments, the internucleotide linkage between N _-1 and N _-2 is n001. In some embodiments, the internucleotide linkage between N _-1 and N _-2 is not chirally controlled. In some embodiments, the internucleotide linkage between N _-1 and N _-2 is chirally controlled. In some embodiments, the internucleotide linkage between N _-1 and N _-2 is R p. In some embodiments, the internucleotide linkage between N _-1 and N _-2 is Sp . In some embodiments, N ₂ comprises a modified sugar. In some embodiments, N _-2 comprises a modified sugar. In some embodiments, each of N ₂ and N _-2 independently comprises a modified sugar. In some embodiments, a modified sugar is a 2'-modified sugar. In some embodiments, a modified sugar is a 2'-OR modified sugar, where R is an optionally substituted C _1-6 aliphatic. In some embodiments, the 2'-modified sugar is a 2'-OMe modified sugar. In some embodiments, a 2'-modified sugar is a 2'-MOE modified sugar. In some embodiments, the modified sugar is a bicyclic sugar, e.g., an LNA sugar, a cEt sugar, and the like.

In some embodiments, there are at least 2, 3, 4, 5, 6, 7, 8, 9 or more nucleosides (eg, N 0 ) to the 3' side of the opposite nucleoside (eg, N ₀ ) of the target adenosine. , 2~30, 3~30, 4~30, 5~30, 2~20, 3~20, 4~20, 5~20, 2~15, 3~15, 4~15, 5~15, 2 ~10, 3~10, 4~10, 5~10 pieces, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, etc. "3' side nucleosides"). In some embodiments, there are at least two 3' side nucleosides. In some embodiments, there are at least three 3' side nucleosides. In some embodiments, there are at least 4 3' side nucleosides. In some embodiments, there are at least 5 3' side nucleosides (e.g., an oligonucleotide comprising 5'-N ₀ N _-1 N _-2 N _-3 N _-4 N _-5 -3'; wherein each of N ₀ , N _-1 , N _-2 , N _-3 , N _-4 , and N _-5 is independently a nucleoside. In some embodiments, there are at least six 3' side nucleosides (e.g., including 5'-N ₀ N _-1 N _-2 N _-3 N _-4 N _-5 N _-6 -3' oligonucleotides, wherein each N ₀ , N _-1 , N _-2 , N _-3 , N _-4 , N _-5 , and N _-6 are each independently a nucleoside. In some embodiments, there are at least 7 3' side nucleosides. In some embodiments, there are at least 8 3' side nucleosides. In some embodiments, there are at least 9 3' side nucleosides. In some embodiments, there are at least 10 3' side nucleosides. In some embodiments, there are two 3' side nucleosides. In some embodiments, there are three 3' side nucleosides. In some embodiments, there are four 3' side nucleosides. In some embodiments, there are 5 3' side nucleosides. In some embodiments, there are six 3' side nucleosides. In some embodiments, there are 7 3' side nucleosides. In some embodiments, there are 8 3' side nucleosides. In some embodiments, there are 9 3' side nucleosides. In some embodiments, there are 10 3' side nucleosides. In some embodiments, at least 15, 16, 17, 18, 19, ₂₀ , 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 or more nucleosides (e.g., 15-50, 20-50, 21-50, 22-50, 23-50, 24-50, 25-50, 26-50, 27 ~50, 28~50, 29~50, 30~50, 15~40, 20~40, 21~40, 22~40, 23~40, 24~40, 25~40, 26~40, 27~40 , 28~40, 29~40, 30~40, 15~30, 20~30, 21~30, 22~30, 23~30, 24~30, 25~30, 26~30, 27~30, 28 ~30, 29-30, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30, etc.). In some embodiments, there are at least 15 5' side nucleosides. In some embodiments, there are at least 16 5' side nucleosides. In some embodiments, there are at least 17 5' side nucleosides. In some embodiments, there are at least 18 5' side nucleosides. In some embodiments, there are at least 19 5' side nucleosides. In some embodiments, there are at least 20 5' side nucleosides. In some embodiments, there are at least 21 5' side nucleosides. In some embodiments, there are at least 22 5' side nucleosides. In some embodiments, there are at least 23 5' side nucleosides. In some embodiments, there are at least 24 5' side nucleosides. In some embodiments, there are at least 25 5' side nucleosides. In some embodiments, there are at least 26 5' side nucleosides. In some embodiments, there are at least 27 5' side nucleosides. In some embodiments, there are at least 28 5' side nucleosides. In some embodiments, there are at least 29 5' side nucleosides. In some embodiments, there are at least 30 5' side nucleosides. In some embodiments, there are 15 5' side nucleosides. In some embodiments, there are 16 5' side nucleosides. In some embodiments, there are 17 5' side nucleosides. In some embodiments, there are 18 5' side nucleosides. In some embodiments, there are 19 5' side nucleosides. In some embodiments, there are 20 5' side nucleosides. In some embodiments, there are 21 5' side nucleosides. In some embodiments, there are 22 5' side nucleosides. In some embodiments, there are 23 5' side nucleosides. In some embodiments, there are 24 5' side nucleosides. In some embodiments, there are 25 5' side nucleosides. In some embodiments, there are 26 5' side nucleosides. In some embodiments, there are 27 5' side nucleosides. In some embodiments, there are 28 5' side nucleosides. In some embodiments, there are 29 5' side nucleosides. In some embodiments, there are 30 5' side nucleosides. In some embodiments, there are at least 4 3' side nucleosides and at least 22 5' side nucleosides. In some embodiments, there are at least 4 3' side nucleosides and at least 23 5' side nucleosides. In some embodiments, there are at least 4 3' side nucleosides and at least 24 5' side nucleosides. In some embodiments, there are at least 4 3' side nucleosides and at least 25 5' side nucleosides. In some embodiments, there are at least 5 3' side nucleosides and at least 22 5' side nucleosides. In some embodiments, there are at least 5 3' side nucleosides and at least 23 5' side nucleosides. In some embodiments, there are at least 5 3' side nucleosides and at least 24 5' side nucleosides. In some embodiments, there are at least 5 3' side nucleosides and at least 25 5' side nucleosides. In some embodiments, there are at least 6 3' side nucleosides and at least 21 5' side nucleosides. In some embodiments, there are at least 6 3' side nucleosides and at least 22 5' side nucleosides. In some embodiments, there are at least 6 3' side nucleosides and at least 23 5' side nucleosides. In some embodiments, there are at least 6 3' side nucleosides and at least 24 5' side nucleosides. In some embodiments, there are at least 7 3' side nucleosides and at least 20 5' side nucleosides. In some embodiments, there are at least 7 3' side nucleosides and at least 21 5' side nucleosides. In some embodiments, there are at least 7 3' side nucleosides and at least 22 5' side nucleosides. In some embodiments, there are at least 7 3' side nucleosides and at least 23 5' side nucleosides. In some embodiments, there are at least 8 3' side nucleosides and at least 19 5' side nucleosides. In some embodiments, there are at least 8 3' side nucleosides and at least 20 5' side nucleosides. In some embodiments, there are at least 8 3' side nucleosides and at least 21 5' side nucleosides. In some embodiments, there are at least 8 3' side nucleosides and at least 22 5' side nucleosides. In some embodiments, there are at least 9 3' side nucleosides and at least 18 5' side nucleosides. In some embodiments, there are at least 9 3' side nucleosides and at least 19 5' side nucleosides. In some embodiments, there are at least 9 3' side nucleosides and at least 20 5' side nucleosides. In some embodiments, there are at least 9 3' side nucleosides and at least 21 5' side nucleosides. In some embodiments, there are at least 10 3' side nucleosides and at least 17 5' side nucleosides. In some embodiments, there are at least 10 3' side nucleosides and at least 18 5' side nucleosides. In some embodiments, there are at least 10 3' side nucleosides and at least 19 5' side nucleosides. In some embodiments, there are at least 10 3' side nucleosides and at least 20 5' side nucleosides. In some embodiments, there are at least 11 3' side nucleosides and at least 16 5' side nucleosides. In some embodiments, there are at least 11 3' side nucleosides and at least 17 5' side nucleosides. In some embodiments, there are at least 11 3' side nucleosides and at least 18 5' side nucleosides. In some embodiments, there are at least 11 3' side nucleosides and at least 19 5' side nucleosides. In some embodiments, there are at least 12 3' side nucleosides and at least 15 5' side nucleosides. In some embodiments, there are at least 12 3' side nucleosides and at least 16 5' side nucleosides. In some embodiments, there are at least 12 3' side nucleosides and at least 17 5' side nucleosides. In some embodiments, there are at least 12 3' side nucleosides and at least 18 5' side nucleosides. In some embodiments, there are at least 13 3' side nucleosides and at least 14 5' side nucleosides. In some embodiments, there are at least 13 3' side nucleosides and at least 15 5' side nucleosides. In some embodiments, there are at least 13 3' side nucleosides and at least 16 5' side nucleosides. In some embodiments, there are at least 13 3' side nucleosides and at least 17 5' side nucleosides. In some embodiments, certain useful lengths and/or positions on the 5' and/or 3' side of the nucleoside opposite the target adenosine (e.g., the C of the UCI of the oligonucleotide described in Figure 2(a)) is described in Figures 2 and 3.

As described herein, modifications including sugar modifications, nucleobase modifications, and the like may be used for N ₁ . In some embodiments, N ₁ contains a natural DNA sugar. In some embodiments, N ₁ contains a native RNA sugar. In some embodiments, N ₁ contains a modified sugar as described herein. In some embodiments, a modified sugar is a 2'-modified sugar. In some embodiments, a modified sugar is a 2'-F modified sugar. In some embodiments, a modified sugar is a 2'-OR modified sugar, where R is an optionally substituted C _1-6 alkyl. In some embodiments, the modified sugar is a 2'-OMe modified sugar. In some embodiments, a modified sugar is a 2'-MOE modified sugar. In some embodiments, the sugar is a UNA sugar. In some embodiments, the sugar is a GNA sugar. In some embodiments, the sugar of N ₁ is sm01. In some embodiments, it is sm11. In some embodiments, it is sm12. In some embodiments, it is sm18. In some embodiments, a modified sugar, e.g., a 2'-F modified sugar or a DNA sugar, is compared to a reference sugar (eg, native RNA sugar, different modified sugars, etc.) in a system (e.g., cell, tissue, organism). etc.) provides higher editing efficiency. In some embodiments, N ₁ contains a natural nucleobase, eg U. In some embodiments, N ₁ contains a modified nucleobase as described herein. In some embodiments, the nucleobase of N ₁ is A, T, C, G, U, hypoxanthine, b001U, b002U, b003U, b004U, b005U, b006U, b007U, b008U, b009U, b011U, b012U, b013U, b001A, b002A, b003A, b001G, b002G, b001C, b002C, b003C, b004C, b005C, b006C, b007C, b008C, b009C, b002I, b003I, b004I, b014I or zdnp. In some embodiments, the nucleobase of N ₁ is T. In some embodiments, it is U. In some embodiments, this is b002A. In some embodiments, this is b003A. In some embodiments, this is b008U. In some embodiments, this is b010U. In some embodiments, this is b011U. In some embodiments, this is b012U. In some embodiments, this is b001C. In some embodiments, this is b004C. In some embodiments, this is b007C. In some embodiments, this is b008C. In some embodiments, N ₁ is a natural nucleoside. In some embodiments, N ₁ is a modified nucleoside. In some embodiments, N ₁ is fU, dU, fA, dA, fT, dT, fC, dC, fG, dG, dI, fI, aC, b001U, b002U, b003U, b004U, b005U, b006U, b007U, b008U, b009U, b010U, b011U, b012U, b013U, b001A, b001rA, b002A, b003A, b001G, b002G, b001C, b002C, b003C, b003mC, b004C, b005C, b006C, b007C, b008C, b002I, b003I, b004I, b014I, Asm01, Gsm01, 5MSfC, Usm04, 5MRdT, Csm04, Csm11, Gsm11, Tsm11, b009Csm11, b009Csm12, Gsm12, Tsm12, Csm12, rCsm13, rCsm14, Csm15, Csm16, Csm17, L034, zdnp and Tsm18. In some embodiments, N ₁ is fU, dU, fA, dA, fT, dT, fC, dC, fG, dG, dI, or fI. In some embodiments, N ₁ is fU, dU, fA, dA, fT, dT, fC, dC, fG, or dG. In some embodiments, N ₁ is dT. In some embodiments, N ₁ is b001A. In some embodiments, N ₁ is b002A. In some embodiments, N ₁ is b003A. In some embodiments, N ₁ is fU. In some embodiments, N ₁ is b008U. In some embodiments, N ₁ is b001C. In some embodiments, N ₁ is b004C. In some embodiments, N ₁ is b007C. In some embodiments, N ₁ is b008C. In some embodiments, N ₁ is b001U. In some embodiments, N ₁ is b008U. In some embodiments, N ₁ is b010U. In some embodiments, N ₁ is b011U. In some embodiments, N ₁ is b012U. In some embodiments, N ₁ is Csm11. In some embodiments, N ₁ is Gsm11. In some embodiments, N ₁ is Tsm11. In some embodiments, N ₁ is b009Csm11. In some embodiments, N ₁ is Csm12. In some embodiments, N ₁ is Gsm12. In some embodiments, N ₁ is Tsm12. In some embodiments, N ₁ is b009Csm12. In some embodiments, N ₁ is Gsm01. In some embodiments, N ₁ is Tsm01. In some embodiments, N ₁ is Csm17. In some embodiments, N ₁ is Tsm18. In some embodiments, N ₁ is b014I. In some embodiments, N ₁ is abasic. In some embodiments, N ₁ is L010. As described herein, in some embodiments at position N ₁ , an oligonucleotide is coincident when it forms a duplex with a nucleic acid (eg, its target transcript for adenosine editing). In some embodiments, this is a mismatch. In some embodiments, it is a wobble. In some embodiments, N ₁ is linked to a natural phosphate linkage. In some embodiments, N ₁ is linked to a modified internucleotide linkage as described herein in various embodiments with defined stereochemistry. In some embodiments, N ₁ is linked to a natural phosphate linkage and a modified internucleotide linkage. In some embodiments, N ₁ is linked to two natural phosphate linkages. In some embodiments, N ₁ is linked to two modified internucleotide linkages, each of which may be independently and optionally stereocontrolled and may be R p or Sp .

As described herein, modifications including sugar modifications, nucleobase modifications, and the like can be used for N _-1 . In some embodiments, N _-1 contains a natural DNA sugar. In some embodiments, N _-1 contains a native RNA sugar. In some embodiments, N _-1 contains a modified sugar as described herein. In some embodiments, a modified sugar is a 2'-modified sugar. In some embodiments, a modified sugar is a 2'-F modified sugar. In some embodiments, a modified sugar is a 2'-OR modified sugar, where R is an optionally substituted C _-1-6 alkyl. In some embodiments, the modified sugar is a 2'-OMe modified sugar. In some embodiments, a modified sugar is a 2'-MOE modified sugar. In some embodiments, the sugar is a UNA sugar. In some embodiments, the sugar is a GNA sugar. In some embodiments, the sugar of N _-1 is sm01. In some embodiments, it is sm11. In some embodiments, it is sm12. In some embodiments, it is sm18. In some embodiments, a modified sugar, e.g., a 2'-F modified sugar or a DNA sugar, is compared to a reference sugar (eg, native RNA sugar, different modified sugars, etc.) in a system (e.g., cell, tissue, organism). etc.) provides higher editing efficiency. In some embodiments, N _-1 contains a natural nucleobase, for example U. In some embodiments, N _-1 contains a modified nucleobase as described herein. In some embodiments, the nucleobase of N _-1 is A, T, C, G, U, hypoxanthine, b001U, b002U, b003U, b004U, b005U, b006U, b007U, b008U, b009U, b011U, b012U, b013U, b001A , b002A, b003A, b001G, b002G, b001C, b002C, b003C, b004C, b005C, b006C, b007C, b008C, b009C, b002I, b003I, b004I, b014I or zdnp. In some embodiments, the nucleobase of N _-1 is T. In some embodiments, it is U. In some embodiments, this is b001A. In some embodiments, this is b002A. In some embodiments, this is b003A. In some embodiments, this is b008U. In some embodiments, this is b011U. In some embodiments, this is b012U. In some embodiments, this is b001C. In some embodiments, this is b004C. In some embodiments, this is b007C. In some embodiments, this is b008C. In some embodiments, this is b009C. In some embodiments, this is b002G. In some embodiments, this is b014I. In some embodiments, N _-1 is a natural nucleoside. In some embodiments, N _-1 is a modified nucleoside. In some embodiments, N ₋₁ is fU, dU, fA, dA, fT, dT, fC, dC, fG, dG, dI, fI, aC, b001U, b002U, b003U, b004U, b005U, b006U, b007U, b008U , b009U, b010U, b011U, b012U, b013U, b001A, b001rA, b002A, b003A, b001G, b002G, b001C, b002C, b003C, b003mC, b004C, b005C, b006C, b007C, b008 C, b002I, b003I, b004I, b014I, Asm01 , Gsm01, 5MSfC, Usm04, 5MRdT, Csm04, Csm11, Gsm11, Tsm11, b009Csm11, b009Csm12, Gsm12, Tsm12, Csm12, rCsm13, rCsm14, Csm15, Csm16, Csm17, L03 4, zdnp and Tsm18. In some embodiments, N ₋₁ is fU, dU, fA, dA, fT, dT, fC, dC, fG, dG, dI, or fI. In some embodiments, N ₋₁ is fU, dU, fA, dA, fT, dT, fC, dC, fG, or dG. In some embodiments, N ₋₁ is dI. In some embodiments, N ₋₁ is rI. In some embodiments, N ₋₁ is dT. In some embodiments, N _-1 is b001A. In some embodiments, N ₋₁ is b002A. In some embodiments, N _-1 is b003A. In some embodiments, N ₋₁ is fU. In some embodiments, N _-1 is b001C. In some embodiments, N ₋₁ is b004C. In some embodiments, N _-1 is b007C. In some embodiments, N ₋₁ is b008C. In some embodiments, N _-1 is b009Csm12. In some embodiments, N ₋₁ is b001U. In some embodiments, N ₋₁ is b008U. In some embodiments, N _-1 is b010U. In some embodiments, N _-1 is b011U. In some embodiments, N _-1 is b012U. In some embodiments, N _-1 is Csm11. In some embodiments, N _-1 is b009Csm11. In some embodiments, N _-1 is Gsm11. In some embodiments, N _-1 is Tsm11. In some embodiments, N _-1 is Csm12. In some embodiments, N _-1 is b009Csm12. In some embodiments, N ₋₁ is Gsm12. In some embodiments, N _-1 is Tsm12. In some embodiments, N _-1 is Gsm01. In some embodiments, N _-1 is Tsm01. In some embodiments, N _-1 is Tsm18. In some embodiments, N ₋₁ is abasic. In some embodiments, N _-1 is L010. In some embodiments, N _-1 is Csm17. In some embodiments, N ₋₁ is b002G. In some embodiments, N _-1 is b014I. As described herein, in some embodiments, at position N-1, an oligonucleotide is coincident when it forms a duplex with a nucleic acid (eg, its target transcript for adenosine editing). In some embodiments, this is a mismatch. In some embodiments, it is a wobble. In some embodiments, N ₋₁ is linked to a natural phosphate linkage. In some embodiments, N _-1 is linked to a modified internucleotide linkage as described herein in various embodiments with defined stereochemistry. In some embodiments, N _-1 is linked to a natural phosphate linkage and a modified internucleotide linkage. In some embodiments, N ₋₁ is linked to two natural phosphate linkages. In some embodiments, N _-1 is linked to two modified internucleotide linkages, each of which may be independently and optionally sterically controlled and may be R p or Sp .

In some embodiments, N ₂ contains a natural sugar. In some embodiments, the sugar of N ₂ is a natural DNA sugar. In some embodiments, it is a native RNA sugar. In some embodiments, it is a modified sugar. In some embodiments, it is a 2'-F modified sugar. In some embodiments, it is a 2'-OR modified sugar (R is C _1-6 aliphatic as described herein). In some embodiments, R is an optionally substituted C _1-6 alkyl. In some embodiments, it is a 2'-OMe modification. In some embodiments, it is a 2'-MOE modified sugar.

In some embodiments, the internucleotide linkage between N ₁ and N ₂ is a natural phosphate linkage. In some embodiments, it is a modified internucleotide linkage. In some embodiments, it is a phosphorothioate internucleotidic linkage. In some embodiments, it is a non-negatively charged internucleotidic linkage. In some embodiments, it is a neutral internucleotide linkage. In some embodiments, it is a phosphoryl guanidine internucleotide linkage. In some embodiments, this is n001. In some embodiments, it is Sp . In some embodiments, it is R p. In some embodiments, it is an S p phosphorothioate internucleotidic linkage. In some embodiments, it is S p n001. In some embodiments, it is R p n001.

In some embodiments, N ₃ contains a natural sugar. In some embodiments, the sugar of N ₃ is a natural DNA sugar. In some embodiments, it is a native RNA sugar. In some embodiments, it is a modified sugar. In some embodiments, it is a 2'-F modified sugar. In some embodiments, it is a 2′-OR modified sugar (R is C _1-6 aliphatic as described herein). In some embodiments, R is an optionally substituted C _1-6 alkyl. In some embodiments, it is a 2'-OMe modification. In some embodiments, it is a 2'-MOE modified sugar.

In some embodiments, the internucleotide linkage between N ₂ and N ₃ is a natural phosphate linkage. In some embodiments, it is a modified internucleotide linkage. In some embodiments, it is a phosphorothioate internucleotidic linkage. In some embodiments, it is an internucleotidic linkage that is not negatively charged. In some embodiments, it is a neutral internucleotide linkage. In some embodiments, it is a phosphoryl guanidine internucleotide linkage. In some embodiments, this is n001. In some embodiments, it is Sp . In some embodiments, it is R p. In some embodiments, it is an S p phosphorothioate internucleotidic linkage. In some embodiments, it is S p n001. In some embodiments, it is R p n001. In some embodiments, N ₄ contains a natural sugar. In some embodiments, the sugar of N ₄ is a natural DNA sugar. In some embodiments, it is a native RNA sugar. In some embodiments, it is a modified sugar. In some embodiments, it is a 2'-F modified sugar. In some embodiments, it is a 2'-OR modified sugar (R is C _1-6 aliphatic as described herein). In some embodiments, R is an optionally substituted C _1-6 alkyl. In some embodiments, it is a 2'-OMe modification. In some embodiments, it is a 2'-MOE modified sugar.

In some embodiments, the internucleotide linkage between N ₃ and N ₄ is a natural phosphate linkage. In some embodiments, it is a modified internucleotide linkage. In some embodiments, it is a phosphorothioate internucleotidic linkage. In some embodiments, it is an internucleotidic linkage that is not negatively charged. In some embodiments, it is a neutral internucleotide linkage. In some embodiments, it is a phosphoryl guanidine internucleotide linkage. In some embodiments, this is n001. In some embodiments, it is Sp . In some embodiments, it is R p. In some embodiments, it is an S p phosphorothioate internucleotidic linkage. In some embodiments, it is S p n001. In some embodiments, it is R p n001.

In some embodiments, N ₅ contains a natural sugar. In some embodiments, the sugar of N ₅ is a natural DNA sugar. In some embodiments, it is a native RNA sugar. In some embodiments, it is a modified sugar. In some embodiments, it is a 2'-F modified sugar. In some embodiments, it is a 2'-OR modified sugar (R is C _1-6 aliphatic as described herein). In some embodiments, R is an optionally substituted C _1-6 alkyl. In some embodiments, it is a 2'-OMe modification. In some embodiments, it is a 2'-MOE modified sugar.

In some embodiments, the internucleotide linkage between N ₄ and N ₅ is a natural phosphate linkage. In some embodiments, it is a modified internucleotide linkage. In some embodiments, it is a phosphorothioate internucleotidic linkage. In some embodiments, it is an internucleotidic linkage that is not negatively charged. In some embodiments, it is a neutral internucleotide linkage. In some embodiments, it is a phosphoryl guanidine internucleotide linkage. In some embodiments, this is n001. In some embodiments, it is Sp . In some embodiments, it is R p. In some embodiments, it is an S p phosphorothioate internucleotidic linkage. In some embodiments, it is S p n001. In some embodiments, it is R p n001.

In some embodiments, N ₆ contains a natural sugar. In some embodiments, the sugar of N ₆ is a natural DNA sugar. In some embodiments, it is a native RNA sugar. In some embodiments, it is a modified sugar. In some embodiments, it is a 2'-F modified sugar. In some embodiments, it is a 2'-OR modified sugar (R is C _1-6 aliphatic as described herein). In some embodiments, R is an optionally substituted C _1-6 alkyl. In some embodiments, it is a 2'-OMe modification. In some embodiments, it is a 2'-MOE modified sugar.

In some embodiments, the internucleotide linkage between N ₅ and N ₆ is a natural phosphate linkage. In some embodiments, it is a modified internucleotide linkage. In some embodiments, it is a phosphorothioate internucleotidic linkage. In some embodiments, it is an internucleotidic linkage that is not negatively charged. In some embodiments, it is a neutral internucleotide linkage. In some embodiments, it is a phosphoryl guanidine internucleotide linkage. In some embodiments, this is n001. In some embodiments, it is Sp . In some embodiments, it is R p. In some embodiments, it is an S p phosphorothioate internucleotidic linkage. In some embodiments, it is S p n001. In some embodiments, it is R p n001.

As described herein, an oligonucleotide or portion thereof, e.g., a first domain, a second domain, etc., may contain one or more, e.g., 1-20, 1-15, 1-14, 1-13, 1-12, 1-11, 1-10, 2-20, 3-15, 4-15, 5-15, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 , 14, 15, 16, 17, 18, 19, may include or consist of 20 blocks, and each of these blocks is independently one or more (eg, 1 to 50, 1 to 40, 1 to 30, 1~25, 1~24, 1~23, 1~22, 1~21, 1~20, 1~10, 1~5, 1, 2, 3, 4, 5, 6, 7, 8 , 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, etc.) and each sugar in the block shares the same structure. In some embodiments, an oligonucleotide or portion thereof, e.g., a first domain, a second domain, etc., contains one or more, e.g., 1-20, 1-15, 1-14, 1-13, 1-12, 1 ~11, 1~10, 2~20, 3~15, 4~15, 5~15 pieces, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, It may include or consist of 14, 15, 16, 17, 18, 19, 20 blocks, etc., each of which is independently one or more (eg, 1 to 50, 1 to 40, 1 to 30 blocks). , 1~25, 1~24, 1~23, 1~22, 1~21, 1~20, 1~10, 1~5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, etc.) and each sugar in the block is the same modified sugar. In some embodiments, each block independently contains 1-10 sugars, eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 sugars. In some embodiments, each block independently contains 1-5 sugars. In some embodiments, each block independently contains 1, 2, or 3 sugars. In some embodiments, one or more blocks, e.g., 1-15, 1-10, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more independently contain 2 or 3 or more sugars. In some embodiments, one or more blocks, e.g., 1-15, 1-10, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more independently contain 2 or 3 sugars. In some embodiments, about or at least about 30%, 40% or 50% of the blocks in an oligonucleotide or portion thereof independently contain two or more (eg, two or three) sugars. In some embodiments, about 50% of the blocks in the oligonucleotides of the first domain independently contain two or more (eg, two or three) sugars. In some embodiments, the block is a 2'-F block, wherein each sugar in the block is a 2'-F modified block. In some embodiments, a block is a 2'-OR block, where R is an optionally substituted C _1-6 aliphatic, wherein each sugar in the block is the same 2'-OR modified sugar. In some embodiments, the block is a 2'-OMe block. In some embodiments, the block is a 2'-MOE block. In some embodiments, a block is a heterocyclic sugar block, wherein each sugar in the block is the same heterocyclic sugar (eg, LNA sugar, cEt, etc.). In some embodiments, the two or more blocks are 2'-F blocks. In some implementations, the every-two block is a 2'-F block. In some embodiments, each 2'-F block independently contains no more than 2, 3, 4, 5, 6, 7, 8, 9, or 10 sugars. In some embodiments, the 2'-F block contains 5 or fewer sugars. In some embodiments, the 2'-F block contains no more than 4 sugars. In some embodiments, the 2'-F block contains 3 or fewer sugars. In some embodiments, between every two 2'-F blocks in an oligonucleotide or portion thereof there is at least one 2'-OR block (R is an optionally substituted C _1-6 aliphatic) or one bicyclic block. there is. In some embodiments, between every two 2′-F blocks in some there is at least one 2′-OR block (R is an optionally substituted C _1-6 aliphatic) or one bicyclic block. In some embodiments, there is at least one 2'-OR block (R is an optionally substituted C _1-6 aliphatic) between every two 2'-F blocks in the oligonucleotide. In some embodiments, between every two 2'-F blocks in the first domain is at least one 2'-OR block, where R is an optionally substituted C _1-6 aliphatic. In some embodiments, there is at least one 2'-OMe block between every two 2'-F blocks in the first domain. In some embodiments, between two 2'-F blocks in the first domain is a 2'-OMe block. In some embodiments, between two 2'-F blocks in the first domain is a 2'-MOE block. In some embodiments, between the two 2'-F blocks in the first domain is a 2'-MOE block and a 2'-OMe block. In some embodiments, there is a 2'-MOE block and a 2'-OMe block between two 2'-F blocks in the first domain, and no 2'-F block. In some embodiments, each 2'-F block is independently linked to a 2'-OR block (R is C _1-6 aliphatic) or a bicyclic sugar block. In some embodiments, each 2'-F block is independently linked to a 2'-OR block, wherein R is C _1-6 aliphatic. In some embodiments, each block to which a 2'-F block binds is independently a 2'-OR block (R is C _1-6 aliphatic) or a bicyclic sugar block. In some embodiments, each block to which a 2'-F block binds is independently a 2'-OR block (R is C _1-6 aliphatic). In some embodiments, each block of the first domain to which a 2'-F block of the first domain binds is independently a 2'-OR block (R is C _1-6 aliphatic) or a heterocycle block. In some embodiments, each block of the first domain to which a 2'-F block of the first domain binds is independently a 2'-OR block (R is C _1-6 aliphatic). In some embodiments, each block of the first domain to which a 2'-OR block (R is C _1-6 aliphatic) or dissociated block binds independently a different 2'-OR block (R is C _1-6 is an aliphatic) 2'-F block or block per ring. In some embodiments, each block of the first domain to which a 2'-OR block (R is C _1-6 aliphatic) binds independently of a different 2'-OR block (R is C _1-6 aliphatic). 2'-F block. In some embodiments, a 2'-OR block is a 2'-OMe block. In some embodiments, a 2'-OR block is a 2'-MOE block. In some embodiments, at least one block is a 2'-OMe block. In some embodiments, about or about at least 2, 3, 4, or 5 blocks are independently 2'-OMe blocks. In some embodiments, at least one block is a 2'-MOE block. In some embodiments, about or about at least 2, 3, 4 or 5 blocks are independently 2'-MOE blocks. In some embodiments, an oligonucleotide or portion thereof, e.g., a first domain, a second domain, etc., contains one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 one or more) 2'-MOE blocks and one or more (eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more) 2'-MOE blocks. In some embodiments, an oligonucleotide or portion thereof, e.g., a first domain, a second domain, etc., contains one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) 2'-MOE blocks and one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) 2'-MOE blocks and one or more (e.g., For example, there are 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) 2'-F blocks. In some embodiments, the first domain includes one or more (eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more) 2'-OMe blocks and one or more (eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more) 2'-OMe blocks. For example, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more) 2'-MOE blocks. In some embodiments, the first domain includes one or more (eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more) 2'-OMe blocks and one or more (eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more) 2'-OMe blocks. For example, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more) 2'-F blocks. In some embodiments, the first domain includes one or more (eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more) 2'-F blocks and one or more (eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more) 2'-F blocks. For example, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more) 2'-MOE blocks. In some embodiments, the first domain includes one or more (eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more) 2'-OMe blocks and one or more (eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more) 2'-OMe blocks. For example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more) 2'-MOE blocks and one or more (eg, 1, 2, 3, 4, 5, 6 , 7, 8, 9 or 10) 2'-F blocks. In some embodiments, the percentage of 2'-F modified sugars in an oligonucleotide or portion thereof, e.g., a first domain, a second domain, etc., is about 20%-80%, 30-70%, 30%-60%, 30% -50%, 40% -60%, 20%, 30%, 40%, 50%, 60%, 70% or 80%, each independently per 2'-OR variant (R is an optionally substituted C _{1 -6} aliphatic) is about 20%-80%, 30-70%, 30%-60%, 30%-50%, 40%-60%, 20%, 30%, 40%, 50%, 60%, 70% or 80%. In some embodiments, the percentage of 2'-F variant sugars in the first domain is about 20%-80%, 30-70%, 30%-60%, 30%-50%, 40%-60%, 20%, Percentages of 2'-OR modified sugars that are 30%, 40%, 50%, 60%, 70% or 80%, each independently 2'-OR modified sugar, where R is an optionally substituted C _1-6 aliphatic Silver is about 20%-80%, 30-70%, 30%-60%, 30%-50%, 40%-60%, 20%, 30%, 40%, 50%, 60%, 70% or 80 %am. In some embodiments, the difference between the percentage of 2'-F variant sugars and the percentage of 2'-OR variant sugars that are each independently 2'-OR variant sugars, where R is optionally substituted C _1-6 aliphatic, is about 50 %, 40%, 30%, 20% or less than 10% (calculated by subtracting the smaller of the two percentages from the larger of the two percentages). In some embodiments, each 2'-OR modified sugar is independently a 2'-OMe or 2'-MOE modified sugar.

For example, in some embodiments, each sugar of N ₂ , N ₅ and N ₆ is independently a 2′-F modified sugar, and each sugar of N ₃ and N ₄ is independently a 2′-OR modified sugar ( R is an optionally substituted C _1-6 aliphatic) or a bicyclic sugar. In some embodiments, each sugar of N ₂ , N ₅ and N ₆ is independently a 2′-F modified sugar, and each sugar of N ₃ and N ₄ is independently a 2′-OR modified sugar. In some embodiments, each sugar of N ₂ , N ₅ and N ₆ is independently a 2′-F modified sugar, and each sugar of N ₃ and N ₄ is independently a 2′-OMe or 2′-MOE modified sugar. am. In some embodiments, each sugar of N ₂ , N ₅ and N ₆ is independently a 2′-F modified sugar, and each sugar of N ₃ and N ₄ is independently a 2′-OMe modified sugar. In some embodiments, at least one sugar is a 2'-MOE modified sugar. In some embodiments, the sugar of N ₃ is a 2'-MOE modified sugar. In some embodiments, the sugar of N ₃ is a 2'-OMe modified sugar. In some embodiments, the sugar of N ₄ is a 2′-MOE modified sugar. In some embodiments, the sugars of N ₃ and N ₄ are 2′-MOE modified sugars. In some embodiments N ₂ forms a 2′-F block. In some embodiments, N ₃ and N ₄ form a 2′-OMe block. In some embodiments, N ₃ and N ₄ form a 2′-MOE block. In some embodiments, N ₅ , N ₆ and/or N ₇ form a 2′-F block. As demonstrated herein, oligonucleotides comprising modified sugars at various positions, e.g., 2'-F modified sugars, 2'-OMe modified sugars, 2'-MOE modified sugars, etc., possess high levels of adenosine editing among other things. can provide. For example, 2'-MOE modified sugars can be incorporated at various positions to provide oligonucleotides capable of adenosine editing; In some embodiments, the sugar of N ₁ is a 2'-MOE modified sugar; In some embodiments, the sugar of N ₂ is a 2′-MOE modified sugar; In some embodiments, the sugar of N ₃ is a 2′-MOE modified sugar; In some embodiments, the sugar of N ₄ is a 2′-MOE modified sugar; In some embodiments, the sugar of N ₅ is a 2′-MOE modified sugar; In some embodiments, the sugar of N ₆ is a 2′-MOE modified sugar; In some embodiments, the sugar of N ₇ is a 2′-MOE modified sugar; In some embodiments, the sugar of N ₈ is a 2′-MOE modified sugar; In some embodiments, the sugar of N _-1 is a 2'-MOE modified sugar; In some embodiments, the sugar of N _-2 is a 2'-MOE modified sugar; In some embodiments, the sugar of N _-3 is a 2'-MOE modified sugar; In some embodiments, the sugar of N _-4 is a 2'-MOE modified sugar; In some embodiments, the sugar of N _-5 is a 2'-MOE modified sugar; In some embodiments, the sugar of N _-6 is a 2'-MOE modified sugar.

As described herein, a variety of internucleotidic linkages may be utilized in an oligonucleotide or portion thereof, eg, a first domain, a second domain, etc. For example, various connections may be used in the first domain. In some embodiments, the first domain is one or more, for example 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 , 19, 20 or more natural phosphate linkages. In some embodiments, the first domain is one or more, for example 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 , 19, 20 or more modified internucleotide linkages. In some embodiments, the first domain comprises one or more native phosphate linkages and one or more modified internucleotide linkages. In some embodiments, the one or more modified internucleotidic linkages are phosphorothioate internucleotidic linkages. In some embodiments, each phosphorothioate internucleotidic linkage is chirally controlled. In some embodiments, each phosphorothioate internucleotidic linkage in an oligonucleotide or portion thereof, eg, a first domain, a second domain, etc., is Sp . In some embodiments, each phosphorothioate internucleotidic linkage in an oligonucleotide is Sp . In some embodiments, one or more modified internucleotidic linkages are independently non-negatively charged internucleotidic linkages. In some embodiments, one or more modified internucleotidic linkages are independently non-negatively charged internucleotidic linkages. In some embodiments, the one or more modified internucleotide linkages are independently phosphoryl guanidine internucleotide linkages. In some embodiments, each phosphorylguanidine internucleotide linkage is independently n001. In some embodiments, the first domain contains about 1-5, eg 1, 2, 3, 4 or 5 negatively charged internucleotide linkages. In some embodiments, each of these non-negatively charged internucleotidic linkages is independently a phosphoryl guanidine internucleotidic linkage. In some embodiments, each of these is independently n001. In some embodiments, one or more of these are independently chirally controlled. In some embodiments, each of these is chirally controlled. In some embodiments, each of these is R p n001. In some embodiments, at least one sugar that is a 2'-OR modified sugar (R is an optionally substituted C _1-6 aliphatic) is linked to a natural phosphate linkage. In some embodiments, one or more 2'-OMe sugars are linked to natural phosphate linkages. In some embodiments, one or more 2'-MOE sugars are linked to a natural phosphate linkage. In some embodiments, one or more 2'-F modified sugars are linked to a natural phosphate linkage. In some embodiments, about or at least about 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% of an oligonucleotide or portion thereof, e.g., a first domain, a second domain, etc. The 2′-OR modified sugar (R is an optionally substituted C _1-6 aliphatic) is independently linked to a natural phosphate linkage. In some embodiments, about or at least about 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% of an oligonucleotide or portion thereof, e.g., a first domain, a second domain, etc. 2'-OMe modified sugars are independently linked to native phosphate linkages. In some embodiments, about or at least about 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% of an oligonucleotide or portion thereof, e.g., a first domain, a second domain, etc. 2'-MOE modified sugars are independently linked to native phosphate linkages. In some embodiments, about or at least about 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% of the 2'-OR variant in the first domain, the second domain (R is optionally substituted C _1-6 aliphatic) are independently linked to natural phosphate linkages. In some embodiments, about or at least about 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% of the 2'-OMe modified sugars in the first domain are independently linked to native phosphate linkages. are combined In some embodiments, about or at least about 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% of the 2'-MOE modified sugars in the first domain are independently linked to native phosphate linkages. are combined In some embodiments, about or at least about 1-10, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 2′- in the first domain, the second domain. The OR modified sugar (R is an optionally substituted C _1-6 aliphatic) is independently linked to a natural phosphate linkage. In some embodiments, about or at least about 1-10, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 2'-OMe modifications in the first domain are Independently linked to natural phosphate linkages. In some embodiments, about or at least about 1-10, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 2'-MOE modifications in the first domain are Independently linked to natural phosphate linkages. In some embodiments, one or more, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more natural phosphate linkages linked to a 2'-F modified sugar, are independently 2'- OR is attached to a modified sugar (R is an optionally substituted C _1-6 aliphatic) or bicyclic sugar. In some embodiments, each native phosphate linkage linked to a 2'-F modified sugar is independently linked to a 2'-OR modified sugar (R is an optionally substituted C _1-6 aliphatic) or bicyclic sugar. In some embodiments, one or more, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more natural phosphate linkages linked to a 2'-F modified sugar, are independently 2'- Binds to the MOE modified sugar. In some embodiments, each native phosphate linkage linked to a 2'-F modified sugar is independently linked to a 2'-MOE modified sugar.

Among other things, the present invention provides various blocks and patterns as described herein, such as 2'-F blocks, 2'-OMe blocks, 2'-MOE blocks, etc. and/or various internucleotidic linkages and Reference oligonucleotides to which oligonucleotides comprising this pattern are compared, for example WO 2016/097212, WO 2017/220751, WO 2018/041973, WO 2018/134301A1, WO 2019/158475, WO 2019/219581, WO 2020/ 157008, WO 2020/165077, WO 2020/201406 or WO 2020/252376 compared to those previously reported, demonstrating that it can provide improved pharmacodynamics, pharmacokinetics and/or adenosine editing levels, etc. In some embodiments, the reference oligonucleotide is an oligonucleotide as reported in WO 2021071858.

In some embodiments, N _-2 contains a natural sugar. In some embodiments, the sugar of N _-2 is a natural DNA sugar. In some embodiments, it is a native RNA sugar. In some embodiments, it is a modified sugar. In some embodiments, it is a 2'-F modified sugar. In some embodiments, it is a 2'-OR modified sugar (R is C _1-6 aliphatic as described herein). In some embodiments, R is an optionally substituted C _1-6 alkyl. In some embodiments, it is a 2'-OMe modification. In some embodiments, it is a 2'-MOE modified sugar.

In some embodiments, the internucleotide linkage between N _-1 and N _-2 is a modified internucleotide linkage. In some embodiments, it is a phosphorothioate internucleotidic linkage. In some embodiments, it is an internucleotidic linkage that is not negatively charged. In some embodiments, it is a neutral internucleotide linkage. In some embodiments, it is a phosphoryl guanidine internucleotide linkage. In some embodiments, this is n001. In some embodiments, it is Sp . In some embodiments, it is R p. In some embodiments, it is an S p phosphorothioate internucleotidic linkage. In some embodiments, it is S p n001. In some embodiments, it is R p n001. In some embodiments, N _-1 is dI and the linkage between N _-1 and N _-2 is an Sp phosphoryl guanidine internucleotide linkage. In some embodiments, N _-1 is dI and the linkage between N _-1 and N _-2 is Sp n001.

In some embodiments, N _-3 contains a natural sugar. In some embodiments, the sugar of N _-3 is a natural DNA sugar. In some embodiments, it is a native RNA sugar. In some embodiments, it is a modified sugar. In some embodiments, it is a 2'-F modified sugar. In some embodiments, it is a 2'-OR modified sugar (R is C _1-6 aliphatic as described herein). In some embodiments, R is an optionally substituted C _1-6 alkyl. In some embodiments, it is a 2'-OMe modification. In some embodiments, it is a 2'-MOE modified sugar.

In some embodiments, the internucleotide linkage between N _-2 and N _-3 is a natural phosphate linkage. In some embodiments, it is a modified internucleotide linkage. In some embodiments, it is a phosphorothioate internucleotidic linkage. In some embodiments, it is an internucleotidic linkage that is not negatively charged. In some embodiments, it is a neutral internucleotide linkage. In some embodiments, it is a phosphoryl guanidine internucleotide linkage. In some embodiments, this is n001. In some embodiments, it is Sp . In some embodiments, it is R p. In some embodiments, it is an S p phosphorothioate internucleotidic linkage. In some embodiments, it is S p n001. In some embodiments, it is R p n001.

In some embodiments, N _-4 contains a natural sugar. In some embodiments, the sugar of N _-4 is a natural DNA sugar. In some embodiments, it is a native RNA sugar. In some embodiments, it is a modified sugar. In some embodiments, it is a 2'-F modified sugar. In some embodiments, it is a 2'-OR modified sugar (R is C _1-6 aliphatic as described herein). In some embodiments, R is an optionally substituted C _1-6 alkyl. In some embodiments, it is a 2'-OMe modification. In some embodiments, it is a 2'-MOE modified sugar.

In some embodiments, the internucleotide linkage between N _-3 and N _-4 is a natural phosphate linkage. In some embodiments, it is a modified internucleotide linkage. In some embodiments, it is a phosphorothioate internucleotidic linkage. In some embodiments, it is a non-negatively charged internucleotidic linkage. In some embodiments, it is a neutral internucleotide linkage. In some embodiments, it is a phosphoryl guanidine internucleotide linkage. In some embodiments, this is n001. In some embodiments, it is Sp . In some embodiments, it is R p. In some embodiments, it is an S p phosphorothioate internucleotidic linkage. In some embodiments, it is S p n001. In some embodiments, it is R p n001.

In some embodiments, N _-5 contains a natural sugar. In some embodiments, the sugar of N _-5 is a natural DNA sugar. In some embodiments, it is a native RNA sugar. In some embodiments, it is a modified sugar. In some embodiments, it is a 2'-F modified sugar. In some embodiments, it is a 2'-OR modified sugar (R is C _1-6 aliphatic as described herein). In some embodiments, R is an optionally substituted C _1-6 alkyl. In some embodiments, it is a 2'-OMe modification. In some embodiments, it is a 2'-MOE modified sugar.

In some embodiments, the internucleotide linkage between N _-4 and N _-5 is a natural phosphate linkage. In some embodiments, it is a modified internucleotide linkage. In some embodiments, it is a phosphorothioate internucleotidic linkage. In some embodiments, it is an internucleotidic linkage that is not negatively charged. In some embodiments, it is a neutral internucleotide linkage. In some embodiments, it is a phosphoryl guanidine internucleotide linkage. In some embodiments, this is n001. In some embodiments, it is Sp . In some embodiments, it is R p. In some embodiments, it is an S p phosphorothioate internucleotidic linkage. In some embodiments, it is S p n001. In some embodiments, it is R p n001.

In some embodiments, N _-6 contains a natural sugar. In some embodiments, the sugar of N _-6 is a natural DNA sugar. In some embodiments, it is a native RNA sugar. In some embodiments, it is a modified sugar. In some embodiments, it is a 2'-F modified sugar. In some embodiments, it is a 2′-OR modified sugar (R is C _1-6 aliphatic as described herein). In some embodiments, R is an optionally substituted C _1-6 alkyl. In some embodiments, it is a 2'-OMe modification. In some embodiments, it is a 2'-MOE modified sugar.

In some embodiments, the internucleotide linkage between N _-5 and N _-6 is a natural phosphate linkage. In some embodiments, it is a modified internucleotide linkage. In some embodiments, it is a phosphorothioate internucleotidic linkage. In some embodiments, it is a non-negatively charged internucleotidic linkage. In some embodiments, it is a neutral internucleotide linkage. In some embodiments, it is a phosphoryl guanidine internucleotide linkage. In some embodiments, this is n001. In some embodiments, it is Sp . In some embodiments, it is R p. In some embodiments, it is an S p phosphorothioate internucleotidic linkage. In some embodiments, it is S p n001. In some embodiments, it is R p n001.

In some embodiments, at least one of N _-1 , N _-2 , N _-3 , N _-4 , N _-5 and N _-6 is a natural DNA sugar. In some embodiments, at least one of N _-2 , N _-3 , N _-4 , N _-5 and N _-6 is a 2′-F modified sugar. In some embodiments, at least one of N _-2 , N _-3 , N _-4 , N _-5 and N _-6 is a 2'-OR modified sugar (R is an optionally substituted C _1-6 aliphatic). . In some embodiments, at least one of N _-2 , N _-3 , N _-4 , N _-5 and N _-6 is a 2'-OMe modified sugar. In some embodiments, at least one of N _-2 , N _-3 , N _-4 , N _-5 and N _-6 is a 2'-MOE modified sugar. In some embodiments, at least one of N _-2 , N _-3 , N _-4 , N _-5 and N _-6 is a bicyclic sugar, eg, an LNA sugar, a cEt sugar, and the like. In some embodiments, one of N _-2 , N _-3 , N _-4 , N _-5 and N _-6 is a 2'-F modified sugar, and each other sugar is independently a 2'-OR modified sugar ( R is an optionally substituted C _1-6 aliphatic (eg, 2'-OMe modified sugar, 2'-MOE modified sugar, etc.) or a bicyclic sugar as described herein. In some embodiments, one of N _-2 , N _-3 , N _-4 , N _-5 and N _-6 is a 2'-F variant sugar and each other sugar is independently a 2'-OR variant sugar. (R is an optionally substituted C _1-6 aliphatic). In some embodiments, one of N _-2 , N _-3 , N _-4 , N _-5 and N _-6 is a 2'-F modified sugar and each other sugar is independently 2'-OMe or 2 '- is per MOE strain. In some embodiments, one of N _-2 , N _-3 , N _-4 , N _-5 and N _-6 is a 2'-F modified sugar and each other sugar is independently a 2'-OMe modified sugar. am. In some embodiments, the sugar of N _-3 is a 2'-F modified sugar. In some embodiments, the sugar of N _-1 is a DNA sugar, the sugar of N _-3 is a 2'-F modified sugar, and each sugar of N _-2 , N _-4 , N _-5 and N _-6 is independently 2′-OR modified sugars (R is an optionally substituted C _1-6 aliphatic) or bicyclic sugars as described herein (eg LNA sugars, ENA sugars, etc.). In some embodiments, the sugar of N _-1 is a DNA sugar, the sugar of N _-3 is a 2'-F modified sugar, and each sugar of N _-2 , N _-4 , N _-5 and N _-6 is independently 2'-OR modified sugar (R is an optionally substituted C _1-6 aliphatic). In some embodiments, the sugar of N _-1 is a DNA sugar, the sugar of N _-3 is a 2'-F modified sugar, and each sugar of N _-2 , N _-4 , N _-5 and N _-6 is independently as per 2'-OMe or 2'-MOE modifications. In some embodiments, the sugar of N _-1 is a DNA sugar, the sugar of N _-3 is a 2'-F modified sugar, and each sugar of N _-2 , N _-4 , N _-5 and N _-6 is independently as per the 2'-OMe variant. In some embodiments N ₋₂ forms a 2′-OMe block. In some embodiments N _-3 forms a 2'-F block. In some embodiments, N _-4 , N _-5 and N _-6 form a 2'-OMe block.

In some embodiments, at least one of N _-2 , N _-3 , N _-4 , N _-5 and N _-6 is bound to a natural phosphate linkage. In some embodiments, the linkage between N _-2 and N _-3 is a natural phosphate linkage. In some embodiments, N _-2 is linked to a non-negatively charged internucleotidic linkage. In some embodiments, at least one of N _-3 , N _-4 , N _-5 and N _-6 is linked to a non-negatively charged internucleotide linkage. In some embodiments, the linkage between N _-5 and N _-6 is a non-negatively charged internucleotidic linkage. In some embodiments, at least one of N _-3 , N _-4 , N _-5 and N _-6 is linked to a phosphorothioate internucleotidic linkage. In some embodiments, each of N _-3 , N _-4 and N _-5 is independently linked to a phosphorothioate internucleotidic linkage. In some embodiments, the non-negatively charged internucleotidic linkages are neutral internucleotidic linkages. In some embodiments, the non-negatively charged internucleotidic linkages are phosphoryl guanidine internucleotidic linkages. In some embodiments, the non-negatively charged internucleotide linkage is n001. In some embodiments, it is R p. In some embodiments, it is Sp . In some embodiments, the phosphorothioate internucleotidic linkage is R p. In some embodiments, the phosphorothioate internucleotidic linkage is Sp . In some embodiments, each phosphorothioate internucleotidic linkage is Sp . In some embodiments, the linkage between N _-2 and v-3 is a natural phosphate linkage, the linkage between N _-3 and N _-4 is a Sp phosphorothioate internucleotidic linkage, and N _-4 and N _-5 The linkage between is an Sp phosphorothioate internucleotidic linkage, and the linkage between N _-5 and N _-6 is a negatively charged R p internucleotidic linkage (eg, R p phosphoryl such as R p n001). guanidine internucleotide linkages). In some embodiments, a native phosphate linkage is linked to at least one modified sugar. In some embodiments, the native phosphate linkage is linked to at least one 2′-OR modified sugar (R is an optionally substituted C _1-6 aliphatic) or bicyclic sugar. In some embodiments, a native phosphate linkage is linked to the 2'-OMe modified sugar. In some embodiments, a native phosphate linkage is linked to a 2'-MOE modified sugar. In some embodiments, the two sugars linked to the native phosphate linkage are independently modified sugars as described herein.

In some embodiments, an oligonucleotide comprises a first domain as described herein (e.g., a first domain in which many or most or all sugars are 2'-F modified sugars) and a second domain as described herein ( eg, domains in which many or most or all sugars are non-2'-F modified sugars (eg, 2'-OMe modified sugars)). In some embodiments, the first domain is on the 5' side of the second domain (eg, various oligonucleotides in Figure 2(a)). In some embodiments, the first domain is on the 3' side of the second domain (eg, various oligonucleotides in Figure 2(b)). In some embodiments, when the first domain is on the 3' side of the second domain (e.g., the various oligonucleotides of Figure 2(b)), at least 1, 2, 3 of the nucleoside opposite the target adenosine , 4, 5, 6, 7, 8, 9, or 10 or more (e.g., 1-20, 2-20, 3-20, 4-20, 5-20, 6-20, 7-20, 7 to 11, etc.) of 5' side nucleosides. In some embodiments, there are at least three. In some embodiments, there are at least four. In some embodiments, there are at least 5. In some embodiments, there are at least 6. In some embodiments, there are at least 7. In some embodiments, there are at least 8. In some embodiments, there are at least 9. In some embodiments, there are at least 10. In some embodiments, there are three. In some embodiments, there are four. In some embodiments, there are five. In some embodiments, there are six. In some embodiments, there are seven. In some embodiments, there are 8. In some embodiments, there are 9. In some embodiments, there are 10. In some embodiments, there are 11. In some embodiments, there are 7-11. In some embodiments, there are 9-11. In some embodiments, there are 10 or 11. In some embodiments, additionally or alternatively, at least 15, 16, 17, 18, 19, 20 or more (e.g., 15-30, 16-30, 17-30 , 18-30, 18-25, 18-22, etc.) on the 5' side of the nucleoside. In some embodiments, there are at least 15. In some embodiments, there are at least 16. In some embodiments, there are at least 17. In some embodiments, there are at least 18. In some embodiments, as described above, at least about 5 (e.g., 5-50, 5-40, 5-30, 5-20, 5-10, 5-9, 5, 6, 7, 8 , 9, or 10, etc.) and at least about 15 (e.g., 15-50, 15-40, 15-30, 15-20, 20-30, 20-25, 15 , 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30, etc.) In some embodiments, independently about 1-10 (e.g., 2-10, 3-10, 3-5, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) ) is independently on the 5' side, or on the 3' side, or on both sides of the editing region (eg, N ₁ N ₀ N _-1 ), R is an optionally substituted C _{1- 6} is aliphatic. In some embodiments, independently about 1-10 (e.g., 2-10, 3-10, 3-5, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) ) are independently on the 5' side, or on the 3' side, or on both sides of the editing region, and R is an optionally substituted C _1-6 aliphatic. In some embodiments, independently about 1-10 (e.g., 2-10, 3-10, 3-5, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) ) are independently on the 5' side, or on the 3' side, or on both sides of the editing region. In some embodiments, they are on the 5' side. In some embodiments, they are on the 3' sides. In some embodiments, they are on both sides. In some embodiments, around the editing region, eg, N ₁ N ₀ N _-1 , for example, about 1-10 (eg, 2 independently ~10, 3-10, 3-5, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10), where R is optionally substituted C _1-6 aliphatic. do. In some embodiments, around the editing region, eg, N ₁ N ₀ N _-1 , eg independently about 1-10 (eg, 2-10) per 2'-OR variant on either side. , 3-10, 3-5, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10), where R is optionally substituted C _1-6 aliphatic. In some embodiments, there are at least two on each side. In some embodiments, each 2'-OR modified sugar is a 2'-OMe modified sugar. In some embodiments, each 2'-OR modified sugar is a 2'-MOE modified sugar. Specific examples are illustrated in FIGS. 2 ((a) and/or (b)) and FIG. 3 .

One of the many advantages of the provided technology is that much shorter oligonucleotides can provide similar or higher levels of adenosine editing compared to other traditional technologies. One of ordinary skill in the art reading this invention will be able to understand one or more structural elements (eg, sugar modifications, nucleobase modifications, internucleotidic linkage modifications, stereochemistry and/or its longer oligonucleotides (e.g., 5', 3', or bilateral extensions of a target adenosine) may also be beneficial, for example, for adenine editing and editing of a target adenosine. It will be appreciated that it may be useful for a variety of uses described herein including prevention and/or treatment of disease.

In some embodiments, ADAR1 p150 may allow for more changes in length on the 5' side and/or 3' side than ADAR1 p110 and/or positioning of nucleosides opposite the target adenosine. In some embodiments, the present invention provides particularly useful lengths on the 5' side and/or 3' side and/or of the nucleoside opposite the target adenosine for editing (e.g., by ADAR1 p110 and/or ADAR1 p150). provide location. In some embodiments, certain useful lengths on the 5' side and/or 3' side and/or of an oligonucleotide providing the same editing; in some embodiments of WV-42027; in some embodiments of WV-42028; in some embodiments of WV-42029; in some embodiments of WV-42030; in some embodiments of WV -42031) is useful for editing in cells expressing ADAR1, eg ADAR1 p110 and/or p150.

In some embodiments, each phosphorothioate linked to the nucleoside opposite the target adenosine is independently a phosphorothioate internucleoside linkage. In some embodiments, the internucleotide linkage between N ₀ and N ₋₁ is an R p phosphorothioate internucleotide linkage. In some embodiments, the internucleotide linkage between N _-1 and N _-2 is an R p phosphorothioate internucleotide linkage.

In some embodiments, the present invention provides oligonucleotides comprising an editing region capable of providing high editing efficiency. In some embodiments, a provided editing region is or comprises 5'-N ₁ N ₀ N _-1 -3' as described herein.

In some embodiments, the present invention provides oligonucleotides comprising 5'-N ₁ N ₀ N _-1 -3' as described herein.

In some embodiments, N ₀ is as described herein. In some embodiments, N ₀ comprises a sugar and a nucleobase as described herein. In some embodiments, N ₀ has a natural DNA sugar. In some embodiments, N ₀ has a native RNA sugar. In some embodiments, N ₀ has a modification sugar, eg, a 2′-F modification sugar. In some embodiments, the sugar of the opposite nucleobase of the target adenosine or N ₀ is arabinofuranose. In some embodiments, the sugar of the opposite nucleobase of the target adenosine or the sugar of N ₀ is , wherein C1' binds to a nucleobase as described herein. In some embodiments, N ₀ has a natural nucleobase. In some embodiments, the nucleobase of N ₀ is C. In some embodiments, the nucleobase of N ₀ is b001A. In some embodiments, N ₀ is deoxycytidine. In some embodiments, N ₀ is cytidine. In some embodiments, N ₀ is 2'-FC (the 2'-OH of cytidine is replaced by -F). In some embodiments, N ₀ is b001A. In some embodiments, N ₀ is Csm15. In some embodiments, N ₀ is b001rA. In some embodiments, N ₀ is b008U. In some embodiments, the nucleobase of N ₀ is not T or U. In some embodiments, the nucleobase of N ₀ is not T. In some embodiments, the nucleobase of N ₀ is not U. In some embodiments, N ₀ does not match A.

In some embodiments, the nucleobase of N ₁ is U and the nucleobase of N _-1 is hypoxanthine. In some embodiments, N ₁ is 2′-FU and N ₋₁ is deoxyinosine. In some embodiments, the nucleobase of N ₀ is as described herein, such as cytosine, b001A, b008U, and the like. In some embodiments, N ₀ is as described herein, such as deoxycytidine, b001A, Csm15, b001rA, b008U, and the like. In some embodiments, N ₀ is deoxycytidine. In some embodiments, N ₀ is b001A. In some embodiments, N ₀ is Csm15. In some embodiments, N ₀ is b001rA. In some embodiments, N ₀ is b008U. In some embodiments, such 5'-N ₁ N ₀ N _-1 -3' is particularly useful for targeting RNA comprising 5'-C A A -3' to edit target adenosine A.

In some embodiments, the nucleobase of N ₁ is T and the nucleobase of N _-1 is hypoxanthine. In some embodiments, N ₁ is thymidine and N ₋₁ is deoxyinosine. In some embodiments, the nucleobase of N ₀ is as described herein, such as cytosine, b001A, b008U, and the like. In some embodiments, N ₀ is as described herein, such as deoxycytidine, b001A, Csm15, b001rA, b008U, and the like. In some embodiments, N ₀ is deoxycytidine. In some embodiments, N ₀ is b001A. In some embodiments, N ₀ is Csm15. In some embodiments, N ₀ is b001rA. In some embodiments, N ₀ is b008U. In some embodiments, such 5'-N ₁ N ₀ N _-1 -3' is particularly useful for targeting RNA comprising 5'-C A A -3' to edit the target adenosine A.

In some embodiments, the nucleobase of N ₁ is C and the nucleobase of N _-1 is hypoxanthine. In some embodiments, N ₁ is 2′-FC and N ₋₁ is deoxyinosine. In some embodiments, the nucleobase of N ₀ is as described herein, such as cytosine, b001A, b008U, and the like. In some embodiments, N ₀ is as described herein, such as deoxycytidine, b001A, Csm15, b001rA, b008U, and the like. In some embodiments, N ₀ is deoxycytidine. In some embodiments, N ₀ is b001A. In some embodiments, N ₀ is Csm15. In some embodiments, N ₀ is b001rA. In some embodiments, N ₀ is b008U. In some embodiments, such 5'-N ₁ N ₀ N _-1 -3' is particularly useful for targeting RNA comprising 5'-C A A -3' to edit the target adenosine A.

In some embodiments, the nucleobase of N ₁ is U and the nucleobase of N _-1 is guanine. In some embodiments, N ₁ is 2′-FU and N ₋₁ is deoxyguanosine. In some embodiments, the nucleobase of N ₀ is as described herein, such as cytosine, b001A, b008U, and the like. In some embodiments, N ₀ is as described herein, such as deoxycytidine, b001A, Csm15, b001rA, b008U, and the like. In some embodiments, N ₀ is deoxycytidine. In some embodiments, N ₀ is b001A. In some embodiments, N ₀ is Csm15. In some embodiments, N ₀ is b001rA. In some embodiments, N ₀ is b008U. In some embodiments, such 5'-N ₁ N ₀ N _-1 -3' is particularly useful for targeting RNA comprising 5'-C A A -3' to edit the target adenosine A. In some embodiments, there are 6 or at least 6 nucleosides on the 3′ side of N ₀ (eg, where there are 6, N ₋₁ to N ₋₆ ).

In some embodiments, the nucleobase of N ₁ is C and the nucleobase of N _-1 is guanine. In some embodiments, N ₁ is 2′-FC and N ₋₁ is deoxyguanosine. In some embodiments, the nucleobase of N ₀ is as described herein, such as cytosine, b001A, b008U, and the like. In some embodiments, N ₀ is as described herein, such as deoxycytidine, b001A, Csm15, b001rA, b008U, and the like. In some embodiments, N ₀ is deoxycytidine. In some embodiments, N ₀ is b001A. In some embodiments, N ₀ is Csm15. In some embodiments, N ₀ is b001rA. In some embodiments, N ₀ is b008U. In some embodiments, such 5'-N ₁ N ₀ N _-1 -3' is particularly useful for targeting RNA comprising 5'-C A A -3' to edit the target adenosine A. In some embodiments, there are 6 or at least 6 nucleosides on the 3′ side of N ₀ (eg, where there are 6, N ₋₁ to N ₋₆ ).

In some embodiments, the nucleobase of N ₁ is U and the nucleobase of N _-1 is hypoxanthine. In some embodiments, N ₁ is 2′-FU and N ₋₁ is deoxyinosine. In some embodiments, the nucleobase of N ₁ is C and the nucleobase of N _-1 is hypoxanthine. In some embodiments, N ₁ is 2′-FC and N ₋₁ is deoxyinosine. In some embodiments, the nucleobase of N ₁ is U and the nucleobase of N _-1 is G. In some embodiments, N ₁ is 2′-FU and N ₋₁ is dG. In some embodiments, the nucleobase of N ₁ is C and the nucleobase of N _-1 is G. In some embodiments, N ₁ is 2′-FC and N ₋₁ is dG. In some embodiments, the nucleobase of N ₁ is G and the nucleobase of N _-1 is hypoxanthine. In some embodiments, N ₁ is 2′-FG and N ₋₁ is deoxyinosine. In some embodiments, the nucleobase of N ₀ is as described herein, such as cytosine, b001A, b008U, and the like. In some embodiments, N ₀ is as described herein, such as deoxycytidine, b001A, Csm15, b001rA, b008U, and the like. In some embodiments, N ₀ is deoxycytidine. In some embodiments, N ₀ is b001A. In some embodiments, N ₀ is Csm15. In some embodiments, N ₀ is b001rA. In some embodiments, N ₀ is b008U. In some embodiments, such 5'-N ₁ N ₀ N _-1 -3' is particularly useful for targeting RNA comprising 5'-C A A -3' to edit the target adenosine A.

In some embodiments, the nucleobase of N ₁ is U and the nucleobase of N _-1 is T. In some embodiments, N ₁ is 2′-FU and N ₋₁ is dT. In some embodiments, the nucleobase of N ₁ is C and the nucleobase of N _-1 is thymine. In some embodiments, N ₁ is 2′-FC and N ₋₁ is dT. In some embodiments, the nucleobase of N ₁ is U and the nucleobase of N _-1 is hypoxanthine. In some embodiments, N ₁ is 2′-FU and N ₋₁ is deoxyinosine. In some embodiments, the nucleobase of N ₁ is U and the nucleobase of N _-1 is G. In some embodiments, N ₁ is 2′-FU and N ₋₁ is dG. In some embodiments, the nucleobase of N ₁ is C and the nucleobase of N _-1 is hypoxanthine. In some embodiments, N ₁ is 2′-FU and N ₋₁ is deoxyinosine. In some embodiments, N ₀ is as described herein, such as deoxycytidine, b001A, Csm15, b001rA, b008U, and the like. In some embodiments, N ₀ is deoxycytidine. In some embodiments, N ₀ is b001A. In some embodiments, N ₀ is Csm15. In some embodiments, N ₀ is b001rA. In some embodiments, N ₀ is b008U. In some embodiments, such 5'-N ₁ N ₀ N _-1 -3' is particularly useful for targeting RNA comprising 5'-A A A -3' to edit the target adenosine A.

In some embodiments, the nucleobase of N ₁ is A and the nucleobase of N _-1 is T. In some embodiments, N ₁ is 2′-FA and N ₋₁ is dT. In some embodiments, the nucleobase of N ₁ is G and the nucleobase of N _-1 is T. In some embodiments, N ₁ is 2′-FG and N ₋₁ is dT. In some embodiments, the nucleobase of N ₁ is A and the nucleobase of N _-1 is hypoxanthine. In some embodiments, N ₁ is 2′-FA and N ₋₁ is deoxyinosine. In some embodiments, the nucleobase of N ₁ is A and the nucleobase of N _-1 is G. In some embodiments, N ₁ is 2′-FA and N ₋₁ is dG. In some embodiments, the nucleobase of N ₁ is A and the nucleobase of N _-1 is C. In some embodiments, N ₁ is 2′-FA and N ₋₁ is dC. In some embodiments, N ₀ is as described herein, such as deoxycytidine, b001A, Csm15, b001rA, b008U, and the like. In some embodiments, N ₀ is deoxycytidine. In some embodiments, N ₀ is b001A. In some embodiments, N ₀ is Csm15. In some embodiments, N ₀ is b001rA. In some embodiments, N ₀ is b008U. In some embodiments, such 5'-N ₁ N ₀ N _-1 -3' is particularly useful for targeting RNA comprising 5'-A A U -3' to edit the target adenosine A.

In some embodiments, the nucleobase of N ₁ is U and the nucleobase of N _-1 is T. In some embodiments, N ₁ is 2′-FU and N ₋₁ is dT. In some embodiments, the nucleobase of N ₁ is C and the nucleobase of N _-1 is T. In some embodiments, N ₁ is 2′-FC and N ₋₁ is dT. In some embodiments, the nucleobase of N ₁ is C and the nucleobase of N _-1 is A. In some embodiments, N ₁ is 2′-FC and N ₋₁ is dA. In some embodiments, the nucleobase of N ₁ is C and the nucleobase of N _-1 is C. In some embodiments, N ₁ is 2′-FC and N ₋₁ is dC. In some embodiments, the nucleobase of N ₁ is U and the nucleobase of N _-1 is hypoxanthine. In some embodiments, N ₁ is 2′-FU and N ₋₁ is deoxyinosine. In some embodiments, the nucleobase of N ₁ is C and the nucleobase of N _-1 is hypoxanthine. In some embodiments, N ₁ is 2′-FC and N ₋₁ is deoxyinosine. In some embodiments, the nucleobase of N ₁ is C and the nucleobase of N _-1 is G. In some embodiments, N ₁ is 2′-FC and N ₋₁ is dG. In some embodiments, the nucleobase of N ₁ is U and the nucleobase of N _-1 is G. In some embodiments, N ₁ is 2′-FU and N ₋₁ is dG. In some embodiments, N ₀ is as described herein, such as deoxycytidine, b001A, Csm15, b001rA, b008U, and the like. In some embodiments, N ₀ is deoxycytidine. In some embodiments, N ₀ is b001A. In some embodiments, N ₀ is Csm15. In some embodiments, N ₀ is b001rA. In some embodiments, N ₀ is b008U. In some embodiments, such 5'-N ₁ N ₀ N _-1 -3' is particularly useful for targeting RNA comprising 5'-A A G -3' to edit the target adenosine A.

In some embodiments, the nucleobase of N ₁ is G and the nucleobase of N _-1 is T. In some embodiments, N ₁ is 2′-FG and N ₋₁ is dT. In some embodiments, the nucleobase of N ₁ is G and the nucleobase of N _-1 is A. In some embodiments, N ₁ is 2′-FG and N ₋₁ is dA. In some embodiments, the nucleobase of N ₁ is G and the nucleobase of N _-1 is C. In some embodiments, N ₁ is 2′-FG and N ₋₁ is dC. In some embodiments, the nucleobase of N ₁ is G and the nucleobase of N _-1 is hypoxanthine. In some embodiments, N ₁ is 2′-FG and N ₋₁ is deoxyinosine. In some embodiments, the nucleobase of N ₁ is G and the nucleobase of N _-1 is G. In some embodiments, N ₁ is 2′-FG and N ₋₁ is dG. In some embodiments, N ₀ is as described herein, such as deoxycytidine, b001A, Csm15, b001rA, b008U, and the like. In some embodiments, N ₀ is deoxycytidine. In some embodiments, N ₀ is b001A. In some embodiments, N ₀ is Csm15. In some embodiments, N ₀ is b001rA. In some embodiments, N ₀ is b008U. In some embodiments, such 5'-N ₁ N ₀ N _-1 -3' is particularly useful for targeting RNA comprising 5'-A A C -3' to edit the target adenosine A.

In some embodiments, the nucleobase of N ₁ is U and the nucleobase of N _-1 is A. In some embodiments, N ₁ is 2′-FU and N ₋₁ is dA. In some embodiments, the nucleobase of N ₁ is C and the nucleobase of N _-1 is A. In some embodiments, N ₁ is 2′-FC and N ₋₁ is dA. In some embodiments, the nucleobase of N ₁ is A and the nucleobase of N _-1 is A. In some embodiments, N ₁ is 2′-FA and N ₋₁ is dA. In some embodiments, the nucleobase of N ₁ is G and the nucleobase of N _-1 is A. In some embodiments, N ₁ is 2′-FG and N ₋₁ is dA. In some embodiments, the nucleobase of N ₁ is U and the nucleobase of N _-1 is hypoxanthine. In some embodiments, N ₁ is 2'-FU and N _-1 is deoxyinosine. In some embodiments, the nucleobase of N ₁ is C and the nucleobase of N _-1 is hypoxanthine. In some embodiments, N ₁ is 2′-FC and N ₋₁ is deoxyinosine. In some embodiments, N ₀ is as described herein, such as deoxycytidine, b001A, Csm15, b001rA, b008U, and the like. In some embodiments, N ₀ is deoxycytidine. In some embodiments, N ₀ is b001A. In some embodiments, N ₀ is Csm15. In some embodiments, N ₀ is b001rA. In some embodiments, N ₀ is b008U. In some embodiments, such 5'-N ₁ N ₀ N _-1 -3' is particularly useful for targeting RNA comprising 5'-U A A -3' to edit the target adenosine A.

In some embodiments, the nucleobase of N ₁ is A and the nucleobase of N _-1 is A. In some embodiments, N ₁ is 2′-FA and N ₋₁ is dA. In some embodiments, the nucleobase of N ₁ is G and the nucleobase of N _-1 is A. In some embodiments, N ₁ is 2′-FG and N ₋₁ is dA. In some embodiments, the nucleobase of N ₁ is C and the nucleobase of N _-1 is A. In some embodiments, N ₁ is 2′-FC and N ₋₁ is dA. In some embodiments, the nucleobase of N ₁ is U and the nucleobase of N _-1 is A. In some embodiments, N ₁ is 2′-FU and N ₋₁ is dA. In some embodiments, the nucleobase of N ₁ is A and the nucleobase of N _-1 is hypoxanthine. In some embodiments, N ₁ is 2′-FA and N ₋₁ is deoxyinosine. In some embodiments, the nucleobase of N ₁ is G and the nucleobase of N _-1 is hypoxanthine. In some embodiments, N ₁ is 2′-FG and N ₋₁ is deoxyinosine. In some embodiments, N ₀ is as described herein, such as deoxycytidine, b001A, Csm15, b001rA, b008U, and the like. In some embodiments, N ₀ is deoxycytidine. In some embodiments, N ₀ is b001A. In some embodiments, N ₀ is Csm15. In some embodiments, N ₀ is b001rA. In some embodiments, N ₀ is b008U. In some embodiments, such 5'-N ₁ N ₀ N _-1 -3' is particularly useful for targeting RNA comprising 5'-U A U -3' to edit the target adenosine A.

In some embodiments, the nucleobase of N ₁ is C and the nucleobase of N _-1 is A. In some embodiments, N ₁ is 2′-FC and N ₋₁ is dA. In some embodiments, the nucleobase of N ₁ is U and the nucleobase of N _-1 is A. In some embodiments, N ₁ is 2′-FU and N ₋₁ is dA. In some embodiments, the nucleobase of N ₁ is C and the nucleobase of N _-1 is T. In some embodiments, N ₁ is 2′-FC and N ₋₁ is dT. In some embodiments, the nucleobase of N ₁ is A and the nucleobase of N _-1 is A. In some embodiments, N ₁ is 2′-FA and N ₋₁ is dA. In some embodiments, the nucleobase of N ₁ is G and the nucleobase of N _-1 is A. In some embodiments, N ₁ is 2′-FG and N ₋₁ is dA. In some embodiments, the nucleobase of N ₁ is C and the nucleobase of N _-1 is C. In some embodiments, N ₁ is 2′-FC and N ₋₁ is dC. In some embodiments, the nucleobase of N ₁ is U and the nucleobase of N _-1 is hypoxanthine. In some embodiments, N ₁ is 2′-FU and N ₋₁ is deoxyinosine. In some embodiments, the nucleobase of N ₁ is C and the nucleobase of N _-1 is hypoxanthine. In some embodiments, N ₁ is 2′-FC and N ₋₁ is deoxyinosine. In some embodiments, the nucleobase of N ₁ is C and the nucleobase of N _-1 is G. In some embodiments, N ₁ is 2′-FC and N ₋₁ is dG. In some embodiments, N ₀ is as described herein, such as deoxycytidine, b001A, Csm15, b001rA, b008U, and the like. In some embodiments, N ₀ is deoxycytidine. In some embodiments, N ₀ is b001A. In some embodiments, N ₀ is Csm15. In some embodiments, N ₀ is b001rA. In some embodiments, N ₀ is b008U. In some embodiments, such 5'-N ₁ N ₀ N _-1 -3' is particularly useful for targeting RNA comprising 5'-U A G -3' to edit the target adenosine A.

In some embodiments, the nucleobase of N ₁ is G and the nucleobase of N _-1 is T. In some embodiments, N ₁ is 2′-FG and N ₋₁ is dT. In some embodiments, the nucleobase of N ₁ is U and the nucleobase of N _-1 is A. In some embodiments, N ₁ is 2′-FU and N ₋₁ is dA. In some embodiments, the nucleobase of N ₁ is A and the nucleobase of N _-1 is A. In some embodiments, N ₁ is 2′-FA and N ₋₁ is dA. In some embodiments, the nucleobase of N ₁ is C and the nucleobase of N _-1 is A. In some embodiments, N ₁ is 2′-FC and N ₋₁ is dA. In some embodiments, the nucleobase of N ₁ is G and the nucleobase of N _-1 is A. In some embodiments, N ₁ is 2′-FG and N ₋₁ is dA. In some embodiments, the nucleobase of N ₁ is G and the nucleobase of N _-1 is C. In some embodiments, N ₁ is 2′-FG and N ₋₁ is dC. In some embodiments, the nucleobase of N ₁ is G and the nucleobase of N _-1 is hypoxanthine. In some embodiments, N ₁ is 2′-FG and N ₋₁ is deoxyinosine. In some embodiments, the nucleobase of N ₁ is G and the nucleobase of N _-1 is G. In some embodiments, N ₁ is 2′-FG and N ₋₁ is dG. In some embodiments, N ₀ is as described herein, such as deoxycytidine, b001A, Csm15, b001rA, b008U, and the like. In some embodiments, N ₀ is deoxycytidine. In some embodiments, N ₀ is b001A. In some embodiments, N ₀ is Csm15. In some embodiments, N ₀ is b001rA. In some embodiments, N ₀ is b008U. In some embodiments, such 5'-N ₁ N ₀ N _-1 -3' is particularly useful for targeting RNA comprising 5'-U A C -3' to edit the target adenosine A.

In some embodiments, the nucleobase of N ₁ is C and the nucleobase of N _-1 is C. In some embodiments, N ₁ is 2′-FC and N ₋₁ is dC. In some embodiments, the nucleobase of N ₁ is U and the nucleobase of N _-1 is hypoxanthine. In some embodiments, N ₁ is 2′-FU and N ₋₁ is deoxyinosine. In some embodiments, the nucleobase of N ₁ is C and the nucleobase of N _-1 is hypoxanthine. In some embodiments, N ₁ is 2′-FC and N ₋₁ is deoxyinosine. In some embodiments, the nucleobase of N ₁ is U and the nucleobase of N _-1 is G. In some embodiments, N ₁ is 2′-FU and N ₋₁ is dG. In some embodiments, the nucleobase of N ₁ is C and the nucleobase of N _-1 is G. In some embodiments, N ₁ is 2′-FC and N ₋₁ is dG. In some embodiments, N ₀ is as described herein, such as deoxycytidine, b001A, Csm15, b001rA, b008U, and the like. In some embodiments, N ₀ is deoxycytidine. In some embodiments, N ₀ is b001A. In some embodiments, N ₀ is Csm15. In some embodiments, N ₀ is b001rA. In some embodiments, N ₀ is b008U. In some embodiments, such 5'-N ₁ N ₀ N _-1 -3' is particularly useful for targeting RNA comprising 5'-GA A A -3' to edit the target adenosine A.

In some embodiments, the nucleobase of N ₁ is A and the nucleobase of N _-1 is hypoxanthine. In some embodiments, N ₁ is 2′-FA and N ₋₁ is deoxyinosine. In some embodiments, the nucleobase of N ₁ is G and the nucleobase of N _-1 is hypoxanthine. In some embodiments, N ₁ is 2′-FG and N ₋₁ is deoxyinosine. In some embodiments, the nucleobase of N ₁ is A and the nucleobase of N _-1 is G. In some embodiments, N ₁ is 2′-FA and N ₋₁ is dG. In some embodiments, the nucleobase of N ₁ is G and the nucleobase of N _-1 is G. In some embodiments, N ₁ is 2′-FG and N ₋₁ is dG. In some embodiments, the nucleobase of N ₁ is A and the nucleobase of N _-1 is A. In some embodiments, N ₁ is 2′-FA and N ₋₁ is dA. In some embodiments, N ₀ is as described herein, such as deoxycytidine, b001A, Csm15, b001rA, b008U, and the like. In some embodiments, N ₀ is deoxycytidine. In some embodiments, N ₀ is b001A. In some embodiments, N ₀ is Csm15. In some embodiments, N ₀ is b001rA. In some embodiments, N ₀ is b008U. In some embodiments, such 5'-N ₁ N ₀ N _-1 -3' is particularly useful for targeting RNA comprising 5'-GA U -3' to edit the target adenosine A.

In some embodiments, the nucleobase of N ₁ is C and the nucleobase of N _-1 is T. In some embodiments, N ₁ is 2′-FC and N ₋₁ is dT. In some embodiments, the nucleobase of N ₁ is C and the nucleobase of N _-1 is A. In some embodiments, N ₁ is 2′-FC and N ₋₁ is dA. In some embodiments, the nucleobase of N ₁ is C and the nucleobase of N _-1 is C. In some embodiments, N ₁ is 2′-FC and N ₋₁ is dC. In some embodiments, the nucleobase of N ₁ is C and the nucleobase of N _-1 is hypoxanthine. In some embodiments, N ₁ is 2′-FC and N ₋₁ is deoxyinosine. In some embodiments, the nucleobase of N ₁ is U and the nucleobase of N _-1 is G. In some embodiments, N ₁ is 2′-FU and N ₋₁ is dG. In some embodiments, the nucleobase of N ₁ is C and the nucleobase of N _-1 is G. In some embodiments, N ₁ is 2′-FC and N ₋₁ is dG. In some embodiments, N ₀ is as described herein, such as deoxycytidine, b001A, Csm15, b001rA, b008U, and the like. In some embodiments, N ₀ is deoxycytidine. In some embodiments, N ₀ is b001A. In some embodiments, N ₀ is Csm15. In some embodiments, N ₀ is b001rA. In some embodiments, N ₀ is b008U. In some embodiments, such 5'-N ₁ N ₀ N _-1 -3' is particularly useful for targeting RNA comprising 5'-GA G -3' to edit the target adenosine A.

In some embodiments, the nucleobase of N ₁ is G and the nucleobase of N _-1 is A. In some embodiments, N ₁ is 2′-FG and N ₋₁ is dA. In some embodiments, the nucleobase of N ₁ is G and the nucleobase of N _-1 is hypoxanthine. In some embodiments, N ₁ is 2′-FG and N ₋₁ is deoxyinosine. In some embodiments, the nucleobase of N ₁ is G and the nucleobase of N _-1 is G. In some embodiments, N ₁ is 2′-FG and N ₋₁ is dG. In some embodiments, the nucleobase of N ₁ is G and the nucleobase of N _-1 is T. In some embodiments, N ₁ is 2′-FG and N ₋₁ is dT. In some embodiments, the nucleobase of N ₁ is G and the nucleobase of N _-1 is C. In some embodiments, N ₁ is 2′-FG and N ₋₁ is dC. In some embodiments, N ₀ is as described herein, such as deoxycytidine, b001A, Csm15, b001rA, b008U, and the like. In some embodiments, N ₀ is deoxycytidine. In some embodiments, N ₀ is b001A. In some embodiments, N ₀ is Csm15. In some embodiments, N ₀ is b001rA. In some embodiments, N ₀ is b008U. In some embodiments, such 5'-N ₁ N ₀ N _-1 -3' is particularly useful for targeting RNA comprising 5'-GA C -3' to edit the target adenosine A.

In some embodiments, the nucleobase of N ₁ is U and the nucleobase of N _-1 is hypoxanthine. In some embodiments, N ₁ is 2′-FU and N ₋₁ is deoxyinosine. In some embodiments, the nucleobase of N ₁ is A and the nucleobase of N _-1 is hypoxanthine. In some embodiments, N ₁ is 2′-FA and N ₋₁ is deoxyinosine. In some embodiments, the nucleobase of N ₁ is A and the nucleobase of N _-1 is G. In some embodiments, N ₁ is 2′-FA and N ₋₁ is dG. In some embodiments, the nucleobase of N ₁ is G and the nucleobase of N _-1 is hypoxanthine. In some embodiments, N ₁ is 2′-FG and N ₋₁ is deoxyinosine. In some embodiments, the nucleobase of N ₁ is C and the nucleobase of N _-1 is hypoxanthine. In some embodiments, N ₁ is 2′-FC and N ₋₁ is deoxyinosine. In some embodiments, N ₀ is as described herein, such as deoxycytidine, b001A, Csm15, b001rA, b008U, and the like. In some embodiments, N ₀ is deoxycytidine. In some embodiments, N ₀ is b001A. In some embodiments, N ₀ is Csm15. In some embodiments, N ₀ is b001rA. In some embodiments, N ₀ is b008U. In some embodiments, such 5'-N ₁ N ₀ N _-1 -3' is particularly useful for targeting RNA comprising 5'-C A U -3' to edit the target adenosine A.

In some embodiments, the nucleobase of N ₁ is U and the nucleobase of N _-1 is hypoxanthine. In some embodiments, N ₁ is 2′-FU and N ₋₁ is deoxyinosine. In some embodiments, the nucleobase of N ₁ is C and the nucleobase of N _-1 is hypoxanthine. In some embodiments, N ₁ is 2′-FC and N ₋₁ is deoxyinosine. In some embodiments, the nucleobase of N ₁ is G and the nucleobase of N _-1 is hypoxanthine. In some embodiments, N ₁ is 2′-FG and N ₋₁ is deoxyinosine. In some embodiments, the nucleobase of N ₁ is A and the nucleobase of N _-1 is hypoxanthine. In some embodiments, N ₁ is 2′-FA and N ₋₁ is deoxyinosine. In some embodiments, the nucleobase of N ₁ is U and the nucleobase of N _-1 is G. In some embodiments, N ₁ is 2′-FU and N ₋₁ is dG. In some embodiments, the nucleobase of N ₁ is C and the nucleobase of N _-1 is G. In some embodiments, N ₁ is 2′-FC and N ₋₁ is dG. In some embodiments, the nucleobase of N ₁ is G and the nucleobase of N _-1 is G. In some embodiments, N ₁ is 2′-FG and N ₋₁ is dG. In some embodiments, N ₀ is as described herein, such as deoxycytidine, b001A, Csm15, b001rA, b008U, and the like. In some embodiments, N ₀ is deoxycytidine. In some embodiments, N ₀ is b001A. In some embodiments, N ₀ is Csm15. In some embodiments, N ₀ is b001rA. In some embodiments, N ₀ is b008U. In some embodiments, such 5'-N ₁ N ₀ N _-1 -3' is particularly useful for targeting RNA comprising 5'-C A G -3' to edit the target adenosine A.

In some embodiments, the nucleobase of N ₁ is A and the nucleobase of N _-1 is hypoxanthine. In some embodiments, N ₁ is 2′-FA and N ₋₁ is deoxyinosine. In some embodiments, the nucleobase of N ₁ is G and the nucleobase of N _-1 is hypoxanthine. In some embodiments, N ₁ is 2′-FG and N ₋₁ is deoxyinosine. In some embodiments, the nucleobase of N ₁ is G and the nucleobase of N _-1 is G. In some embodiments, N ₁ is 2′-FG and N ₋₁ is dG. In some embodiments, the nucleobase of N ₁ is U and the nucleobase of N _-1 is hypoxanthine. In some embodiments, N ₁ is 2′-FU and N ₋₁ is deoxyinosine. In some embodiments, N ₀ is as described herein, such as deoxycytidine, b001A, Csm15, b001rA, b008U, and the like. In some embodiments, N ₀ is deoxycytidine. In some embodiments, N ₀ is b001A. In some embodiments, N ₀ is Csm15. In some embodiments, N ₀ is b001rA. In some embodiments, N ₀ is b008U. In some embodiments, such 5'-N ₁ N ₀ N _-1 -3' is particularly useful for targeting RNA comprising 5'-C A C -3' to edit the target adenosine A.

In some embodiments, the nucleobase of N ₁ is U and the nucleobase of N _-1 is A. In some embodiments, N ₁ is 2′-FU and N ₋₁ is dA. In some embodiments, the nucleobase of N ₁ is C and the nucleobase of N _-1 is A. In some embodiments, N ₁ is 2′-FC and N ₋₁ is dA. In some embodiments, the nucleobase of N ₁ is G and the nucleobase of N _-1 is A. In some embodiments, N ₁ is 2′-FG and N ₋₁ is dA. In some embodiments, the nucleobase of N ₁ is C and the nucleobase of N _-1 is hypoxanthine. In some embodiments, N ₁ is 2′-FC and N ₋₁ is deoxyinosine. In some embodiments, the nucleobase of N ₁ is U and the nucleobase of N _-1 is hypoxanthine. In some embodiments, N ₁ is 2′-FU and N ₋₁ is deoxyinosine. In some embodiments, the nucleobase of N ₁ is A and the nucleobase of N _-1 is A. In some embodiments, N ₁ is 2′-FA and N ₋₁ is dA. In some embodiments, N ₀ is b001rA. In some embodiments, N ₀ is b008U. In some embodiments, such 5'-N ₁ N ₀ N _-1 -3' is particularly useful for targeting RNA comprising 5'-U A G -3' to edit the target adenosine A.

In some embodiments, nucleobase U can be replaced with T without lowering the level of editing. In some embodiments, the nucleobase U may be replaced with T to increase the level of editing. In some embodiments, 2'-FU can be replaced with thymidine. See FIG. 19 for example. In some embodiments, N ₁ is thymidine. In some embodiments, N ₁ is thymidine and N ₀ is as described herein, eg b001A, b008U, etc. In some embodiments, N ₁ is thymidine, N ₀ is as described herein, eg b001A, b008U, etc., and N ₋₁ is I.

In some embodiments, N ₀ is a wobble or mismatch with respect to A when aligned to a target sequence and/or hybridized to a target nucleic acid. In some embodiments, N ₁ is not identical to its opposite nucleobase. In some embodiments, N ₋₁ is not a match to its opposite nucleobase. In some embodiments, two of N ₋₁ , N ₀ and N ₁ are not independently identical to their opposite nucleobases. In some embodiments, N ₀ and N ₁ are not independently identical to their opposite nucleobases. In some embodiments, N ₀ and N ₋₁ are not independently identical to their opposite nucleobases. In some implementations, if there is no match, it is a wobble. In some implementations, if it is not a match, it is a match. In some embodiments, the nucleobase of N ₁ is C and its opposite nucleobase is A. In some embodiments, more nucleosides (eg, 6 or more) to the 3' side of N ₀ may allow for more mismatches/wobbles of the 5'-N ₁ N ₀ N _-1 -3' .

In some embodiments, each internucleotidic linkage linked to N ₀ is independently an Sp phosphorothioate internucleotidic linkage. In some embodiments, each internucleotide linkage linked to N ₁ is independently an S p phosphorothioate internucleotide linkage. In some embodiments, the internucleotidic linkage linked to N _-1 is a non-negatively charged internucleotidic linkage. In some embodiments, the internucleotide linkage linked to N _-1 is a neutral internucleotide linkage. In some embodiments, the internucleotide linkage linked to N _-1 is a phosphoryl guanidine internucleotide linkage. In some embodiments, the internucleotide linkage linked to N _-1 is n001. In some embodiments, the phosphoryl guanidine internucleotide linkage linked to N _-1 (eg, at position 3' thereof), eg n001, is chirally controlled and is R p . In some embodiments, the phosphorylguanidine internucleotidic linkage linked to N _-1 (eg, at position 3' thereof), eg, n001, is chirally controlled and is Sp (eg, in some embodiments , where N _-1 is dI).

base sequence

As will be appreciated by those skilled in the art, the structural features of the present invention, such as nucleobase modifications, sugar modifications, internucleotidic linkage modifications, linkage phosphorus stereochemistry, etc., and combinations thereof, can be used with a variety of suitable base sequences to achieve the desired properties and/or Alternatively, oligonucleotides and compositions having an activity may be provided. For example, an oligonucleotide for adenosine modification (eg, conversion to I in the presence of an ADAR protein) generally has a sequence sufficiently complementary to the sequence of the target nucleic acid comprising the target adenosine. The nucleoside opposite the target adenosine can be present at various positions of the oligonucleotide. In some embodiments, the one or more opposing nucleosides are in the first domain. In some embodiments, the one or more opposing nucleosides are in the second domain. In some embodiments, the one or more opposing nucleosides are in the first subdomain. In some embodiments, the one or more opposing nucleosides are in a second subdomain. In some embodiments, the one or more opposing nucleosides are in a third subdomain. The oligonucleotides of the present invention may target one or more target adenosines. In some embodiments, the one or more opposing nucleosides are each independently in a portion having structural features of the second subdomain, and each independently has one or more or all structural features of the opposing nucleosides as described herein. In many embodiments, eg for targeting G to A mutations, the oligonucleotide can selectively target only one target adenosine for modification, eg converted to I by ADAR. In some embodiments, the opposite nucleoside is closer to the 3'-end than to the 5'-end of the oligonucleotide.

In some embodiments, the oligonucleotide is a base sequence described herein (eg, in a table) or a portion thereof (eg, 10-50, 10-40, 10-30, 10-20, or 10, 11, has a span of 12, 13, 14, 15, 16, 17, 18, 19, 20, or at least 10, at least 15, at least 20, at least 25 contiguous nucleobases (0-5 (e.g. , 0, 1, 2, 3, 4, or 5 mismatches), each T may be independently substituted for U and vice versa. In some embodiments, an oligonucleotide comprises a base sequence described herein or a portion thereof, wherein the portion spans at least 10 contiguous nucleobases, or at least 15 contiguous nucleobases with 0-5 mismatches. In some embodiments, a provided oligonucleotide has a base sequence described herein, or a portion thereof, wherein the portion is a span of at least 10 contiguous nucleobases, or a span of at least 10 contiguous nucleobases with 1-5 mismatches; Each T may be independently substituted with U and vice versa.

In some embodiments, the nucleotide sequence of the oligonucleotide is 10 to 60, optionally concatenated, sequences identical to or complementary to the nucleotide sequence of a nucleic acid, eg, a gene or a transcript thereof (eg, mRNA). For example, about or at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32 , 33, 34, 35, 36, 37, 38, 39, 40, 45, 50, 55, 60; in some embodiments, at least 15; in some embodiments, at least 16; in some embodiments, at least 17; in some embodiments, at least 18; in some embodiments, at least 19; in some embodiments, at least 20; in some embodiments, at least 21; in some embodiments, at least 22; in some embodiments, at least 22; in some embodiments In examples, at least 23; in some embodiments, at least 24; in some embodiments, at least 25; in some embodiments, at least 26; in some embodiments, at least 27; in some embodiments, at least 28 in some embodiments, at least 29; in some embodiments, at least 30; in some embodiments, at least 31; in some embodiments, at least 32; in some embodiments, at least 33; in some embodiments , at least 34; in some embodiments, at least 35) bases. In some embodiments, the base sequence of the oligonucleotide is or comprises a sequence complementary to a target sequence of a gene or transcript thereof. In some embodiments, the sequence is 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 45, 50, 55, 60 or more nucleobases in length.

In some embodiments, a target sequence is or comprises a characteristic sequence of a nucleic acid sequence in that it defines a nucleic acid sequence compared to others in a related organism (e.g., a characteristic sequence is another genomic nucleic acid sequence (e.g., , gene) or transcripts thereof or have at least various discrepancies therefrom). In some embodiments, a characteristic sequence of a transcript defines that transcript compared to other transcripts in a related organism (e.g., in some embodiments, a characteristic sequence is from a different nucleic acid sequence (e.g., a different gene)). absent in transcribed transcripts). In some embodiments, transcript variants of a nucleic acid sequence (eg, mRNA variants of a gene) may share a common characteristic sequence that defines them, for example from transcripts of other genes. In some embodiments, the characteristic sequence comprises a target adenosine. In some embodiments, the oligonucleotide selectively forms a duplex with a nucleic acid comprising a target adenosine, and the target adenosine is within the duplex region and can be modified by a protein such as ADAR1 or ADAR2.

The base sequence of a given oligonucleotide is generally a target nucleic acid, e.g., an RNA transcript (e.g., pre-mRNA, mature mRNA, etc.) It has sufficient length and complementarity for In some embodiments, the oligonucleotide is complementary to a portion of the target RNA sequence that includes the target adenosine (as understood by those skilled in the art, in many cases the target nucleic acid is longer than an oligonucleotide of the invention, and complementarity is the greater of the two). can be appropriately evaluated based on short oligonucleotides). In some embodiments, the base sequence of the oligonucleotide has 90% or more identity with the base sequence of the oligonucleotide disclosed in the table, and each T may be independently substituted with U and vice versa. In some embodiments, the base sequence of the oligonucleotide has 95% or more identity with the base sequence of the oligonucleotide disclosed in the table, and each T may be independently substituted with U and vice versa. In some embodiments, the base sequence of the oligonucleotide is 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 of the oligonucleotides disclosed in the table. . A base is free (eg, no nucleobases are present in a nucleotide).

In some embodiments, the present invention relates to an oligonucleotide having a base sequence comprising the base sequence of any oligonucleotide disclosed herein, wherein each T may be independently replaced with a U or vice versa.

In some embodiments, the present invention relates to an oligonucleotide having a base sequence that is the base sequence of any oligonucleotide disclosed herein, wherein each T may be independently replaced with a U or vice versa.

In some embodiments, the present invention relates to an oligonucleotide having a base sequence comprising at least 15 contiguous bases of the base sequence of any oligonucleotide disclosed herein, wherein each T may be independently replaced with a U and The reverse is also possible.

In some embodiments, the present invention relates to an oligonucleotide having a base sequence that is at least 90% identical to the base sequence of any oligonucleotide disclosed herein, wherein each T may be independently replaced with a U and vice versa .

In some embodiments, the present invention relates to an oligonucleotide having a base sequence that is at least 95% identical to the base sequence of any oligonucleotide disclosed herein, wherein each T may be independently replaced with a U and vice versa .

In some embodiments, the base sequence of the oligonucleotide is, comprises, or is 10 to 40 base sequences of any oligonucleotide described herein, such as 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40 contiguous bases, each T independently can be replaced by U and vice versa.

In some embodiments, the oligonucleotide is an oligonucleotide set forth in the tables herein.

In some embodiments, the base sequence of the oligonucleotide is complementary to the base sequence of a target nucleic acid, eg, a portion comprising a target adenosine.

In some embodiments, an oligonucleotide has a base sequence comprising at least 15 contiguous bases (e.g., 15, 16, 17, 18, 19, or 20) of oligonucleotides in the table, wherein each T is may be independently substituted with U and vice versa.

In some embodiments, the oligonucleotide is a sequence of bases listed in any table or a portion thereof (e.g., 10-40, e.g., 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 , 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40 nucleobases) (each T can be independently replaced by U) and vice versa), and/or sugar, nucleobase, and/or internucleotide linkage modifications and/or stereochemistry, and/or patterns thereof, and/or additional chemical properties listed in any Table. moieties (in addition to oligonucleotide chains, eg, targeting moieties, lipid moieties, carbohydrate moieties, etc.).

In some embodiments, the terms “complementary,” “fully complementary,” and “substantially complementary” refer to a base match between an oligonucleotide and a target sequence, as would be understood by one skilled in the art in the context in which they are used. can be used Note that substituting T for U and vice versa generally does not change the amount of complementarity. As used herein, an oligonucleotide that is “substantially complementary” to a target sequence is largely or mostly complementary, but need not be 100% complementary. In some embodiments, a substantially complementary sequence (eg an oligonucleotide) has one or more, for example 1, 2, 3, 4, or 5 mismatches when maximally aligned to a target sequence. In some embodiments, an oligonucleotide has a base sequence that is substantially complementary to a target sequence of a target nucleic acid. In some embodiments, an oligonucleotide has a base sequence that is substantially complementary to the complement of an oligonucleotide sequence disclosed herein. As will be appreciated by those skilled in the art, in some embodiments, the sequence of an oligonucleotide need not be 100% complementary to a target for the oligonucleotide to perform its function (eg, converting an A to an I in a nucleic acid). In some embodiments, mismatches are tolerated at the 5' and/or 3' ends or middle of the oligonucleotide. In some embodiments, as shown herein, one or more mismatches in adenosine modifications are preferred. In some embodiments, the oligonucleotide comprises a portion for complementarity to the target nucleic acid, and optionally a portion not primarily for complementarity to the target nucleic acid (e.g., in some embodiments, the oligonucleotide comprises a portion for protein binding) may include). In some embodiments, the base sequence of a provided oligonucleotide is completely complementary to the target sequence (A-T/U and C-G base pairing). In some embodiments, the base sequence of a provided oligonucleotide is completely complementary to the target sequence except at the nucleoside opposite the target nucleoside (eg, adenosine) (A-T/U and C-G base pairing).

In some embodiments, the present invention provides oligonucleotides comprising sequences found in the oligonucleotides listed in the table, wherein one or more U's are independently and optionally replaced with T's, and vice versa. In some embodiments, an oligonucleotide can include at least one T and/or at least one U. In some embodiments, the present invention provides oligonucleotides comprising sequences found in oligonucleotides listed in the tables herein, wherein the sequences have greater than 50% identity to the sequences of oligonucleotides listed in the tables. In some embodiments, the present invention provides an oligonucleotide whose base sequence is the sequence of an oligonucleotide set forth in the table, wherein each T may be independently replaced with a U and vice versa. In some embodiments, the present invention provides oligonucleotides comprising sequences found in oligonucleotides in the Tables, wherein the oligonucleotides have backbone linkage patterns, backbone chiral center patterns, and/or backbones of the same oligonucleotide or other oligonucleotides in the Tables herein. It has a phosphorus deformation pattern.

In some embodiments, the present invention provides an oligonucleotide having a base sequence that is, comprises, or comprises a portion of an oligonucleotide sequence disclosed herein (eg, a table), wherein each T is independently U may be replaced with U and vice versa, and optionally the oligonucleotide is a chemical modification, stereochemistry, format, additional chemical moieties (e.g., targeting moieties, lipid moieties, carbohydrate moieties, etc.) described herein. , and/or other structural features.

In some embodiments, "some" (e.g., a portion of a sequence or variation pattern or other structural element) is at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 , 17, 18, 19, or 20 monomer units in length.

One skilled in the art upon reading this disclosure will understand that the techniques herein can be used to target a variety of target nucleic acids, including target adenosine, for editing. In some embodiments, a target nucleic acid is a transcript of a PiZZ allele. In some embodiments, the target adenosine... It is atcgac A agaaagggactgaagc... In some embodiments, an oligonucleotide of the invention has a suitable base sequence to have sufficient complementarity to selectively form a duplex with a portion of a transcript comprising a target adenosine for editing.

As described herein, the nucleoside opposite the target nucleoside (eg, A) can be located at a variety of positions. In some embodiments, the opposite nucleoside is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 from the 5'-end of the oligonucleotide. , 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 or more positions. In some embodiments, it is at a position three or more times from the 5'-end of the oligonucleotide. In some embodiments, it is at a position 4 or more times from the 5'-end of the oligonucleotide. In some embodiments, it is at a position 5 or more times from the 5'-end of the oligonucleotide. In some embodiments, it is located 6 or more times from the 5'-end of the oligonucleotide. In some embodiments, it is at position 7 or more from the 5'-end of the oligonucleotide. In some embodiments, it is at position 8 or more from the 5'-end of the oligonucleotide. In some embodiments, it is at position 9 or more from the 5'-end of the oligonucleotide. In some embodiments, it is at a position 10 or more times from the 5'-end of the oligonucleotide. In some embodiments, the opposite nucleoside is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 from the 3'-end of the oligonucleotide. , 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 or more positions. In some embodiments, it is at a position three or more times from the 3'-end of the oligonucleotide. In some embodiments, it is at a position 4 or more times from the 3'-end of the oligonucleotide. In some embodiments, it is at position 5 or more from the 3'-end of the oligonucleotide. In some embodiments, it is at position 6 or more from the 3'-end of the oligonucleotide. In some embodiments, it is at position 7 or more from the 3'-end of the oligonucleotide. In some embodiments, it is at position 8 or more from the 3'-end of the oligonucleotide. In some embodiments, it is at position 9 or more from the 3'-end of the oligonucleotide. In some embodiments, it is at position 10 or more from the 3'-end of the oligonucleotide. In some embodiments, the nucleobase at position 1 from the 5'-end and/or 3'-end is complementary to the corresponding nucleobase of the target sequence when aligned for maximal complementarity. In some implementations, a specific position (eg, position 6, 7, or 8) may provide higher editing efficiency.

Examples include certain exemplary base sequences, nucleobase modifications and patterns thereof, sugar modifications and patterns thereof, internucleotidic linkages and patterns thereof, linkage phosphorus stereochemistry and patterns thereof, linkers, and/or additional chemical moieties, and the like. Specific oligonucleotides that do are shown in Table 1 below. In particular, such oligonucleotides can be used to correct a G to A mutation in a gene or gene product (eg, by converting A to I). In some embodiments, stereorandom oligonucleotide compositions are listed in the table. In some embodiments, the present invention provides chirally controlled oligonucleotide compositions.

In some embodiments, a base sequence is or comprises a specific sequence. In some embodiments, the nucleotide sequence is a nucleotide sequence complementary to or complementary to a nucleotide sequence comprising the same. In some embodiments, the base sequence is or comprises a sequence that differs from the specific sequence at no more than 1, 2, 3, 4 or 5 positions. In some embodiments, the base sequence is about 15-30 (e.g., 15-25, 15-20, 20-30, 15, 16) of a particular sequence in no more than 1, 2, 3, 4 or 5 positions. , 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30) contiguous nucleobases. In some embodiments, the base sequence is or comprises a sequence that differs from the specified sequence at no more than one position. In some embodiments, the base sequence is or comprises a sequence that differs from the specified sequence at no more than two positions. In some embodiments, the base sequence is or comprises a sequence that differs from the specified sequence at no more than 3 positions. In some embodiments, the base sequence is or comprises a sequence that differs from the specified sequence at no more than 4 positions. In some embodiments, the base sequence is or comprises a sequence that differs from the specified sequence at no more than 5 positions. In some embodiments, a particular sequence is or comprises a base sequence selected from Table 1 (eg, any of Tables 1A-1I, 1J-10, etc.). In some embodiments, the specific sequence is 5-30, 10-30, 15-30, 20-30, or 25-30 (eg, 5, 6, 7, 8, 9 , 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30) consecutive bases, or include In some embodiments, a particular sequence is or comprises 10 contiguous bases within a sequence of bases selected from Table 1. In some embodiments, a particular sequence is or comprises 11 contiguous bases within a base sequence selected from Table 1. In some embodiments, a particular sequence is or comprises 12 contiguous bases within a base sequence selected from Table 1. In some embodiments, a particular sequence is or comprises 13 contiguous bases within a sequence selected from Table 1. In some embodiments, a particular sequence is or comprises 14 contiguous bases within a base sequence selected from Table 1. In some embodiments, a particular sequence is or comprises 15 contiguous bases within a base sequence selected from Table 1. In some embodiments, a particular sequence is or comprises 16 contiguous bases within a base sequence selected from Table 1. In some embodiments, a particular sequence is or comprises 17 contiguous bases within a base sequence selected from Table 1. In some embodiments, a particular sequence is or comprises 18 contiguous bases within a base sequence selected from Table 1. In some embodiments, a particular sequence is or comprises 19 contiguous bases within a sequence selected from Table 1. In some embodiments, a particular sequence is or comprises 20 contiguous bases within a sequence of bases selected from Table 1. In some embodiments, a particular sequence is or comprises 21 contiguous bases within a sequence selected from Table 1. In some embodiments, a particular sequence is or comprises 22 contiguous bases within a base sequence selected from Table 1. In some embodiments, a particular sequence is or comprises 23 contiguous bases within a sequence selected from Table 1. In some embodiments, a particular sequence is or comprises 24 contiguous bases within a base sequence selected from Table 1. In some embodiments, a particular sequence is or comprises 25 contiguous bases within a sequence selected from Table 1. In some embodiments, a particular sequence is or comprises 26 contiguous bases within a base sequence selected from Table 1. In some embodiments, a particular sequence is or comprises 27 contiguous bases within a sequence selected from Table 1. In some embodiments, a particular sequence is or comprises 28 contiguous bases within a sequence selected from Table 1. In some embodiments, a particular sequence is or comprises 29 contiguous bases within a sequence selected from Table 1. In some embodiments, a particular sequence is or comprises 30 contiguous bases within a sequence selected from Table 1. In some embodiments, the base sequence selected from Table 1 is a base sequence selected from Table 1A. In some embodiments, the base sequence selected from Table 1 is a base sequence selected from Table 1B. In some embodiments, the base sequence selected from Table 1 is a base sequence selected from Table 1C. In some embodiments, the base sequence selected from Table 1 is a base sequence selected from Table ID. In some embodiments, the base sequence selected from Table 1 is a base sequence selected from Table 1E. In some embodiments, the base sequence selected from Table 1 is a base sequence selected from Table 1F. In some embodiments, the base sequence selected from Table 1 is a base sequence selected from Table 1G. In some embodiments, the base sequence selected from Table 1 is a base sequence selected from Table 1H. In some embodiments, the base sequence selected from Table 1 is a base sequence selected from Table 1I. In some embodiments, the base sequence selected from Table 1 is a base sequence selected from Table 1J. In some embodiments, the base sequence selected from Table 1 is a base sequence selected from Table 1K. In some embodiments, the base sequence selected from Table 1 is a base sequence selected from Table 1L. In some embodiments, the base sequence selected from Table 1 is a base sequence selected from Table 1M. In some embodiments, the base sequence selected from Table 1 is a base sequence selected from Table 1N. In some embodiments, the base sequence selected from Table 1 is a base sequence selected from Table 10. In some embodiments, the base sequence is from Table 1 (e.g., 1A, 1B, 1C, 1D, 1E, 1F, 1G, 1H and/or 1I) of WO 2021/071858, which is incorporated herein by reference in its entirety. is selected from In some embodiments, a particular sequence is or comprises UCCCUUUCTCIUCGA, and each U may independently be replaced with a T or vice versa. In some embodiments, a particular sequence is or comprises UCCCUUUCTCIUCGA. In some embodiments, a particular sequence is or comprises UCCCUUUCTCGUCGA, and each U may independently be replaced with a T and vice versa. In some embodiments, a particular sequence is or comprises UCCCUUUCTCGUCGA. In some embodiments, a particular sequence is or comprises UUCAGUCCCUUUCTCIUCGA, and each U may independently be replaced with a T or vice versa. In some embodiments, a particular sequence is or comprises UUCAGUCCCUUUCTCIUCGA. In some embodiments, a particular sequence is or comprises UUCAGUCCCUUUCTCGUCGA, and each U may independently be replaced with a T and vice versa. In some embodiments, a particular sequence is or comprises UUCAGUCCCUUUCTCGUCGA. In some embodiments, a particular sequence is or comprises CCCCAGCAGCUUCAGUCCCUUUCTCIUCGA, and each U may independently be replaced with a T or vice versa. In some embodiments, a particular sequence is or comprises CCCCAGCAGCUUCAGUCCCUUUCTCIUCGA. In some embodiments, a particular sequence is or comprises CCCCAGCAGCUUCAGUCCCUUUCTCGUCGA, and each U may be independently replaced with a T or vice versa. In some embodiments, a particular sequence is or comprises CCCCAGCAGCUUCAGUCCCUUUCTCGUCGA. In some embodiments, a particular sequence is or comprises CCCAGCAGCUUCAGUCCCUUUCUAAIUCGAU, and each U can be independently replaced with a T or vice versa. In some embodiments, a particular sequence is or comprises CCCAGCAGCUUCAGUCCCUUUCUAAIUCGAU. In some embodiments, a particular sequence is or comprises ACAUAAUUUACACGAAAGCAAUGCCAUCAC, and each U can be independently replaced with a T or vice versa. In some embodiments, a particular sequence is or comprises ACAUAAUUUACACGAAAGCAAUGCCAUCAC. In some embodiments, a particular sequence is or comprises AUCCACUGUGGCACCCAGAUUAUCCAUGUU, and each U can be independently replaced with a T or vice versa. In some embodiments, a particular sequence is or comprises AUCCACUGUGGCACCCAGAUUAUCCAUGUU. In some embodiments, a particular sequence is or comprises CCCAGCAGCUUCAGUCCCUUUCTUIUCGAU. In some embodiments, a particular sequence is or comprises CCCAGCAGCUUCAGUCCCUUTCTUIUCGAU.

Specific oligonucleotides and/or compositions are described in Table 1 below, which contains several sections, e.g., 1A, 1B, 1C, etc., which may be individually referred to as Tables 1A, 1B, 1C, etc. Specific oligonucleotides and/or compositions referred to in the present invention are described in WO 2021/071858, eg Table 1 of WO 2021/071858. All oligonucleotides and/or compositions of WO 2021/071858 are incorporated herein by reference.

Table 1: Exemplary oligonucleotides and/or compositions

Table 1A: Exemplary oligonucleotides and/or compositions targeting UGP2

Table 1B: Exemplary oligonucleotides and/or compositions targeting ACTB

Table 1C: Exemplary oligonucleotides and/or compositions targeting LUC.

Table 1D: Exemplary oligonucleotides and/or compositions targeting SERPINA1

Table 1E: Exemplary oligonucleotides and/or compositions targeting SERPINA1

Table 1F: Exemplary oligonucleotides and/or compositions targeting SERPINA1

Table 1G: Exemplary oligonucleotides and/or compositions targeting KEAP1

Table 1H: Exemplary oligonucleotides and/or compositions targeting NRF2

Table 1I: Exemplary oligonucleotides and/or compositions targeting UGP2

Table 1J: Exemplary oligonucleotides and/or compositions targeting ACTB

Table 1K: Exemplary oligonucleotides and/or compositions targeting EEEF

Table 1L: Exemplary oligonucleotides and/or compositions targeting SRSF

Table 1M: Exemplary oligonucleotides and/or compositions targeting EEF1A1

Table 1N: Exemplary oligonucleotides and/or compositions targeting UGP2

Table 10: Exemplary oligonucleotides and/or compositions targeting SERPINA1

main:

Descriptions, base sequences, and stereochemistry/linkage may be split into multiple lines in Table 1 (eg, Table 1A, Table 1B, Table 1C, etc.) due to their length. Unless otherwise specified, all oligonucleotides in Table 1 are single stranded. As will be appreciated by those skilled in the art, nucleoside units are unmodified and, unless indicated otherwise (e.g., r, m, m5, eo, etc.), unmodified nucleobases and 2'-deoxy sugars contains; Linkages are natural phosphate linkages unless otherwise indicated; Acidic/basic groups can independently exist in their salt form. When a sugar is not specified, the sugar is a natural DNA sugar; When internucleotidic linkages are not specified, they are natural phosphate linkages. Moieties and Variants:

a: 2′-NH ₂ (eg aC: );

m: 2'-OMe;

m5: methyl at position 5 of C (nucleobase is 5-methylcytosine);

m5lC: methyl at position 5 of C (nucleobase is 5-methylcytosine) and sugar is LNA sugar;

l: per LNA;

I: nucleobase is hypoxanthine;

f: 2'-F;

r: 2'-OH;

eo: 2'-MOE (2'-OCH ₂ CH ₂ OCH ₃ );

m5Ceo: 5-methyl 2'-O-methoxyethyl C;

O, PO: phosphodiester (phosphate). It can be a linker or terminal group (or a component thereof), eg, a linkage between a linker and an oligonucleotide chain, an internucleotide linkage (natural phosphate linkage), and the like. Phosphodiesters are usually denoted by an "O" in the stereochemistry/linkage column, usually not in the description column (end groups, e.g. 5'-terminal groups, are indicated in the description and are usually not shown in stereochemistry/linkages). not shown); When a linkage is not indicated in the Description column, it is usually a phosphodiester unless otherwise indicated. Note that the phosphate linkage between the linker (e.g., L001) and the oligonucleotide chain may not appear in the Description column, but may appear as an "O" in the Stereochemistry/Linkage column;

*, PS: phosphorothioate. This may be a terminal group (if a terminal group, e.g. 5'-end group, indicated in the description and generally not in the stereochemistry/linkage), or a linkage, e.g., a linker (e.g., L001) and an oligonucleotide may be interchain linkages, internucleotidic linkages (phosphorothioate internucleotidic linkages), and the like;

R , R p : phosphorothioates of the R p configuration. * Note that R in the description represents a single phosphorothioate linkage in the R p configuration;

S , S p: phosphorothioates of the S p configuration. Note that *S in the description represents a single phosphorothioate linkage in the S p configuration;

X: steric random phosphorothioate;

n001: ;

nX (when used for n001): steric random n001;

nR (when used for n001) or n001R: n001 in the Rp configuration;

nS (when used for n001) or n001S: n001 in the Sp array;

*n001: ;

n*X: three-dimensional random *n001;

n002: ;

nX (when used for n002): steric random n002;

nR (when used for n002) or n002R: n002 in the Rp configuration;

nS (when used for n002) or n002S: n002 in the Sp array;

n003: ;

nX (when used for n003): steric random n003;

nR (when used for n003) or n003R: n003 in the Rp configuration;

nS (when used for n003) or n003S: n003 in the Sp array;

n004: ;

nX (when used with n004): steric random n004;

nR (when used for n004) or n004R: n004 in the Rp configuration;

nS (when used for n004) or n004S: n004 in the Sp array;

n006: ;

nX (when used with n006): steric random n006;

nR (when used for n006) or n006R: n006 in the Rp configuration;

nS (when used for n006) or n006S: n006 in the Sp array;

n008: ;

nX (when used with n008): stereo random n008;

nR (when used for n008) or n008R: n008 in the Rp configuration;

nS (when used for n008) or n008S: n008 in the Sp array;

n020: ;

nX (when used for n020): Stereo Random n020;

nR (when used for n020) or n020R: n020 in the Rp configuration;

nS (when used with n020) or n020S: n020 in the Sp array;

n025: ;

nX (when used with n025): steric random n025;

nR (when used for n025) or n025R: n025 in the Rp configuration;

nS (when used with n025) or n025S: n025 in the Sp array;

n026 ;

nX (when used with n026): steric random n026;

nR (when used with n026) or n026R: n026 in the Rp configuration;

nS (when used with n026) or n026S: n026 in the Sp array;

n051: ;

nX (when used with n051): steric random n051;

nR (when used for n051) or n051R: n051 in the Rp configuration;

nS (when used for n051) or n051S: n051 in Sp configuration;

n057:

;

nX (when used with n057): steric random n057;

nR (when used for n057) or n057R: n057 in the Rp configuration;

nS (when used with n057) or n057S: n057 in the Sp configuration;

wherein -C(O)- is bonded to nitrogen; As used in the table, n013 can be represented by O in stereochemistry/linkage;

L001: linked to Mod (e.g. Mod001) via -NH- and, for example in the case of WV-27457, -NH-(linked to the 5'-end of the oligonucleotide chain via a phosphate linkage (O or PO)) CH ₂ ) ₆ - linker (C6 linker, C6 amine linker or C6 amino linker). For example in WV-27457, L001 is linked to Mod001 via -NH- (forming an amide group -C(O)-NH-) and linked to the oligonucleotide chain via a phosphate linkage (O);

L010: . In some embodiments, when L010 is present in the middle of the oligonucleotide, for example a linkage (e.g., a phosphate linkage (O or PO) or a phosphorothioate linkage (which may not be chirally controlled or may be chirally controlled ( S via p or R p))), independently linked to internucleotide linkages as other sugars (e.g., DNA sugars) (e.g., the 5'-carbon is joined to another unit (e.g., the 3' of the sugar) and the 3'-carbon is linked to another unit (eg, the 5'-carbon of carbon);

L012:-CH ₂ CH ₂ OCH ₂ CH ₂ OCH ₂ CH ₂ -. When L012 is present in the middle of the oligonucleotide, each of the two ends is independently an internucleotidic linkage (e.g., a phosphate linkage (O or PO) or a phosphorothioate linkage (which may be unchirally controlled or chirally controlled (Sp or Rp)));

L022: , unless otherwise indicated, L022 is linked to the rest of the molecule via a phosphate, for example, via R p phosphorothioate in WV-42488;

L023: HO-(CH ₂ ) ₆ -, unless otherwise indicated, CH ₂ is linked to the rest of the molecule via a phosphate (e.g., O in WV-39202(OnRnRnRnRSSSSSSSSSSSSSSSSSSSnRSSSSSnRSSnR is a phosphate linkage linking L023 to the rest of the molecule) indicated);

L025: , -CH ₂ -linkage is used as the C5 linkage of the sugar (eg DNA sugar) and linked to another unit (eg 3' of the sugar), and the linkage on the ring is used as the C3 linkage and the other unit (eg, the 5'-carbon of carbon), each of which is independently, for example, a linkage (eg, a phosphate linkage (O or PO) or a phosphorothioate linkage (chiral uncontrolled or chiral can be controlled (Sp or Rp))). When L025 is at the 5'-end without any modification, the -CH ₂ - linking site is bonded to -OH. For example, in various oligonucleotides, L025L025L025- has the structure of (can exist in various salt forms) and has a linkage as indicated (e.g., a phosphate linkage (O or PO) or a phosphorothioate linkage (which may be unchirally controlled or chirally controlled (Sp or Rp)) ))) to the 5'-carbon of the oligonucleotide chain;

L028:-CH ₂ CH ₂ OCH ₂ CH ₂ OCH ₂ CH ₂ OCH ₂ CH ₂ -. When L028 is present in the middle of the oligonucleotide, each of the two ends is independently an internucleotidic linkage (e.g., a phosphate linkage (O or PO) or a phosphorothioate linkage (which may be unchirally controlled or chirally controlled (Sp or Rp)));

sm04: . A linking nucleobase is indicated before sm04; For example in WV-28787, "Usm04" indicates that U is bound to sm04 ( ); In WV-44238, "Csm04" indicates that C is bound to sm04 (

sm11: . A linking nucleobase is indicated before sm11; For example, in WV-47403, "Csm11" indicates that C is bound to sm11 ( );

sm12: . A linking nucleobase is preceded by sm12; For example, in WV-47402, "Csm12" indicates that C is bound to sm12 ( );

a: 2'-NH ₂ ;

b001U: base number phosphorus nucleosides;

b001rU: base number , wherein the sugar is the natural RNA sugar (r);

b002U: base number phosphorus nucleosides;

b003U: base value phosphorus nucleosides;

b004U: base number phosphorus nucleosides;

b005U: base value phosphorus nucleosides;

b006U: base number phosphorus nucleosides;

b007U: base number phosphorus nucleosides;

b008U: base number phosphorus nucleosides;

b009U: base number phosphorus nucleosides;

b010U: A nucleoside having the structure of;

b011U: base number phosphorus nucleosides;

b012U: base number phosphorus nucleosides;

b003I: base value phosphorus nucleosides;

b004I: base number phosphorus nucleosides;

b014I: base number phosphorus nucleosides;

b001G: base number phosphorus nucleosides;

b002G: base value phosphorus nucleosides;

b001A: base value phosphorus nucleosides;

b002A: base number phosphorus nucleosides;

b003A: base number phosphorus nucleosides;

zdnp: base number phosphorus nucleosides;

b001C: base number phosphorus nucleosides;

b002C: base value phosphorus nucleosides;

b003C: base number phosphorus nucleosides;

b004C: base number phosphorus nucleosides;

b007C: base number phosphorus nucleosides;

b008C: base number phosphorus nucleosides;

b009C: base number phosphorus nucleosides;

5MR: 5'-Me modification to sugar and configuration of 5'-carbon - sugar is R (e.g., 5MRdT ; 5MRm5dC: );

5MS: 5'-Me modification to the sugar and configuration of the 5'-carbon - the sugar is S (e.g., 5MSdT: ; 5MSm5dC: );

rNxsm13: , where Nx is a nucleobase (eg rCsm13: );

rNxsm14: , where Nx is a nucleobase (eg rCsm14: );

sm15: . A linking nucleobase is indicated before sm15 (e.g., Csm15: );

sm16: . A linking nucleobase is preceded by sm16 (e.g., Csm16: ); and

sm17: . A linking nucleobase is preceded by sm17 (e.g., Csm17: ).

In some embodiments, the sugar is linked to the internucleotidic linkage via an oxygen atom, eg, the oxygen atom of a natural phosphate linkage as in typical natural DNA molecules. In some embodiments, the sugar is attached to the internucleotidic linkage through an atom other than oxygen. In some embodiments, the sugar is linked to an internucleotidic linkage through the nitrogen atom of the sugar. In some embodiments, the sugar is linked to an internucleotidic linkage via a ring nitrogen atom of the sugar (eg, in sm01); In this case, the ring nitrogen atom of the sugar can form a direct bond with the linking phosphorus atom (see eg sm01n001), and one skilled in the art will understand that the oxygen atom can be removed from the linkage (see eg sm01n001) . See also, for example, sm18, which can bind directly to the linking phosphorus through a nitrogen atom as shown in the oligonucleotides in the table (eg, sm18n001). Methods using specific reagents (e.g., phosphoramidites, nucleosides, etc.) and various modifications, such as those exemplified in the tables herein, e.g., modified sugars, modified nucleobases, etc., are described in the Examples or WO 2021/071858, incorporated herein by reference.

oligonucleotide composition

In particular, the present invention provides various oligonucleotide compositions. In some embodiments, the present invention provides oligonucleotide compositions of the oligonucleotides described herein. In some embodiments, an oligonucleotide composition comprises a plurality of oligonucleotides described herein. In some embodiments, the oligonucleotide composition is chirally controlled. In some embodiments, the oligonucleotide composition is not chirally controlled (sterically random).

Linkages of natural phosphate linkages are achiral. Many modified internucleotidic linkages, for example phosphorothioate internucleotidic linkages, are chiral. In some embodiments, during manufacture of an oligonucleotide composition (e.g., in conventional phosphoramidite oligonucleotide synthesis), the arrangement of chiral linking phosphorus is not intentionally designed or controlled, resulting in a complex random mixture of various stereoisomers ( diastereomers) to generate non-chiral controlled (stereorandom) oligonucleotide compositions (practically racemic) (for oligonucleotides with n chiral internucleotidic linkages (the linkages are chiral), typically 2 ⁿ two stereoisomers (eg, when n equals 10, 2 ¹⁰ = 1,032; when n equals 20, 2 ²⁰ = 1,048,576). These stereoisomers have the same constitution but differ with respect to the stereochemical pattern of the linking phosphorus.

In some embodiments, a stereorandom oligonucleotide composition has properties and/or activity sufficient for a particular purpose and/or application. In some embodiments, preparation of stereorandom oligonucleotide compositions may be cheaper, easier, and/or simpler than chirally controlled oligonucleotide compositions. However, stereoisomers within a stereorandom composition may have different properties, activities, and/or toxicity, resulting in inconsistent therapeutic effects and/or Or it may cause unintended side effects.

In some embodiments, the present invention includes techniques for designing and manufacturing chirally controlled oligonucleotide compositions. In some embodiments, the present invention provides a chirally controlled oligonucleotide composition of many of the oligonucleotides of Table 1, for example comprising S and/or R in stereochemistry/linkage. In some embodiments, a chirally controlled oligonucleotide composition comprises a controlled/predetermined (not random as in a stereorandom composition) level of a plurality of oligonucleotides, wherein the oligonucleotides have one or more chiral internucleotidic linkages (chiral control nucleotides). linkage) share the same linkage, stereochemistry. In some embodiments, oligonucleotides share the same backbone chiral center pattern (stereochemistry of linking phosphorus). In some embodiments, the backbone chiral center pattern is as described herein. In some embodiments, the plurality of oligonucleotides are structurally identical.

In some embodiments, the present invention provides an oligonucleotide composition comprising a plurality of oligonucleotides, wherein the plurality of oligonucleotides

1) common base sequence and

2) one or more (e.g., about 1-50, 1-40, 1-30, 1-25, 1-20, 1-15, 1-10, 5-50, 5-40, 5-30; 5-25, 5-20, 5-15, 5-10, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 or more) chiral internucleotide linkages (“chiral controlled internucleotide linkages”) that share stereochemistry, which is independently the same linkage.

In some embodiments, the present invention provides an oligonucleotide composition comprising a plurality of oligonucleotides, wherein the plurality of oligonucleotides

1) common base sequence and

2) one or more (e.g., about 1-50, 1-40, 1-30, 1-25, 1-20, 1-15, 1-10, 5-50, 5-40, 5-30; 5-25, 5-20, 5-15, 5-10, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 or more) chiral internucleotide linkages ("chiral controlled internucleotide linkages") that share stereochemistry, which is the same linkage independently;

The composition is enriched in a plurality of oligonucleotides relative to a substantially racemic preparation of oligonucleotides that share a common base sequence.

In some embodiments, the oligonucleotide composition is a chirally controlled oligonucleotide composition comprising a plurality of oligonucleotides, wherein the oligonucleotides

consensus Sequence,

a common backbone connection pattern; and

One or more (e.g., 1-50, 1-40, 1-30, 1-25, 1-20, 1-15, 1-10, 5-50, 5-40, 5-30, 5-25 , 5~20, 5~15, 5~10, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 , 20 or more) share the same linkage stereochemistry in chiral internucleotidic linkages (chiral controlled internucleotidic linkages),

The composition is enriched in a plurality of oligonucleotides relative to a substantially racemic preparation of oligonucleotides that share a common base sequence and backbone linkage pattern.

In some embodiments, the oligonucleotide composition is a chirally controlled oligonucleotide composition comprising a plurality of oligonucleotides, wherein the oligonucleotides

consensus Sequence,

a common backbone connection pattern; and

share a common backbone chiral center pattern comprising at least one Sp;

The composition is enriched in a plurality of oligonucleotides relative to a substantially racemic preparation of oligonucleotides that share a common base sequence and backbone linkage pattern.

In some embodiments, the oligonucleotide composition is a chirally controlled oligonucleotide composition comprising a plurality of oligonucleotides, wherein the oligonucleotides

consensus Sequence,

a common backbone connection pattern; and

share a common backbone chiral center pattern comprising at least one Rp;

The composition is enriched in a plurality of oligonucleotides relative to a substantially racemic preparation of oligonucleotides that share a common base sequence and backbone linkage pattern.

In some embodiments, the present invention provides a chirally controlled oligonucleotide composition comprising a plurality of oligonucleotides, wherein the oligonucleotides

1) common configuration and

2) one or more (e.g., 1-50, 1-40, 1-30, 1-25, 1-20, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more) chiral internucleotide linkages (chiral controlled internucleotide linkages) that are identical linkages stereochemistry share,

The composition is enriched in a plurality of oligonucleotides relative to a substantially racemic preparation of oligonucleotides of common composition.

In some embodiments, the present invention provides an oligonucleotide composition comprising a plurality of oligonucleotides, wherein the plurality of oligonucleotides

1) common base sequence and

2) one or more (e.g., about 1-50, 1-40, 1-30, 1-25, 1-20, 1-15, 1-10, 5-50, 5-40, 5-30; 5-25, 5-20, 5-15, 5-10, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 or more) chiral internucleotide linkages ("chiral controlled internucleotide linkages") that share stereochemistry, which is the same linkage independently;

The stereochemical purity of the linking phosphorus of each chiral controlled internucleotide linkage is independently 80%-100% (e.g., 85-100%, 90-100%, about or at least about 90%, 91%, 92%, 93 %, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5%).

In some embodiments, the oligonucleotide composition is a chirally controlled oligonucleotide composition comprising a plurality of oligonucleotides, wherein the oligonucleotides

consensus Sequence,

a common backbone connection pattern; and

One or more (e.g., 1-50, 1-40, 1-30, 1-25, 1-20, 1-15, 1-10, 5-50, 5-40, 5-30, 5-25 , 5~20, 5~15, 5~10, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 , 20 or more) share the same linkage stereochemistry in chiral internucleotidic linkages (chiral controlled internucleotidic linkages),

The stereochemical purity of the linking phosphorus of each chiral controlled internucleotide linkage is independently 80%-100% (e.g., 85-100%, 90-100%, about or at least about 90%, 91%, 92%, 93 %, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5%).

In some embodiments, the present invention provides a chirally controlled oligonucleotide composition comprising a plurality of oligonucleotides, wherein the oligonucleotides

1) common configuration and

2) one or more (e.g., 1-50, 1-40, 1-30, 1-25, 1-20, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more) chiral internucleotide linkages (chiral controlled internucleotide linkages) that are identical linkages stereochemistry share,

The stereochemical purity of the linking phosphorus of each chiral controlled internucleotide linkage is independently 80%-100% (e.g., 85-100%, 90-100%, about or at least about 90%, 91%, 92%, 93 %, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5%).

In some embodiments, the present invention provides an oligonucleotide composition comprising a plurality of oligonucleotides, wherein the plurality of oligonucleotides

1) common base sequence and

2) one or more (e.g., about 1-50, 1-40, 1-30, 1-25, 1-20, 1-15, 1-10, 5-50, 5-40, 5-30; 5-25, 5-20, 5-15, 5-10, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 or more) chiral internucleotide linkages ("chiral controlled internucleotide linkages") that share stereochemistry, which is the same linkage independently;

The consensus nucleotide sequence is complementary to the nucleotide sequence of the nucleic acid portion containing the target adenosine.

In some embodiments, the present invention provides an oligonucleotide composition comprising one or more of a plurality of oligonucleotides, each of the plurality of oligonucleotides independently

1) common base sequence and

2) one or more (e.g., about 1-50, 1-40, 1-30, 1-25, 1-20, 1-15, 1-10, 5-50, 5-40, 5-30; 5-25, 5-20, 5-15, 5-10, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 or more) chiral internucleotide linkages ("chiral controlled internucleotide linkages") that share stereochemistry, which is the same linkage independently;

Each of the plurality of consensus nucleotide sequences is independently complementary to a nucleotide sequence of a nucleic acid portion including a target adenosine.

In some embodiments, the present invention provides a composition comprising a plurality of oligonucleotides of a particular oligonucleotide type, wherein the particular oligonucleotide type is

a) consensus sequence;

b) a common backbone connection pattern;

c) a common backbone chiral center pattern;

d) characterized by a deformation pattern that is a common backbone;

The composition is enriched in oligonucleotides of a particular oligonucleotide type relative to a substantially racemic preparation of oligonucleotides having the same common base sequence, backbone linkage pattern, and backbone phosphorus modification pattern, or within a composition that shares a common base sequence. chirally controlled in that all oligonucleotides at nonrandom levels are a plurality of oligonucleotides;

The consensus nucleotide sequence is complementary to the nucleotide sequence of the nucleic acid portion containing the target adenosine.

In some embodiments, as described herein, some are about or at least about 10-40, 15-40, 20-40, for example 10, 11, 12, 13, 14, 15, 16, 17, 18 , 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 or more nucleobases in length. In some embodiments, a portion is about or at least about or up to about 1% to 50% of a nucleic acid. In some embodiments, a portion is the full length of a nucleic acid. In some embodiments, a consensus base sequence is complementary to a sequence of bases in a portion of a nucleic acid as described herein. In some embodiments, it is completely complementary across its length except for the nucleobase opposite the target adenosine. In some embodiments, it is fully complementary over its length. In some embodiments, the target adenosine is associated with a disease, disorder or disease. In some embodiments, the target adenosine is a G to A mutation associated with a condition, disorder or disease. In some embodiments, the target adenosine is edited to I by a provided oligonucleotide or composition. In some embodiments, editing as described herein increases the expression, level, and/or activity of a transcript or its product (eg, mRNA, protein, etc.). In some embodiments, editing as described herein reduces expression, level, and/or activity of a transcript or its product (eg, mRNA, protein, etc.).

In some embodiments, the plurality of oligonucleotides share the same nucleobase modifications and/or sugar modifications. In some embodiments, the plurality of oligonucleotides share the same internucleotidic linkage modification (an internucleotide linkage can be in various acid, base, and/or salt forms). In some embodiments, the plurality of oligonucleotides share the same nucleobase modifications, sugar modifications, and internucleotide linkage modifications, if any. In some embodiments, the plurality of oligonucleotides are in the same form, eg an acid form, a base form, or particularly a salt form (eg, a pharmaceutically acceptable salt form, eg a salt form). In some embodiments, oligonucleotides in a composition can exist in more than one form, such as an acid form, a base form, and/or one or more salt forms. In some embodiments, in an aqueous solution (eg, when dissolved in a buffer such as PBS), anions and cations can be separated. In some embodiments, the plurality of oligonucleotides have the same composition. In some embodiments, the plurality of oligonucleotides are structurally identical. In some embodiments, the present invention provides a chirally controlled oligonucleotide composition comprising a plurality of oligonucleotides, wherein the oligonucleotides have a common composition and contain one or more (e.g., 1-60, 1-50, 1-40 , 1~30, 1~25, 1~20, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 , 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44 , 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60 or more) chiral internucleotide linkages (chiral controlled internucleotide linkages) share phosphorus stereochemistry, and the composition is enriched in a plurality of oligonucleotides compared to a substantially racemic preparation of oligonucleotides of common composition.

In some embodiments, at least one chiral internucleotide linkage is chirally controlled. In some embodiments, at least two internucleotide linkages are independently chirally controlled. In some embodiments, the number of chirally controlled internucleotide linkages is at least three. In some embodiments, the number is at least 4. In some embodiments, the number is at least 5. In some embodiments, the number is at least 6. In some embodiments, the number is at least 7. In some embodiments, the number is at least 8. In some embodiments, the number is at least 9. In some embodiments, the number is at least 10. In some embodiments, the number is at least 11. In some embodiments, the number is at least 12. In some embodiments, the number is at least 13. In some embodiments, the number is at least 14. In some embodiments, the number is at least 15. In some embodiments, the number is at least 20. In some embodiments, the number is at least 25. In some embodiments, the number is at least 30.

In some embodiments, at least 5%-100% (e.g., about 10%-100%, 20-100%, 30%-100%, 40%-100%, 50%-80%) of all internucleotide linkages , 50%~85%, 50%~90%, 50%~95%, 60%~80%, 60%~85%, 60%~90%, 60%~95%, 60%~100%, 65 %~80%, 65%~85%, 65%~90%, 65%~95%, 65%~100%, 70%~80%, 70%~85%, 70%~90%, 70%~ 95%, 70% to 100%, 75% to 80%, 75% to 85%, 75% to 90%, 75% to 95%, 75% to 100%, 80% to 85%, 80% to 90% , 80%~95%, 80%~100%, 85%~90%, 85%~95%, 85%~100%, 90%~95%, 90%~100%, 10%, 20%, 30 %, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%, etc.) are chirally controlled. In some embodiments, at least 5%-100% (e.g., about 10%-100%, 20-100%, 30%-100%, 40%-100%, 50%-80%) of all chiral internucleotide linkages %, 50%~85%, 50%~90%, 50%~95%, 60%~80%, 60%~85%, 60%~90%, 60%~95%, 60%~100%, 65%~80%, 65%~85%, 65%~90%, 65%~95%, 65%~100%, 70%~80%, 70%~85%, 70%~90%, 70% ~95%, 70%~100%, 75%~80%, 75%~85%, 75%~90%, 75%~95%, 75%~100%, 80%~85%, 80%~90 %, 80%~95%, 80%~100%, 85%~90%, 85%~95%, 85%~100%, 90%~95%, 90%~100%, 10%, 20%, 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%, etc.) are chirally controlled. In some embodiments, at least 5%-100% (e.g., about 10%-100%, 20-100%, 30%-100%, 40%-100%, 50%-100%) of all phosphorothioate internucleotide linkages %~80%, 50%~85%, 50%~90%, 50%~95%, 60%~80%, 60%~85%, 60%~90%, 60%~95%, 60%~ 100%, 65%~80%, 65%~85%, 65%~90%, 65%~95%, 65%~100%, 70%~80%, 70%~85%, 70%~90% , 70%~95%, 70%~100%, 75%~80%, 75%~85%, 75%~90%, 75%~95%, 75%~100%, 80%~85%, 80 %~90%, 80%~95%, 80%~100%, 85%~90%, 85%~95%, 85%~100%, 90%~95%, 90%~100%, 10%, 20%, 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%, etc.) are chirally controlled. In some embodiments, the percentage is at least 50%. In some embodiments, the percentage is at least 60%. In some embodiments, the percentage is at least 70%. In some embodiments, the percentage is at least 80%. In some embodiments, the percentage is at least 90%. In some embodiments, the percentage is at least 90%. In some embodiments, each chiral internucleotidic linkage is chirally controlled. In some embodiments, each phosphorothioate internucleotidic linkage is chirally controlled.

In some embodiments, at most 1-10, eg at most 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 chiral internucleotide linkages are not chirally controlled. In some embodiments, at most one chiral internucleotide linkage is not chirally controlled. In some embodiments, up to two chiral internucleotidic linkages are not chirally controlled. In some embodiments, up to three chiral internucleotide linkages are not chirally controlled. In some embodiments, up to 4 chiral internucleotide linkages are not chirally controlled. In some embodiments, up to 5 chiral internucleotide linkages are not chirally controlled. In some embodiments, the number of non-chiral controlled internucleotide linkages is one. In some embodiments, the number is two. In some embodiments, the number is three. In some embodiments, the number is four. In some embodiments, the number is five.

In some embodiments, the present invention provides a composition comprising a plurality of oligonucleotides, wherein each oligonucleotide of the plurality is independently a particular oligonucleotide or salt thereof. In some embodiments, the present invention provides a composition comprising a plurality of oligonucleotides, wherein each oligonucleotide of the plurality is independently a particular oligonucleotide or a pharmaceutically acceptable salt thereof. In some embodiments, such compositions are enriched relative to a substantially racemic preparation of a particular oligonucleotide. As will be appreciated by those skilled in the art, the plurality of oligonucleotides share a common sequence, which is the sequence of bases of a particular oligonucleotide. In some embodiments, at least about 5%-100%, 10%-100%, 20-100%, 30%-100%, 40%-100% of all oligonucleotides in the composition share the base sequence of a particular oligonucleotide. , 50%~100%, 5%~90%, 10%~90%, 20~90%, 30%~90%, 40%~90%, 50%~90%, 5%~85%, 10% ~85%, 20~85%, 30%~85%, 40%~85%, 50%~85%, 5%~80%, 10%~80%, 20~80%, 30%~80%, 40%~80%, 50%~80%, 5%~75%, 10%~75%, 20~75%, 30%~75%, 40%~75%, 50%~75%, 5%~ 70%, 10%~70%, 20~70%, 30%~70%, 40%~70%, 50%~70%, 5%~65%, 10%~65%, 20~65%, 30 %~65%, 40%~65%, 50%~65%, 5%~60%, 10%~60%, 20~60%, 30%~60%, 40%~60%, 50%~60 %, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% are a plurality of oligonucleotides. In some embodiments, at least about 5%-100%, 10%-100%, 20-100%, 30%-100%, 40%-100% of all oligonucleotides in the composition share the composition of a particular oligonucleotide or salt thereof. %, 50%~100%, 5%~90%, 10%~90%, 20~90%, 30%~90%, 40%~90%, 50%~90%, 5%~85%, 10 %~85%, 20~85%, 30%~85%, 40%~85%, 50%~85%, 5%~80%, 10%~80%, 20~80%, 30%~80% , 40%~80%, 50%~80%, 5%~75%, 10%~75%, 20~75%, 30%~75%, 40%~75%, 50%~75%, 5% ~70%, 10%~70%, 20~70%, 30%~70%, 40%~70%, 50%~70%, 5%~65%, 10%~65%, 20~65%, 30%~65%, 40%~65%, 50%~65%, 5%~60%, 10%~60%, 20~60%, 30%~60%, 40%~60%, 50%~ 60%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95% , 96%, 97%, 98%, or 99% are a plurality of oligonucleotides. In some embodiments, the percentage is at least 10%. In some embodiments, the percentage is at least 20%. In some embodiments, the percentage is at least 30%. In some embodiments, the percentage is at least 40%. In some embodiments, the percentage is at least 50%. In some embodiments, the percentage is at least 60%. In some embodiments, the percentage is at least 70%. In some embodiments, the percentage is at least 80%. In some embodiments, the percentage is at least 90%. In some embodiments, the percentage is at least 95%. In some embodiments, the percentage is between about 5 and 100%. In some embodiments, the percentage is between about 10 and 100%. In some embodiments, the percentage is about 20-100%. In some embodiments, the percentage is about 30-90%. In some embodiments, the percentage is about 30-80%. In some embodiments, the percentage is about 30-70%. In some embodiments, the percentage is about 40-90%. In some embodiments, the percentage is about 40-80%. In some embodiments, the percentage is about 40-70%. In some embodiments, a particular oligonucleotide is an oligonucleotide exemplified herein, eg, an oligonucleotide in Table 1 or another table.

In some embodiments, the enrichment relative to the substantially racemic preparation is at least of all oligonucleotides in the composition, or all oligonucleotides in the composition that share a plurality of common base sequences, or all oligonucleotides in the composition that share a plurality of common configurations. Approx. 5% to 100%, 10% to 100%, 20 to 100%, 30% to 100%, 40% to 100%, 50% to 100%, 5% to 90%, 10% to 90%, 20% to 90%, 30%~90%, 40%~90%, 50%~90%, 5%~85%, 10%~85%, 20~85%, 30%~85%, 40%~85%, 50%~85%, 5%~80%, 10%~80%, 20~80%, 30%~80%, 40%~80%, 50%~80%, 5%~75%, 10%~ 75%, 20-75%, 30%-75%, 40%-75%, 50%-75%, 5%-70%, 10%-70%, 20-70%, 30%-70%, 40 %~70%, 50%~70%, 5%~65%, 10%~65%, 20~65%, 30%~65%, 40%~65%, 50%~65%, 5%~60 %, 10%~60%, 20~60%, 30%~60%, 40%~60%, 50%~60%, 5%, 10%, 20%, 30%, 40%, 50%, 60 %, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% are a plurality of oligonucleotides. In some embodiments, the percentage is at least 10%. In some embodiments, the percentage is at least 20%. In some embodiments, the percentage is at least 30%. In some embodiments, the percentage is at least 40%. In some embodiments, the percentage is at least 50%. In some embodiments, the percentage is at least 60%. In some embodiments, the percentage is at least 70%. In some embodiments, the percentage is at least 80%. In some embodiments, the percentage is at least 90%. In some embodiments, the percentage is at least 95%. In some embodiments, the percentage is between about 5 and 100%. In some embodiments, the percentage is between about 10 and 100%. In some embodiments, the percentage is about 20-100%. In some embodiments, the percentage is about 30-90%. In some embodiments, the percentage is about 30-80%. In some embodiments, the percentage is about 30-70%. In some embodiments, the percentage is about 40-90%. In some embodiments, the percentage is about 40-80%. In some embodiments, the percentage is about 40-70%.

In some embodiments, at least about 5%-100%, 10%-100%, 20-100%, 30%-100%, 40%-100% of all oligonucleotides in the composition that share a plurality of common base sequences, 50%~100%, 5%~90%, 10%~90%, 20~90%, 30%~90%, 40%~90%, 50%~90%, 5%~85%, 10%~ 85%, 20-85%, 30%-85%, 40%-85%, 50%-85%, 5%-80%, 10%-80%, 20-80%, 30%-80%, 40 %~80%, 50%~80%, 5%~75%, 10%~75%, 20~75%, 30%~75%, 40%~75%, 50%~75%, 5%~70 %, 10%~70%, 20~70%, 30%~70%, 40%~70%, 50%~70%, 5%~65%, 10%~65%, 20~65%, 30% ~65%, 40%~65%, 50%~65%, 5%~60%, 10%~60%, 20~60%, 30%~60%, 40%~60%, 50%~60% , 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96 %, 97%, 98%, or 99% is a plurality of oligonucleotides. In some embodiments, the percentage is at least 10%. In some embodiments, the percentage is at least 20%. In some embodiments, the percentage is at least 30%. In some embodiments, the percentage is at least 40%. In some embodiments, the percentage is at least 50%. In some embodiments, the percentage is at least 60%. In some embodiments, the percentage is at least 70%. In some embodiments, the percentage is at least 80%. In some embodiments, the percentage is at least 90%. In some embodiments, the percentage is at least 95%. In some embodiments, the percentage is between about 5 and 100%. In some embodiments, the percentage is between about 10 and 100%. In some embodiments, the percentage is about 20-100%. In some embodiments, the percentage is about 30-90%. In some embodiments, the percentage is about 30-80%. In some embodiments, the percentage is about 30-70%. In some embodiments, the percentage is about 40-90%. In some embodiments, the percentage is about 40-80%. In some embodiments, the percentage is about 40-70%.

Chirally Controlled Oligonucleotide The levels of the plurality of oligonucleotides in the composition are controlled. In contrast, in a non-chiral controlled (or stereorandom, racemic) oligonucleotide composition (or formulation), the level of oligonucleotide is random and uncontrolled. In some embodiments, the enrichment relative to a substantially racemic preparation is at a level described herein.

In some embodiments, the level (e.g., controlled level, predetermined level, abundance) as a percentage is (DS) ^nc or greater, and the DS (diastereopurity of individual internucleotide linkages) is between 90% and 100%; nc is the number of chirally controlled internucleotide linkages as described herein (e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more). In some embodiments, each chiral internucleotidic linkage is chirally controlled and nc is the number of chiral internucleotidic linkages. In some embodiments, the DS is greater than or equal to 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5%. In some embodiments, the DS is greater than 90%. In some embodiments, the DS is greater than or equal to 91%. In some embodiments, the DS is greater than or equal to 92%. In some embodiments, the DS is greater than or equal to 93%. In some embodiments, the DS is 94% or greater. In some embodiments, the DS is 95% or greater. In some embodiments, the DS is 96% or greater. In some embodiments, the DS is 97% or greater. In some embodiments, the DS is greater than 98%. In some embodiments, the DS is greater than 99%. In some embodiments, a level (eg, controlled level, predetermined level, enrichment) is the percentage of all oligonucleotides in a composition that share the same composition, and the percentage is greater than or equal to (DS) ^nc . For example, if DS is 99% and nc is 10, the percentage is greater than 90% ((99%) ¹⁰ 0.90 = 90%). As will be appreciated by those skilled in the art, in stereorandom formulations the percentage is generally about 1/2 ^nc (when nc is 10, the percentage is about 1/2 ¹⁰ 0.001 = 0.1%). In some embodiments, the abundance (e.g., relative to a substantially racemic preparation), level, etc., is determined by all oligonucleotides in the composition, or all oligonucleotides in the composition that share a plurality of common base sequences, or all oligonucleotides that share a plurality of common configurations. that at least about (DS) ^nc of all oligonucleotides in a composition comprising a plurality of oligonucleotides. In some embodiments, it is among all oligonucleotides in the composition. In some embodiments, it is among all oligonucleotides in a composition that share a plurality of common base sequences. In some embodiments, it is among all oligonucleotides in a composition that share a plurality of common configurations. In some embodiments, different forms of an oligonucleotide (eg, different salt forms) may be properly considered to have the same composition.

In some embodiments, oligonucleotides have a diastereomeric excess (d.e) of the linking phosphorus independently of about or at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96% , 97%, 98% or 99% of one or more (e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more) ) of the chiral control chiral internucleotidic linkage. In some embodiments, about or at least about 50%, 60%, 70%, 75%, 80%, 85%, 90%, or 95% of all chiral internucleotidic linkages comprising a chiral linking phosphorus are independently such chiral It is a control internucleotide linkage. In some embodiments, about or at least about 50%, 60%, 70%, 75%, 80%, 85%, 90% or 95% of the phosphorothioate internucleotide linkages are independently such chirally controlled internucleotide linkages. . In some embodiments, each phosphorothioate internucleotidic linkage is independently such a chirally controlled internucleotidic linkage. In some embodiments, each chiral internucleotidic linkage comprising a chiral linking phosphorus is independently such a chiral control internucleotidic linkage. In some embodiments, d.e. is about or at least about 80%. In some embodiments, d.e. is about or at least about 85%. In some embodiments, d.e. is about or at least about 90%. In some embodiments, d.e. is about or at least about 95%. In some embodiments, d.e. is about or at least about 96%. In some embodiments, d.e. is about or at least about 97%. In some embodiments, d.e. is about or at least about 98%.

In some embodiments, an oligonucleotide composition (also referred to as an oligonucleotide composition) is a chirally controlled oligonucleotide composition comprising a plurality of oligonucleotides, wherein the oligonucleotides

consensus Sequence,

a common backbone connection pattern; and

share the same linkage stereochemistry in one or more chiral internucleotidic linkages (chiral controlled internucleotidic linkages);

The percentage of a plurality of oligonucleotides within all oligonucleotides of a composition that share a common base sequence and backbone linkage pattern is greater than (DS) ^nc , where DS is between 90% and 100%, and nc is the number of chiral control internucleotide linkages.

In some embodiments, an oligonucleotide composition (also referred to as an oligonucleotide composition) is a chirally controlled oligonucleotide composition comprising a plurality of oligonucleotides, wherein the oligonucleotides

consensus Sequence,

a common backbone connection pattern; and

share a common backbone chiral center pattern comprising at least one Sp ,

The percentage of a plurality of oligonucleotides within all oligonucleotides of a composition that share a common base sequence and backbone linkage pattern is greater than (DS) ^nc , where DS is between 90% and 100%, and nc is the number of chiral control internucleotide linkages.

In some embodiments, the level of diastereopurity of a plurality of oligonucleotides in a composition can be determined as the product of the diastereopurity of each chiral control internucleotide linkage in the oligonucleotide. In some embodiments, the diastereopurity of an internucleotide linkage linking two nucleosides in an oligonucleotide (or nucleic acid) is represented by the diastereopurity of an internucleotide linkage of a dimer linking the two nucleosides. and these dimers are prepared using comparable conditions, in some cases identical synthetic cycle conditions (e.g., for a linkage between Nx and Ny in an oligonucleotide...NxNy..., the dimer is NxNy).

In some embodiments, a chirally controlled oligonucleotide composition comprises two or more of a plurality of oligonucleotides, each plurality independently of a plurality of oligonucleotides as described herein (e.g., of various chirally controlled oligonucleotide compositions). am. For example, in some embodiments, each ascites independently shares a common base sequence and stereochemistry that is the same linkage at one or more chiral internucleotidic linkages, and each ascites is independently enriched or enriched relative to a stereorandom agent of the plurality. Each plurality independently has a level as described herein. In some embodiments, at least two ascites or each ascites independently targets a different adenosine. In some embodiments, at least two or each plurality independently targets different transcripts of the same or different nucleic acids. In some embodiments, at least two ascites or each ascites independently targets transcripts of different genes. In particular, such compositions can be used to target two or more targets simultaneously and in the same system in some embodiments.

In some embodiments, all chiral internucleotidic linkages are chirally controlled and the composition is a fully chirally controlled oligonucleotide composition. In some embodiments, not all chiral internucleotide linkages are chirally controlled internucleotide linkages, and the composition is a partially chirally controlled oligonucleotide composition.

Oligonucleotides may contain or consist of various backbone chiral center patterns (stereochemical patterns of chiral linking phosphorus). Certain useful backbone chiral center patterns are described herein. In some embodiments, the plurality of oligonucleotides share a common backbone chiral center pattern, which is described herein (e.g., "Linking Phosphorus Stereochemistry and Patterns Thereof," Table 1, backbone chiral centers of the chiral control oligonucleotides (as in a pattern, etc.) is or includes a pattern.

In some embodiments, a chirally controlled oligonucleotide composition is a chirally pure (or stereopure, stereochemically pure) oligonucleotide composition, wherein the oligonucleotide composition comprises a plurality of oligonucleotides, wherein the oligonucleotides are identical [each chiral linkage Including that each chiral element of the oligonucleotide comprising is independently defined (stereodefined)], the composition does not contain other stereoisomers. Chirally pure (or stereopure, stereochemically pure) oligonucleotide compositions of oligonucleotide stereoisomers do not contain other stereoisomers (as understood by those skilled in the art, one or more unintended stereoisomers may be present as impurities) .

Chirally controlled oligonucleotide compositions can exhibit many advantages over stereorandom oligonucleotide compositions. In particular, a chirally controlled oligonucleotide composition is more uniform with respect to oligonucleotide structure than a corresponding sterically random oligonucleotide composition. By controlling the stereochemistry, compositions of individual stereoisomers can be prepared and evaluated so that stereoisomerically controlled oligonucleotide compositions having desired properties and/or activities can be developed. In some embodiments, a chirally controlled oligonucleotide composition provides a superior delivery, stability, elimination, activity, selectivity, and/or toxicity profile compared to, for example, a corresponding stereorandom oligonucleotide composition. In some embodiments, a chirally controlled oligonucleotide composition provides greater efficacy, fewer side effects, and/or a more convenient and effective dosing regimen. In particular, backbone chiral center patterns as described herein, optionally in combination with other structural features described herein, such as nucleobases, sugars, modifications of internucleotidic linkages, etc., can be used to provide highly efficient directed adenosine editing. .

In some embodiments, the oligonucleotide composition comprises one or more internucleotide linkages that are stereocontrolled (chirally controlled; in some embodiments, stereopure) and one or more internucleotide linkages that are stereorandom. In some embodiments, the oligonucleotide composition comprises one or more internucleotide linkages that are stereocontrolled (chirally controlled; in some embodiments, stereopure) and one or more internucleotide linkages that are stereorandom.

In some embodiments, the oligonucleotide composition comprises one or more internucleotide linkages that are stereocontrolled (eg, chirally controlled or stereopure) and one or more internucleotide linkages that are stereorandom. Such oligonucleotides can target a variety of nucleic acids, can have a variety of base sequences, and can provide efficient adenosine editing (eg, A to I conversion).

In some embodiments, the present invention provides chirally controlled oligonucleotide compositions. In some embodiments, a provided chirally controlled oligonucleotide composition comprises a plurality of oligonucleotides of the same composition and has one or more internucleotidic linkages. In some embodiments, for example, the plurality of oligonucleotides in a chirally controlled oligonucleotide composition are a plurality of oligonucleotides selected from Table 1 (and/or one or more various salt forms thereof), the oligonucleotides being capable of chirally controlled internucleotide linkages and at least one R p or Sp linking phosphorus. In some embodiments, for example, the plurality of oligonucleotides in a chirally controlled oligonucleotide composition are a plurality of oligonucleotides selected from Table 1 (and/or one or more various salt forms thereof), each phosphorothio Eight internucleotide linkages are independently chirally controlled (each phosphorothioate internucleotide linkage is independently R p or Sp ). In some embodiments, an oligonucleotide composition, e.g., an oligonucleotide composition, is a single oligonucleotide in that the oligonucleotides in the composition are impurities that arise in the manufacturing process of the single oligonucleotide, and in some cases after certain purification procedures. It is a substantially pure preparation of oligonucleotides. In some embodiments, the single oligonucleotide is an oligonucleotide of Table 1, and each chiral internucleotidic linkage of the oligonucleotide is chirally controlled (e.g., designated as S or R rather than X in "stereochemistry/linkage"). .

In some embodiments, a chirally controlled oligonucleotide composition has increased activity and/or stability, increased delivery, and/or reduced ability to induce adverse effects such as complement, TLR9 activation, etc., relative to a corresponding stereorandom oligonucleotide composition. can In some embodiments, a sterically random (non-chiral controlled) oligonucleotide composition is such that the corresponding plurality of oligonucleotides do not contain chiral controlled internucleotide linkages, but the sterically randomized oligonucleotide composition is otherwise identical to the chirally controlled oligonucleotide composition. It is different from the chiral control oligonucleotide composition in

In some embodiments, the present invention relates to chirally controlled oligonucleotide compositions capable of modulating the level, activity or expression of a gene or its gene product. Reference conditions (e.g., absence of oligonucleotides and/or compositions of the invention, and/or reference oligonucleotides and/or oligonucleotide compositions (e.g., oligonucleotides with the same base sequence but different modifications, comparable structures) In some embodiments, the level, activity or expression of a gene or its gene product is increased ( For example, correction of mutation restoration from G to A through conversion of A to I, increasing the level of protein translation, increasing the production of certain protein isoforms, increasing the level of certain splicing products and proteins encoded thereby. regulation of splicing, etc.), in some embodiments, the level, activity or expression of a gene or its gene product is reduced (e.g., generation of stop codons via A to I conversion and/or codon alteration, protein translation reduction in levels, reduction in production of specific protein isoforms, modulation of splicing to reduce levels of specific splicing products and proteins encoded thereby, etc.).

In some embodiments, the present invention may increase the level, activity or expression of a gene or its gene product, and herein (e.g., Table 1 (each T may be independently replaced with U and vice versa) ) providing a chirally controlled oligonucleotide composition comprising a plurality of oligonucleotides sharing a common base sequence that is, includes, or spans (eg, at least 10 or 15 contiguous bases) the base sequence disclosed in do. In some embodiments, the present invention may increase the level, activity or expression of a gene or its gene product, and herein (e.g., Table 1 (each T may be independently replaced with U and vice versa) )), or a chirally controlled oligonucleotide composition comprising a plurality of oligonucleotides sharing a common base sequence including the same. In some embodiments, the present invention may increase the level, activity or expression of a gene or its gene product, and herein (e.g., Table 1 (each T may be independently replaced with U and vice versa) )) provides a chirally controlled oligonucleotide composition comprising a plurality of oligonucleotides sharing a common nucleotide sequence, which is the nucleotide sequence disclosed in

In some embodiments, the present invention can reduce the level, activity or expression of a gene or its gene product, and herein (e.g., Table 1 (each T can be independently replaced by a U and vice versa) ) providing a chirally controlled oligonucleotide composition comprising a plurality of oligonucleotides sharing a common base sequence that is, includes, or spans (eg, at least 10 or 15 contiguous bases) the base sequence disclosed in do. In some embodiments, the present invention can reduce the level, activity or expression of a gene or its gene product, and herein (e.g., Table 1 (each T can be independently replaced by a U and vice versa) )), or a chirally controlled oligonucleotide composition comprising a plurality of oligonucleotides sharing a common base sequence including the same. In some embodiments, the present invention can reduce the level, activity or expression of a gene or its gene product, and herein (e.g., Table 1 (each T can be independently replaced by a U and vice versa) )) provides a chirally controlled oligonucleotide composition comprising a plurality of oligonucleotides sharing a common nucleotide sequence, which is the nucleotide sequence disclosed in

In some embodiments, a provided chirally controlled oligonucleotide composition is a chirally controlled oligonucleotide composition comprising a plurality of oligonucleotides. In some embodiments, a chirally controlled oligonucleotide composition is a chirally pure (or “stereochemically pure”) oligonucleotide composition. In some embodiments, the present invention provides a chirally pure oligonucleotide composition of the oligonucleotides of Table 1, wherein each chiral internucleotidic linkage of the oligonucleotide is independently chirally controlled (e.g., R p or Sp is " can be determined from R or S but not X in "stereochemistry/linkage"). As will be appreciated by those skilled in the art, chemical selectivity rarely achieves completeness (absolute 100%). In some embodiments, a chiral pure oligonucleotide composition comprises a plurality of oligonucleotides that are structurally identical and all of the same structure (same stereoisomeric form; with respect to oligonucleotides, oligonucleotides generally have multiple chiral have the same diastereomeric form because the center is present), and a chiral pure oligonucleotide composition is generally diastereomeric because of the presence of multiple chiral centers in any other stereoisomer (with respect to oligonucleotides, oligonucleotides generally have multiple chiral centers). isomers; eg, to the extent achievable by stereoselective preparation). As will be appreciated by those skilled in the art, a stereorandom (or "racemic", "non-chiral controlled") oligonucleotide composition is a random mixture of many stereoisomers (e.g., 2 ⁿ diastereomers (n is another Chiral centers (e.g., carbon chiral centers in a sugar) are chirally controlled so that each independently exists in a configuration and only the chiral linking phosphorus centers are the number of chiral linking phosphorus for oligonucleotides that are not chiral controlled)) .

Certain data indicative of the properties and/or activity of a chirally controlled oligonucleotide composition, e.g., in modulating the level, activity and/or expression of a target gene and/or its product, are disclosed herein. Examples of the invention are illustrated.

In some embodiments, the present invention provides an oligonucleotide composition comprising an oligonucleotide comprising at least one chiral linking phosphorus. In some embodiments, the present invention provides an oligonucleotide composition comprising an oligonucleotide comprising at least one chiral linking phosphorus. In some embodiments, the present invention provides an oligonucleotide composition wherein the oligonucleotide comprises chirally controlled phosphorothioate internucleotidic linkages, wherein the linkages have the R p configuration. In some embodiments, the present invention provides oligonucleotide compositions wherein the oligonucleotide comprises chirally controlled phosphorothioate internucleotidic linkages, wherein the linkage phosphorus has an S p configuration. In some embodiments, the present invention provides an oligonucleotide composition wherein the oligonucleotide comprises chirally controlled phosphorothioate internucleotidic linkages, wherein the linking phosphorus has an R p configuration and the linking phosphorus has an Sp configuration. In some embodiments, such oligonucleotide compositions are chirally controlled, and the R p and/or S p internucleotide linkages are independently chirally controlled internucleotidic linkages.

In some embodiments, a provided oligonucleotide or oligonucleotide composition (eg, a chirally controlled oligonucleotide composition) is surprisingly effective compared to a reference oligonucleotide or oligonucleotide composition. In some embodiments, a desired biological effect (e.g., where an increase in level is targeted, measured by an increase (where an increase is desired) and/or a decrease (where a decrease is desired) the level of an mRNA, protein, etc. can be enhanced more than 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, or 100 fold (e.g., the desired mRNA, measured by levels of protein, etc.). In some embodiments, the change is measured by an increase in desired mRNA and/or protein levels, or a decrease in undesirable mRNA and/or protein levels, compared to a baseline condition. In some embodiments, change is measured by an increase in the level of a desired mRNA compared to a baseline condition. In some embodiments, change is measured by a decrease in undesirable mRNA levels compared to a baseline condition. In some embodiments, each reference condition is the absence of a provided oligonucleotide or oligonucleotide composition and/or the presence of a reference oligonucleotide or oligonucleotide composition. In some embodiments, reference oligonucleotides share the same base sequence, but have different nucleobase modifications, sugar modifications, internucleotide linkage modifications, and/or linkage phosphorus stereochemistry. In some embodiments, a reference oligonucleotide composition is a composition of oligonucleotides having the same base sequence but different nucleobase modifications, sugar modifications, internucleotidic linkage modifications, and/or linkage stereochemistry. In some embodiments, a reference composition for a chirally controlled oligonucleotide composition has identical base sequences, nucleobase modifications, sugar modifications, and/or internucleotidic linkage modifications (provided that there is a lack and/or low level of linkage phosphorus stereochemical control). or a corresponding stereorandom composition of oligonucleotides having the same configuration.

In some embodiments, the present invention provides chirally controlled oligonucleotide compositions wherein the linkage phosphorus of at least one chirally controlled internucleotidic linkage is Sp . In some embodiments, the present invention provides chirally controlled oligonucleotide compositions in which most of the linkages of the chirally controlled internucleotidic linkages are Sp . In some embodiments, about 50%-100%, 55%-100%, 60%-100%, 65%-100% of all chiral controlled internucleotide linkages (or all chiral internucleotide linkages, or all internucleotide linkages) , 70%-100%, 75%-100%, 80%-100%, 85%-100%, 90%-100%, 55%-95%, 60%-95%, 65%-95%, or At least about 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99% is Sp . In some embodiments, about 50%-100%, 55%-100%, 60%-100%, 65%-100%, 70%-100%, 75%-100% of all chirally controlled phosphorothioate internucleotidic linkages 100%, 80% to 100%, 85% to 100%, 90% to 100%, 55% to 95%, 60% to 95%, 65% to 95%, or about 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99% or more is Sp. In some embodiments, at most 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 of the phosphorothioate internucleotide linkages are not chirally controlled or are chirally controlled and R p . In some embodiments, at most 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 of the phosphorothioate internucleotide linkages are chirally controlled and Rp. In some embodiments, the number is at most one. In some embodiments, the number is at most two. In some embodiments, the number is up to three. In some embodiments, the number is up to four. In some embodiments, the number is up to 5. In some embodiments, each phosphorothioate internucleotidic linkage is independently chirally controlled. In some embodiments, the present invention provides chirally controlled oligonucleotide compositions in which a majority of the chiral internucleotidic linkages are chirally controlled and their linkage phosphorus is Sp . In some embodiments, the present invention provides chirally controlled oligonucleotide compositions wherein each chiral internucleotidic linkage is chirally controlled and each chiral linkage phosphorus is Sp . In some embodiments, the present invention provides a chirally controlled oligonucleotide composition, eg, a chirally controlled oligonucleotide composition having at least one chirally controlled internucleotide linkage being an R p linkage. In some embodiments, the present invention provides a chirally controlled oligonucleotide composition wherein at least one chirally controlled internucleotide linkage comprises an R p linkage phosphorus and at least one chirally controlled internucleotide linkage comprises an S p linkage phosphorus.

In some embodiments, the present invention provides a chirally controlled oligonucleotide composition wherein at least two chirally controlled internucleotidic linkages have stereochemistry and/or other P-modifications with respect to each other, wherein the P-modification is a modification at the linkage phosphorus. provides In some embodiments, the present invention provides a chirally controlled oligonucleotide composition wherein at least two chirally controlled internucleotide linkages have different stereochemistry with respect to each other, and the backbone chiral central pattern of the oligonucleotide is characterized by a repeating pattern of alternating stereochemistry. to provide.

In certain embodiments, the present invention provides a chirally controlled oligonucleotide composition comprising a plurality of oligonucleotides, wherein at least two individual internucleotide linkages within each oligonucleotide have different P-modifications relative to each other. In certain embodiments, the invention provides a chiral oligonucleotide comprising a plurality of oligonucleotides wherein at least two individual internucleotide linkages within each oligonucleotide have different P-modifications relative to each other and each oligonucleotide comprises a native phosphate linkage. A control oligonucleotide composition is provided. In certain embodiments, the invention provides a plurality of oligonucleotides wherein at least two individual internucleotide linkages within each oligonucleotide have different P-modifications relative to each other and each oligonucleotide comprises a phosphorothioate internucleotide linkage. Provides a chirally controlled oligonucleotide composition comprising a. In certain embodiments, the invention provides a method wherein at least two individual internucleotide linkages within each oligonucleotide have different P-modifications relative to each other and each oligonucleotide comprises a natural phosphate linkage and a phosphorothioate internucleotide linkage. A chirally controlled oligonucleotide composition comprising a plurality of oligonucleotides is provided. In certain embodiments, the invention provides a plurality of oligonucleotides wherein at least two individual internucleotide linkages within each oligonucleotide have different P-modifications relative to each other and each oligonucleotide comprises a phosphorothioate triester internucleotide linkage. A chirally controlled oligonucleotide composition comprising an oligonucleotide is provided. In certain embodiments, the invention provides that at least two individual internucleotidic linkages within each oligonucleotide have different P-modifications relative to each other and each oligonucleotide contains a natural phosphate linkage and a phosphorothioate triester internucleotidic linkage. A chirally controlled oligonucleotide composition comprising a plurality of oligonucleotides comprising In certain embodiments, the invention provides that within each oligonucleotide at least two individual internucleotidic linkages have different P-modifications relative to each other and each oligonucleotide comprises a phosphorothioate internucleotidic linkage and a phosphorothioate triester A chirally controlled oligonucleotide composition comprising a plurality of oligonucleotides comprising internucleotide linkages is provided.

In some embodiments, the present invention provides a chirally controlled oligonucleotide composition comprising a plurality of oligonucleotides sharing a common base sequence, which is the base sequence of the oligonucleotides disclosed herein, wherein at least one internucleotide linkage is chirally controlled.

Linkage phosphorus stereochemistry and backbone chiral center patterns

In particular, the present invention provides various oligonucleotide compositions. In some embodiments, the present invention provides oligonucleotide compositions of the oligonucleotides described herein. In some embodiments, an oligonucleotide composition comprises a plurality of oligonucleotides described herein. In some embodiments, the oligonucleotide composition is chirally controlled. In some embodiments, the oligonucleotide composition is not chirally controlled (sterically random).

In contrast to natural phosphate linkages, chiral modified internucleotide linkages, such as phosphorothioate internucleotide linkages, are chiral. In particular, the invention provides techniques (eg, oligonucleotides, compositions, methods, etc.) involving control of the stereochemistry of chiral linking phosphorus in chiral internucleotidic linkages. In some embodiments, as shown herein, control of stereochemistry achieves desired stability, reduced toxicity, improved modification of target nucleic acids, improved levels of transcripts and/or their encoded products (eg, mRNAs, proteins, etc.) may provide improved properties and/or activity, including modified regulation and the like. In some embodiments, the present invention provides a useful backbone chiral center pattern for an oligonucleotide and/or region thereof, which pattern comprises each chiral linkage phosphorus ( R p or Sp ) of the chiral linkage phosphorus in the 5' to 3' direction. combination of stereochemistry, indication of each achiral linking phosphorus (Op, if present), and the like. Specific patterns are provided in various tables (eg, stereochemistry/linkage as an example; such patterns can be applied to various oligonucleotides having various base sequences and modifications (eg, those described herein including patterns thereof) may apply).

Useful backbone chiral center patterns, such as those for oligonucleotides, first domains, second domains, first subdomains, second subdomains, third subdomains, etc., are extensively described herein. For example, in some embodiments, an oligonucleotide or one or more portions thereof (e.g., a first domain, a second domain, a first subdomain, a second subdomain, and/or a third subdomain therein, and / or 5'-end and/or 3'-end) high levels of Sp internucleotidic linkages provide high stability and/or activity. In some embodiments, the first domain comprises a high level of Sp internucleotidic linkages. In some embodiments, the second domain comprises a high level of Sp internucleotidic linkages (number and/or percentage relative to native phosphate linkages and/or R p internucleotidic linkages). In some embodiments, the first subdomain comprises a high level of S p internucleotidic linkages. In some embodiments, the second subdomain comprises a high level of S p internucleotidic linkages. In some embodiments, the third subdomain comprises a high level of S p internucleotidic linkages. In some embodiments, as shown herein, R p internucleotidic linkages can be used at various positions and/or moieties. For example, in some embodiments, the first domain comprises one or more or high levels of R p internucleotidic linkages, and in some embodiments, the second subdomain comprises one or more or high levels of R p internucleotidic linkages. do.

In some embodiments, multiple linkages in a chirally controlled internucleotidic linkage are Sp . In some embodiments, at least 10%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75% of the chiral control internucleotide linkages , 80%, 85%, 90%, or 95% have Sp linked phosphorus. In some embodiments, at least 10%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75% of all chiral internucleotide linkages , 80%, 85%, 90%, or 95% are chirally controlled internucleotidic linkages with an Sp linkage phosphorus. In some embodiments, at least 10%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75% of all internucleotide linkages 80%, 85%, 90%, or 95% are chirally controlled internucleotidic linkages with Sp linkage phosphorus. In some embodiments, at least 10%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70% of all phosphorothioate internucleotide linkages , 75%, 80%, 85%, 90%, or 95% have Sp linked phosphorus. In some embodiments, the percentage is at least 20%. In some embodiments, the percentage is at least 30%. In some embodiments, the percentage is at least 40%. In some embodiments, the percentage is at least 50%. In some embodiments, the percentage is at least 60%. In some embodiments, the percentage is at least 65%. In some embodiments, the percentage is at least 70%. In some embodiments, the percentage is at least 75%. In some embodiments, the percentage is at least 80%. In some embodiments, the percentage is at least 90%. In some embodiments, the percentage is at least 95%. In some embodiments, all chiral control internucleotidic linkages have an Sp linkage phosphorus. In some embodiments, all chirally controlled phosphorothioate internucleotidic linkages have an Sp linkage phosphorus. In some embodiments, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, Internucleotidic linkages of 23, 24, or 25 are chirally controlled internucleotidic linkages with an S p linkage phosphorus. In some embodiments, at least 5 internucleotidic linkages are chirally controlled internucleotidic linkages with Sp linkages phosphorus. In some embodiments, at least 6 internucleotidic linkages are chirally controlled internucleotidic linkages with an Sp linkage phosphorus. In some embodiments, at least 7 internucleotidic linkages are chirally controlled internucleotidic linkages with an Sp linkage phosphorus. In some embodiments, at least 8 internucleotidic linkages are chirally controlled internucleotidic linkages with an Sp linkage phosphorus. In some embodiments, at least 9 internucleotidic linkages are chirally controlled internucleotidic linkages with an Sp linkage phosphorus. In some embodiments, at least 10 internucleotidic linkages are chirally controlled internucleotidic linkages with an Sp linkage phosphorus. In some embodiments, at least 11 internucleotidic linkages are chirally controlled internucleotidic linkages with an Sp linkage phosphorus. In some embodiments, at least 12 internucleotidic linkages are chirally controlled internucleotidic linkages with an Sp linkage phosphorus. In some embodiments, at least 13 internucleotidic linkages are chirally controlled internucleotidic linkages with Sp linkages phosphorus. In some embodiments, at least 14 internucleotidic linkages are chirally controlled internucleotidic linkages with an Sp linkage phosphorus. In some embodiments, at least 15 internucleotidic linkages are chirally controlled internucleotidic linkages with an Sp linkage phosphorus. In some embodiments, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, Internucleotidic linkages of 23, 24, or 25 are chirally controlled internucleotidic linkages with an R p linkage phosphorus. In some embodiments, at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, The 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 internucleotide linkages are chirally controlled internucleotide linkages with an R p linkage phosphorus. In some embodiments, at most one internucleotidic linkage in an oligonucleotide is a chirally controlled internucleotidic linkage with an R p linkage phosphorus. In some embodiments, no more than two internucleotidic linkages in an oligonucleotide are chirally controlled internucleotidic linkages with R p linkage phosphorus. In some embodiments, no more than 3 internucleotidic linkages in an oligonucleotide are chirally controlled internucleotidic linkages with R p linkage phosphorus. In some embodiments, no more than 4 internucleotidic linkages in an oligonucleotide are chirally controlled internucleotidic linkages with R p linkage phosphorus. In some embodiments, no more than 5 internucleotidic linkages in an oligonucleotide are chirally controlled internucleotidic linkages with R p linkage phosphorus.

In some embodiments, all, essentially all, or most of the internucleotide linkages in an oligonucleotide or portion thereof (e.g., all chiral controlled internucleotide linkages, or all chiral internucleotidic linkages, or all internucleotide linkages in an oligonucleotide) Approximately 50% to 100%, 55% to 100%, 60% to 100%, 65% to 100%, 70% to 100%, 75% to 100%, 80% to 100%, 85% to 100% of connections , 90% to 100%, 55% to 95%, 60% to 95%, 65% to 95%, or about 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% , 95%, 97%, 99% or more) is one or a few internucleotide linkages (e.g., all chiral control internucleotide linkages in an oligonucleotide, or all chiral internucleotide linkages, or 1 of all internucleotide linkages) , 2, 3, 4, or 5, and/or less than 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, or 5%) of the R p configuration. , except that S p is the configuration. In some embodiments, all, essentially all, or most of the internucleotide linkages in the first domain (e.g., all chiral controlled internucleotide linkages, or all chiral internucleotide linkages, or all internucleotide linkages in the first domain) About 50% to 100%, 55% to 100%, 60% to 100%, 65% to 100%, 70% to 100%, 75% to 100%, 80% to 100%, 85% to 100%, 90% to 100%, 55% to 95%, 60% to 95%, 65% to 95%, or about 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99% or more) are in the S p configuration. In some embodiments, each internucleotide linkage in the first domain is a phosphorothioate in the S p configuration. In some embodiments, each internucleotide linkage in a domain is a phosphorothioate in the S p configuration. In some embodiments, all, essentially all, or most of the internucleotide linkages in the second domain (e.g., all chiral controlled internucleotide linkages, or all chiral internucleotide linkages, or all internucleotide linkages in the second domain) About 50% to 100%, 55% to 100%, 60% to 100%, 65% to 100%, 70% to 100%, 75% to 100%, 80% to 100%, 85% to 100%, 90% to 100%, 55% to 95%, 60% to 95%, 65% to 95%, or about 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99% or more) are in the S p configuration. In some embodiments, each internucleotide linkage in the second domain is a phosphorothioate in the S p configuration. In some embodiments, each internucleotide linkage in the second domain is a phosphorothioate in the S p configuration, except for one phosphorothioate in the R p configuration. In some embodiments, all, essentially all, or most of the internucleotide linkages in a subdomain of the second domain (e.g., all chiral control internucleotide linkages in a first subdomain of the second domain, or all chiral nucleotides) internucleotide linkages, or about 50% to 100%, 55% to 100%, 60% to 100%, 65% to 100%, 70% to 100%, 75% to 100%, 80% to 100% of all internucleotide linkages %, 85% to 100%, 90% to 100%, 55% to 95%, 60% to 95%, 65% to 95%, or about 55%, 60%, 65%, 70%, 75%, 80 %, 85%, 90%, 95%, 97%, 99% or more) is the Sp configuration. In some embodiments, each internucleotide linkage in the first subdomain of the second domain is a phosphorothioate in the S p configuration. In some embodiments, each internucleotide linkage in the first subdomain of the second domain is a phosphorothioate in the S p configuration, except for one phosphorothioate in the R p configuration. In some embodiments, all, essentially all or most of the internucleotide linkages in a second subdomain of the second domain (e.g., all chirally controlled internucleotide linkages in a second subdomain of the second domain, or all chiral internucleotide linkages, or about 50% to 100%, 55% to 100%, 60% to 100%, 65% to 100%, 70% to 100%, 75% to 100%, 80% of all internucleotide linkages ~100%, 85%~100%, 90%~100%, 55%~95%, 60%~95%, 65%~95%, or about 55%, 60%, 65%, 70%, 75% , 80%, 85%, 90%, 95%, 97%, 99% or more) are in the Sp configuration, except that one or a few internucleotide linkages are in the R p configuration. In some embodiments, each internucleotide linkage in the second subdomain of the second domain is a phosphorothioate in the S p configuration, except for one phosphorothioate in the R p configuration. In some embodiments, each internucleotide linkage in the second subdomain of the second domain is a phosphorothioate in the S p configuration, except for one phosphorothioate in the R p configuration. In some embodiments, all, essentially all or most of the internucleotide linkages in a third subdomain of the second domain (e.g., all chirally controlled internucleotide linkages in a third subdomain of the second domain, or all chiral internucleotide linkages, or about 50% to 100%, 55% to 100%, 60% to 100%, 65% to 100%, 70% to 100%, 75% to 100%, 80% of all internucleotide linkages ~100%, 85%~100%, 90%~100%, 55%~95%, 60%~95%, 65%~95%, or about 55%, 60%, 65%, 70%, 75% , 80%, 85%, 90%, 95%, 97%, 99% or more) is the Sp configuration. In some embodiments, each internucleotide linkage in the third subdomain of the second domain is a phosphorothioate in the S p configuration, except for one phosphorothioate in the R p configuration. In some embodiments, each internucleotide linkage in the third subdomain of the second domain is a phosphorothioate in the S p configuration, except for one phosphorothioate in the R p configuration.

In some embodiments, an oligonucleotide comprises one or more R p internucleotidic linkages. In some embodiments, an oligonucleotide comprises no more than one R p internucleotidic linkage. In some embodiments, an oligonucleotide comprises 5 or more R p internucleotidic linkages. In some embodiments, about 5% to 50% of all chirally controlled internucleotide linkages in an oligonucleotide are R p . In some embodiments, about 5% to 40% of all chirally controlled internucleotide linkages in an oligonucleotide are Rp. In some embodiments, a particular portion (eg, a domain, subdomain, etc.) may contain (eg, in number and/or percentage) relatively more R p internucleotidic linkages (eg, a second subdomain).

In some embodiments, the oligonucleotide comprises one or more R p phosphorothioate internucleotidic linkages at one or more positions, e.g., positions -1, -2, +1, +2, +7, +8, etc. In some embodiments, the internucleotide linkage at position -1 is an R p phosphorothioate internucleotidic linkage. In some embodiments, the internucleotidic linkage at position -2 is an R p phosphorothioate internucleotidic linkage. In some embodiments, the internucleotidic linkage at position +1 is an R p phosphorothioate internucleotidic linkage. In some embodiments, the internucleotidic linkage at position +2 is an R p phosphorothioate internucleotidic linkage. In some embodiments, the 2 or 3 internucleotide linkages at positions -1, -2, +1 and +2 are R p phosphorothioate internucleotide linkages. In some embodiments, positions are -1 and -2. In some embodiments, positions are +1 and +2. In some embodiments, positions are -1 and +1. In some embodiments, positions are -1, +1 and +2. In some embodiments, positions are -1, -2 and +1. In some embodiments, only one internucleotidic linkage is an R p phosphorothioate internucleotidic linkage. In some embodiments, only one internucleotide linkage is an R p phosphorothioate internucleotide linkage and is at position +2, +1, 1 or 2. In some implementations, the position is +1. In some implementations, the position is +2 times. In some embodiments, position is -1. In some embodiments, position is -2. In some embodiments, it is observed that use of R p internucleotidic linkages can improve editing efficiency by ADAR1 (p110 and/or p150) and/or ADAR2. In some embodiments, the improvement in editing by ADAR1 (p110 and/or p150) is greater than that by ADAR2 (no improvement or little editing compared to absence of Rp ).

In some embodiments, the present invention provides a chirally controlled oligonucleotide composition, wherein the composition comprises non-random or controlled levels of a plurality of oligonucleotides, wherein the plurality of oligonucleotides share a common base sequence and have a range of 1-60; 1~50, 1~40, 1~30, 1~25, 1~20, 1~15, 1~10, 5~50, 5~40, 5~30, 5~25, 5~20, 5~ 15, 5~10, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 or more chiral internucleotidic linkages independently share the same sequence of linkages.

In some embodiments, provided oligonucleotides contain 2-30 chirally controlled internucleotide linkages. In some embodiments, provided oligonucleotide compositions include 5-30 chirally controlled internucleotide linkages. In some embodiments, provided oligonucleotide compositions include 10-30 chirally controlled internucleotide linkages. In some embodiments, provided oligonucleotide compositions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, or 25 or more chiral controlled internucleotide linkages.

In some embodiments, about 1-100% of all internucleotide linkages are chirally controlled internucleotide linkages. In some embodiments, about 1-100% of all chiral internucleotide linkages are chiral controlled internucleotide linkages. In some embodiments, the percentage is between about 5% and 100%. In some embodiments, the percentage is at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 965, 96%, 98%, or 99%. In some embodiments, the percentage is about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 965, 96%, 98%, or 99%.

In some embodiments, the internucleotidic linkages of the Sp configuration (with the Sp linking phosphorus) are phosphorothioate internucleotidic linkages. In some embodiments, achiral internucleotidic linkages are natural phosphate linkages. In some embodiments, the internucleotidic linkages of the Rp configuration (with Rp linkage phosphorus) are phosphorothioate internucleotidic linkages. In some embodiments, each internucleotidic linkage of the Sp configuration is a phosphorothioate internucleotidic linkage. In some embodiments, each achiral internucleotidic linkage is a natural phosphate linkage. In some embodiments, each internucleotidic linkage of the R p configuration is a phosphorothioate internucleotidic linkage. In some embodiments, each internucleotide linkage of the S p arrangement is a phosphorothioate internucleotide linkage, each achiral internucleotide linkage is a natural phosphate linkage, and each internucleotidic linkage of the R p arrangement is a phosphorothioate internucleotide linkage. It is an eight internucleotide linkage.

In some embodiments, each provided oligonucleotide in a chirally controlled oligonucleotide composition comprises a different type of internucleotidic linkage. In some embodiments, a provided oligonucleotide comprises at least one native phosphate linkage and at least one modified internucleotidic linkage. In some embodiments, provided oligonucleotides have at least one natural phosphate linkage and 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, Between 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 modified nucleotides Include connections. In some embodiments, the modified internucleotidic linkage is a phosphorothioate internucleotidic linkage. In some embodiments, each modified internucleotide linkage is a phosphorothioate internucleotide linkage. In some embodiments, each modified internucleotide linkage is independently a chiral internucleotide linkage and is independently chirally controlled.

In some embodiments, each of the oligonucleotides in a chirally controlled oligonucleotide composition comprises at least two internucleotidic linkages with different stereochemistry and/or different P-modifications relative to each other. In some embodiments, at least two internucleotidic linkages have different stereochemistry with respect to each other. In some embodiments, each oligonucleotide comprises a pattern of backbone chiral centers comprising a stereochemistry that is alternating linkages.

In some embodiments, the phosphorothioate triester linkage includes a chiral auxiliary, which is used, eg, to control the stereoselectivity of a reaction, eg, a coupling reaction in an oligonucleotide synthesis cycle. In some embodiments, the phosphorothioate triester linkage does not include a chiral auxiliary. In some embodiments, the phosphorothioate triester linkage is intentionally maintained until and/or during administration of the oligonucleotide composition to the subject.

In some embodiments, oligonucleotides are linked to a solid support. In some embodiments, the solid support is a support for oligonucleotide synthesis. In some embodiments, the solid support comprises glass. In some embodiments, the solid support is CPG (controlled pore glass). In some embodiments, the solid support is a polymer. In some embodiments, the solid support is polystyrene. In some embodiments, the solid support is highly cross-linked polystyrene (HCP). In some embodiments, the solid support is a hybrid support of controlled pore glass (CPG) and highly cross-linked polystyrene (HCP). In some embodiments, the solid support is a metal foam. In some embodiments, the solid support is a resin. In some embodiments, oligonucleotides are cleaved from the solid support.

In some embodiments, all other chiral centers of an oligonucleotide are stereodefined (e.g., carbon chiral centers of sugars, which are defined, for example, in phosphoramidites for oligonucleotide synthesis), except for chiral linkage phosphorus centers. The purity of many oligonucleotides and compositions thereof, particularly stereochemical purity, particularly diastereomeric purity, depends on the stereoselectivity at the chiral linkage phosphorus in the coupling step when forming chiral internucleotidic linkages (as understood by those skilled in the art, Many cases of oligonucleotide synthesis in which a nucleotide contains more than one chiral center can be controlled by diastereoselectivity). In some embodiments, the coupling step has a stereoselectivity of 60% (diastereoselectivity if there are other chiral centers) at the linking phosphorus. After this coupling step, the new internucleotidic linkages formed can be said to have a stereochemical purity of 60% (in the case of oligonucleotides, generally diastereomeric purity given the presence of other chiral centers). In some embodiments, each coupling step is independently at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, It has a stereoselectivity of 98%, 99%, or 99.5%. In some embodiments, chiral control internucleotide linkages are generally at least 85%, 87%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99.5% or Nearly 100% (in some embodiments, at least 85%; in some embodiments, at least 87%; in some embodiments, at least 90%; in some embodiments, at least 95%; in some embodiments, at least 96%; in some embodiments, at least 96%; some In embodiments, it is formed with a stereoselectivity of at least 97%; in some embodiments, at least 98%; in some embodiments, at least 99%). In some embodiments, stereoselectivity is at least 85%. In some embodiments, stereoselectivity is at least 87%. In some embodiments, stereoselectivity is at least 90%. In some embodiments, each coupling step independently has a stereoselectivity of approximately 100%.

In some embodiments, the stereopurity of chiral centers, e.g., chiral linking phosphorus in the composition, is at least 60%, 70%, 80%, 85%, 87%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5%. In some embodiments, the stereopurity is at least 80%. In some embodiments, the stereopurity is at least 85%. In some embodiments, the stereopurity is at least 87%. In some embodiments, the stereopurity is at least 90%. In some embodiments, the stereopurity is approximately 100%. In some embodiments, each chiral control internucleotide linkage is independently at least 85%, 87%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99.5% or nearly 100% (in some embodiments, at least 85%; in some embodiments, at least 87%; in some embodiments, at least 90%; in some embodiments, at least 95%; in some embodiments, at least 95%; in some embodiments , at least 96%; in some embodiments, at least 97%; in some embodiments, at least 98%; in some embodiments, at least 99%) of stereochemical purity (generally diastereomeric for oligonucleotides with multiple chiral centers) isomeric purity). In some embodiments, chirally controlled internucleotidic linkages have a stereochemical purity of at least 90%. In some embodiments, the majority of the chirally controlled internucleotidic linkages independently have a stereochemical purity of at least 90%. In some embodiments, each chiral control internucleotidic linkage independently has a stereochemical purity of at least 90%. In some embodiments, each phosphorothioate internucleotidic linkage is independently chirally controlled. In some embodiments, at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%, or all chiral control internucleotide linkages are Sp . In some embodiments, at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%, or all chirally controlled phosphorothioate internucleotidic linkages are Sp. In some embodiments, at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%, or all phosphorothioate internucleotidic linkages are chirally controlled and Sp.

Stereoselectivity and stereopurity can be evaluated by various techniques. In some embodiments, stereoselectivity and/or stereopurity is approximately 100% in that nearly all detectable stereoisomers have the intended stereochemistry when the composition is analyzed by an analytical method (eg, NMR, HPLC, etc.) .

In some embodiments, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 2, 3, 4, 5, 6, 7, 8, 9, or 10 couplings independently have less than about 60%, 70%, 80%, 85%, or 90% stereoselectivity [in the case of oligonucleotide synthesis, in general, a diastereomer for the chiral center(s) that is the linkage formed selectivity].

In some embodiments, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 oligonucleotides in a sterically random (or racemic) preparation (or sterically random/non-chiral controlled oligonucleotide composition) , 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 chiral internucleotidic linkages are independently linked to the chiral linkage of the internucleotide linkage(s) and less than about 60%, 65%, 70%, 75%, 80%, or 85% stereochemical purity (generally diastereomeric purity for oligonucleotides containing multiple chiral centers). In some embodiments, the stereochemical purity (stereopurity) is less than about 60%. In some embodiments, the stereochemical purity (stereopurity) is less than about 65%. In some embodiments, the stereochemical purity (stereopurity) is less than about 70%. In some embodiments, the stereochemical purity (stereopurity) is less than about 75%. In some embodiments, the stereochemical purity (stereopurity) is less than about 80%.

In some embodiments, a compound of the invention (e.g., an oligonucleotide, a chiral auxiliary, etc.) comprises multiple chiral elements (e.g., multiple carbons and/or phosphorus (e.g., a linking phosphorus of a chiral internucleotidic linkage)). chiral centers). In some embodiments, at least 1, 2, 3, 4, 5, 6, 7, 8, 9 or more chiral elements of a provided compound (eg oligonucleotide) are each independently a diastereomer as described herein. have purity In some embodiments, the diastereomeric purity is at least 85%. In some embodiments, the diastereomeric purity is at least 86%. In some embodiments, the diastereomeric purity is at least 87%. In some embodiments, the diastereomeric purity is at least 88%. In some embodiments, the diastereomeric purity is at least 89%. In some embodiments, the diastereomeric purity is at least 90%. In some embodiments, the diastereomeric purity is at least 91%. In some embodiments, the diastereomeric purity is at least 92%. In some embodiments, the diastereomeric purity is at least 93%. In some embodiments, the diastereomeric purity is at least 94%. In some embodiments, the diastereomeric purity is at least 95%. In some embodiments, the diastereomeric purity is at least 96%. In some embodiments, the diastereomeric purity is at least 97%. In some embodiments, the diastereomeric purity is at least 98%. In some embodiments, the diastereomeric purity is at least 99%.

As will be appreciated by those of ordinary skill in the art, in some embodiments, the diastereoselectivity of coupling or the diastereomeric purity of the chiral linkage phosphorus center is dependent on the diastereoselectivity of dimer formation or a portion of a dimer prepared under identical or comparable conditions. Can be assessed via stereoisomeric purity, these dimers have identical 5'- and 3'-nucleoside and internucleoside linkages.

A variety of techniques are used to determine the stereochemistry of chiral elements (e.g., arrangement of chiral linking phosphorus) and/or identification or confirmation of backbone chiral center patterns, and/or stereoselectivity (e.g., diastereometry of coupling steps in oligonucleotide synthesis). selectivity) and/or stereochemical purity (eg, internucleotide linkages, diastereomeric purity of compounds (eg, oligonucleotides), etc.). Exemplary techniques include NMR (e.g., 1D (one-dimensional) and/or 2D (two-dimensional) ¹ H- ³¹ P HETCOR (heteronuclear correlation spectroscopy)), HPLC, RP-HPLC, mass spectrometry, LC-MS, and cleavage of internucleotidic linkages by stereospecific nucleases and the like, which may be used individually or in combination. Examples of useful nucleases are benzonase, micrococcal nuclease, and svPDE (snake venom phosphodiesterase), which have specific internucleotidic linkages with Rp linkage phosphorus (e.g., Rp phosphorothioate linkages). specific); and nuclease P1, mung bean nuclease, and nuclease S1, which are specific for internucleotide linkages with Sp linked phosphorus (eg, Sp phosphorothioate linkages). Without wishing to be bound by any particular theory, the present invention believes that, at least in some cases, cleavage of an oligonucleotide by a particular nuclease can lead to structural elements, such as chemical modifications (e.g., 2'-modifications of sugars), bases Note that it may be influenced by sequence, or stereochemical context. For example, in some cases, Benzonase and micrococcal nucleases specific for internucleotidic linkages with Rp linkage phosphorus form isolated Rp phosphorothioate internucleotides flanked by Sp phosphorothioate internucleotide linkages. It is observed that the connection could not be severed.

In some embodiments, oligonucleotides that share a common base sequence, a common backbone linkage pattern, and a common backbone chiral center pattern share a common backbone phosphorus modification pattern and a common base modification pattern. In some embodiments, oligonucleotide compositions that share a common base sequence, a common backbone linkage pattern, and a common backbone chiral center pattern share a common backbone phosphorus modification pattern and a common nucleoside modification pattern. In some embodiments, oligonucleotides that share a common base sequence, a common backbone linkage pattern, and a common backbone chiral center pattern have the same structure.

In some embodiments, the present invention provides an oligonucleotide composition comprising a plurality of oligonucleotides capable of directing the deamination of a target adenosine in a target nucleic acid, wherein the plurality of oligonucleotides are of a particular oligonucleotide type, the composition comprising: Chirally controlled in that oligonucleotides of a particular oligonucleotide type are enriched relative to a substantially racemic preparation of oligonucleotides having the same sequence.

In some embodiments, a plurality of oligonucleotides or a particular oligonucleotide type of oligonucleotide in a provided oligonucleotide composition is an oligonucleotide. In some embodiments, the present invention provides a chirally controlled oligonucleotide composition comprising a plurality of oligonucleotides, wherein the oligonucleotides

consensus Sequence,

a common backbone connection pattern; and

share the same linkage stereochemistry in one or more chiral internucleotidic linkages (chiral controlled internucleotidic linkages);

The composition is enriched in a plurality of oligonucleotides relative to a substantially racemic preparation of oligonucleotides that share a common base sequence and backbone linkage pattern.

In some embodiments, the present invention provides a chirally controlled oligonucleotide composition comprising a plurality of oligonucleotides, wherein the oligonucleotides

consensus Sequence,

a common backbone connection pattern; and

share a common backbone chiral center pattern, and the composition comprises at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60% of the oligonucleotides in the composition , 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 93%, 95%, 96%, 97%, 98%, or 99% of the consensus sequence , a substantially pure preparation of a single oligonucleotide in that it has a common backbone linkage pattern, and a common backbone chiral center pattern.

In some embodiments, the oligonucleotide composition type is further defined by 4) additional chemical moieties, if present.

In some embodiments, the percentage is at least about 10%. In some embodiments, the percentage is at least about 20%. In some embodiments, the percentage is at least about 30%. In some embodiments, the percentage is at least about 40%. In some embodiments, the percentage is at least about 50%. In some embodiments, the percentage is at least about 60%. In some embodiments, the percentage is at least about 70%. In some embodiments, the percentage is at least about 75%. In some embodiments, the percentage is at least about 80%. In some embodiments, the percentage is at least about 85%. In some embodiments, the percentage is at least about 90%. In some embodiments, the percentage is at least about 91%. In some embodiments, the percentage is at least about 92%. In some embodiments, the percentage is at least about 93%. In some embodiments, the percentage is at least about 94%. In some embodiments, the percentage is at least about 95%. In some embodiments, the percentage is at least about 96%. In some embodiments, the percentage is at least about 97%. In some embodiments, the percentage is at least about 98%. In some embodiments, the percentage is at least about 99%. In some embodiments, the percentage is greater than or equal to (DS) ^nc , and DS and nc are each independently as described herein.

In some embodiments, a plurality of oligonucleotides share the same composition. In some embodiments, the plurality of oligonucleotides are identical (identical stereoisomers). In some embodiments, a chirally controlled oligonucleotide composition is a stereopure oligonucleotide composition in which the plurality of oligonucleotides are identical (identical stereoisomers) and the composition does not contain any other stereoisomers. One skilled in the art will understand that processing, selectivity, purification, etc. may not achieve completeness and therefore one or more other stereoisomers may be present as impurities.

In some embodiments, a provided composition, when contacted with a target nucleic acid (e.g., a transcript (e.g., pre-mRNA hybridized with an oligonucleotide of the composition, mature mRNA, other type of RNA, etc.)) is a target nucleic acid. and/or the level of the product encoded thereby is reduced compared to that observed in baseline conditions. In some embodiments, the level of a nucleic acid and/or a product thereof (a nucleic acid is the product of A to I editing of a target nucleic acid) is increased. In some embodiments, the reference condition is selected from the group consisting of absence of the composition, presence of the reference composition, and combinations thereof. In some embodiments, the reference condition is the absence of a composition. In some embodiments, the reference condition is the presence of a reference composition. In some embodiments, a reference composition is a composition in which the oligonucleotide does not hybridize with the target nucleic acid. In some embodiments, a reference composition is a composition in which the oligonucleotide does not contain sequences sufficiently complementary to the target nucleic acid. In some embodiments, a reference composition is a composition in which the oligonucleotides share the same base sequence but do not share the same nucleobases, sugars, and/or internucleotide linkage modifications. In some embodiments, a provided composition is a chirally controlled oligonucleotide composition and a reference composition is the same non-chirally controlled oligonucleotide composition except that it is not chirally controlled (e.g., a composition identical to a plurality of oligonucleotides in a chirally controlled oligonucleotide composition). is a racemic preparation of oligonucleotides of

In some embodiments, the present invention provides a chirally controlled oligonucleotide composition comprising a plurality of oligonucleotides capable of directing the deamination of a target adenosine in a target nucleic acid, wherein the oligonucleotides

consensus Sequence,

a common backbone connection pattern; and

share the same linkage stereochemistry in one or more chiral internucleotidic linkages (chiral controlled internucleotidic linkages);

The composition is enriched in a plurality of oligonucleotides relative to a substantially racemic preparation of oligonucleotides that share a common base sequence and backbone linkage pattern;

wherein, when the oligonucleotide composition is contacted with the target sequence, the deamination of the target adenosine in the target nucleic acid is improved compared to that observed under reference conditions selected from the group consisting of the absence of the composition, the presence of the reference composition, and combinations thereof. to be

As will be appreciated by those skilled in the art, deamination of a target adenosine can be assessed using a variety of techniques. In some embodiments, the technique is sequencing in which deaminated adenosine is detected as G or I. In some embodiments, deamination is assessed by the level of product (eg, RNA, protein (eg, target A is replaced by I, but otherwise encoded by the same sequence as the target nucleic acid), etc.).

As shown herein, oligonucleotide structural elements (e.g., sugar modifications, backbone linkages, backbone chiral centers, backbone phosphorus modifications, patterns thereof, etc.) and combinations thereof may surprisingly provide improved properties and/or bioactivity. can

In some embodiments, an oligonucleotide composition is a single oligonucleotide stereoisomer in that the oligonucleotides in the composition that are of the same composition but are not stereoisomers are impurities that arise in the manufacturing process of the oligonucleotide, and in some cases after certain purification procedures. It is a substantially pure formulation.

In some embodiments, the present invention provides oligonucleotides and oligonucleotide compositions that are chirally controlled and in some embodiments stereopure. For example, in some embodiments, provided compositions include non-random or controlled levels of one or more individual oligonucleotide types. In some embodiments, oligonucleotides of the same oligonucleotide type are identical.

nucleobase

A variety of nucleobases in a given oligonucleotide may be used in accordance with the present invention. In some embodiments, the nucleobases are natural nucleobases, the most commonly occurring being A, T, C, G, and U. In some embodiments, a nucleobase is a modified nucleobase in that it is not A, T, C, G, or U. In some embodiments, the nucleobase is an optionally substituted A, T, C, G, or U, or a substituted tautomer of A, T, C, G, or U. In some embodiments, a nucleobase is an optionally substituted A, T, C, G, or U, such as 5mC, 5-hydroxymethyl C, and the like. In some embodiments, a nucleobase is an alkyl-substituted A, T, C, G, or U. In some embodiments, the nucleobase is A. In some embodiments, the nucleobase is T. In some embodiments, the nucleobase is C. In some embodiments, the nucleobase is G. In some embodiments, the nucleobase is U. In some embodiments, the nucleobase is 5mC. In some embodiments, a nucleobase is a substituted A, T, C, G, or U. In some embodiments, a nucleobase is a substituted tautomer of A, T, C, G, or U. In some embodiments, substitutions protect certain functional groups within the nucleobase to minimize undesirable reactions during oligonucleotide synthesis. Techniques suitable for nucleobase protection in oligonucleotide synthesis are well known in the art and can be used in accordance with the present invention. In some embodiments, the modified nucleobase improves the properties and/or activity of the oligonucleotide. For example, in many cases, 5mC can be used instead of C to modulate certain undesirable biological effects (eg immune response). In some embodiments, when determining sequence identity, substituted nucleobases with identical hydrogen bonding patterns are treated identically to unsubstituted nucleobases, e.g., 5mC can be treated identically to C [e.g., An oligonucleotide having 5mC instead of C (eg, AT5mCG) is considered to have the same base sequence as an oligonucleotide having a C at the corresponding position(s) (eg, ATCG)]. In some embodiments, a nucleobase is or comprises an optionally substituted ring having at least one nitrogen atom. In some embodiments, the nucleobase comprises ring BA as described herein, and at least one monocyclic ring of ring BA comprises a nitrogen ring atom.

In some embodiments, an oligonucleotide comprises one or more A, T, C, G, or U. In some embodiments, an oligonucleotide comprises one or more optionally substituted A, T, C, G, or U. In some embodiments, the oligonucleotide comprises one or more of 5-methylcytidine, 5-hydroxymethylcytidine, 5-formylcytosine, or 5-carboxylcytosine. In some embodiments, the oligonucleotide comprises one or more 5-methylcytidine. In some embodiments, each nucleobase in an oligonucleotide is selected from the group consisting of optionally substituted A, T, C, G and U, and optionally substituted tautomers of A, T, C, G and U. In some embodiments, each nucleobase in an oligonucleotide is optionally protected A, T, C, G and U. In some embodiments, each nucleobase in an oligonucleotide is an optionally substituted A, T, C, G or U. In some embodiments, each nucleobase in an oligonucleotide is selected from the group consisting of A, T, C, G, U and 5mC.

As shown herein, the use of a particular nucleobase at a particular position (e.g., the nucleoside opposite to the target adenosine and/or its adjacent nucleoside(s)) provides properties and/or activities (e.g., adenosine to I). editing) can provide improved oligonucleotides. In some embodiments, a useful nucleobase is or comprises ring BA as described herein. In some embodiments, the nucleobase of the nucleoside is BA-I, BA-I-a, BA-I-b, BA-II, BA-II-a, BA-II-b, BA-III, BA-III-a, Ring BA or tautomers of Ring BA having structures BA-III-b, BA-IV, BA-IV-a, BA-IV-b, BA-V, BA-V-a, BA-V-b, or BA-VI is or comprises, wherein the nucleobase is optionally substituted or protected. In some embodiments, a nucleobase is an optionally substituted or protected below, or an optionally substituted or protected tautomer thereof.

In some embodiments, the modified nucleobases are b001U, b002U, b003U, b004U, b005U, b006U, b007U, b008U, b009U, b011U, b012U, b013U, b001A, b002A, b003A, b001G, b002G, b001C, b002C , b003C, b004C , b005C, b006C, b007C, b008C, b009C, b002I, b003I, b004I, b014I or zdnp. In some embodiments, the modified nucleobases are zdnp, b001U, b002U, b003U, b004U, b005U, b006U, b008U, b002A, b001G, b004C, b007U, b001A, b001C, b002C, b003C, b002I, b003I, b009U, b003A or b007C am. In some embodiments, the present invention provides oligonucleotides comprising one or more such nucleobases. In some embodiments, the invention provides compounds comprising such nucleobases. In some embodiments, the present invention provides monomers comprising such nucleobases (eg, those useful in oligonucleotide synthesis). In some embodiments, the present invention provides phosphoramidites comprising such nucleobases. In some embodiments, the phosphoramidite is a CED phosphoramidite. In some embodiments, monomers include auxiliary moieties as described herein (eg, including P forming bonds to O and N, O and S, S and S, etc.). In some embodiments, the phosphoramidites include chiral auxiliary moieties as described herein (eg, including P forming bonds to O and N). In some embodiments, R ^NS comprises such nucleobases. In some embodiments, nucleobases are protected for oligonucleotide synthesis.

In some embodiments, the present invention provides various nucleosides. In some embodiments, b001U, b002U, b003U, b004U, b005U, b006U, b008U, b002A, b001G, b004C, b007U, b001A, b001C, b002C, b003C, b002I, b003I, b009U, b003A, or b0 07C is also its nucleobase b001U, b002U, b003U, b004U, b005U, b006U, b008U, b002A, b001G, b004C, b007U, b001A, b001C, b002C, b003C, b002I, b003I, b009U, b003A, or b007 can refer to a nucleoside that is C. there is. For example, b001A has its nucleobase and may refer to a nucleoside whose sugar is a natural DNA sugar; Sugar variants may also be indicated. For example, the "r" in b001rA indicates the presence of a 2'-OH on the sugar (natural RNA sugar). In some embodiments, the present invention , or A compound having the structure of, or a salt thereof, wherein BA ^s is as described herein. In some embodiments, a provided compound, e.g., a nucleoside, or a salt thereof, wherein “*” indicates linkages to internucleotidic linkages when present in various oligonucleotides, and BA ^s are as described herein. In some embodiments, BA ^s is a nucleobase, eg, BA as described herein. In some embodiments, BA is protected for oligonucleotide synthesis. In some embodiments, a provided nucleoside is

) or Tsm18( ), or salts thereof, wherein "*" indicates a linkage to an internucleotidic linkage when present in various oligonucleotides. In some embodiments, an oligonucleotide comprises a nucleoside described herein. In some embodiments, a nucleoside is linked to an internucleotidic linkage via a nitrogen atom (eg, sm01, sm18, etc.), wherein the nitrogen atom is linked directly to the linking phosphorus atom. In some embodiments, the present invention provides monomers of nucleosides (eg, Asm01, Gsm01, Tsm18, etc.) as described herein. In some embodiments, the present invention provides phosphoramidites of nucleosides as described herein. In some embodiments, such monomers or phosphoramidites include a protected hydroxyl (eg, DMTrO-) and/or a protected nucleobase (eg, for oligonucleotide synthesis). In some embodiments, such monomers or phosphoramidites include a protected hydroxyl (eg, DMTrO-), optionally a protected nucleobase (eg, useful in oligonucleotide synthesis), and/or a chiral auxiliary group. . Specific reagents useful for incorporating various nucleosides and/or compounds into oligonucleotides, such as various phosphoramidites, and specific techniques (e.g., cycles, conditions, etc.) for utilizing these reagents for oligonucleotide preparation are It is described in the examples or WO 2021/071858. Certain oligonucleotides and compositions thereof comprising modified nucleosides are prepared using these reagents and techniques and are set forth herein as examples in various tables, including, for example, those in Table 1.

In some embodiments, the present invention provides oligonucleotides comprising one or more modified nucleobases as described herein. In some embodiments, the present invention provides a compound comprising a modified nucleobase as described herein. In some embodiments, the present invention provides monomers comprising modified nucleobases as described herein (eg, those useful for oligonucleotide synthesis). In some embodiments, the present invention provides phosphoramidites comprising modified nucleobases as described herein. In some embodiments, the phosphoramidite is a CED phosphoramidite. In some embodiments, monomers include auxiliary moieties as described herein (eg, including P forming bonds to O and N, O and S, S and S, etc.). In some embodiments, the phosphoramidites include chiral auxiliary moieties as described herein (eg, including P forming bonds to O and N). In some embodiments, R ^NS comprises a nucleobase as described herein. In some embodiments, R ^NS comprises a modified nucleobase as described herein. In some embodiments, nucleobases are protected for oligonucleotide synthesis.

In some embodiments, the oligonucleotide comprises one or more structures independently selected from pseudoisocytidine, Benner base Z, 5-hydroxyC, 5-aminoC, and 8-oxoA.

In some embodiments, the nucleobase is an optionally substituted 2AP (2-amino purine, ) or DAP (2,6-diamino purine, )am. In some embodiments, a nucleobase is an optionally substituted 2AP. In some embodiments, a nucleobase is an optionally substituted DAP. In some embodiments, the nucleobase is 2AP. In some embodiments, the nucleobase is DAP.

As will be appreciated by those skilled in the art, a variety of nucleobases are known in the art and can be used in accordance with the present invention (e.g. US 9394333, US 9744183, US 9605019, US 9982257, US 20170037399, US 20180216108, US 20180216107, US 9598458, WO 2017/062862, WO 2018/067973, WO 2017/160741, WO 2017/192679, WO 2017/210647, WO 2018/098264, WO 2018/022473, WO 2018/223056, WO 2018/223073, WO 2018 /223081, WO 2018/237194, WO 2019/032607, WO 2019/032612, WO 2019/055951, WO 2019/075357, WO 2019/200185, WO 2019/217784, WO 2019/032612, WO 2020/191252 and/or as described in WO 2021/071858), each sugar, base, and internucleotide linkage modification is independently incorporated herein by reference. In some embodiments, nucleobases are protected and useful for oligonucleotide synthesis.

In some embodiments, the nucleobase is a natural nucleobase or a modified nucleobase derived from a natural nucleobase. Examples include uracil, thymine, adenine, cytosine, and guanine (optionally each amino group is protected by an acyl protecting group), 2-fluorouracil, 2-fluorocytosine, 5-bromouracil, 5-iodouracil, 2, including 6-diaminopurine, azacytosine, pyrimidine analogs such as pseudoisocytosine and pseudouracil, and other modified nucleobases such as 8-substituted purines, xanthines, or hypoxanthines (the latter two being natural degradation products) do. Specific examples of modified nucleobases are described in Chiu and Rana, RNA, 2003, 9, 1034-1048, Limbach et al. Nucleic Acids Research, 1994, 22, 2183-2196 and Revankar and Rao, Comprehensive Natural Products Chemistry, vol. 7, 313. In some embodiments, the modified nucleobase is a substituted uracil, thymine, adenine, cytosine, or guanine. In some embodiments, the modified nucleobase is a functional replacement for uracil, thymine, adenine, cytosine, or guanine, eg, in terms of hydrogen bonding and/or base pairing. In some embodiments, the nucleobase is optionally substituted uracil, thymine, adenine, cytosine, 5-methylcytosine, or guanine. In some embodiments, the nucleobase is uracil, thymine, adenine, cytosine, 5-methylcytosine, or guanine.

In some embodiments, provided oligonucleotides include one or more 5-methylcytosines. In some embodiments, the present invention provides oligonucleotides whose base sequences are disclosed herein (eg, Table 1), wherein each T may independently be replaced with a U and vice versa, and each cytosine is optionally and independently replaced by 5-methylcytosine and vice versa. As will be appreciated by those skilled in the art, in some embodiments, 5mC can be treated as a C relative to the base sequence of an oligonucleotide (such oligonucleotide contains a nucleobase modification at the C position) (e.g., the table 1, various oligonucleotides). In the description of oligonucleotides, generally the nucleobases, sugars, and internucleotidic linkages are unmodified, unless otherwise stated.

In some embodiments, the modified base is an optionally substituted adenine, cytosine, guanine, thymine, or uracil, or a tautomer thereof. In some embodiments, a modified nucleobase is a modified adenine, cytosine, guanine, thymine, or uracil modified by one or more of the following modifications:

Nucleobases are acyl, halogen, amino, azide, alkyl, alkenyl, alkynyl, aryl, heteroalkyl, heteroalkenyl, heteroalkynyl, heterocyclyl, heteroaryl, carboxyl, hydroxyl, biotin, avidin, strep modified with one or more optionally substituted groups independently selected from tabidine, substituted silyl, and combinations thereof;

one or more atoms of the nucleobase are independently replaced with a different atom selected from carbon, nitrogen, and sulfur;

one or more double bonds in the nucleobase are independently hydrogenated; or

One or more aryl or heteroaryl rings are independently inserted into the nucleobase.

In some embodiments, the base is optionally substituted A, T, C, G, or U, one or more -NH ₂ are independently and optionally replaced with -C(-LR ¹ ) ₃ , and one or more -NH- is independently and optionally replaced by -C(-LR ¹ ) ₂ -, one or more =N- is independently and optionally replaced by -C(-LR ¹ )-, and one or more =CH- is independently and optionally replaced by = N-, one or more =O are independently and optionally replaced by =S, =N(-LR ¹ ), or =C(-LR ¹ ) ₂ , and two or more -LR ¹ are optionally replaced between them. together with the intervening atoms to form a 3-30 membered bicyclic or polycyclic ring having 0-10 heteroatom ring atoms. In some embodiments, the modifying base is optionally substituted A, T, C, G, or U, one or more -NH ₂ are independently and optionally replaced with -C(-LR ¹ ) ₃ , and one or more -NH- is independently and optionally replaced by -C(-LR ¹ ) ₂ -, one or more =N- is independently and optionally replaced by -C(-LR 1 )-, and one or more =CH- is independently and optionally replaced by -C(-LR ¹ )- =N-, one or more =O are independently and optionally replaced by =S, =N(-LR ¹ ), or =C(-LR ¹ ) ₂ , and two or more -LR ¹ are optionally Together with the intervening atoms, it forms a 3-30 membered bicyclic or polycyclic ring having 0-10 heteroatomic ring atoms, and the modified bases are different from natural A, T, C, G, and U. In some embodiments, a base is an optionally substituted A, T, C, G, or U. In some embodiments, a modified base is a substitution A, T, C, G, or U, and a modified base is different from native A, T, C, G, and U.

In some embodiments, the modified nucleobase is a modified nucleobase known in the art (eg, WO2017/210647). In some embodiments, a modified nucleobase is an extended-size nucleobase to which one or more aryl and/or heteroaryl rings, such as a phenyl ring, have been added. Specific examples of modified nucleobases, including nucleobase substitutions, include the Glen Research catalog (Glen Research, Sterling, VA); See Krueger AT et al, Acc. Chem. Res., 2007, 40, 141-150]; See Kool, ET, Acc. Chem. Res., 2002, 35, 936-943]; See Benner S.A., et al., Nat. Rev. Genet., 2005, 6, 553-543]; See Romesberg, F.E., et al., Curr. Opin. Chem. Biol., 2003, 7, 723-733]; or Hirao, I., Curr. Opin. Chem. Biol., 2006, 10, 622-627. In some embodiments, the extended size nucleobase is an extended size nucleobase described, for example, in WO2017/210647. In some embodiments, the modified nucleobases are moieties such as chorine- or porphyrin-derived rings. Certain porphyrin-induced base substitutions are described, for example, in Morales-Rojas, H and Kool, ET, Org. Lett., 2002, 4, 4377-4380. In some embodiments, the porphyrin-derived ring is a porphyrin-derived ring described, for example, in WO2017/219647. In some embodiments, the modified nucleobase is a modified nucleobase described, for example, in WO2017/219647. In some embodiments, a modified nucleobase is fluorescent. Examples of such fluorescent modified nucleobases include phenanthrene, pyrene, stilbene, isoxanthine, isoxanthopterin, terphenyl, terthiophene, benzoterthiophene, coumarin, lumazine, tethered stilbene, benzo -uracil, naphtho-uracil, etc., and those described for example in WO2017/210647. In some embodiments, the nucleobase or modified nucleobase is selected from: C5-propyne T, C5-propyne C, C5-thiazole, phenoxazine, 2-thio-thymine, 5-triazolylphenyl-thymine , diaminopurine, and N2-aminopropylguanine.

In some embodiments, the modified nucleobases are selected from 5-substituted pyrimidines, 6-azapyrimidines, alkyl or alkynyl substituted pyrimidines, alkyl substituted purines, and N-2, N-6 and O-6 substituted purines. . In certain embodiments, the modified nucleobase is 2-aminopropyladenine, 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-N-methylguanine, 6-N-methyladenine, 2-propyladenine , 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-propynyl (-C≡C-CH ₃ ) uracil, 5-propynylcytosine, 6-azouracil, 6-azocytosine, 6-azo Thymine, 5-ribosyluracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl, 8-aza and other 8-substituted purines, 5 -halo, especially 5-bromo, 5-trifluoromethyl, 5-haluracil, and 5-halocytosine, 7-methylguanine, 7-methyladenine, 2-F-adenine, 2-aminoadenine, 7- Deazaguanine, 7-deazaadenine, 3-deazaguanine, 3-deazaadenine, 6-N-benzoyladenine, 2-N-isobutyrylguanine, 4-N-benzoylcytosine, 4-N-benzoyl uracil, 5-methyl 4-N-benzoylcytosine, 5-methyl 4-N-benzoyluracil, universal bases, hydrophobic bases, promiscuous bases, size-extended bases, and fluorinated bases. In some embodiments, the modified nucleobase is a tricyclic pyrimidine, such as 1,3-diazaphenoxazin-2-one, 1,3-diazaphenothiazin-2-one, or 9-(2-aminoethoxy) -1,3-diazaphenoxazin-2-one (G-clamp). In some embodiments, a modified nucleobase is one in which a purine or pyrimidine base is replaced with another heterocycle, e.g., 7-deaza-adenine, 7-deazaguanosine, 2-aminopyridine, or 2-pyridone. am. In some embodiments, modified nucleobases are described in US 3687808, The Concise Encyclopedia Of Polymer Science And Engineering, Kroschwitz, JI, Ed., John Wiley & Sons, 1990, 858-859; Englisch et al., Angewandte Chemie, International Edition, 1991, 30, 613; Sanghvi, YS, Chapter 15, Antisense Research and Applications, Crooke, ST and Lebleu, B., Eds., CRC Press, 1993, 273-288; or Chapters 6 and 15, Antisense Drug Technology, Crooke ST, Ed., CRC Press, 2008, 163-166 and 442-443.

In some embodiments, modified nucleobases and methods thereof are described in US 20030158403, US 3687808, US 4845205, US 5130302, US 5134066, US 5 175273, US 5367066, US 5432272, US 5434257, US 5457187, US 5 459255, US 5484908, US 5502177, US 5525711, US 5552540, US 5587469, US 5594 121, US 5596091, US 5614617, US 5645985, US 5681941, US 5750692, US 5763588, US 5830653, or US 6005096.

In some embodiments, a modified nucleobase is substituted. In some embodiments, the modified nucleobase is substituted to contain, for example, a heteroatom, an alkyl group, or a linking moiety (a fluorescent moiety, a biotin or avidin moiety, or linked to another protein or peptide). In some embodiments, a modified nucleobase is a "universal base" that is not a nucleobase in the most classical sense, but functions similarly to a nucleobase. One example of a universal base is 3-nitropyrrole.

In some embodiments, nucleosides that may be used in provided technologies include modified nucleobases and/or modified sugars (e.g., 4-acetylcytidine; 5-(carboxyhydroxylmethyl)uridine; 2'- O-methylcytidine; 5-carboxymethylaminomethyl-2-thiouridine; 5-carboxymethylaminomethyluridine; dihydrouridine; 2'-O-methylpseudouridine; beta,D-galactosylq eosine; 2'-O-methylguanosine; N ⁶ -isopentenyladenosine; 1-methyladenosine; 1-methylpseudouridine; 1-methylguanosine; 1-methylinosine; 2,2-dimethylguanosine ; 2-methyladenosine; 2-methylguanosine; N ⁷ -methylguanosine; 3-methyl-cytidine; 5-methylcytidine; 5-hydroxymethylcytidine; 5-formylcytosine; 5-carboxylcytosine ; N ⁶ -methyladenosine; 7-methylguanosine; 5-methylaminoethyluridine; 5-methoxyaminomethyl-2-thiouridine; beta,D-mannosylqueosine; 5-methoxycarbonyl methyluridine; 5-methoxyuridine; 2-methylthio-N ⁶ -isopentenyladenosine; N-((9-beta,D-ribofuranosyl-2-methylthiopurin-6-yl)carbamoyl )Threonine; N-((9-beta,D-ribofuranosylpurin-6-yl)-N-methylcarbamoyl)threonine; Uridine-5-oxyacetic acid methyl ester; Uridine-5-oxyacetic acid (v ); pseudouridine; cueosin; 2-thiocytidine; 5-methyl-2-thiouridine; 2-thiouridine; 4-thiouridine; 5-methyluridine; 2'-O-methyl -5-methyluridine; and 2'-O-methyluridine).

In some embodiments, a nucleobase, e.g., a modified nucleobase, comprises one or more biomolecule binding moieties, e.g., antibodies, antibody fragments, biotin, avidin, streptavidin, receptor ligands, or chelating moieties. do. In another embodiment, the nucleobase is 5-bromouracil, 5-iodouracil, or 2,6-diaminopurine. In some embodiments, a nucleobase comprises substitution with a fluorescent or biomolecule binding moiety. In some embodiments, a substituent is a fluorescent moiety. In some embodiments, the substituent is biotin or avidin.

Specific examples of nucleobases and related methods are described in US 3687808, 4845205, US 513030, US 5134066, US 5175273, US 5367066, US 5432272, US 5457187, US 5457191, US 5459255, US 5484908, US 55 02177, US 5525711, US 5552540, US 5587469, US 5594121, US 5596091, US 5614617, US 5681941, US 5750692, US 6015886, US 6147200, US 6166197, US 6222025, US 6235887, US 6380368, US 6528640, US 6639062, US 6617438, US 7045610, US 7427672 , or US 7495088.

In some embodiments, an oligonucleotide comprises a nucleobase, sugar, nucleoside, and/or internucleotide linkage described in any of the following: Gryaznov, S; Chen, J.-K. J. Am. Chem. Soc. 1994, 116, 3143]; See Hendrix et al. 1997 Chem. Eur. J. 3: 110]; See Hyrup et al. 1996 Bioorg. Med. Chem. 4: 5]; See Jepsen et al. 2004 Oligo. 14: 130-146]; See Jones et al. J. Org. Chem. 1993, 58, 2983]; See Koizumi et al. 2003 Nuc. Acids Res. 12: 3267-3273]; See Koshkin et al. 1998 Tetrahedron 54: 3607-3630]; See Kumar et al. 1998 Bioo. Med. Chem. Let. 8: 2219-2222]; See Lauritsen et al. 2002 Chem. Comm. 5: 530-531]; See Lauritsen et al. 2003 Bioo. Med. Chem. Lett. 13: 253-256]; See Mesmaeker et al. Angew. Chem., Int. Ed. Engl. 1994, 33, 226]; See Morita et al. 2001 Nucl. Acids Res. Supp. 1: 241-242]; See Morita et al. 2002 Bioo. Med. Chem. Lett. 12: 73-76]; See Morita et al. 2003 Bioo. Med. Chem. Lett. 2211-2226]; See Nielsen et al. 1997 Chem. Soc. Rev. 73]; See Nielsen et al. 1997 J. Chem. Soc. Perkins Transl. 1: 3423-3433]; See Obika et al. 1997 Tetrahedron Lett. 38 (50): 8735-8]; See Obika et al. 1998 Tetrahedron Lett. 39: 5401-5404]; See Pallan et al. 2012 Chem. Comm. 48: 8195-8197]; See Petersen et al. 2003 TRENDS Biotech. 21: 74-81]; See Rajwanshi et al. 1999 Chem. Commun. 1395-1396]; See Schultz et al. 1996 Nucleic Acids Res. 24: 2966]; See Seth et al. 2009 J. Med. Chem. 52: 10-13]; See Seth et al. 2010 J. Med. Chem. 53: 8309-8318]; See Seth et al. 2010 J. Org. Chem. 75: 1569-1581]; See Seth et al. 2012 Bioo. Med. Chem. Lett. 22: 296-299]; See Seth et al. 2012 Mol. Ther-Nuc. Acids. 1, e47]; See Seth, Punit P; Siwkowski, Andrew; Allerson, Charles R; Vasquez, Guillermo; Lee, Sam; Prakash, Tazha P; Kinberger, Garth; Migawa, Michael T; Gaus, Hans; Bhat, Balkrishen; et al. From Nucleic Acids Symposium Series (2008), 52(1), 553-554; See Singh et al. 1998 Chem. Comm. 1247-1248]; See Singh et al. 1998 J. Org. Chem. 63: 10035-39]; See Singh et al. 1998 J. Org. Chem. 63: 6078-6079]; See Sorensen 2003 Chem. Comm. 2130-2131]; See Ts'o et al. Ann. N.Y. Acad. Sci. 1988, 507, 220]; See Van Aerschot et al. 1995 Angew. Chem. Int. Ed. Engl. 34: 1338]; See Vasseur et al. J. Am. Chem. Soc. 1992, 114, 4006]; WO 2007090071; or WO 2016/079181.

In some embodiments, an oligonucleotide comprises a modified nucleobase, nucleoside, or nucleotide described in any of Feldman et al. 2017 J. Am. Chem. Soc. 139: 11427-11433], Feldman et al. 2017 Proc. Natl. Acad. Sci. USA 114: E6478-E6479], Hwang et al. 2009 Nucl. Acids Res. 37: 4757-4763], Hwang et al. 2008 J. Am. Chem. Soc. 130: 14872-14882], Lavergne et al. 2012 Chem. Eur. J. 18: 1231-1239], Lavergne et al. 2013 J. Am. Chem. Soc. 135: 5408-5419], Ledbetter et al. 2018 J. Am. Chem. Soc. 140: 758-765], Malyshev et al. 2009 J. Am. Chem. Soc. 131: 14620-14621], Seo et al. 2009 Chem. Bio. Chem. 10: 2394-2400] (e.g., d3FB, d2Py analogues, d2Py, d3MPy, d4MPy, d5MPy, d34DMPy, d35DMPy, d45DMPy, d5FM, d5PrM, d5SICS, dFEMO, dMMO2, dNaM, dNM01, dTPT3, 2'-Azi nucleotides containing 2'-chloro, 2'-amino or arabinose sugars, isocarbostyryl-, naphthyl- and azaindole-nucleotides, and modified and derivative and functionalized versions thereof, e.g. sugars containing 2'-modifications and/or other modifications, and dMMO2 derivatives having meta-chlorine, -bromine, -iodine, -methyl or -propynyl substituents).

In some embodiments, the nucleobase comprises at least one optionally substituted ring comprising heteroatomic ring atoms. In some embodiments, the nucleobase comprises at least one optionally substituted ring comprising a nitrogen ring atom. In some embodiments, these rings are aromatic. In some embodiments, the nucleobase is linked to the sugar through a heteroatom. In some embodiments, the nucleobase is linked to the sugar through a nitrogen atom. In some embodiments, the nucleobase is attached to the sugar through a ring nitrogen atom.

In some embodiments, oligonucleotides are described in WO 2018/022473, WO 2018/098264, WO 2018/223056, WO 2018/223073, WO 2018/223081, WO 2018/237194, WO 2019/032607, WO 2019/055951, WO 2019 /075357, WO 2019/200185, WO 2019/217784, WO 2019/032612, WO 2020/191252 and/or WO 2021/071858, comprising nucleobases or modified nucleobases, wherein each base and modified nucleobase are independently incorporated herein by reference.

In some embodiments, a nucleobase is an optionally substituted purine base residue. In some embodiments, a nucleobase is a protected purine base residue. In some embodiments, the nucleobase is an optionally substituted adenine residue. In some embodiments, the nucleobase is a protected adenine residue. In some embodiments, the nucleobase is an optionally substituted guanine residue. In some embodiments, the nucleobase is a protected guanine residue. In some embodiments, a nucleobase is an optionally substituted cytosine residue. In some embodiments, a nucleobase is a protected cytosine residue. In some embodiments, the nucleobase is an optionally substituted thymine residue. In some embodiments, the nucleobase is a protected thymine residue. In some embodiments, the nucleobase is an optionally substituted uracil residue. In some embodiments, the nucleobase is a protected uracil residue. In some embodiments, the nucleobase is an optionally substituted 5-methylcytosine residue. In some embodiments, the nucleobase is a protected 5-methylcytosine residue.

In some embodiments, provided oligonucleotides are disclosed in, for example, US 5552540, US 6222025, US 6528640, US 4845205, US 5681941, US 5750692, US 6015886, US 5614617, US 6147200, US 5457187, US 6639062, US 7427672, US 5459255 , US 5484908, US 7045610, US 3687808, US 5502177, US 5525711 6235887, US 5175273, US 6617438, US 5594121, US 6380368, US 5367066, US 5587469, US 6166197, US 5432272, US 7495088, US 5134066, or US 5596091 It includes the modified nucleobases described in. In some embodiments, the nucleobase is described in WO 2020/154344, WO 2020/154343, WO 2020/154342, WO 2020/165077, WO 2020/201406, WO 2020/216637, or WO 2020/252376; can be used according to

In some embodiments, a nucleobase is a protected base moiety as used in oligonucleotide preparation. In some embodiments, the nucleobase is described in US 2011/0294124, US 2015/0211006, US 2015/0197540, WO 2015/107425, WO 2017/192679, WO 2018/022473, WO 2018/098264, WO 2018/223056 , WO 2018 /223073, WO 2018/223081, WO 2018/237194, WO 2019/032607, WO 2019/055951, WO 2019/075357, WO 2019/200185, WO 2019/217784, WO 2019/032612, WO 2020/191252 and/or base residues exemplified in WO 2021/071858, each base residue being independently incorporated herein by reference.

sugar

A variety of sugars can be used in accordance with the present invention, including modified sugars. In some embodiments, the present invention provides sugar modifications and their patterns (optionally other structural elements (e.g., internucleotide linkage modifications and their patterns, their combined with backbone chiral central patterns, etc.).

The most common naturally occurring nucleosides are ribose sugars (e.g., in RNA) or deoxyribose linked to the nucleobases adenosine (A), cytosine (C), guanine (G), thymine (T), or uracil (U). sugars (eg in DNA). In some embodiments, sugars, e.g., various sugars in many of the oligonucleotides of Table 1 (unless otherwise noted) are is a natural DNA sugar (in DNA nucleic acids or oligonucleotides) having the structure When at the 5'-end of a nucleotide, the 5' position may be linked to a 5'-end group (e.g., -OH), when at the 3'-end of an oligonucleotide, the 3' position may be linked to the 3'-end group. may be linked to a terminal group (eg -OH). In some embodiments, the sugar is It is a natural RNA sugar (in RNA nucleic acids or oligonucleotides) having the structure of: a nucleobase is attached at the 1' position and the 3' and 5' positions are linked to internucleotidic linkages (as understood by those skilled in the art, oligo When at the 5'-end of a nucleotide, the 5' position may be linked to a 5'-end group (e.g., -OH), when at the 3'-end of an oligonucleotide, the 3' position may be linked to the 3'-end group. may be linked to a terminal group (eg -OH). In some embodiments, the sugar is a modified sugar in that it is not a natural DNA sugar or a natural RNA sugar. In particular, modified sugars can provide improved stability. In some embodiments, a modifying sugar may be used to alter and/or optimize one or more hybridization properties. In some embodiments, modifying sugars can be used to alter and/or optimize target nucleic acid recognition. In some embodiments, a modified sugar can be used to optimize the Tm. In some embodiments, modified sugars can be used to improve oligonucleotide activity.

Among other things, the present invention demonstrates that a variety of non-natural RNA sugars, such as natural DNA sugars, various modified sugars, and the like, can be used in accordance with the present invention. For example, one or more natural DNA sugars may be tolerated at various positions. In some embodiments, incorporation of one or more natural DNA sugars provides increased levels of editing, or increased levels of editing by ADAR1 (p110, p150 or both), ADAR2, or both. In some embodiments, editing by ADAR1 is improved. In some embodiments, N _-3 , N _-1 , N ₁ , N 4 _, N ₅ , N ₇ , N ₈ , N ₁₀ , N ₁₂ , N 13 , N ₁₄ , N ₁₅ , N ₁₆ , _{N 17 ,} N One or more sugars of ₁₈ , N ₂₀ and N ₂₁ are independently natural DNA sugars (- (eg N _-1 ): count from N ₀ to the 3'-end of the oligonucleotide; + or just a number (eg N _{1 )} . ): Count from N ₀ to the 5'-end of the oligonucleotide; each N _NZ is independently a nucleoside, where NZ is for example about -100, -90, -80, -70, -60, - An integer from 50, -40, -30, -20, -10, -9, -8, -7, -6, -5, -4, etc. to __). In some embodiments, N _-3 , N _-1 , N 0 , N ₁ , N ₄ , N ₅ , N ₇ , N ₈ , N ₁₀ , N ₁₂ , N ₁₃ , N ₁₄ , N ₁₅ , _{N 16 ,} N One or more sugars of ₁₇ , N ₁₈ , N ₂₀ and N ₂₁ are independently natural DNA sugars. In some embodiments, one or more sugars of N ₋₁ , N ₅ , N ₁₁ , N ₁₂ and N ₂₀ are independently natural DNA sugars. In some embodiments, the sugar of N _-1 is a natural DNA sugar. In some embodiments, the sugar of N ₀ is a natural DNA sugar. In some embodiments, the sugar of N ₁ is a natural DNA sugar. In some embodiments, the sugar of N ₅ is a natural DNA sugar. In some embodiments, the sugar of N ₁₁ is a natural DNA sugar. In some embodiments, the sugar of N ₁₂ is a natural DNA sugar. In some embodiments, modified sugars are allowed at more than one position. In some embodiments, a 2'-modified sugar, eg, a 2'-F and/or 2'-OR modified sugar, is used at one or more or most positions, where R is an optionally substituted C _1-6 aliphatic (e.g., methyl). In some embodiments, a modified sugar is used at one or more or most or all of the 5'-N ₁ N ₀ N _-1 -3' positions. In some embodiments, a 2'-OR modified sugar is used at one or more or most or all positions of 5'-N ₁ N ₀ N _-1 -3', wherein R is an optionally substituted C _1-6 aliphatic (eg For example, methyl). In some embodiments, a modified sugar is used at one or more or most or all of the 5'-N ₁ N ₀ N _-1 -3' positions, and one or more 2'-F modified sugars, natural DNA sugars and/or native RNA A sugar is used at 5'-N ₁ N ₀ N _-1 -3'. In some embodiments, a modifying sugar is used at one or more or most or all positions of 5'-N ₁ N ₀ N _-1 -3', and each sugar of 5'-N ₁ N ₀ N _-1 -3' is independently a 2'-F modified sugar, a native DNA sugar, or a native RNA sugar. In some embodiments, a modifying sugar is used at one or more or most or all positions of 5'-N ₁ N ₀ N _-1 -3', and each sugar of 5'-N ₁ N ₀ N _-1 -3' is independently a 2'-F modified sugar or a natural DNA sugar. In some embodiments, a modifying sugar is used at one or more or most or all positions of 5'-N ₁ N ₀ N _-1 -3', and each sugar of 5'-N ₁ N ₀ N _-1 -3' is independently a natural DNA sugar. In some embodiments, a modified sugar, for example a 2'-OR modified sugar, where R is an optionally substituted C _1-6 alkyl, has increased levels of editing, or ADAR1 (p110, p150 or both), ADAR2, or provide an increased level of editing by both. In some embodiments, editing by ADAR2 is improved. In some embodiments, the modified sugar is a bicyclic sugar (eg LNA sugar, cEt sugar, etc.). In some embodiments, a bicyclic sugar can be used at one or more or all positions where a 2'-OR sugar is used, where R is an optionally substituted C _1-6 alkyl. In some embodiments, 2'-OR is 2'-OMe. In some embodiments, a 2'-OR is a 2'-MOE. In some embodiments, the majority is at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% (e.g., 55%-100%, 60%-100%). %, 70~100%, 75%~100%, 80%~100%, 90%~100%, 95%~100%, 60%~95%, 70%~95%, 75~95%, 80~ 95%, 85-95%, 90-95%, 51%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or 99%.

In some embodiments, the first few (e.g., 1-10, 1, 2, 3, 4, or 5) sugars (unless otherwise specified, 5'-end) of a provided oligonucleotide or first domain from) is independently a modified sugar. In some embodiments, each of the first few sugars is independently a modified sugar. In some embodiments, the first 1, 2 or 3 sugars of a provided oligonucleotide or first domain are independently modified sugars. In some embodiments, the first sugar is a modified sugar. In some embodiments, the first two sugars are independently modified sugars. In some embodiments, the first three sugars are independently modified sugars (eg, WV-27458). In some embodiments, a modified sugar is a bicyclic sugar. In some embodiments, a modified sugar is a 2'-modified sugar. In some embodiments, each modified sugar is independently a 2'-modified sugar. In some embodiments, the modified sugar is a 2'-OMe modified sugar. In some embodiments, each modified sugar is a 2'-OMe modified sugar. In some embodiments, a modified sugar is a 2'-MOE modified sugar. In some embodiments, each modified sugar is a 2'-MOE modified sugar. In some embodiments, each modified sugar is independently a 2'-OMe or 2'-MOE modified sugar.

In some embodiments, the last few (e.g., 1-10, 1, 2, 3, 4, or 5) sugars (unless otherwise specified) of a provided oligonucleotide or second domain or third subdomain , from the 5'-end) is independently a modified sugar. In some embodiments, each of the last few sugars is independently a modified sugar. In some embodiments, the last 1, 2 or 3 sugars of a provided oligonucleotide or second domain or third subdomain are independently modified sugars. In some embodiments, the last sugar is a modified sugar. In some embodiments, the last two sugars are independently modified sugars. In some embodiments, the last three sugars are independently modified sugars. In some embodiments, the last four sugars are independently modified sugars (eg, WV-27458). In some embodiments, a modified sugar is a bicyclic sugar. In some embodiments, a modified sugar is a 2'-modified sugar. In some embodiments, each modified sugar is independently a 2'-modified sugar. In some embodiments, the modified sugar is a 2'-OMe modified sugar. In some embodiments, each modified sugar is a 2'-OMe modified sugar. In some embodiments, a modified sugar is a 2'-MOE modified sugar. In some embodiments, each modified sugar is a 2'-MOE modified sugar. In some embodiments, each modified sugar is independently a 2'-OMe or 2'-MOE modified sugar.

Sugars can be linked to internucleotidic linkages at various positions. As a non-limiting example, the internucleotidic linkage may be attached to the 2', 3', 4', or 5' position of the sugar. In some embodiments, as is most common in natural nucleic acids, the internucleotidic linkage is linked with one sugar at the 5' position and with another sugar at the 3' position.

In some embodiments, the sugar is an optionally substituted natural DNA or RNA sugar. In some embodiments, the sugar is optionally substituted am. In some embodiments, the 2' position is optionally substituted. In some embodiments, the sugar is am. In some embodiments, the sugar is or and each of R ^1s , R ^2s , R ^3s , R ^4s , and R ^5s is independently -H, a suitable substituent, or a suitable sugar modification (eg US 9394333, US 9744183, US 9605019, US 9982257 , US 20170037399, US 20180216108, US 20180216107, US 9598458, WO 2017/062862, WO 2018/067973, WO 2017/160741, WO 2017/192679, WO 2017/21064 7, WO 2018/098264, WO 2018/022473, WO 2018 /223056, WO 2018/223073, WO 2018/223081, WO 2018/237194, WO 2019/032607, WO 2019/032612, WO 2019/055951, WO 2019/075357, WO 2019/200185, WO 2019/217784, WO 2019 /032612, WO 2020/191252 and/or WO 2021/071858, the description of each substituent, sugar modification, R ^1s , R ^2s , R ^3s , R ^4s , and R ^5s , and the modified sugar independently incorporated herein by reference). In some embodiments, each of R ^1s , R ^2s , R ^3s , R ^4s , and R ^5s is independently R ^s , and each R ^s is independently -F, -Cl, -Br, -I, -CN, -N ₃ , -NO, -NO ₂ , -L ^s -R', -L ^s -OR', -L ^s -SR', -L ^s -N(R') ₂ , -OL ^s -OR', -OL ^s -SR', or -OL ^s -N(R') ₂ , each R' independently as described herein, and each L ^s independently a covalent bond or an optionally substituted divalent C _{1 -6} aliphatic or heteroaliphatic with 1 to 4 heteroatoms; Two R ^s together form a cross-linked product -L ^s -. In some embodiments, R' is an optionally substituted C _1-10 aliphatic. In some embodiments, the sugar is has the structure of In some embodiments, the sugar is has the structure of In some embodiments, the sugar is has the structure of In some embodiments, the sugar is has the structure of In some embodiments, the sugar is has the structure of In some embodiments, the sugar is has the structure of In some embodiments, the sugar is has the structure of In some embodiments, the sugar is has the structure of In some embodiments, the sugar is has the structure of In some embodiments, R ^5s is an optionally substituted C _1-6 aliphatic. In some embodiments, R ^5s is an optionally substituted C _1-6 alkyl. In some embodiments, R ^5s is optionally substituted methyl. In some embodiments, R ^5s is methyl. In some embodiments, the sugar is has the structure of In some embodiments, the sugar is has the structure of In some embodiments, the sugar is has the structure of A variety of these sugars are used in Table 1. In some embodiments, the sugar is has the structure of In some embodiments, the 2'-modified sugar is has the structure of, and R ^2s is a 2'-modification. In some embodiments, the sugar is and R ^2s is -H, halogen, or -OR (R is an optionally substituted C _1-6 aliphatic). In some embodiments, R ^2s is -H. In some embodiments, R ^2s is -F. In some embodiments, R ^2s is -OMe. In some embodiments, the modified nucleoside is mA, mT, mC, m5mC, mG, mU, etc., and R ^2s is -OMe. In some embodiments, R ^2s is -OCH ₂ CH ₂ OMe. In some embodiments, the modified nucleoside is Aeo, Teo, Ceo, m5Ceo, Geo, Ueo, etc., and R ^2s is -OCH ₂ CH ₂ OMe. In some embodiments, R ^2s is -OCH ₂ CH ₂ OH. In some embodiments, an oligonucleotide (eg, as in fA, fT, fC, f5mC, fG, fU, etc.) It includes a 2'-F modified sugar having the structure of In some embodiments, an oligonucleotide is (eg, as in mA, mT, mC, m5mC, mG, mU, etc.) It includes a 2'-OMe modified sugar having a structure of In some embodiments, oligonucleotides (eg, as in Aeo, Teo, Ceo, m5Ceo, Geo, Ueo, etc.) It includes a 2'-MOE modified sugar having the structure of

In some embodiments, the sugar is has the structure of, R ^2s and R ^4s together form -L ^s -, L ^s is a covalent bond or optionally substituted divalent C _1-6 aliphatic or heteroaliphatic having 1 to 4 heteroatoms. In some embodiments, each heteroatom is independently selected from nitrogen, oxygen, or sulfur. In some embodiments, L ^s is an optionally substituted C2-O-CH ₂ -C4. In some embodiments, L ^s is C2-O-CH ₂ -C4. In some embodiments, L ^s is C2-O-( R )-CH(CH ₂ CH ₃ )-C4. In some embodiments, L ^s is C2-O-( S )-CH(CH ₂ CH ₃ )-C4.

In some embodiments, the sugar is , where each variable is independently as described herein. In some embodiments, the sugar is , where each variable is independently as described herein. In some embodiments, R ^5s is -H. In some embodiments, the sugar is , where each variable is independently as described herein. In some embodiments, R ^3s is -OH. In some embodiments, R ^3s is -H. In some embodiments, the sugar is am. In some embodiments, the sugar is am.

In some embodiments, the sugar is

In some embodiments, a nucleoside comprising a modified sugar is or a salt form thereof, BA ^s is -H or an optionally substituted or protected nucleobase (eg BA), and R ^2s is as described herein. In some embodiments, R ^2s is —OH, halogen, or optionally substituted C ₁ -C ₆ alkoxy. In some embodiments, BA ^s is -H. In some embodiments, BA ^s is an optionally substituted or protected nucleobase. In some embodiments, BA ^s is BA. In some embodiments, R ^2s is -F. In some embodiments, a nucleoside comprising a modified sugar is or a salt form thereof, and each variable is independently as described herein. In some embodiments, R ^2s is -H, -OH, halogen, or optionally substituted C ₁ -C ₆ alkoxy. In some embodiments, R ^2s is -H. In some embodiments, R ^2s is -F. In some embodiments, a nucleoside comprising a modified sugar is It has the structure of, and each variable is as described herein. In some embodiments, a nucleoside comprising a modified sugar is or a salt form thereof, and each variable is independently as described herein. In some embodiments, R ^2s is -H, -OH, halogen, or optionally substituted C ₁ -C ₆ alkoxy. In some embodiments, R ^2s is -H. In some embodiments, R ^2s is -F. In some embodiments, a nucleoside comprising a modified sugar is or a salt form thereof, R ^2s' is R ^s , R ^s , R ^2s , and BA ^s are each independently as described herein. In some embodiments, each of R ^2s and R ^2s' is independently -H, -OH, halogen, or optionally substituted C ₁ -C ₆ alkoxy. In some embodiments, R ^2s is -H. In some embodiments, R ^2s is -OH. In some embodiments, R ^2s is halogen. In some embodiments, R ^2s is -F. In some embodiments, R ^2s is an optionally substituted C ₁ -C ₆ alkoxy. In some embodiments, R ^2s' is -H. In some embodiments, R ^2s' is -OH. In some embodiments, R ^2s' is halogen. In some embodiments, R ^2s' is -F. In some embodiments, R ^2s' is an optionally substituted C ₁ -C ₆ alkoxy. In some embodiments, BA ^s is -H. In some embodiments, BA ^s is an optionally substituted or protected nucleobase. In some embodiments, BA ^s is BA. In some embodiments, nucleobases such as BA are optionally substituted or protected for oligonucleotide synthesis. These specific nucleosides, including sugars and nucleobases, and their use are described in WO 2020/154342. In some embodiments, the oligonucleotide is arabinoside, 2'-deoxy-2'-fluoro-arabinoside, 2'-OR arabinoside, adeoxycytidine, DNA-basic, RNA-basic or 2'-OR abasic, where R is not hydrogen (eg, optionally substituted C _1-6 aliphatic). In some embodiments, 2'-OR is 2'-OMe. In some embodiments, a 2'-OR is a 2'-MOE. In some embodiments, the oligonucleotide comprises 2'-O-methyl-arabinocytidine (amC). In some embodiments, oligonucleotides include such nucleosides. In some embodiments, a monomer comprises such nucleosides. In some embodiments, the phosphoramidite comprises such a nucleoside (in some embodiments, one linkage (eg, -CH ₂ - linkage) is optionally substituted -OH, for example (-ODMTr ), and one linkage (eg, ring linkage) is bound to O, which is also bound to P of phosphoramidite). In some embodiments, one or more or each of the 5' contiguous nucleosides (eg, N ₁ ), opposite nucleosides (N ₀ ), and 3' contiguous nucleosides (eg, N _-1 ) are independently as such a nucleoside. In some embodiments, 5'-N ₁ N ₀ N _-1 -3' is amCCA. In some embodiments, the sugar is has the structure of where each variable is as described herein and C1' is bound to a nucleobase. In some embodiments, the sugar is arabinose. In some embodiments, the sugar is It has the structure of, where C1' is bound to a nucleobase.

In some embodiments, the sugar is , wherein the nucleobase is bound at position 1'.

In some embodiments, a nucleoside comprising a modified sugar is or a salt form thereof, wherein each of R ^6s and R ^7s is independently R ^s , BA ^s is -H or an optionally substituted or protected nucleobase (eg BA), and R ^s are independently described herein. same as bar In some embodiments, R ^6s is -H, -OH, or halogen, and R ^7s is -H, -OH, halogen, or optionally substituted C ₁ -C ₆ alkoxy. In some embodiments, BA ^s is -H. In some embodiments, BA ^s is an optionally substituted or protected nucleobase. In some embodiments, BA ^s is BA. In some embodiments, a nucleoside comprising a modified sugar is or a salt form thereof, R ^8s and R ^9s are each independently R ^s , and R ^s and BA ^s are each independently as described herein. In some embodiments, R ^8s is -H or halogen, and R ^9s is -H, -OH, halogen, or optionally substituted C ₁ -C ₆ alkoxy. In some embodiments, a nucleoside comprising a modified sugar is or a salt form thereof, R ^10s and R ^11s are each independently R ^s , and R ^s and BA ^s are each independently as described herein. In some embodiments, R ^10s is -H or halogen, and R ^11s is -H, -OH, halogen, or optionally substituted C ₁ -C ₆ alkoxy. In some embodiments, a nucleoside comprising a modified sugar is or a salt form thereof, and BA ^s are as described herein. In some embodiments, a nucleoside comprising a modified sugar is or a salt form thereof, and BA ^s are as described herein. One skilled in the art understands that in some embodiments nitrogen may be bonded directly to the linking phosphorus. In some embodiments, halogen is -F. In some embodiments, BA ^s is -H. In some embodiments, BA ^s is an optionally substituted or protected nucleobase. In some embodiments, BA ^s is BA. In some embodiments, nucleobases such as BA are optionally substituted or protected for oligonucleotide synthesis. In some embodiments, the oligonucleotide comprises alpha-homo-DNA, beta-homo-DNA moieties. These specific nucleosides, including sugars and nucleobases, and their use are described in WO 2020/154343. In some embodiments, oligonucleotides include such nucleosides. In some embodiments, a monomer comprises such nucleosides. In some embodiments, the phosphoramidite comprises such a nucleoside (in some embodiments, one linkage (eg, -CH ₂ -linkage) is attached to an optionally substituted -OH, eg -ODMTr and one linkage (e.g. ring linkage) is bonded to P of the phosphoramidite (e.g. when the linking ring atom is N) or phosphoamidite (e.g. when the linking ring atom is C) bonded to O, which is also bonded to P of formidite). In some embodiments, one or more or each of the 5' contiguous nucleoside (eg, N ₁ ), the opposite nucleoside (N ₀ ), and the 3' contiguous nucleoside (eg, N _-1 ) is independently as such a nucleoside.

In some embodiments, a nucleoside comprising a modified sugar is or a salt form thereof, and each variable is as described herein. In some embodiments, a nucleoside comprising a modified sugar is or a salt form thereof, and each variable is as described herein. In some embodiments, a nucleoside comprising a modified sugar is or a salt form thereof, and each variable is as described herein. In some embodiments, a nucleoside comprising a modified sugar is or a salt form thereof, R ^12s is R ^s , and each of R ^s and BA ^s is independently as described herein. In some embodiments, R ^12s is -H, -OH, halogen, optionally substituted C _1-6 alkyl, optionally substituted C _1-6 heteroalkyl, or optionally substituted C _1-6 alkoxy. In some embodiments, halogen is -F. In some embodiments, a nucleoside comprising a modified sugar is or a salt form thereof, and each variable is as described herein. In some embodiments, a nucleotide comprising a modified sugar is or a salt form thereof, R ^13s is R ^s , and each of R ^s and BA ^s is independently as described herein. In some embodiments, R ^13s is -H or an optionally substituted C ₁ -C ₆ alkyl. In some embodiments, a nucleoside comprising a modified sugar is or a salt form thereof, and each variable is as described herein. In some embodiments, a nucleotide comprising a modified sugar is or a salt form thereof, and each variable is as described herein. In some embodiments, a linkage is an amide linkage. In some embodiments, BA ^s is -H. In some embodiments, BA ^s is an optionally substituted or protected nucleobase. In some embodiments, BA ^s is BA. In some embodiments, nucleobases such as BA are optionally substituted or protected for oligonucleotide synthesis. These specific nucleosides and nucleotides, including sugars and nucleobases, and their uses are described in WO 2020/154344. In some embodiments, oligonucleotides include such nucleosides. In some embodiments, an oligonucleotide comprises such a nucleoside (in some embodiments, one linkage (eg, -CH ₂ - linkage) is an optionally substituted -OH, eg (-ODMTr) and one linkage (e.g. ring linkage) is bound to O which is also bound to P of phosphoramidite). In some embodiments, one or more or each of the 5' contiguous nucleosides (eg, N ₁ ), opposite nucleosides (N ₀ ), and 3' contiguous nucleosides (eg, N _-1 ) are independently as such a nucleoside.

In some embodiments, the sugar is an acyclic sugar, such as a UNA sugar. In some embodiments, the sugar is optionally substituted am. In some embodiments, the 2' position is optionally substituted. In some embodiments, the sugar is am. In some embodiments, the sugar is has the structure of In some embodiments, R ^2s is -OH. In some embodiments, the sugar is , where "*" represents a carbon atom bonded to a nucleobase. In some embodiments, the sugar is , where "*" represents a carbon atom bonded to a nucleobase. In some embodiments, the carbon atom bonded to the nitrogen atom of the nucleobase is in the R configuration (eg, sm18). In some embodiments, an oligonucleotide comprises a sugar described herein.

In some embodiments, sugars are linked without going through the 5' and 3' positions. One skilled in the art recognizes that for such sugars, 5' can refer to the side/direction towards the 5'-end of the oligonucleotide and 3' can refer to the side/direction towards the 3'-end of the oligonucleotide. .

In some embodiments, each of R ^1s , R ^2s , R ^3s , R ^4s , and R ^5s is independently R ^s , and R ^s are independently -H, halogen, -CN, -N ₃ , -NO, -NO ₂ , -L ^s -R', -L ^s -Si(R') ₃ , -L ^s -OR', -L ^s -SR', -L ^s -N(R') ₂ , -OL ^s -R ', -OL ^s -Si(R) ₃ , -OL ^s -OR', -OL ^s -SR', or -OL ^s -N(R') ₂ , and L ^s is ^LB as described herein; , each other variable independently as described herein. In some embodiments, each of R ^1s and R ^2s is independently R ^s . In some embodiments, R ^s is -H. In some embodiments, R ^s is not -H. In some embodiments, L ^s is a covalent bond. In some embodiments, each of R ^2s and R ^4s is independently -H, -F, -OR, -N(R) ₂ . In some embodiments, R ^2s is -H, -F, -OR, -N(R) ₂ . In some embodiments, R ^4s is -H. In some embodiments, R ^2s and R ^4s form 2'-OL ^s - and L ^s is an optionally substituted C _1-6 alkylene. In some embodiments, L ^s is optionally substituted -CH ₂ -. In some embodiments, L ^s is optionally substituted -CH ₂ -.

In some embodiments, R is hydrogen. In some embodiments, R is not hydrogen. In some embodiments, R is C _1-10 aliphatic, C _1-10 heteroaliphatic having 1-10 heteroatoms (independently selected from oxygen, nitrogen, sulfur, phosphorus, and silicon), C _6-20 aryl, A 5-20 membered heteroaryl ring having 1-10 heteroatoms (independently selected from oxygen, nitrogen, sulfur, phosphorus, and silicon), and 1-10 heteroatoms (oxygen, nitrogen, sulfur, phosphorus, and silicon) independently selected from) is an optionally substituted group selected from 3 to 20 membered heterocyclic rings having

In some embodiments, R is an optionally substituted C _1-30 aliphatic. In some embodiments, R is an optionally substituted C _1-20 aliphatic. In some embodiments, R is an optionally substituted C _1-15 aliphatic. In some embodiments, R is an optionally substituted C _1-10 aliphatic. In some embodiments, R is an optionally substituted C _1-6 aliphatic. In some embodiments, R is an optionally substituted C _1-6 alkyl. In some embodiments, R is optionally substituted hexyl, pentyl, butyl, propyl, ethyl, or methyl. In some embodiments, R is optionally substituted hexyl. In some embodiments, R is an optionally substituted pentyl. In some embodiments, R is optionally substituted butyl. In some embodiments, R is optionally substituted propyl. In some embodiments, R is optionally substituted ethyl. In some embodiments, R is optionally substituted methyl. In some embodiments, R is hexyl. In some embodiments, R is pentyl. In some embodiments, R is butyl. In some embodiments, R is propyl. In some embodiments, R is ethyl. In some embodiments, R is methyl. In some embodiments, R is isopropyl. In some embodiments, R is n-propyl. In some embodiments, R is tert-butyl. In some embodiments, R is sec-butyl. In some embodiments, R is n-butyl. In some embodiments, R is -(CH ₂ ) ₂ OCH ₃ .

In some embodiments, R is an optionally substituted phenyl. In some embodiments, R is phenyl.

In some embodiments, R ^2s is a 2'-modification as described herein and R ^4s is -H. In some embodiments, R ^2s is -OR and R is not hydrogen. In some embodiments, R ^2s is -F. In some embodiments, R ^2s is -OMe. In some embodiments, for example, in the various X _eo used in Table 1 (where X is m5C, T, G, A, etc.) R ^2s is -OCH ₂ CH ₂ CH ₃ . In some embodiments, R ^2s is selected from -H, -F, and -OR, and R is an optionally substituted C _1-6 alkyl. In some embodiments, R ^2s is selected from -H, -F, and -OMe.

In some embodiments, the sugar is a bicyclic sugar, eg, a sugar where R ^2s and R ^4s together form an optionally substituted ring as described herein. In some embodiments, the sugar is selected from LNA sugars, BNA sugars, cEt sugars, and the like. In some embodiments, the crosslinker is between 2' and 4'-carbon atoms (corresponding to R ^2s and R ^4s forming an optionally substituted ring as described herein, together with the intervening atoms). In some embodiments, a crosslinker is 2'-L ^a -L ^b -4', L ^a is -O-, -S-, or N(R), and L ^b is an optionally substituted C _1-4 2 is an aliphatic chain (eg methylene).

In some embodiments, the sugar is 2'-OMe, 2'-MOE, 2'-F, LNA (locked nucleic acid) sugar, ENA (ethylene bridged nucleic acid) sugar, BNA (NMe) (methylamino bridged nucleic acid) sugar, 2 '-F ANA (2'-F arabinose), alpha-DNA (alpha-D-ribose), 2'/5' ODN (eg 2'/5' linked oligonucleotide), Inv (inverted sugar ), eg, inverted deoxyribose), AmR (amino-ribose), ThioR (thio-ribose), HNA (hexose nucleic acid), CeNA (cyclohexene nucleic acid), or MOR (morpholino) sugar.

Those skilled in the art after reading this disclosure will understand that various types of sugar modifications are known and can be used in accordance with the present invention. In some embodiments, the sugar modification is a 2'-modification (eg, R ^2s ). In some embodiments, a 2'-modification is 2'-F. In some embodiments, a 2'-modification is 2'-OR and R is not hydrogen. In some embodiments, a 2'-modification is 2'-OR and R is an optionally substituted C _1-6 aliphatic. In some embodiments, the 2'-modification is 2'-OR and R is an optionally substituted C _1-6 alkyl. In some embodiments, the 2'-modification is 2'-OMe. In some embodiments, a 2'-modification is a 2'-MOE. In some embodiments, the 2'-modification is -OL ^b - or -L ^b -L ^b - linking the 2'-carbon of the sugar moiety to another carbon of the sugar moiety. In some embodiments, a 2'-modification is a 2'-OL ^b -4' or 2'-L ^b -L ^b -4' linking the 2'-carbon of a sugar moiety to the 4'-carbon of a sugar moiety. am. In some embodiments, the 2'-modification is S-cEt. In some embodiments, the modified sugar is an LNA sugar. In some embodiments, -L ^b - is -C(R) ₂ -. In some embodiments, the 2'-modification is (C2-OC(R) ₂ -C4), and each R is independently as described herein. In some embodiments, the 2'-modification is a modification per LNA (C2-O-CH ₂ -C4). In some embodiments, the 2'-modification is (C2-O-CHR-C4) and R is as described herein. In some embodiments, the 2'-modification is (C2-O-( R )-CHR-C4) and R is as described herein and is not hydrogen. In some embodiments, the 2'-modification is (C2-O-( S )-CHR-C4) and R is as described herein and is not hydrogen. In some embodiments, R is an optionally substituted C _1-6 aliphatic. In some embodiments, R is an optionally substituted C _1-6 alkyl. In some embodiments, R is unsubstituted C _1-6 alkyl. In some embodiments, R is methyl. In some embodiments, R is ethyl. In some embodiments, the 2'-modification is (C2-O-CHR-C4) and R is an optionally substituted C _1-6 aliphatic. In some embodiments, the 2'-modification is (C2-O-CHR-C4) and R is an optionally substituted C _1-6 alkyl. In some embodiments, the 2'-modification is (C2-O-CHR-C4) and R is methyl. In some embodiments, the 2'-modification is (C2-O-CHR-C4) and R is ethyl. In some embodiments, the 2'-modification is (C2-O-( R )-CHR-C4) and R is an optionally substituted C _1-6 aliphatic. In some embodiments, the 2'-modification is (C2-O-( R )-CHR-C4) and R is an optionally substituted C _1-6 alkyl. In some embodiments, the 2'-modification is (C2-O-( R )-CHR-C4) and R is methyl. In some embodiments, the 2'-modification is (C2-O-( R )-CHR-C4) and R is ethyl. In some embodiments, the 2'-modification is (C2-O-( S )-CHR-C4) and R is an optionally substituted C _1-6 aliphatic. In some embodiments, the 2'-modification is (C2-O-( S )-CHR-C4) and R is an optionally substituted C _1-6 alkyl. In some embodiments, the 2'-modification is (C2-O-( S )-CHR-C4) and R is methyl. In some embodiments, the 2'-modification is (C2-O-( S )-CHR-C4) and R is ethyl. In some embodiments, the 2'-modification is C2-O-( R )-CH(CH ₂ CH ₃ )-C4. In some embodiments, the 2'-modification is C2-O-( S )-CH(CH ₂ CH ₃ )-C4. In some embodiments, the sugar is a natural DNA sugar. In some embodiments, the sugar is a natural RNA sugar. In some embodiments, the sugar is an optionally substituted natural DNA sugar. In some embodiments, the sugar is a natural DNA sugar optionally substituted at the 2'. In some embodiments, the sugar is a 2'-substituted (2'-modified) natural DNA sugar. In some embodiments, the sugar is a natural DNA sugar modified at 2'(2'-modified).

In some embodiments, the sugar is optionally substituted ribose or deoxyribose. In some embodiments, the sugar is optionally modified ribose or deoxyribose, and one or more hydroxyl groups of the ribose or deoxyribose moiety are optionally and independently halogen, R', -N(R') ₂ , -OR ', or -SR', and each R' is as described herein. In some embodiments, the sugar is optionally substituted deoxyribose, and the 2' position of the deoxyribose is optionally substituted. In some embodiments, the sugar is optionally substituted deoxyribose, wherein the 2' position of deoxyribose is optionally substituted with halogen, R', -N(R') ₂ , -OR', or -SR', respectively R' of are independently described herein. In some embodiments, the sugar is optionally substituted deoxyribose, and the 2' position of deoxyribose is optionally substituted with a halogen. In some embodiments, the sugar is optionally substituted deoxyribose, and the 2' position of deoxyribose is optionally substituted with one or more -F. In some embodiments, the sugar is optionally substituted deoxyribose, optionally substituted at the 2' position of deoxyribose with -OR', and each R' is independently described herein. In some embodiments, the sugar is optionally substituted deoxyribose, optionally substituted at position 2' of deoxyribose with -OR', and each R' independently is an optionally substituted C ₁ -C ₆ aliphatic. In some embodiments, the sugar is optionally substituted deoxyribose, optionally substituted at the 2' position of deoxyribose with -OR', and each R' independently is an optionally substituted C ₁ -C ₆ alkyl. In some embodiments, the sugar is optionally substituted deoxyribose, and the 2' position of deoxyribose is optionally substituted with -OMe. In some embodiments, the sugar is optionally substituted deoxyribose, and the 2' position of deoxyribose is optionally substituted with -O-methoxyethyl.

In some embodiments, provided oligonucleotides include one or more modified sugars. In some embodiments, provided oligonucleotides include one or more modified sugars and one or more natural sugars.

Examples of bicyclic sugars are alpha-L-methyleneoxy (4'-CH ₂ -O-2') LNA, beta-D-methyleneoxy (4'-CH ₂ -O-2') LNA, ethyleneoxy (4'- (CH ₂ ) ₂ -O-2') LNA, aminooxy (4'-CH ₂ -ON(R)-2') LNA, and oxyamino(4'-CH ₂ -N(R)-O-2 ') contains the sugar of LNA. In some embodiments, a bicyclic sugar, such as an LNA or BNA sugar, is a sugar having at least one bridge between two sugar carbons. In some embodiments, a bicyclic sugar of a nucleoside can have the stereochemical configuration of alpha-L-ribofuranose or beta-D-ribofuranose.

In some embodiments, a bicyclic sugar can be further defined by an isomeric configuration. For example, a sugar comprising a 4'-(CH ₂ )-O-2' crosslinker may exist in an alpha-L configuration or a beta-D configuration. In some embodiments, a 4'-2' crosslink is -L-4'-(CH ₂ )-O-2', bD-4'-CH ₂ -O-2', 4'-(CH ₂ ) ₂ -O-2', 4'-CH ₂ -ON(R')-2', 4'-CH ₂ -N(R')-O-2', 4'-CH(R')-O-2 ', 4'-CH(CH ₃ )-O-2', 4'-CH ₂ -S-2', 4'-CH ₂ -N(R')-2', 4'-CH ₂ -CH( R′)-2′, 4′-CH ₂ -CH(CH ₃ )-2′, and 4′-(CH ₂ ) _3-2 ′, each R′ as described herein. In some embodiments, R' is -H, a protecting group, or an optionally substituted C ₁ -C ₁₂ alkyl. In some embodiments, R' is -H or an optionally substituted C ₁ -C ₁₂ alkyl.

In some embodiments, the bicyclic sugar is alpha-L-methyleneoxy (4'-CH ₂ -O-2') BNA, beta-D-methyleneoxy (4'-CH ₂ -O-2') BNA, ethyleneoxy (4'-(CH ₂ ) ₂ -O-2') BNA, aminooxy (4'-CH ₂ -ON(R)-2') BNA, oxyamino(4'-CH ₂ -N(R)- O-2') BNA, methyl(methyleneoxy)(4'-CH(CH ₃ )-O-2') BNA (also known as constrained ethyl or cEt), methylene-thio(4'-CH ₂ -S-2 ') BNA, methylene-amino(4'-CH ₂ -N(R)-2') BNA, methyl carbocyclic (4'-CH ₂ -CH(CH ₃ )-2') BNA, propylene carbocyclic (4'-(CH ₂ ) ₃ -2') BNA, or the sugar of vinyl BNA.

In some embodiments, the sugar modification is a modification described in US 9006198. In some embodiments, modified sugars are described in US 9006198. In some embodiments, sugar modifications are disclosed in US 9394333, US 9744183, US 9605019, US 9982257, US 20170037399, US 20180216108, US 20180216107, US 9598458, WO 2017/062862, WO 2018/0 67973, WO 2017/160741, WO 2017/ 192679, WO 2017/210647, WO 2018/098264, WO 2018/022473, WO 2018/223056, WO 2018/223073, WO 2018/223081, WO 2018/237194, WO 2019/032607, WO2 019/032612, WO 2019/055951 , WO 2019/075357, WO 2019/200185, WO 2019/217784, WO 2019/032612, WO 2020/191252 and/or WO 2021/071858, wherein each sugar modification and modified sugar are independently referenced herein. included as

In some embodiments, the modified sugar is US 5658873, US 5118800, US 5393878, US 5514785, US 5627053, US 7034133;7084125, US 7399845, US 5319080, US 5591722, US 5597909, US 5466 786, US 6268490, US 6525191, US 5519134, US 5576427, US 6794499, US 6998484, US 7053207, US 4981957, US 5359044, US 6770748, US 7427672, US 5446137, US 6670461, US 7569686, US 7 741457, US 8022193, US 8030467, US 8278425, US 5610300, US 5646265, US 8278426, US 5567811, US 5700920, US 8278283, US 5639873, US 5670633, US 8314227, US 2008/0039618, US 2009/0012281, WO 2021/03077 8, WO 2020/154344, WO 2020/154343, WO 2020/154342, WO 2020/165077, WO 2020/201406, WO 2020/216637, or WO 2020/252376.

In some embodiments, the sugar modification is 2'-OMe, 2'-MOE, 2'-LNA, 2'-F, 5'-vinyl, or S-cEt. In some embodiments, the modified sugar is a sugar of FRNA, FANA, or morpholino. In some embodiments, the oligonucleotide is a nucleic acid analog, e.g., GNA, LNA, PNA, TNA, F-HNA (F-THP or 3'-fluoro tetrahydropyran), MNA (mannitol nucleic acid, e.g., (Leumann 2002 Bioorg. Med. Chem. 10: 841-854), ANA (anitol nucleic acid), or morpholino, or parts thereof. In some embodiments, the sugar is as in flexible nucleic acids or serinol nucleic acids. In some embodiments, a sugar modification replaces a natural sugar with another cyclic or acyclic moiety. Examples of such moieties are well known in the art (eg, those used in morpholino, glycol nucleic acids, etc.) and may be used in accordance with the present invention. As will be appreciated by those skilled in the art, when used with modified sugars, in some embodiments internucleotidic linkages may be modified, such as in morpholino, PNA, and the like. In some embodiments, the sugar is a (R)-GNA sugar. In some embodiments, the sugar is an (S)-GNA sugar. In some embodiments, nucleosides with GNA sugars are used as N ₋₁ , N ₀ and/or N ₁ . In some embodiments, N ₀ is a nucleoside with a GNA sugar. In some embodiments, the sugar is a bicyclic sugar. In some embodiments, the sugar is an LNA sugar. In some embodiments, the sugar is an acyclic sugar. In some embodiments, the sugar is a UNA sugar. In some embodiments, nucleosides with UNA sugars are used as N ₋₁ , N ₀ and/or N ₁ . In some embodiments, N ₀ is a nucleoside with a UNA sugar. In some embodiments, nucleosides are abasic. In some embodiments, an abasic sugar is used as N ₋₁ , N ₀ and/or N ₁ . In some embodiments, N ₀ is a nucleoside with an abasic sugar.

In some embodiments, the sugar is a 6'-modified bicyclic sugar having (R) or (S)-chirality at the 6-position (eg, as described in US 7399845). In some embodiments, the sugar is a 5'-modified bicyclic sugar with (R) or (S)-chirality at the 5-position (eg, as described in US 20070287831).

In some embodiments, the modified sugar is -F; -CF ₃ , -CN, -N ₃ , -NO, -NO ₂ , -OR', -SR', or -N(R') ₂ (each R' is independently described herein); -O-(C ₁ -C ₁₀ alkyl), -S-(C ₁ -C ₁₀ alkyl), -NH-(C ₁ -C ₁₀ alkyl), or -N(C ₁ -C ₁₀ alkyl) ₂ ; -O-(C ₂ -C ₁₀ alkenyl), -S-(C ₂ -C ₁₀ alkenyl), -NH-(C ₂ -C ₁₀ alkenyl), or -N(C ₂ -C ₁₀ alkenyl) ) ₂ ; -O-(C ₂ -C ₁₀ alkynyl), -S-(C ₂ -C ₁₀ alkynyl), -NH-(C ₂ -C ₁₀ alkynyl), or -N(C ₂ -C ₁₀ alkynyl) ) ₂ ; or -O--(C ₁ -C ₁₀ alkylene)-O--(C ₁ -C ₁₀ alkyl), -O-(C ₁ -C ₁₀ alkylene)-NH-(C ₁ -C ₁₀ alkyl) or -O-(C ₁ -C ₁₀ alkylene)-NH(C ₁ -C ₁₀ alkyl) ₂ , -NH-(C ₁ -C ₁₀ alkylene)-O-(C ₁ -C ₁₀ alkyl), or -N(C ₁ -C ₁₀ alkyl)-(C ₁ -C ₁₀ alkylene)-O-(C ₁ -C ₁₀ alkyl) at least one substituent at the 2' position (usually one substituent and often located on the longitudinal axis), wherein each of alkyl, alkylene, alkenyl, and alkynyl is independently and optionally substituted. In some embodiments, the substituent is -O(CH ₂ ) _n OCH ₃ , -O(CH ₂ ) _n NH ₂ , MOE, DMAOE, or DMAEOE, and n is from 1 to about 10. In some embodiments, modified sugars are described in WO 2001/088198; and Martin et al., Helv. Chim. Acta, 1995, 78, 486-504]. In some embodiments, the modified sugar is a substituted silyl group, an RNA cleavage group, a reporter group, a fluorescent label, an intercalator, a group for improving the pharmacokinetic properties of a nucleic acid, a group for improving the pharmacodynamic properties of a nucleic acid , or other substituents with similar properties. In some embodiments, the modification is at one or more of the 2', 3', 4', or 5' positions, including the 3' position of the sugar on the 3'-terminal nucleoside or the 5' position of the 5'-terminal nucleoside. takes place in

In some embodiments, the 2'-OH of ribose is -H, -F; -CF ₃ , -CN, -N ₃ , -NO, -NO ₂ , -OR', -SR', or -N(R') ₂ (each R' is independently described herein); -O-(C ₁ -C ₁₀ alkyl), -S-(C ₁ -C ₁₀ alkyl), -NH-(C ₁ -C ₁₀ alkyl), or -N(C ₁ -C ₁₀ alkyl) ₂ ; -O-(C ₂ -C ₁₀ alkenyl), -S-(C ₂ -C ₁₀ alkenyl), -NH-(C ₂ -C ₁₀ alkenyl), or -N(C ₂ -C ₁₀ alkenyl) ) ₂ ; -O-(C ₂ -C ₁₀ alkynyl), -S-(C ₂ -C ₁₀ alkynyl), -NH-(C ₂ -C ₁₀ alkynyl), or -N(C ₂ -C ₁₀ alkynyl) ) ₂ ; or -O-(C ₁ -C ₁₀ alkylene)-O--(C ₁ -C ₁₀ alkyl), -O-(C ₁ -C ₁₀ alkylene)-NH-(C ₁ -C ₁₀ alkyl), or -O-(C ₁ -C ₁₀ alkylene)-NH(C ₁ -C ₁₀ alkyl) ₂ , -NH-(C ₁ -C ₁₀ alkylene)-O-(C ₁ -C ₁₀ alkyl), or - N(C ₁ -C ₁₀ alkyl)-(C ₁ -C ₁₀ alkylene)-O-(C ₁ -C ₁₀ alkyl), wherein alkyl, alkylene, alkenyl, and alkynyl are each independently and optionally substituted. In some embodiments, 2'-OH is replaced with -H (deoxyribose). In some embodiments, 2'-OH is replaced with -F. In some embodiments, 2'-OH is replaced with -OR'. In some embodiments, 2'-OH is replaced with -OMe. In some embodiments, 2'-OH is replaced with -OCH ₂ CH ₂ OMe.

In some embodiments, the sugar modification is a 2'-modification. Commonly used 2'-modifications include, but are not limited to, 2'-OR, where R is not hydrogen and as described herein. In some embodiments, the strain is 2'-OR and R is an optionally substituted C _1-6 aliphatic. In some embodiments, the strain is 2'-OR and R is an optionally substituted C _1-6 alkyl. In some embodiments, the modification is 2'-OMe. In some embodiments, a modification is a 2'-MOE. In some embodiments, the 2'-modification is S-cEt. In some embodiments, the modified sugar is an LNA sugar. In some embodiments, the 2'-modification is -F. In some embodiments, the 2'-modification is FANA. In some embodiments, the 2'-modification is an FRNA. In some embodiments, the sugar modification is a 5'-modification, eg 5'-Me. In some embodiments, the sugar modification changes the size of the sugar ring. In some embodiments, the sugar modification is a sugar moiety in FHNA.

In some embodiments, a sugar modification replaces a sugar moiety with another cyclic or acyclic moiety. Examples of such moieties are well known in the art and include, but are not limited to, those used in morpholinos (optionally with phosphorodiamidate linkages), glycol nucleic acids, and the like.

In some embodiments, one or more of the sugars of the oligonucleotide are modified. In some embodiments, a modified sugar comprises a 2'-modification. In some embodiments, each modified sugar independently comprises a 2'-modification. In some embodiments, a 2'-modification is a 2'-OR. In some embodiments, the 2'-modification is 2'-OMe. In some embodiments, a 2'-modification is a 2'-MOE. In some embodiments, a 2'-modification is a modification per LNA. In some embodiments, a 2'-modification is 2'-F. In some embodiments, each sugar modification is independently a 2'-modification. In some embodiments, each sugar modification is independently 2'-OR or 2'-F. In some embodiments, each sugar variant is independently 2'-OR or 2'-F, and R is an optionally substituted C _1-6 alkyl. In some embodiments, each sugar modification is independently 2'-OR or 2'-F, and at least one is 2'-F. In some embodiments, each sugar variant is independently 2'-OR or 2'-F, R is an optionally substituted C _1-6 alkyl, and at least one is 2'-OR. In some embodiments, each sugar modification is independently 2'-OR or 2'-F, at least one is 2'-F and at least one is 2'-OR. In some embodiments, each sugar variant is independently 2'-OR or 2'-F, R is an optionally substituted C _1-6 alkyl, at least one is 2'-F, and at least one is 2'- is OR. In some embodiments, each sugar modification is independently a 2'-OR. In some embodiments, each sugar variant is independently 2'-OR, and R is an optionally substituted C _1-6 alkyl. In some embodiments, each sugar modification is 2'-OMe. In some embodiments, each sugar modification is a 2'-MOE. In some embodiments, each sugar modification is independently 2'-OMe or 2'-MOE. In some embodiments, each sugar modification is independently a 2'-OMe, 2'-MOE, or LNA sugar.

Modified sugars include cyclobutyl or cyclopentyl moieties instead of pentofuranosyl sugars. Representative examples of such modified sugars include those described in US 4,981,957, US 5,118,800, US 5,319,080, or US 5,359,044. In some embodiments, an oxygen atom in the ribose ring is replaced with nitrogen, sulfur, selenium, or carbon. In some embodiments, -O- is replaced with -N(R')-, -S-, -Se-, or -C(R') ₂ -. In some embodiments, the modified sugar is a modified ribose in which an oxygen atom in the ribose ring is replaced with a nitrogen, and the nitrogen is optionally substituted with an alkyl group (eg, methyl, ethyl, isopropyl, etc.).

A non-limiting example of a modified sugar is glycerol, which is part of glycerol nucleic acid (GNA), which is described, for example, in Zhang, R et al., J. Am. Chem. Soc., 2008, 130, 5846-5847]; See Zhang L, et al., J. Am. Chem. Soc., 2005, 127, 4174-4175 and Tsai CH et al., PNAS, 2007, 14598-14603.

Flexible nucleic acids (FNA) are based on mixed acetal aminals of formyl glycerol, which are described, for example, in Joyce GF et al., PNAS, 1987, 84, 4398-4402 and in Heuberger BD and Switzer C, J. Am. Chem. Soc., 2008, 130, 412-413].

In some embodiments, oligonucleotides and/or modified nucleosides thereof are described in WO 2018/022473, WO 2018/098264, WO 2018/223056, WO 2018/223073, WO 2018/223081, WO 2018/237194, WO 2019/032607, including sugars or modified sugars described in WO 2019/055951, WO 2019/075357, WO 2019/200185, WO 2019/217784, WO 2019/032612, WO 2020/191252 and/or WO 2021/071858; Modifying sugars are independently incorporated herein by reference.

In some embodiments, one or more hydroxyl groups on the sugar are optionally and independently replaced with halogen, R'-N(R') ₂ , -OR', or -SR', each R' being independently described in the invention.

In some embodiments, modified nucleosides are described in WO 2018/022473, WO 2018/098264, WO 2018/223056, WO 2018/223073, WO 2018/223081, WO 2018/237194, WO 2019/032607, WO 2019/055951 , WO 2019/075357, WO 2019/200185, WO 2019/217784, WO 2019/032612, WO 2020/191252 and/or WO 2021/071858, each modified nucleoside independently described herein. included by reference.

In some embodiments, the sugar modification is 5'-vinyl (R or S), 5'-methyl (R or S), 2'-SH, 2'-F, 2'-OCH ₃ , 2'-OCH ₂ CH ₃ , 2′-OCH ₂ CH ₂ F, or 2′-O(CH ₂ ) ₂₀ CH ₃ . In some embodiments, the substituent at the 2' position, eg, the 2'-modification, is allyl, amino, azido, thio, O-allyl, OC ₁ -C ₁₀ alkyl, OCF ₃ , OCH ₂ F, O(CH ₂ ) ₂ SCH ₃ , O(CH ₂ ) ₂ -ON(R _m )(R _n ), O-CH ₂ -C(=0)-N(R _m )(R _n ), and O-CH ₂ -C (=0)-N(R ₁ )-(CH ₂ ) ₂ -N(R _m )(R _n ), each of allyl, amino, and alkyl being optionally substituted, and R _l , R _m , and R _n each independently R' as described herein. In some embodiments, each of R _l , R _m , and R _n is independently -H or an optionally substituted C ₁ -C ₁₀ alkyl.

In some embodiments, bicyclic sugars include bridges between two sugar carbons, such as between 4' and 2' ribosyl ring carbon atoms, such as -L ^b -L ^b -, -L-, etc. do. In some embodiments, the crosslinker is 4'-(CH ₂ )-O-2' (eg, per LNA), 4'-(CH ₂ )-S-2', 4'-(CH ₂ ) ₂ -O -2' (eg per ENA), 4'-CH(R')-O-2' (eg 4'-CH(CH ₃ )-O-2', 4'-CH(CH ₂ OCH ₃ ) -O-2', and examples from US 7399845, etc.), 4'-CH(R') ₂ -O-2' (eg 4'-C(CH ₃ )(CH ₃ )-O-2' and WO 2009006478, etc.), 4'-CH ₂ -N(OR')-2' (eg, 4'-CH ₂ -N(OCH ₃ )-2', WO 2008150729, etc.), 4'-CH ₂ -ON(R')-2' (eg, 4'-CH ₂ -ON(CH ₃ )-2', US 20040171570, etc.), 4'-CH ₂ -N(R')-O-2' [eg R is -H, C ₁ -C ₁₂ alkyl, or a protecting group (see eg US 7427672)], 4'-C(R') ₂ -C(H)(R')-2 '(eg 4'-CH ₂ -C(H)(CH ₃ )-2', examples such as Chattopadhyaya et al., J. Org. Chem., 2009, 74, 118-134), or 4'-C(R') ₂ -C(=C(R') ₂ )-2' (eg 4'-CH ₂ -C(=CH ₂ )-2', examples such as WO 2008154401).

In some embodiments, the sugar is a tetrahydropyran or THP sugar. In some embodiments, a modified nucleoside is a tetrahydropyran nucleoside or a THP nucleoside, which is a nucleoside having a 6-membered tetrahydropyran sugar substituted for the pentofuranosyl residue of a common natural nucleoside. THP sugars and/or nucleosides include hexitol nucleic acids (HNA), anitol nucleic acids (ANA), mannitol nucleic acids (MNA) (see, e.g., Leumann, Bioorg. Med. Chem., 2002, 10, 841-854). ]) or fluoro HNA (F-HNA).

In some embodiments, sugars include rings with more than 5 atoms and/or more than 1 heteroatom (see, e.g., Braasch et al., Biochemistry, 2002, 41, 4503-4510; US 5698685; US 5166315; US 5185444; US 5034506, etc.).

As will be appreciated by those skilled in the art, modifications of sugars, nucleobases, internucleotidic linkages, etc. can and often are used in combination in oligonucleotides (see, eg, various oligonucleotides in Table 1).

In some embodiments, the nucleoside has a 6-membered cyclohexenyl in place of the pentofuranosyl residue of the naturally occurring nucleoside. Examples of cyclohexenyl nucleosides and their preparation and use are described, for example, in WO 2010036696; See Robeyns et al., J. Am. Chem. Soc., 2008, 130(6), 1979-1984]; Horvath et al., Tetrahedron Letters, 2007, 48, 3621-3623; See Nauwelaerts et al., J. Am. Chem. Soc., 2007, 129(30), 9340-9348; Gu et al., Nucleosides, Nucleotides & Nucleic Acids, 2005, 24(5-7), 993-998; Nauwelaerts et al., Nucleic Acids Research, 2005, 33(8), 2452-2463; Robeyns et al., Acta Crystallographica, Section F: Structural Biology and Crystallization Communications, 2005, F61(6), 585-586; Gu et al., Tetrahedron, 2004, 60(9), 2111-2123; Gu et al., Oligonucleotides, 2003, 13(6), 479-489; See Wang et al., J. Org. Chem., 2003, 68, 4499-4505]; Verbeure et al., Nucleic Acids Research, 2001, 29(24), 4941-4947; See Wang et al., J. Org. Chem., 2001, 66, 8478-82]; Wang et al., Nucleosides, Nucleotides & Nucleic Acids, 2001, 20(4-7), 785-788; See Wang et al., J. Am. Chem., 2000, 122, 8595-8602]; WO 2006047842; WO 2001049687 and the like are described.

Many monocyclic, bicyclic, and tricyclic ring systems are suitable as sugar substitutes (modified sugars) and can be used in accordance with the present invention. See, eg, Leumann, Christian J. Bioorg. & Med. Chem., 2002, 10, 841-854. These ring systems can be further improved in properties and/or activity through various additional substitutions.

In some embodiments, a 2'-modified sugar is a furanosyl sugar modified at the 2' position. In some embodiments, a 2'-modification is halogen, -R'(R' is not -H), -OR'(R' is not -H), -SR', -N(R') ₂ , optionally substituted -CH ₂ -CH=CH ₂ , optionally substituted alkenyl, or optionally substituted alkynyl. In some embodiments, the 2'-modification is -O[(CH ₂ ) _n O] _m CH ₃ , -O(CH ₂ ) _n NH ₂ , -O(CH ₂ ) _n CH ₃ , -O(CH ₂ ) _n F, -O(CH ₂ ) _n ONH ₂ , -OCH ₂ C(=O)N(H)CH ₃ , and -O(CH ₂ ) _n ON[(CH ₂ ) _n CH ₃ ] ₂ ; , each n and m is independently 1 to about 10. In some embodiments, the 2′-strain is an optionally substituted C ₁ -C ₁₂ alkyl, an optionally substituted alkenyl, an optionally substituted alkynyl, an optionally substituted alkaryl, an optionally substituted aralkyl, an optionally substituted —O— Alkaryl, optionally substituted -O-aralkyl, -SH, -SCH ₃ , -OCN, -Cl, -Br, -CN, -F, -CF ₃ , -OCF ₃ , -SOCH ₃ , -SO ₂ CH ₃ , -ONO ₂ , -NO ₂ , -N ₃ , -NH ₂ , optionally substituted heterocycloalkyl, optionally substituted heterocycloalkaryl, optionally substituted aminoalkylamino, optionally substituted polyalkylamino, substituted silyl , reporter groups, intercalators, groups for improving pharmacokinetic properties, groups for improving pharmacodynamic properties, and other substituents. In some embodiments, the 2'-modification is a 2'-MOE modification (see, eg, Baker et al., J. Biol. Chem., 1997, 272, 11944-12000). In some cases, 2'-MOE modifications have improved binding affinity compared to unmodified sugars and some other modified nucleosides, such as 2'-O-methyl, 2'-O-propyl, and 2'-O-aminopropyl. Reported to have Mars. Oligonucleotides with 2'-MOE modifications have also been reported to be able to inhibit gene expression, with promising properties for in vivo use (see, e.g., Martin, Helv. Chim. Acta, 1995, 78 , 486-504; Altmann et al., Chimia, 1996, 50, 168-176; Altmann et al., Biochem. Altmann et al., Nucleosides Nucleotides, 1997, 16, 917-926; and the like).

In some embodiments, a 2'-modified or 2'-substituted sugar or nucleoside is a sugar or nucleoside comprising a substituent other than -H (generally not considered a substituent) or -OH at the 2' position of the sugar. . In some embodiments, a 2'-modified sugar is a bicyclic sugar comprising a bridge connecting two carbon atoms of the sugar ring, one of which is the 2' carbon. In some embodiments, the 2'-modification is non-bridged, for example allyl, amino, azido, thio, optionally substituted -O-allyl, optionally substituted -OC ₁ -C ₁₀ alkyl, -OCF ₃ , -O (CH ₂ ) ₂ OCH ₃ , 2′-O(CH ₂ ) ₂ SCH ₃ , -O(CH ₂ ) ₂ ON(R _m )(R _n ), or -OCH ₂ C(=O)N(R _m )(R _n ), and each R _m and R _n is independently -H or an optionally substituted C ₁ -C ₁₀ alkyl.

As per specific variants, their manufacture and use are described in US 4981957, US 5118800, US 5319080, US 5359044, US 5393878, US 5446137, US 5466786, US 5514785, US 5519134, US 5567811, US 5576427, US 5591722, US 5597909, US 5610300 , US 5627053, US 5639873, US 5646265, US 5670633, US 5700920, US 5792847, US 6600032, and WO 2005121371.

In some embodiments, the sugar is N-methanocarba, LNA, cMOE BNA, cEt BNA, α-L-LNA or related analogs, HNA, Me-ANA, MOE-ANA, Ara-FHNA, FHNA, R-6 is the sugar of '-Me-FHNA, S-6'-Me-FHNA, ENA, or c-ANA. In some embodiments, the modified internucleotide linkage is a C3-amide (e.g., a sugar having an amide modification attached to C3', Mutisya et al. 2014 Nucleic Acids Res. 2014 Jun 1; 42(10): 6542 -6551]), formacetal, thioformacetal, MMI [eg methylene (methylimino), Peoc'h et al. 2006 Nucleosides and Nucleotides 16 (7-9)]], PMO (phosphorodiamidate linked morpholino) linkage (which links two sugars), or PNA (peptide nucleic acid) linkage. In some embodiments, examples of internucleotidic linkages and/or sugars are described in Allerson et al. 2005 J. Med. Chem. 48: 901-4]; BMCL 2011 21: 1122; BMCL 2011 21: 588]; BMCL 2012 22: 296; See Chattopadhyaya et al. 2007 J. Am. Chem. Soc. 129: 8362]; See Chem. Bio. Chem. 2013 14:58]; See Curr. Prot. Nucl. Acids Chem. 2011 1.24.1]; See Egli et al. 2011 J. Am. Chem. Soc. 133: 16642]; See Hendrix et al. 1997 Chem. Eur. J. 3: 110]; See Hyrup et al. 1996 Bioorg. Med. Chem. 4: 5]; See Imanishi 1997 Tet. Lett. 38: 8735]; Literature [J. Am. Chem. Soc. 1994, 116, 3143]; Literature [J. Med. Chem. 2009 52: 10]; Literature [J. Org. Chem. 2010 75: 1589]; See Jepsen et al. 2004 Oligo. 14: 130-146]; See Jones et al. J. Org. Chem. 1993, 58, 2983]; See Jung et al. 2014 ACIEE 53: 9893]; See Kodama et al. 2014 AGDS]; Koizumi 2003 BMC 11: 2211; See Koizumi et al. 2003 Nuc. Acids Res. 12: 3267-3273]; See Koshkin et al. 1998 Tetrahedron 54: 3607-3630]; See Kumar et al. 1998 Bioo. Med. Chem. Let. 8: 2219-2222]; See Lauritsen et al. 2002 Chem. Comm. 5: 530-531]; See Lauritsen et al. 2003 Bioo. Med. Chem. Lett. 13: 253-256]; See Lima et al. 2012 Cell 150: 883-894; See Mesmaeker et al. Angew. Chem., Int. Ed. Engl. 1994, 33, 226]; See Migawa et al. 2013 Org. Lett. 15: 4316]; See Mol. Ther. Nucl. Acids 2012 1: e47]; See Morita et al. 2001 Nucl. Acids Res. Supp. 1: 241-242]; See Morita et al. 2002 Bioo. Med. Chem. Lett. 12: 73-76]; See Morita et al. 2003 Bioo. Med. Chem. Lett. 2211-2226]; See Murray et al. 2012 Nucl. Acids Res. 40: 6135]; See Nielsen et al. 1997 Chem. Soc. Rev. 73]; See Nielsen et al. 1997 J. Chem. Soc. Perkins Transl. 1: 3423-3433]; See Obika et al. 1997 Tetrahedron Lett. 38 (50): 8735-8]; See Obika et al. 1998 Tetrahedron Lett. 39: 5401-5404]; See Obika et al. 2008 J. Am. Chem. Soc. 130: 4886]; See Obika et al. 2011 Org. Lett. 13: 6050]; See Oestergaard et al. 2014 JOC 79: 8877]; See Pallan et al. 2012 Biochem. 51:7]; See Pallan et al. 2012 Chem. Comm. 48: 8195-8197]; See Petersen et al. 2003 TRENDS Biotech. 21: 74-81]; See Prakash et al. 2010 J. Med. Chem. 53: 1636]; See Prakash et al. 2015 Nucl. Acids Res. 43: 2993-3011]; See Prakash et al. 2016 Bioorg. Med. Chem. Lett. 26: 2817-2820]; See Rajwanshi et al. 1999 Chem. Commun. 1395-1396]; See Schultz et al. 1996 Nucleic Acids Res. 24: 2966]; See Seth et al. 2008 Nucl. Acid Sym. Ser. 52:553]; See Seth et al. 2009 J. Med. Chem. 52: 10-13]; See Seth et al. 2010 J. Am. Chem. Soc. 132: 14942]; See Seth et al. 2010 J. Med. Chem. 53: 8309-8318]; See Seth et al. 2010 J. Org. Chem. 75: 1569-1581]; See Seth et al. 2011 BMCL 21: 4690]; See Seth et al. 2012 Bioo. Med. Chem. Lett. 22: 296-299]; See Seth et al. 2012 Mol. Ther-Nuc. Acids. 1, e47]; Seth et al., Nucleic Acids Symposium Series (2008), 52(1), 553-554; See Singh et al. 1998 Chem. Comm. 1247-1248]; See Singh et al. 1998 J. Org. Chem. 63: 10035-39]; See Singh et al. 1998 J. Org. Chem. 63: 6078-6079]; See Sorensen 2003 Chem. Comm. 2130-2131]; See Starrup et al. 2010 Nucl. Acids Res. 38: 7100]; See Swayze et al. 2007 Nucl. Acids Res. 35: 687]; See Ts'o et al. Ann. N.Y. Acad. Sci. 1988, 507, 220]; See Van Aerschot et al. 1995 Angew. Chem. Int. Ed. Engl. 34: 1338]; See Vasseur et al. J. Am. Chem. Soc. 1992, 114, 4006]; WO 2007090071; WO 2016079181; US 6326199; US 6066500; or US 6440739.

In some embodiments, oligonucleotides or portions thereof (eg, domains, subdomains, etc.) contain high levels of 2'-F modified sugars. For example, about 10% to 100% (e.g., about 20%, 25%, 30%, 35%, 40%, 45%, 50%) of sugars in an oligonucleotide or portion thereof (e.g., domain, subdomain, etc.) %, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, at least 95%, or about 100%) includes 2'-F. In some embodiments, at least about 50% of the sugars in an oligonucleotide or portion thereof include a 2'-F. In some embodiments, at least about 60% of the sugars in an oligonucleotide or portion thereof include a 2'-F. In some embodiments, at least about 70% of the sugars in an oligonucleotide or portion thereof include a 2'-F. In some embodiments, at least about 80% of the sugars in an oligonucleotide or portion thereof include a 2'-F. In some embodiments, about 90% or more of the sugars in an oligonucleotide or portion thereof include a 2'-F. In some embodiments, the oligonucleotide or portion thereof also includes one or more sugars that do not include a 2'-F (eg, a sugar that does not include modifications and/or a sugar that includes other modifications).

In some embodiments, up to about 1% to 95% (eg, up to about 1%, 5%, 10%, 15%, 20%) of sugars in an oligonucleotide or portion thereof (eg, a domain, subdomain, etc.) , 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, etc.) is 2' -Includes MOE. In some embodiments, up to about 50% of the sugars in an oligonucleotide or portion thereof comprise a 2'-MOE. In some embodiments, a sugar in an oligonucleotide or portion thereof does not include a 2'-MOE. In some embodiments, up to 1, 2, 3, 4, or 5 sugars in an oligonucleotide or portion thereof include a 2'-MOE.

A variety of additional sugars useful for making oligonucleotides or analogs thereof are known in the art and can be used in accordance with the present invention.

internucleotide linkage

Among other things, the present invention provides a variety of internucleotide linkages, including a variety of modified internucleotide linkages that can be used in conjunction with other structural elements, such as various sugars as described herein, to provide oligonucleotides and compositions thereof. .

In some embodiments, oligonucleotides include base modifications, sugar modifications, and/or internucleotide linkage modifications. A variety of internucleotidic linkages can be used in accordance with the present invention to link units comprising nucleobases, such as nucleosides. In some embodiments, provided oligonucleotides include both one or more modified internucleotidic linkages and one or more natural phosphate linkages. As is well known to those skilled in the art, natural phosphate linkages are widely found in natural DNA and RNA molecules; They have the structure -OP(O)(OH)O-, link sugars in nucleosides of DNA and RNA, and can exist in various salt forms (e.g., at physiological pH (about 7.4)). , natural phosphate linkages exist mainly in the form of salts with the anion -OP(O)(O ^- )O-). A modified internucleotide linkage or non-natural phosphate linkage is an internucleotide linkage that is not a natural phosphate linkage or salt form thereof. Modified internucleotide linkages may also exist in salt form depending on the structure. For example, as understood by those skilled in the art, phosphorothioate internucleotidic linkages having the structure -OP(O)(SH)O- can exist in various salt forms (e.g. at physiological pH (about 7.4), the anion is -OP(O)(S ^- )O-).

In some embodiments, the oligonucleotide is an internucleotide linkage that is a modified internucleotide linkage, e.g., phosphorothioate, phosphorodithioate, methylphosphonate, phosphoroamidate, thiophosphate, 3'-thiophosphate, or 5'-thiophosphate.

In some embodiments, the modified internucleotide linkage is a chiral internucleotide linkage comprising a chiral linkage phosphorus. In some embodiments, the chiral internucleotidic linkage is a phosphorothioate linkage. In some embodiments, chiral internucleotidic linkages are non-negatively charged internucleotidic linkages. In some embodiments, chiral internucleotidic linkages are neutral internucleotidic linkages. In some embodiments, chiral internucleotidic linkages are chiral controlled with respect to the chiral linkage phosphorus. In some embodiments, a chiral internucleotidic linkage is stereochemically pure with respect to the chiral linking phosphorus. In some embodiments, chiral internucleotidic linkages are not chirally controlled. In some embodiments, the backbone chiral center pattern comprises or consists of positions of chiral control internucleotidic linkages ( R p or Sp ) and linkage phosphorous arrangements and positions of achiral internucleotidic linkages (eg, natural phosphate linkages). do.

In some embodiments, the internucleotidic linkage comprises a P-modification, and the P-modification is a modification at the linking phosphorus. In some embodiments, the modified internucleotidic linkage is a moiety that does not contain phosphorus but serves to link two sugars or two moieties that each independently contain a nucleobase, as in, for example, a peptide nucleic acid (PNA) am.

In some embodiments, an oligonucleotide is a modified internucleotide linkage, for example, one having the structure of Formula I , Ia , Ib , or Ic and described herein and/or in WO 2018/022473, WO 2018/098264, WO 2018/223056, WO 2018/223073, WO 2018/223081, WO 2018/237194, WO 2019/032607, WO 2019/055951, WO 2019/075357, WO 2019/200185, WO 2019/217784, WO 2019/0326 12, WO 2020/191252 and/or or WO 2021/071858, wherein each internucleotidic linkage (eg, of Formula I , Ia , Ib , Ic, etc.) is independently incorporated herein by reference. In some embodiments, the modified internucleotidic linkage is a chiral internucleotidic linkage. In some embodiments, the modified internucleotidic linkage is a phosphorothioate internucleotidic linkage.

In some embodiments, the modified internucleotidic linkage is a non-negatively charged internucleotidic linkage. In some embodiments, provided oligonucleotides include one or more internucleotidic linkages that are not negatively charged. In some embodiments, the non-negatively charged internucleotidic linkages are positively charged internucleotidic linkages. In some embodiments, the non-negatively charged internucleotidic linkages are neutral internucleotidic linkages. In some embodiments, the present invention provides oligonucleotides comprising one or more neutral internucleotide linkages. In some embodiments, non-negatively charged internucleotide linkages are disclosed herein and/or in US 9394333, US 9744183, US 9605019, US 9982257, US 20170037399, US 20180216108, US 20180216107, US 9598458, WO 2017/062 862, WO 2018/ 067973, WO 2017/160741, WO 2017/192679, WO 2017/210647, WO 2018/098264, WO 2018/022473, WO 2018/223056, WO 2018/223073, WO 2018/223081, WO 2018/237194, WO 2019/ 032607, WO 2019/032612, WO 2019/055951, WO 2019/075357, WO 2019/200185, WO 2019/217784, WO 2019/032612, WO 2020/191252 and/or WO 2021/071858 Formula In- as described 1 , In-2 , In-3 , In-4 , II , II-a-1 , II-a-2 , II-b-1 , II-b-2 , II-c-1 , II-c- 2 , II-d-1 , II-d-2, etc., or a salt form thereof, and each internucleotide linkage that is not negatively charged (eg, formula In-1 , In-2 , In- 3 , In-4 , II , II-a-1 , II-a-2 , II-b-1 , II-b-2 , II-c-1 , II-c-2 , II-d-1 , II-d-2 , etc., or suitable salt forms thereof) are independently incorporated herein by reference.

In some embodiments, non-negatively charged internucleotidic linkages can improve delivery and/or activity (eg, adenosine editing activity).

In some embodiments, a modified internucleotide linkage (eg, a non-negatively charged internucleotide linkage) comprises an optionally substituted triazolyl. In some embodiments, a modified internucleotidic linkage (eg, a non-negatively charged internucleotidic linkage) comprises an optionally substituted alkynyl. In some embodiments, the modified internucleotidic linkage comprises a triazole or alkyne moiety. In some embodiments, a triazole moiety (eg, a triazolyl group) is optionally substituted. In some embodiments, a triazole moiety (eg, a triazolyl group) is substituted. In some embodiments, the triazole moiety is unsubstituted. In some embodiments, the modified internucleotide linkage comprises an optionally substituted cyclic guanidine moiety. In some embodiments, a modified internucleotide linkage is and optionally chirally controlled, R ¹ is -L-R', L is ^LB as described herein, and R' is as described herein. In some embodiments, each R ¹ is independently R'. In some embodiments, each R' is independently R. In some embodiments, two R ¹ are R and together form a ring as described herein. In some embodiments, two R ¹ on two different nitrogen atoms are R and together form a ring as described herein. In some embodiments, R ¹ is independently optionally substituted C _1-6 aliphatic as described herein. In some embodiments, R ¹ is methyl. In some embodiments, two R' on the same nitrogen atom are R and together form a ring as described herein. In some embodiments, a modified internucleotide linkage is It has a structure of and is optionally chiral controlled. In some embodiments, Is am. In some embodiments, the modified internucleotide linkage comprises an optionally substituted cyclic guanidine moiety; , , or has a structure, and W is O or S. In some embodiments, W is O. In some embodiments, W is S. In some embodiments, the non-negatively charged internucleotidic linkages are stereochemically controlled.

In some embodiments, a non-negatively charged internucleotidic linkage or a neutral internucleotidic linkage is an internucleotidic linkage comprising a triazole moiety. In some embodiments, the non-negatively charged internucleotidic linkage or non-negatively charged internucleotidic linkage comprises an optionally substituted triazolyl group. In some embodiments, an internucleotidic linkage comprising a triazole moiety (eg, an optionally substituted triazolyl group) is has the structure of In some embodiments, an internucleotide linkage comprising a triazole moiety is has the structure of In some embodiments, an internucleotide linkage comprising a triazole moiety is Has the chemical formula, W is O or S. In some embodiments, an internucleotidic linkage comprising an alkyne moiety (eg, an optionally substituted alkynyl group) is Has the chemical formula, W is O or S. In some embodiments, an internucleotidic linkage, eg, a non-negatively charged internucleotidic linkage, a neutral internucleotidic linkage comprises a cyclic guanidine moiety. In some embodiments, an internucleotidic linkage comprising a cyclic guanidine moiety is has the structure of In some embodiments, a non-negatively charged internucleotidic linkage or a neutral internucleotidic linkage is , , , or Is or comprises a structure selected from, and W is O or S.

In some embodiments, the internucleotidic linkage is a Tmg group ( ). In some embodiments, an internucleotidic linkage comprises a Tmg group It has the structure of ("Tmg internucleotidic linkages"). In some embodiments, neutral internucleotide linkages include internucleotide linkages of PNA and PMO, and Tmg internucleotide linkages.

In some embodiments, the non-negatively charged internucleotidic linkage is of formula I , Ia , Ib , Ic , In-1 , In-2 , In-3 , In-4 , II , II-a-1 , II-a -2 , II-b-1 , II-b-2 , II-c-1 , II-c-2 , II-d-1 , II-d-2 etc., or a salt form thereof. In some embodiments, the non-negatively charged internucleotidic linkage comprises an optionally substituted 3-20 membered heterocyclyl or heteroaryl group having 1-10 heteroatoms. In some embodiments, the non-negatively charged internucleotidic linkage comprises an optionally substituted 3-20 membered heterocyclyl or heteroaryl group having 1-10 heteroatoms, at least one heteroatom being nitrogen. In some embodiments, such heterocyclyl or heteroaryl groups are five-membered ring groups. In some embodiments, such heterocyclyl or heteroaryl groups are six-membered ring groups.

In some embodiments, the non-negatively charged internucleotidic linkage comprises an optionally substituted 5-20 membered heteroaryl group having 1-10 heteroatoms. In some embodiments, the non-negatively charged internucleotidic linkage comprises an optionally substituted 5-20 membered heteroaryl group having 1-10 heteroatoms, at least one heteroatom being nitrogen. In some embodiments, the non-negatively charged internucleotidic linkage comprises an optionally substituted 5-6 membered heteroaryl group having 1-4 heteroatoms, at least one heteroatom being nitrogen. In some embodiments, the non-negatively charged internucleotidic linkage comprises an optionally substituted 5-membered heteroaryl group having 1-4 heteroatoms, at least one heteroatom being nitrogen. In some embodiments, the heteroaryl group is directly bonded to the linking phosphorus. In some embodiments, the non-negatively charged internucleotidic linkage comprises an optionally substituted triazolyl group. In some embodiments, the non-negatively charged internucleotidic linkage is an unsubstituted triazolyl group, e.g. includes In some embodiments, the non-negatively charged internucleotidic linkage is a substituted triazolyl group, e.g. includes

In some embodiments, the non-negatively charged internucleotidic linkage comprises an optionally substituted 5-20 membered heterocyclyl group having 1-10 heteroatoms. In some embodiments, the non-negatively charged internucleotidic linkage comprises an optionally substituted 5-20 membered heterocyclyl group having 1-10 heteroatoms, wherein at least one heteroatom is nitrogen. In some embodiments, the non-negatively charged internucleotidic linkage comprises an optionally substituted 5-6 membered heterocyclyl group having 1-4 heteroatoms, at least one heteroatom being nitrogen. In some embodiments, the non-negatively charged internucleotidic linkage comprises an optionally substituted 5-membered heterocyclyl group having 1-4 heteroatoms, at least one heteroatom being nitrogen. In some embodiments, at least two heteroatoms are nitrogen. In some embodiments, the heterocyclyl group is directly bonded to the linking phosphorus. In some embodiments, a heterocyclyl group is bonded to the linking phosphorus through a linker, for example, =N- when the heterocyclyl group is part of a guanidine moiety derived and bonded to the linking phosphorus through =N-. In some embodiments, non-negatively charged internucleotidic linkages are optionally substituted includes the flag In some embodiments, non-negatively charged internucleotidic linkages are substituted includes the flag In some embodiments, a non-negatively charged internucleotide linkage is group, and each R ¹ is independently -LR. In some embodiments, each R ¹ is independently an optionally substituted C _1-6 alkyl. In some embodiments, each R ¹ is independently methyl.

In some embodiments, the modified internucleotide linkage, e.g., the non-negatively charged internucleotide linkage, comprises a triazole or alkyne moiety, each of which is optionally substituted. In some embodiments, the modified internucleotidic linkage comprises a triazole moiety. In some embodiments, the modified internucleotidic linkage comprises an unsubstituted triazole moiety. In some embodiments, the modified internucleotidic linkage comprises a substituted triazole moiety. In some embodiments, the modified internucleotidic linkage comprises an alkyl moiety. In some embodiments, the modified internucleotidic linkage comprises an optionally substituted alkynyl group. In some embodiments, the modified internucleotidic linkage comprises an unsubstituted alkynyl group. In some embodiments, the modified internucleotidic linkage comprises a substituted alkynyl group. In some embodiments, an alkynyl group is directly bonded to the linking phosphorus.

In some embodiments, oligonucleotides include different types of internucleotidic phosphorus linkages. In some embodiments, a chiral control oligonucleotide comprises at least one natural phosphate linkage and at least one modified (non-natural) internucleotidic linkage. In some embodiments, the oligonucleotide comprises at least one natural phosphate linkage and at least one phosphorothioate. In some embodiments, an oligonucleotide comprises at least one internucleotidic linkage that is not negatively charged. In some embodiments, an oligonucleotide comprises at least one native phosphate linkage and at least one internucleotidic linkage that is not negatively charged. In some embodiments, the oligonucleotide comprises at least one phosphorothioate internucleotidic linkage and at least one non-negatively charged internucleotidic linkage. In some embodiments, an oligonucleotide comprises at least one phosphorothioate internucleotidic linkage, at least one native phosphate linkage, and at least one non-negatively charged internucleotidic linkage. In some embodiments, the oligonucleotide is one or more non-negatively charged, e.g., 1-50, 1-40, 1-30, 1-20, 1-15, 1-10, 1, 2, 3, 4 , 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more internucleotide linkages. In some embodiments, an oligonucleotide comprises a specified number of non-negatively charged internucleotide linkages, e.g., 1 or less, 2 or less, 3 or less, 4 or less, 5 or less, 6 or less, 7 or less. Not more than 8, not more than 9, not more than 10, not more than 11, not more than 12, not more than 13, not more than 14, not more than 15, not more than 16, not more than 17, not more than 18, not more than 19 , not more than 20, not more than 21, not more than 22, not more than 23, not more than 24, not more than 25, not more than 26, not more than 27, not more than 28, not more than 29, or not more than 30 negatively charged It contains unlinked internucleotide linkages. In some embodiments, an oligonucleotide comprises internucleotidic linkages that are not negatively charged. In some embodiments, the non-negatively charged internucleotidic linkages are less than 50%, 40%, 40%, 30%, 20%, 10%, 5%, or 1% internucleotidic linkages at a given pH in an aqueous solution. It is not negatively charged in that it exists in the form of a negatively charged salt. In some embodiments, the pH is about pH 7.4. In some embodiments, the pH is between about 4-9. In some embodiments, the percentage is less than 10%. In some embodiments, the percentage is less than 5%. In some embodiments, the percentage is less than 1%. In some embodiments, the internucleotide linkage is a non-negatively charged internucleotidic linkage, in that the neutral form of the internucleotidic linkage does not have a pKa less than or equal to about 1, 2, 3, 4, 5, 6, or 7 in water. am. In some embodiments, the pKa is no greater than 7. In some embodiments, the pKa is no greater than 6. In some embodiments, the pKa is not 5 or less. In some embodiments, the pKa is not 4 or less. In some embodiments, the pKa is not less than 3. In some embodiments, the pKa is not less than 2. In some embodiments, the pKa is not less than 1. In some embodiments, the pKa of a neutral form of internucleotide linkage can be represented by the pKa of a neutral form of a compound having the structure CH ₃ -internucleotide linkage-CH ₃ . For example, the pKa of a neutral form of an internucleotidic linkage having structure I is (X, Y, Z are each independently -O-, -S-, -N(R')-; L is L ^B and R ¹ is -L-R') It can be expressed as the pKa of a compound, pKa of It can be expressed as a pKa of In some embodiments, the non-negatively charged internucleotidic linkages are neutral internucleotidic linkages. In some embodiments, the non-negatively charged internucleotidic linkages are positively charged internucleotidic linkages. In some embodiments, the non-negatively charged internucleotidic linkage comprises a guanidine moiety. In some embodiments, the non-negatively charged internucleotidic linkage comprises a heteroaryl base moiety. In some embodiments, the non-negatively charged internucleotidic linkage comprises a triazole moiety. In some embodiments, the non-negatively charged internucleotidic linkage comprises an alkynyl moiety.

In some embodiments, neutral or non-negatively charged internucleotide linkages are described in US 9394333, US 9744183, US 9605019, US 9982257, US 20170037399, US 20180216108, US 20180216107, US 9598458, WO 2017/0628 62, WO 2018/067973 , WO 2017/160741, WO 2017/192679, WO 2017/210647, WO 2018/098264, WO 2018/022473, WO 2018/223056, WO 2018/223073, WO 2018/223081, WO 2018/2 37194, WO 2019/032607 , WO 2019/032612, WO 2019/055951, WO 2019/075357, WO 2019/200185, WO 2019/217784, WO 2019/032612, WO 2020/191252 and/or WO 2021/071858 neutral has a structure of positive or negatively charged internucleotidic linkages, each neutral or negatively charged internucleotidic linkage being incorporated herein by reference.

In some embodiments, each R' is independently an optionally substituted C _1-6 aliphatic. In some embodiments, each R' is independently an optionally substituted C _1-6 alkyl. In some embodiments, each R' is independently -CH ₃ . In some embodiments, each R ^s is -H.

In some embodiments, a non-negatively charged internucleotide linkage is has the structure of In some embodiments, a non-negatively charged internucleotide linkage is has the structure of In some embodiments, a non-negatively charged internucleotide linkage is has the structure of In some embodiments, a non-negatively charged internucleotide linkage is has the structure of In some embodiments, a non-negatively charged internucleotide linkage is has the structure of In some embodiments, a non-negatively charged internucleotide linkage is has the structure of In some embodiments, a non-negatively charged internucleotide linkage is has the structure of In some embodiments, a non-negatively charged internucleotide linkage is has the structure of In some embodiments, a non-negatively charged internucleotide linkage is has the structure of In some embodiments, a non-negatively charged internucleotide linkage is has the structure of In some embodiments, a non-negatively charged internucleotide linkage is has the structure of In some embodiments, a non-negatively charged internucleotide linkage is has the structure of In some embodiments, W is O. In some embodiments, W is S. In some embodiments, neutral internucleotidic linkages are the aforementioned non-negatively charged internucleotidic linkages.

In some embodiments, provided oligonucleotides are disclosed in US 9394333, US 9744183, US 9605019, US 9982257, US 20170037399, US 20180216108, US 20180216107, US 9598458, WO 2017/062862, WO 2018/ 067973, WO 2017/160741, WO 2017 /192679, WO 2017/210647, WO 2018/098264, WO 2018/022473, WO 2018/223056, WO 2018/223073, WO 2018/223081, WO 2018/237194, WO 2019/032607, WO 2019/032612, WO 2019 /055951, WO 2019/075357, WO 2019/200185, WO 2019/217784, WO 2019/032607, WO 2019/032612, WO 2019/055951, WO 2019/075357, WO 2019/200185, WO 2019/217784, WO 2019 /032612, WO 2020/191252 and/or WO 2021/071858 Formulas I, Ia, Ib, Ic, In-1, In-2, In-3, In-4, II, II-a-1, II contains at least one internucleotide linkage of -a-2, II-b-1, II-b-2, II-c-1, II-c-2, II-d-1, or II-d-2 ; , each of the formulas I, Ia, Ib, Ic, In-1, In-2, In-3, In-4, II, II-a-1, II-a-2, II-b-1, II- b-2, II-c-1, II-c-2, II-d-1, or II-d-2 , or salt forms thereof, are independently incorporated herein by reference.

In some embodiments, an oligonucleotide comprises neutral internucleotidic linkages and chirally controlled internucleotidic linkages. In some embodiments, the oligonucleotide comprises neutral internucleotide linkages and chirally controlled internucleotide linkages that are not neutral internucleotide linkages. In some embodiments, the oligonucleotide comprises neutral internucleotidic linkages and chirally controlled phosphorothioate internucleotidic linkages. In some embodiments, the present invention provides oligonucleotides comprising one or more non-negatively charged internucleotide linkages and one or more phosphorothioate internucleotide linkages, wherein each phosphorothioate internucleotide linkage in the oligonucleotide are independently chirally controlled internucleotidic linkages. In some embodiments, the present invention provides oligonucleotides comprising one or more neutral internucleotide linkages and one or more phosphorothioate internucleotide linkages, wherein each phosphorothioate internucleotide linkage in the oligonucleotide is independently chiral It is a control internucleotide linkage. In some embodiments, the oligonucleotide comprises at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more chirally controlled phosphorothioate internucleotides. Include connections. In some embodiments, the non-negatively charged internucleotidic linkages are chirally controlled. In some embodiments, non-negatively charged internucleotidic linkages are not chirally controlled. In some embodiments, neutral internucleotidic linkages are chirally controlled. In some embodiments, neutral internucleotidic linkages are not chirally controlled. In some embodiments, the oligonucleotide comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) chiral controls and one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) non-chiral controlled chiral internucleotidic linkages. In some embodiments, the oligonucleotide comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) chiral controls and one or more (e.g., 1, 2 , 3, 4, 5, 6, 7, 8, 9, or 10 non-chirally controlled negatively charged internucleotide linkages (in some embodiments, each of which is independently n001) . In some embodiments, neutral internucleotidic linkages are chirally controlled. In some embodiments, neutral internucleotidic linkages are not chirally controlled. In some embodiments, the oligonucleotide comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) chiral controls and one or more (e.g., 1, 2 , 3, 4, 5, 6, 7, 8, 9, or 10) non-chirally controlled neutral internucleotide linkages (in some embodiments, each of which is independently n001).

Without wishing to be bound by any particular theory, the present invention notes that neutral internucleotide linkages may be more hydrophobic than phosphorothioate internucleotide linkages (PS), which may be more hydrophobic than natural phosphate linkages (PO). In general, unlike PS or PO, neutral internucleotidic linkages carry less charge. Without wishing to be bound by any particular theory, the present invention notes that the incorporation of one or more neutral internucleotide linkages into an oligonucleotide may increase the ability of the oligonucleotide to be taken up into cells and/or to escape from endosomes. do. Without wishing to be bound by any particular theory, it is noted that the present invention may use incorporation of one or more neutral internucleotide linkages to control the melting temperature of a duplex formed between an oligonucleotide and its target nucleic acid.

Without wishing to be bound by any particular theory, the present invention provides oligonucleotides that mediate functions such as target adenosine editing upon incorporation of one or more non-negatively charged internucleotide linkages (eg, neutral internucleotide linkages) into the oligonucleotide. Note that the ability of nucleotides can be increased.

As will be appreciated by those skilled in the art, natural phosphate linkages and formulas I , Ia , Ib , Ic , In-1 , In-2 , In-3 , In-4 , II , II-a-1 , II-a-2 , II-b-1 , II-b-2 , II-c-1 , II-c-2 , II-d-1 , II-d-2 , or a salt form thereof. , US 9394333, US 9744183, US 9605019, US 9982257, US 20170037399, US 20180216108, US 20180216107, US 9598458; WO 2017/160741, WO 2017/192679, WO 2017/210647, WO 2018/098264, WO 2018/022473, WO 2018/223056, WO 2018/223073, WO 2018/223081, WO 2018/237194, WO 2019/032607, WO 2019/032612, WO 2019/055 951, WO 2019/075357; linking two nucleosides (which may be natural or modified) as described in WO 2019/200185, WO 2019/217784, WO 2019/032612, WO 2020/191252 and/or WO 2021/071858; I , Ia , Ib , Ic , In-1 , In-2 , In-3 , In-4 , II , II-a-1 , II-a-2 , II-b-1 , II-b-2 , II-c-1 , II-c-2 , II-d-1 , II-d-2 , or salt forms thereof are independently incorporated herein by reference. A common linkage, as in natural DNA and RNA, is that an internucleotidic linkage forms a bond with two sugars, which may or may not be modified as described herein. In many embodiments, as exemplified herein, the internucleotidic linkage is with one optionally modified ribose or deoxyribose at the 5' carbon through an oxygen atom or heteroatom (e.g., Y and Z in various formulas). and forms a bond with another optionally modified ribose or deoxyribose at the 3' carbon. In some embodiments, each nucleoside unit linked by an internucleotide linkage is independently optionally substituted A, T, C, G or U, or a substituted tautomer of A, T, C, G or U. nucleobases, or nucleobases comprising optionally substituted heterocyclyl and/or heteroaryl rings having at least one nitrogen atom.

In some embodiments, the linkage has or comprises the structure -YP ^L (-XR ^L )-Z- or a salt form thereof, where

P ^L is P, P(=W), P->B(-L ^L -R ^L ) ₃ , or P ^N ;

W is O, N(-L ^L -R ^L ), S, or Se;

P ^N is P=NC(-L ^L -R')(=L ^N -R') or P=NL ^L -R ^L ;

L ^N is =NL ^L1 -, =CH-L ^L1 -, wherein CH is optionally substituted, =N ⁺ (R')(Q ^- )-L ^L1 -;

Q ^- is an anion,

X, Y, and Z are each independently -O-, -S-, -L ^L -N(-L ^L -R ^L )-L ^L -, -L ^L -N=C(-L ^L -R ^L )-L ^L -, or L ^L ;

Each R ^L is independently -L ^L -N(R') ₂ , -L ^L -R', -N=C(-L ^L -R') ₂ , -L ^L -N(R')C( NR')N(R') ₂ , -L ^L -N(R')C(O)N(R') ₂ , a carbohydrate, or one or more additional chemical moieties optionally linked through a linker;

L ^L1 and L ^L are each independently L;

-Cy ^IL- is -Cy-;

each L is independently a covalent bond or a divalent optionally substituted linear or branched group selected from C _1-30 aliphatic groups and C _1-30 heteroaliphatic groups having 1 to 10 heteroatoms, wherein one or more The methylene units are optionally and independently selected from C _1-6 alkylene, C _1-6 alkenylene, , a divalent C ₁ -C ₆ heteroaliphatic group having 1 to 5 heteroatoms, -C(R')2-, -Cy-, -O-, -S-, -SS-, -N(R' )-, -C(O)-, -C(S)-, -C(NR')-, -C(NR')N(R')-, -N(R')C(NR')N (R')-, -C(O)N(R')-, -N(R')C(O)N(R')-, -N(R')C(O)O-, -S (O)-, -S(O)2-, -S(O) ₂ N(R')-, -C(O)S-, -C(O)O-, -P(O)(OR' )-, -P(O)(SR')-, -P(O)(R')-, -P(O)(NR')-, -P(S)(OR')-, -P( S)(SR')-, -P(S)(R')-, -P(S)(NR')-, -P(R')-, -P(OR')-, -P(SR ')-, -P(NR')-, -P(OR')[B(R') ₃ ]-, -OP(O)(OR')O-, -OP(O)(SR')O -, -OP(O)(R')O-, -OP(O)(NR')O-, -OP(OR')O-, -OP(SR')O-, -OP(NR') selected from O-, -OP(R')O-, -OP(OR')[B(R') ₃ ]O-, or -[C(R') ₂ C(R') ₂ O]n- is optionally substituted with an optionally substituted group, and one or more nitrogen or carbon atoms are optionally and independently replaced with Cy ^L ;

each -Cy- is independently an optionally substituted divalent 3-30 membered monocyclic, bicyclic, or polycyclic ring having 0-10 heteroatoms;

each -Cy- is independently an optionally substituted trivalent or tetravalent 3-30 membered monocyclic, bicyclic, or polycyclic ring having 0-10 heteroatoms;

each R' is independently -R, -C(0)R, -C(0)N(R) ₂ , -C(0)OR, or -S(0) ₂ R;

each R is independently -H, or C _1-30 aliphatic, C _1-30 heteroaliphatic having 1-10 heteroatoms, C _6-30 aryl, C _6-30 arylaliphatic, 1-10 heteroatoms is an optionally substituted group selected from C _6-30 arylheteroaliphatic having , 5-30 membered heteroaryl having 1-10 heteroatoms, and 3-30 membered heterocyclyl having 1-10 heteroatoms;

two R groups optionally and independently together form a covalent bond;

Two or more R groups on the same atom optionally and independently form an optionally substituted 3-30 membered monocyclic, bicyclic, or polycyclic ring having 0-10 heteroatoms in addition to the corresponding atom together with the corresponding atom;

Two or more R groups on two or more atoms are optionally and independently substituted 3 to 30 membered monocyclic, bicyclic or polycyclic rings having 0 to 10 heteroatoms in addition to the intervening atoms together with the atoms intervening therebetween. form

In some embodiments, the internucleotide linkage has the structure -OP ^L (-XR ^L )-O-, and each variable is independently as described herein. In some embodiments, the internucleotide linkage has the structure -OP(=W)(-XR ^L )-O-, each variable independently as described herein. In some embodiments, the internucleotide linkage has the structure -OP(=W)[-N(-L ^L -R ^L )-R ^L ]-O-, each variable independently as described herein. In some embodiments, the internucleotide linkage has the structure -OP(=W)(-NH-L ^L -R ^L )-O-, each variable independently as described herein. In some embodiments, the internucleotide linkage has the structure -OP(=W)[-N(R') ₂ ]-O-, each variable independently as described herein. In some embodiments, the internucleotide linkage has the structure -OP(=W)(-NHR')-O-, and each variable is independently as described herein. In some embodiments, the internucleotide linkage has the structure -OP(=W)(-NHSO ₂ R)-O-, each variable independently as described herein. In some embodiments, R is methyl. In some embodiments, the internucleotide linkage is -OP(=0)(-NHSO ₂ CH ₃ )-O-. In some embodiments, the internucleotide linkage has the structure -OP(=W)[-N=C(-L ^L -R') ₂ ]-O-, each variable independently as described herein. In some embodiments, the internucleotide linkage has the structure -OP(=W)[-N=C[N(R') ₂ ] ₂ ]-O-, each variable independently as described herein. In some embodiments, the internucleotide linkage has the structure -OP(=W)(-N=C(R”) ₂ )-O-, each variable independently as described herein. In some embodiments, the internucleotide linkage has the structure -OP(=W)(-N(R”) ₂ )-O-, each variable independently as described herein. In some embodiments, W is O. In some embodiments, W is S. In some embodiments, such internucleotidic linkages are non-negatively charged internucleotidic linkages. In some embodiments, such internucleotidic linkages are neutral internucleotidic linkages.

In some embodiments, the internucleotide linkage has the structure -P ^L (-XR ^L )-Z-, each variable independently as described herein. In some embodiments, the internucleotide linkage has the structure -P ^L (-XR ^L )-O-, and each variable is independently as described herein. In some embodiments, the internucleotide linkage has the structure -P(=W)(-XR ^L )-O-, each variable independently as described herein. In some embodiments, the internucleotide linkage has the structure -P(=W)[-N(-L ^L -R ^L )-R ^L ]-O-, each variable independently as described herein. In some embodiments, the internucleotide linkage has the structure -P(=W)(-NH-L ^L -R ^L )-O-, each variable independently as described herein. In some embodiments, the internucleotide linkage has the structure -P(=W)[-N(R') ₂ ]-O-, each variable independently as described herein. In some embodiments, the internucleotide linkage has the structure -P(=W)(-NHR')-O-, and each variable is independently as described herein. In some embodiments, the internucleotide linkage has the structure -P(=W)(-NHSO ₂ R)-O-, each variable independently as described herein. In some embodiments, R is methyl. In some embodiments, the internucleotide linkage is -P(=0)(-NHSO ₂ CH ₃ )-O-. In some embodiments, the internucleotide linkage has the structure -P(=W)[-N=C(-L ^L -R') ₂ ]-O-, each variable independently as described herein. In some embodiments, the internucleotide linkage has the structure -P(=W)[-N=C[N(R') ₂ ] ₂ ]-O-, each variable independently as described herein. In some embodiments, the internucleotide linkage has the structure -P(=W)(-N=C(R”) ₂ )-O-, each variable independently as described herein. In some embodiments, the internucleotide linkage has the structure -P(=W)(-N(R”) ₂ )-O-, each variable independently as described herein. In some embodiments, W is O. In some embodiments, W is S. In some embodiments, such internucleotidic linkages are non-negatively charged internucleotidic linkages. In some embodiments, such internucleotidic linkages are neutral internucleotidic linkages. In some embodiments, the P of such an internucleotidic linkage is linked to the N of the sugar.

In some embodiments, the linkage is a phosphoryl guanidine internucleotide linkage. In some embodiments, the linkage is a thiophosphoryl guanidine internucleotide linkage.

In some embodiments, one or more methylene units are optionally and independently replaced with moieties described herein. In some embodiments, L or L ^L is or includes -SO ₂ -. In some embodiments, L or L ^L is or comprises -SO ₂ N(R')-. In some embodiments, L or LL is or comprises -C(O)-. In some embodiments, L or L ^L is or comprises -C(O)O-. In some embodiments, L or L ^L is or comprises -C(0)N(R')-. In some embodiments, L or L ^L is or includes -P(=W)(R')-. In some embodiments, L or L ^L is or comprises -P(=0)(R')-. In some embodiments, L or L ^L is or comprises -P(=S)(R')-. In some embodiments, L or L ^L is or comprises -P(R')-. In some embodiments, L or L ^L is or includes -P(=W)(OR')-. In some embodiments, L or L ^L is or comprises -P(=0)(OR')-. In some embodiments, L or L ^L is or comprises -P(=S)(OR')-. In some embodiments, L or L ^L is or comprises -P(OR')-.

In some embodiments, -XR ^L is -N(R')SO ₂ R ^L . In some embodiments, -XR ^L is -N(R')C(O)R ^L . In some embodiments, -XR ^L is -N(R')P(=0)(R')R ^L .

In some embodiments, a linkage, e.g., a non-negatively charged internucleotide linkage or a neutral internucleotide linkage, is -P(=W)(-N=C(R”) ₂ )-, -P(=W)( -N(R')SO ₂ R”)-, -P(=W)(-N(R')C(O)R”)-, -P(=W)(-N(R”) ₂ ) -, -P(=W)(-N(R')P(O)(R”) ₂ )-, -OP(=W)(-N=C(R”) ₂ )O-, -OP( =W)(-N(R')SO ₂ R”)O-, -OP(=W)(-N(R')C(O)R”)O-, -OP(=W)(-N (R”) ₂ )O-, -OP(=W)(-N(R')P(O)(R”) ₂ )O-, -P(=W)(-N=C(R”) ₂ )O-, -P(=W)(-N(R')SO ₂ R”)O-, -P(=W)(-N(R')C(O)R”)O-, - Structure of P(=W)(-N(R”) ₂ )O-, or -P(=W)(-N(R')P(O)(R”) ₂ )O-, or a salt thereof has or contains, in which case

W is O or S;

each R” is independently R', -OR', -P(=W)(R') ₂ or -N(R') ₂ ;

each R′ is independently -R, -C(O)R, -C(O)N(R) ₂ , -C(O)OR, or -S(O) ₂ R;

each R is independently -H, or C _1-30 aliphatic, C _1-30 heteroaliphatic having 1-10 heteroatoms, C _6-30 aryl, C _6-30 arylaliphatic, 1-10 heteroatoms is an optionally substituted group selected from C _6-30 arylheteroaliphatic having , 5-30 membered heteroaryl having 1-10 heteroatoms, and 3-30 membered heterocyclyl having 1-10 heteroatoms;

two R groups optionally and independently together form a covalent bond;

Two or more R groups on the same atom optionally and independently form an optionally substituted 3-30 membered monocyclic, bicyclic, or polycyclic ring having 0-10 heteroatoms in addition to the corresponding atom together with the corresponding atom;

Two or more R groups on two or more atoms are optionally and independently substituted 3 to 30 membered monocyclic, bicyclic or polycyclic rings having 0 to 10 heteroatoms in addition to the intervening atoms together with the atoms intervening therebetween. form

In some embodiments, W is O. In some embodiments, the internucleotide linkage is -P(=0)(-N=C(R”) ₂ )-, -P(=0)(-N(R′)SO ₂ R”)-, -P (=O)(-N(R')C(O)R”)-, -P(=O)(-N(R”) ₂ )-, -P(=O)(-N(R') P(O)(R”) ₂ )-, -OP(=O)(-N=C(R”) ₂ )O-, -OP(=O)(-N(R')SO ₂ R”) O-, -OP(=O)(-N(R')C(O)R”)O-, -OP(=O)(-N(R”) ₂ )O-, -OP(=O) (-N(R')P(O)(R”) ₂ )O-, -P(=O)(-N=C(R”) ₂ )O-, -P(=O)(-N( R')SO ₂ R”)O-, -P(=O)(-N(R')C(O)R”)O-, -P(=O)(-N(R”) ₂ )O -, or -P(=O)(-N(R')P(O)(R”) ₂ )O-, or a salt form thereof. In some embodiments, the internucleotide linkage is -P(=0)(-N=C(R”) ₂ )- -P(=0)(-N(R”) ₂ )-, -OP(=0) (-N=C(R”) ₂ )-O-, -OP(=O)(-N(R”) ₂ )-O-, -P(=O)(-N=C(R”) ₂ )-O-, or -P(=O)(-N(R”) ₂ )-O-, or a salt thereof. In some embodiments, the internucleotide linkage is -OP(=0)(-N=C(R”) ₂ )-O- or -OP(=0)(-N(R”) ₂ )-O-, or It has a salt form structure. In some embodiments, the internucleotide linkage has the structure -OP(=0)(-N=C(R”) ₂ )-O- or a salt form thereof. In some embodiments, the internucleotidic linkage has the structure -OP(=0)(-N(R”) ₂ )-O- or a salt form thereof. In some embodiments, the internucleotide linkage has the structure -OP(=0)(-N(R')SO ₂ R”)O- or a salt form thereof. In some embodiments, the internucleotide linkage has the structure -OP(=0)(-N(R')C(0)R”)0- or a salt form thereof. In some embodiments, the internucleotide linkage has the structure -OP(=0)(-N(R')P(0)(R”) ₂ )O- or a salt form thereof. In some embodiments, the internucleotide linkage is n001.

In some embodiments, W is S. In some embodiments, the internucleotide linkage is -P(=S)(-N=C(R”) ₂ )-, -P(=S)(-N(R′)SO ₂ R”)-, -P (=S)(-N(R')C(O)R”)-, -P(=S)(-N(R”) ₂ )-, -P(=S)(-N(R') P(O)(R”) ₂ )-, -OP(=S)(-N=C(R”) ₂ )O-, -OP(=S)(-N(R')SO ₂ R”) O-, -OP(=S)(-N(R')C(O)R”)O-, -OP(=S)(-N(R”) ₂ )O-, -OP(=S) (-N(R')P(O)(R”) ₂ )O-, -P(=S)(-N=C(R”) ₂ )O-, -P(=S)(-N( R')SO ₂ R”)O-, -P(=S)(-N(R')C(O)R”)O-, -P(=S)(-N(R”) ₂ )O -, or -P(=S)(-N(R')P(O)(R”) ₂ )O-, or has a structure in the form of a salt thereof. In some embodiments, the internucleotide linkage is -P(=S)(-N=C(R”) ₂ )- -P(=S)(-N(R”) ₂ )-, -OP(=S) (-N=C(R”) ₂ )-O-, -OP(=S)(-N(R”) ₂ )-O-, -P(=S)(-N=C(R”) ₂ )-O-, or -P(=S)(-N(R”) ₂ )-O-, or a salt form thereof. In some embodiments, the internucleotide linkage is -OP(=S)(-N=C(R”) ₂ )-O- or OP(=S)(-N(R”) ₂ )-O-, or It has a salt-like structure. In some embodiments, the internucleotide linkage has the structure -OP(=S)(-N=C(R”) ₂ )-O- or a salt form thereof. In some embodiments, the internucleotide linkage has the structure -OP(=S)(-N(R”) ₂ )-O- or a salt form thereof. In some embodiments, the internucleotide linkage has the structure -OP(=S)(-N(R')SO ₂ R”)O- or a salt form thereof. In some embodiments, the internucleotide linkage has the structure -OP(=S)(-N(R')C(O)R”)O- or a salt form thereof. In some embodiments, the internucleotide linkage has the structure -OP(=S)(-N(R')P(O)(R”) ₂ )O- or a salt form thereof. In some embodiments, the internucleotide linkage is *n001.

In some embodiments, the internucleotide linkage has the structure -P(=0)(-N(R')SO ₂ R”)-, where R” is as described herein. In some embodiments, the internucleotide linkage has the structure -P(=S)(-N(R')SO ₂ R”)-, where R” is as described herein. In some embodiments, the internucleotide linkage has the structure -P(=0)(-N(R')SO ₂ R”)O-, where R” is as described herein. In some embodiments, the internucleotide linkage has the structure -P(=S)(-N(R')SO ₂ R”)O-, where R” is as described herein. In some embodiments, the internucleotide linkage has the structure -OP(=0)(-N(R')SO ₂ R”)O-, where R” is as described herein. In some embodiments, the internucleotide linkage has the structure -OP(=S)(-N(R')SO ₂ R”)O-, where R” is as described herein. In some embodiments, for example, R' of -N(R')- is hydrogen or an optionally substituted C _1-6 aliphatic. In some embodiments, R' is C _1-6 alkyl. In some embodiments, R' is hydrogen. In some embodiments, for example, in -SO ₂ R”, R” is R′ as described herein. In some embodiments, an internucleotide linkage is -P(=0)(-NHSO ₂ R”)- and R" is as described herein. In some embodiments, the internucleotide linkage has the structure -P(=S)(-NHSO ₂ R")-, and R" is as described herein In some embodiments, the internucleotide linkage has the structure -P(=0)(-NHSO ₂ R")O-, and R" is as described herein. In some embodiments, the internucleotide linkage is -P (=S)(-NHSO ₂ R”)O-, and R” is as described herein. In some embodiments, the internucleotide linkage is -OP(=O)(-NHSO ₂ R”)O has the structure -, and R" is as described herein. In some embodiments, the internucleotide linkage has the structure -OP(=S)(-NHSO ₂ R")O-, and R" is as described herein In some embodiments, -XR ^L is -N(R')SO ₂ R ^L , and R' and R ^L are each independently as described herein. In some embodiments, R ^L is R" In some embodiments, R ^L is R'. In some embodiments, -XR ^L is -N(R')SO ₂ R", and R' is as described herein. In some embodiments, XR ^L is -N(R')SO ₂ R' and R' is as described herein. In some embodiments, X-RL is -NHSO ₂ R' and R' is as described herein. In some embodiments wherein R' is R as described herein. In some embodiments, R' is an optionally substituted C _1-6 aliphatic. In some embodiments, R' is an optionally substituted C 1-6 alkyl. In some embodiments, R' is an optionally substituted C _1-6 alkyl. In embodiments, R' is an optionally substituted phenyl. In some embodiments, R' is an optionally substituted heteroaryl. In some embodiments, for example, -SO ₂ R" in R" is R. In some embodiments In an example, R is an optionally substituted group selected from C _1-6 aliphatic, aryl, heterocyclyl, and heteroaryl. In some embodiments, R is an optionally substituted C _1-6 aliphatic. In some embodiments, R is an optionally substituted C _1-6 alkyl. In some embodiments, R' is an optionally substituted C _1-6 alkenyl. In some embodiments, R is an optionally substituted C _1-6 alkynyl. In some embodiments, R is optionally substituted methyl. In some embodiments -XR ^L is -NHSO ₂ CH ₃ . In some embodiments, R is -CF ₃ . In some embodiments, R is methyl. In some embodiments, R is optionally substituted ethyl. In some embodiments, R is ethyl. In some embodiments, R is -CH ₂ CHF ₂ . In some embodiments, R is -CH ₂ CH ₂ OCH ₃ . In some embodiments, R is optionally substituted propyl. In some embodiments, R is optionally substituted butyl. In some embodiments, R is n-butyl. In some embodiments, R is -(CH ₂ ) ₆ NH ₂ . In some embodiments, R is an optionally substituted linear C _2-20 aliphatic. In some embodiments, R is an optionally substituted linear C _2-20 alkyl. In some embodiments, R is a linear C _2-20 alkyl. In some embodiments, R is optionally substituted C ₁ , C ₂ , C ₃ , C ₄ , C ₅ , C ₆ , C ₇ , C 8 , C ₉ , C ₁₀ , C ₁₁ , C ₁₂ , _{C 13 ,} C ₁₄ , C ₁₅ , C ₁₆ , C ₁₇ , C ₁₈ , C ₁₉ , or C ₂₀ aliphatic. In some embodiments, R is optionally substituted C ₁ , C ₂ , C ₃ , C ₄ , C ₅ , C ₆ , C ₇ , C 8 , C ₉ , C ₁₀ , C ₁₁ , C ₁₂ , _{C 13 ,} C ₁₄ , C ₁₅ , C ₁₆ , C ₁₇ , C ₁₈ , C ₁₉ , or C ₂₀ alkyl. In some embodiments, R is an optionally substituted linear C ₁ , C _{2 ,} C ₃ , C ₄ , C ₅ , C ₆ , C ₇ , C ₈ , C ₉ , C 10 , C ₁₁ , C ₁₂ , C ₁₃ , C ₁₄ , C ₁₅ , C ₁₆ , C ₁₇ , C ₁₈ , C ₁₉ , or C ₂₀ alkyl. In some embodiments, R is linear C ₁ , C ₂ , C _{3 ,} C ₄ , C ₅ , C ₆ , C ₇ , C ₈ , C ₉ , C ₁₀ , C 11 , C ₁₂ , C ₁₃ , C ₁₄ , C ₁₅ , C ₁₆ , C ₁₇ , C ₁₈ , C ₁₉ , or C ₂₀ alkyl. In some embodiments, R is an optionally substituted phenyl. In some embodiments, R is phenyl. In some embodiments, R is p-methylphenyl. In some embodiments, R is 4-dimethylaminophenyl. In some embodiments, R is 3-pyridinyl. In some embodiments, R is am. In some embodiments, R is am. In some embodiments, R is benzyl. In some embodiments, R is an optionally substituted heteroaryl. In some embodiments, R is an optionally substituted 1,3-diazolyl. In some embodiments, R is optionally substituted 2-(1,3)-diazolyl. In some embodiments, R is optionally substituted 1-methyl-2-(1,3)-diazolyl. In some embodiments, R is isopropyl. In some embodiments, R” is —N(R′) ₂ . In some embodiments, R” is -N(CH ₃ ) ₂ . In some embodiments, for example, in -SO ₂ R”, R" is -OR' and R' is as described herein. In some embodiments, R' is R as described herein. In some embodiments In an example, R” is -OCH _3. In some embodiments, the linkage is -OP(=O)(-NHSO ₂ R)O-, and R is as described herein. In some embodiments, R is is an optionally substituted linear alkyl as described in. In some embodiments, R is a linear alkyl as described herein In some embodiments, the linkage is -OP(=0)(-NHSO ₂ CH ₃ )O- In some embodiments, the linkage is -OP(=0)(-NHSO ₂ CH ₂ CH ₃ )O- In some embodiments, the linkage is -OP(=0)(-NHSO ₂ CH ₂ CH ₂ OCH ₃ )O- In some embodiments, the linkage is OP(=O)(-NHSO ₂ CH ₂ Ph)O-. In some embodiments, the linkage is -OP(=O)(-NHSO ₂ CH ₂ CHF ₂ )0-.In some embodiments, the linkage is -OP(=0)(-NHSO ₂ (4-methylphenyl))0-.In some embodiments, -XR ^L is am. In some embodiments, the linkage is -OP(=0)(-XR ^L )0-, and -XR ^L is am. In some embodiments, the linkage is -OP(=0)(-NHSO ₂ CH(CH ₃ ) ₂ )O-. In some embodiments, the linkage is -OP(=0)(-NHSO ₂ N(CH ₃ ) ₂ )O-. In some embodiments, the linkage is n002. In some embodiments, linkage is n006. In some embodiments, linkage is n020. In some embodiments, such internucleotide linkages can be used in place of linkages such as n001.

In some embodiments, the internucleotide linkage has the structure -P(=0)(-N(R')C(0)R”)-, where R” is as described herein. In some embodiments, the internucleotide linkage has the structure -P(=S)(-N(R')C(O)R”)-, where R” is as described herein. In some embodiments, the internucleotide linkage has the structure -P(=0)(-N(R')C(0)R")0, where R" is as described herein. In some embodiments, the internucleotide linkage has the structure -P(=S)(-N(R')C(O)R”)O-, where R” is as described herein. In some embodiments, the internucleotide linkage has the structure -OP(=0)(-N(R')C(0)R”)0-, where R” is as described herein. In some embodiments, the internucleotide linkage has the structure -OP(=S)(-N(R')C(O)R”)O-, where R” is as described herein. In some embodiments, for example, R' of -N(R')- is hydrogen or an optionally substituted C _1-6 aliphatic. In some embodiments, R' is C _1-6 alkyl. In some embodiments, R' is hydrogen. In some embodiments, for example, the R” of —C(O)R” is R′ as described herein. In some embodiments, the internucleotide linkage has the structure -P(=0)(-NHC(0)R")-, where R" is as described herein. In some embodiments, the internucleotide linkage has the structure -P(=S)(-NHC(O)R")-, where R" is as described herein. In some embodiments, the internucleotide linkage has the structure -P(=0)(-NHC(0)R")0-, where R" is as described herein. In some embodiments, the internucleotide linkage has the structure -P(=S)(-NHC(O)R")O-, where R" is as described herein. In some embodiments, the internucleotide linkage has the structure -OP(=0)(-NHC(0)R")0-, where R" is as described herein. In some embodiments, the internucleotide linkage has the structure -OP(=S)(-NHC(O)R")O-, where R" is as described herein. In some embodiments, -XR ^L is -N(R')COR ^L , and R ^L is as described herein. In some embodiments, -XR ^L is -N(R')COR” and R” is as described herein. In some embodiments, -XR ^L is -N(R')COR', and R' is as described herein. In some embodiments, -XR ^L is -NHCOR' and R' is as described herein. In some embodiments, R′ is R as described herein. In some embodiments, R' is an optionally substituted C _1-6 aliphatic. In some embodiments, R' is an optionally substituted C _1-6 alkyl. In some embodiments, R' is an optionally substituted phenyl. In some embodiments, R' is an optionally substituted heteroaryl. In some embodiments, for example, in -C(O)R”, R” is R. In some embodiments, R is an optionally substituted group selected from C _1-6 aliphatic, aryl, heterocyclyl, and heteroaryl. In some embodiments, R is an optionally substituted C _1-6 aliphatic. In some embodiments, R is an optionally substituted C _1-6 alkyl. In some embodiments, R' is an optionally substituted C _1-6 alkenyl. In some embodiments, R is an optionally substituted C _1-6 alkynyl. In some embodiments, R is methyl. In some embodiments -XR ^L is -NHC(O)CH ₃ . In some embodiments, R is optionally substituted methyl. In some embodiments, R is -CF ₃ . In some embodiments, R is optionally substituted ethyl. In some embodiments, R is ethyl. In some embodiments, R is -CH ₂ CHF ₂ . In some embodiments, R is -CH ₂ CH ₂ OCH ₃ . In some embodiments, R is an optionally substituted C _1-20 (eg, C _1-6 , C _2-6 , C _3-6 , C _1-10 , C _2-10 , C _3-10 , C _2-20 , C _3-20 , C _10-20 , 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, etc.) is aliphatic. In some embodiments, R is an optionally substituted C _1-20 (eg, C _1-6 , C _2-6 , C _3-6 , C _1-10 , C _2-10 , C _3-10 , C _2-20 , C _3-20 , C _10-20 , 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, etc.) alkyl. In some embodiments, R is an optionally substituted linear C _2-20 aliphatic. In some embodiments, R is an optionally substituted linear C _2-20 alkyl. In some embodiments, R is a linear C _2-20 alkyl. In some embodiments, R is optionally substituted C ₁ , C ₂ , C 3 , C ₄ , C ₅ , C ₆ , C ₇ , C ₈ , C ₉ , C ₁₀ , C ₁₁ , C ₁₂ , _{C 13 ,} C ₁₄ , C ₁₅ , C ₁₆ , C ₁₇ , C ₁₈ , C ₁₉ , or C ₂₀ aliphatic. In some embodiments, R is optionally substituted C ₁ , C ₂ , C 3 , C ₄ , C ₅ , C ₆ , C ₇ , C ₈ , C ₉ , C ₁₀ , C ₁₁ , C ₁₂ , _{C 13 ,} C ₁₄ , C ₁₅ , C ₁₆ , C ₁₇ , C ₁₈ , C ₁₉ , or C ₂₀ alkyl. In some embodiments, R is an optionally substituted linear C ₁ , C _{2 ,} C ₃ , C ₄ , C ₅ , C ₆ , C ₇ , C ₈ , C ₉ , C 10 , C ₁₁ , C ₁₂ , C ₁₃ , C ₁₄ , C ₁₅ , C ₁₆ , C ₁₇ , C ₁₈ , C ₁₉ , or C ₂₀ alkyl. In some embodiments, R is linear C ₁ , C ₂ , C ₃ , C ₄ , C ₅ , C ₆ , C ₇ , C ₈ , C ₉ , C ₁₀ , C ₁₁ , C ₁₂ , C ₁₃ , C ₁₄ , C ₁₅ , C ₁₆ , C ₁₇ , C ₁₈ , C ₁₉ , or C ₂₀ alkyl. In some embodiments, R is an optionally substituted aryl. In some embodiments, R is an optionally substituted phenyl. In some embodiments, R is p-methylphenyl. In some embodiments, R is benzyl. In some embodiments, R is an optionally substituted heteroaryl. In some embodiments, R is an optionally substituted 1,3-diazolyl. In some embodiments, R is optionally substituted 2-(1,3)-diazolyl. In some embodiments, R is optionally substituted 1-methyl-2-(1,3)-diazolyl. In some embodiments, R ^L is -(CH ₂ ) ₅ NH ₂ . In some embodiments, R ^L is am. In some embodiments, R ^L is am. In some embodiments, R” is —N(R′) ₂ . In some embodiments, R” is -N(CH ₃ ) ₂ . In some embodiments, -XR ^L is -N(R')CON(R ^L ) ₂ , and R' and R ^L are each independently as described herein. In some embodiments, -XR ^L is -NHCON(R ^L ) ₂ , and R ^L is as described herein. In some embodiments, two R' or two R ^L , together with the nitrogen atom to which they are attached, form a ring as described herein (eg optionally substituted or ). In some embodiments, for example, in -C(0)R", R" is -OR' and R' is as described herein. In some embodiments, R' is R as described herein. In some embodiments, R' is an optionally substituted C _1-6 aliphatic. In some embodiments, R' is an optionally substituted C _1-6 alkyl. In some embodiments, R" is -OCH 3 . In some embodiments, R' is -OCH ₃ . In embodiments, -XR ^L is -N(R')C(O)OR ^L , and R' and R ^L are each independently as described herein. In some embodiments, R is am. In some embodiments -XR ^L is -NHC(O)OCH ₃ . In some embodiments -XR ^L is -NHC(O)N(CH ₃ ) ₂ . In some embodiments, the linkage is -OP(O)(NHC(O)CH ₃ )O-. In some embodiments, the linkage is -OP(O)(NHC(O)OCH ₃ )O-. In some embodiments, the linkage is -OP(O)(NHC(O)(p-methylphenyl))O-. In some embodiments, the linkage is -OP(O)(NHC(O)N(CH ₃ ) ₂ )O-. In some embodiments, -XR ^L is -N(R')R ^L , and R' and R ^L are each independently as described herein. In some embodiments, -XR ^L is -N(R')R ^L , and R' and R ^L are each independently not hydrogen. In some embodiments, -XR ^L is -NHR ^L and R ^L are as described herein. In some embodiments, R ^L is not hydrogen. In some embodiments, R ^L is an optionally substituted aryl or heteroaryl. In some embodiments, R ^L is optionally substituted aryl. In some embodiments, R ^L is an optionally substituted phenyl. In some embodiments, -XR ^L is -N(R') ₂ , and each R' is independently as described herein. In some embodiments, -XR ^L is -NHR' and R' is as described herein. In some embodiments, -XR ^L is -NHR and R is as described herein. In some embodiments, -XR ^L is R ^L and R ^L are as described herein. In some embodiments, R ^L is -N(R') ₂ and each R' is independently as described herein. In some embodiments, R ^L is -NHR' and R' is as described herein. In some embodiments, R ^L is -NHR and R is as described herein. In some embodiments, R ^L is -N(R') ₂ and each R' is independently as described herein. In some embodiments, none of the R' in -N(R') ₂ is hydrogen. In some embodiments, R ^L is -N(R') ₂ and each R' is independently C _1-6 aliphatic. In some embodiments, R ^L is -L-R', and L and R' are each independently as described herein. In some embodiments, R ^L is -LR, and L and R are each independently as described herein. In some embodiments, R ^L is -N(R')-Cy-N(R')-R'. In some embodiments, R ^L is -N(R')-Cy-C(0)-R'. In some embodiments, R ^L is -N(R')-Cy-O-R'. In some embodiments, R ^L is -N(R')-Cy-SO ₂ -R'. In some embodiments, R ^L is -N(R')-Cy-SO ₂ -N(R') ₂ . In some embodiments, R ^L is -N(R')-Cy-C(0)-N(R') ₂ . In some embodiments, R ^L is -N(R')-Cy-OP(O)(R”) ₂ . In some embodiments, -Cy- is an optionally substituted divalent aryl group. In some embodiments, -Cy- is an optionally substituted phenylene. In some embodiments, -Cy- is an optionally substituted 1,4-phenylene. In some embodiments, -Cy- is 1,4-phenylene. In some embodiments, R ^L is -N(CH ₃ ) ₂ . In some embodiments, R ^L is -N(i-Pr) ₂ . In some embodiments, R ^L is am. In some embodiments, R ^L is am. In some embodiments, R ^L is am. In some embodiments, R ^L is am. In some embodiments, R ^L is am. In some embodiments, R ^L is am. In some embodiments, R ^L is am. In some embodiments, R ^L is am. In some embodiments, R ^L is am. In some embodiments, R ^L is am. In some embodiments, R ^L is am. In some embodiments, R ^L is am. In some embodiments, R ^L is am. In some embodiments, R ^L is am. In some embodiments, R ^L is am. In some embodiments, R ^L is am. In some embodiments, R ^L is am. In some embodiments, R ^L is am. In some embodiments, R ^L is or am. In some embodiments, -XR ^L is -N(R')-C(O)-Cy-R ^L . In some embodiments, -XR ^L is R ^L . In some embodiments, R ^L is -N(R')-C(0)-Cy-0-R'. In some embodiments, R ^L is -N(R')-C(0)-Cy-R'. In some embodiments, R ^L is -N(R')-C(0)-Cy-C(0)-R'. In some embodiments, R ^L is -N(R')-C(O)-Cy-N(R') ₂ . In some embodiments, R ^L is -N(R')-C(O)-Cy-SO ₂ -N(R') ₂ . In some embodiments, RL is -N(R')-C(O)-Cy-C(O)-N(R') ₂ . In some embodiments, R ^L is -N(R')-C(0)-Cy-C(0)-N(R')-SO ₂ -R'. In some embodiments, R′ is R as described herein. In some embodiments, R ^L is am.

As described herein, in some embodiments, one or more methylene units of L, or a variable comprising or being L, are independently -O-, -N(R')-, -C(O)-, -C (O)N(R')-, -SO ₂ -, -SO ₂ N(R')-, or -Cy-. In some embodiments, methylene units are replaced with -Cy-. In some embodiments, -Cy- is an optionally substituted divalent aryl group. In some embodiments, -Cy- is an optionally substituted phenylene. In some embodiments, -Cy- is an optionally substituted 1,4-phenylene. In some embodiments, -Cy- is optionally substituted 2 having 1-10 (eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) heteroatoms. is a 5-20 (eg, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) member heteroaryl group. In some embodiments, -Cy- is monocyclic. In some embodiments, -Cy- is bicyclic. In some embodiments, -Cy- is polycyclic. In some embodiments, each monocyclic unit of -Cy- is independently 3-10 (e.g., 3, 4, 5, 6, 7, 8, 9, or 10) membered, and is independently saturated, partially saturated, or aromatic. In some embodiments, -Cy- is optionally substituted 3-20 (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) membered monocyclic, bicyclic, or polycyclic aliphatic groups. In some embodiments, -Cy- is optionally substituted 3 having 1-10 (eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) heteroatoms. ~20 (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) members monocyclic, bicyclic, or It is a polycyclic heteroaliphatic group.

In some embodiments, the internucleotide linkage has the structure -P(=0)(-N(R')P(0)(R”) ₂ )-, and each R” is independently as described herein . In some embodiments, the internucleotide linkage has the structure -P(=S)(-N(R')P(O)(R”) ₂ )-, and each R” is independently as described herein . In some embodiments, the internucleotide linkage has the structure -P(=0)(-N(R')P(0)(R”) ₂ )0-, wherein each R” is independently as described herein same. In some embodiments, the internucleotide linkage has the structure -P(=S)(-N(R')P(O)(R”) ₂ )O-, wherein each R” is independently as described herein same. In some embodiments, the internucleotide linkage has the structure -OP(=0)(-N(R')P(0)(R”) ₂ )0-, wherein each R” is independently as described herein same. In some embodiments, the internucleotide linkage has the structure -OP(=S)(-N(R')P(O)(R”) ₂ )O-, wherein each R” is independently as described herein same. In some embodiments, for example, R' of -N(R')- is hydrogen or an optionally substituted C _1-6 aliphatic. In some embodiments, R' is C _1-6 alkyl. In some embodiments, R' is hydrogen. In some embodiments, for example, in -P(O)(R”) ₂ , R” is R′ as described herein. In some embodiments, the internucleotide linkage has the structure -P(=0)(-NHP(0)(R”) ₂ )-, and each R” is independently as described herein. In some embodiments, the internucleotide linkage has the structure -P(=S)(-NHP(O)(R”) ₂ )-, and each R” is independently as described herein. In some embodiments, the internucleotide linkage has the structure -P(=0)(-NHP(0)(R") ₂ )0-, and each R" is independently as described herein. In some embodiments, the internucleotide linkage has the structure -P(=S)(-NHP(O)(R”) ₂ )O-, and each R” is independently as described herein. In some embodiments, the internucleotide linkage has the structure -OP(=0)(-NHP(0)(R") ₂ )0-, and each R" is independently as described herein. In some embodiments, the internucleotide linkage has the structure -OP(=S)(-NHP(O)(R”) ₂ )O-, and each R” is independently as described herein. In some embodiments, for example, in —P(O)(R″) ₂ , an occurrence of R″ is R. In some embodiments, R is an optionally substituted group selected from C _1-6 aliphatic, aryl, heterocyclyl, and heteroaryl. In some embodiments, R is an optionally substituted C _1-6 aliphatic. In some embodiments, R is an optionally substituted C _1-6 alkyl. In some embodiments, R' is an optionally substituted C _1-6 alkenyl. In some embodiments, R is an optionally substituted C _1-6 alkynyl. In some embodiments, R is methyl. In some embodiments, R is optionally substituted methyl. In some embodiments, R is -CF ₃ . In some embodiments, R is optionally substituted ethyl. In some embodiments, R is ethyl. In some embodiments, R is -CH ₂ CHF ₂ . In some embodiments, R is -CH ₂ CH ₂ OCH ₃ . In some embodiments, R is an optionally substituted C _1-20 (eg, C _1-6 , C _2-6 , C _3-6 , C _1-10 , C _2-10 , C _3-10 , C _2-20 , C _3-20 , C _10-20 , 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, etc.) is aliphatic. In some embodiments, R is an optionally substituted C _1-20 (eg, C _1-6 , C _2-6 , C _3-6 , C _1-10 , C _2-10 , C _3-10 , C _2-20 , C _3-20 , C _10-20 , 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, etc.) alkyl. In some embodiments, R is an optionally substituted linear C _2-20 aliphatic. In some embodiments, R is an optionally substituted linear C _2-20 alkyl. In some embodiments, R is a linear C _2-20 alkyl. In some embodiments, R is isopropyl. In some embodiments, R is optionally substituted C ₁ , C ₂ , C ₃ , C ₄ , C ₅ , C ₆ , C ₇ , C 8 , C ₉ , C ₁₀ , C ₁₁ , C ₁₂ , _{C 13 ,} C ₁₄ , C ₁₅ , C ₁₆ , C ₁₇ , C ₁₈ , C ₁₉ , or C ₂₀ aliphatic. In some embodiments, R is optionally substituted C ₁ , C ₂ , C 3 , C ₄ , C ₅ , C ₆ , C ₇ , C ₈ , C ₉ , C ₁₀ , C ₁₁ , C ₁₂ , _{C 13 ,} C ₁₄ , C ₁₅ , C ₁₆ , C ₁₇ , C ₁₈ , C ₁₉ , or C ₂₀ alkyl. In some embodiments, R is an optionally substituted linear C ₁ , C _{2 ,} C ₃ , C ₄ , C ₅ , C ₆ , C ₇ , C ₈ , C ₉ , C 10 , C ₁₁ , C ₁₂ , C ₁₃ , C ₁₄ , C ₁₅ , C ₁₆ , C ₁₇ , C ₁₈ , C ₁₉ , or C ₂₀ alkyl. In some embodiments, R is linear C ₁ , C ₂ , C ₃ , C ₄ , C ₅ , C ₆ , C ₇ , C ₈ , C ₉ , C ₁₀ , C ₁₁ , C ₁₂ , C ₁₃ , C ₁₄ , C ₁₅ , C ₁₆ , C ₁₇ , C ₁₈ , C ₁₉ , or C ₂₀ alkyl. In some embodiments, each R" is independently R as described herein, for example in some embodiments, each R" is methyl. In some embodiments, R″ is an optionally substituted aryl. In some embodiments, R is an optionally substituted phenyl. In some embodiments, R is p-methylphenyl. In some embodiments, R is benzyl. In some embodiments, R is an optionally substituted heteroaryl. In some embodiments, R is an optionally substituted 1,3-diazolyl. In some embodiments, R is optionally substituted 2-(1,3)-diazolyl. In some embodiments, R is optionally substituted 1-methyl-2-(1,3)-diazolyl. In some embodiments, the presence of R” is —N(R′) ₂ . In some embodiments, R” is -N(CH ₃ ) ₂ . In some embodiments, for example, in -P(O)(R”) ₂ , the presence of R″ is -OR′ and R′ is as described herein. In some embodiments, R′ is as described herein. is as R. In some embodiments, R' is an optionally substituted C _1-6 aliphatic. In some embodiments, R' is an optionally substituted C _1-6 alkyl. In some embodiments, R" is - OCH _3. In some embodiments, each R" is -OR' as described herein. In some embodiments, each R" is -OCH _3. In some embodiments, each R" is -OR'. OH In some embodiments, the linkage is -OP(O)(NHP(O)(OH) ₂ )O- In some embodiments, the linkage is -OP(O)(NHP(O)(OCH ₃ ) ₂ )O- In some embodiments, the linkage is -OP(O)(NHP(O)(CH ₃ ) ₂ )O-.

In some embodiments, -N(R”) ₂ is -N(R′) ₂ . In some embodiments, -N(R”) ₂ is -NHR. In some embodiments, -N(R”) ₂ is -NHC(O)R. In some embodiments, -N(R”) ₂ is -NHC(O)OR. In some embodiments, -N(R”) ₂ is -NHS(O) ₂ R.

In some embodiments, the internucleotidic linkages are phosphoryl guanidine internucleotidic linkages. In some embodiments, an internucleotide linkage comprises -XR ^L as described herein. In some embodiments, -XR ^L is -N=C(-L ^L -R ^L ) ₂ . In some embodiments, -XR ^L is -N=C[N(R ^L ) ₂ ] ₂ . In some embodiments, -XR ^L is -N=C[NR'R ^L ] ₂ . In some embodiments, -XR ^L is -N=C[N(R′) ₂ ] ₂ . In some embodiments, -XR ^L is -N=C[N(R ^L ) ₂ ](CHR ^L1 R ^L2 ), and R ^L1 and R ^L2 are each independently as described herein. In some embodiments, -XR ^L is -N=C(NR'R ^L )(CHR ^L1 R ^L2 ), and R ^L1 and R ^L2 are each independently as described herein. In some embodiments, -XR ^L is -N=C(NR'R ^L )(CR'R ^L1 R ^L2 ), and R ^L1 and R ^L2 are each independently as described herein. In some embodiments, -XR ^L is -N=C[N(R') ₂ ](CHR'R ^L2 ). In some embodiments, -XR ^L is -N=C[N(R ^L ) ₂ ](R ^L ). In some embodiments, -X-RL is -N=C(NR'R ^L )(R ^L ). In some embodiments, -XR ^L is -N=C(NR'R ^L )(R'). In some embodiments, -XR ^L is -N=C[N(R') ₂ ](R'). In some embodiments, -XR ^L is -N=C(NR'R ^L1 )(NR'R ^L2 ), R ^L1 and R ^L2 are each independently R ^L , and R' and R ^L are each independently as described in In some embodiments, -XR ^L is -N=C(NR'R ^L1 )(NR'R ^L2 ), and the variables are independently as described herein. In some embodiments, -XR ^L is -N=C(NR'R ^L1 )(CHR'R ^L2 ), and the variables are independently as described herein. In some embodiments, -XR ^L is -N=C(NR'R ^L1 )(R'), and the variables are independently as described herein. In some embodiments, each R' is independently R. In some embodiments, R is an optionally substituted C _1-6 aliphatic. In some embodiments, R is methyl. In some embodiments, -XR ^L is am. In some embodiments, two groups selected from R', R ^L , R ^L1 , R ^L2 , etc. (in some embodiments, on the same atom, for example, -N(R') ₂ , or -NR'R ^L , or -N(R ^L ) ² (R' and R ^L can independently be R as described herein), or on another atom, for example -N=C(N R' R ^L ) two R' ) of (C R' R ^L1 R ^L2 ) or -N=C(N R' R ^L1 ) (N R' R ^L2 ) are independently R, together with their intervening atoms, as described herein form a bar-like ring. In some embodiments, R, R', R ^L , R ^L1 , or R ^L2 on the same atom (eg, -N(R') ₂ , -N(R ^L ) ₂ , -NR'R ^L , - NR'R ^L1 , -NR'R ^L2 , -CR'R ^L1 R ^{L2 ,} etc.) together form a ring as described herein. In some embodiments, two of R', R ^L , R ^L1 , or R ^L2 on two different atoms (eg, -N=C(N R' R ^L )(C R' R ^L1 R ^L2 ) , -N=C(N R' R ^L1 )(N R' R ^L2 ), etc. together form a ring as described herein. In some embodiments, the formed ring contains 3 to 20 optionally substituted rings having 0 to 5 additional heteroatoms (e.g., 3 to 15, 3 to 12, 3 to 10, 3 to 9, 3 to 8, 3 ~7, 3~6, 4~15, 4~12, 4~10, 4~9, 4~8 , 4~7, 4~6, 5~15, 5~12, 5~10, 5~9 , 5~8, 5~7, 5~6, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 , 20, etc.) is a monocyclic, bicyclic, or tricyclic ring. In some embodiments, the formed ring is monocyclic as described herein. In some embodiments, the ring formed is an optionally substituted 5-10 membered monocyclic ring. In some embodiments, the rings formed are bicyclic. In some embodiments, the rings formed are polycyclic. In some embodiments, two groups that are or can be R (e.g., -N=C(N R' R ^L )(C R' R ^L1 R ^L2 ) or -N=C(N R' R ^L1 )(N R' R ^L2 ) of two R' , -N=C(N R' R ^L )(C R' R ^L1 R ^L2 ), -N=C(N R' R ^L1 )(N R' Two R' ) such as R ^L2 ) together are an optionally substituted divalent hydrocarbon chain, for example an optionally substituted C _1-20 aliphatic chain, an optionally substituted -(CH ₂ )n (n is 1 to 20 ( For example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20). In some embodiments, the hydrocarbon chain is saturated. In some embodiments, the hydrocarbon chain is partially unsaturated. In some embodiments, the hydrocarbon chain is unsaturated. In some embodiments, the hydrocarbon chain is unsaturated. group (e.g., two R' of -N=C(N R' R ^L )(C R' R ^L1 R ^L2 ) or -N=C(N R' R ^L1 )(N R' R ^L2 ) , -N=C(N R' R ^L )(C R' R ^L1 R ^L2 ), -N=C(N R' R ^L1 )(N R' R ^L2 ), etc. together. Forms an optionally substituted divalent heteroaliphatic chain, for example an optionally substituted C _1-20 heteroaliphatic chain having 1-10 heteroatoms.In some embodiments, the heteroaliphatic chain is saturated. In some embodiments, the heteroaliphatic chain is partially unsaturated. In some embodiments, the heteroaliphatic chain is unsaturated. In some embodiments, the chain is optionally substituted -(CH ₂ )-. In some embodiments, the chain is optionally substituted -(CH ₂ ) ₂ - In some embodiments, the chain is optionally substituted -(CH ₂ )- In some embodiments, the chain is optionally substituted -(CH ₂ ) ₂ -. In an embodiment, the chain is optionally substituted -(CH ₂ ) ₃ -. In some embodiments, the chain is optionally substituted -(CH ₂ ) ₄ -. In some embodiments, the chain is optionally substituted -(CH ₂ ) ₅ -. In some embodiments, the chain is optionally substituted -(CH ₂ ) ₆ -. In some embodiments, the chain is optionally substituted -CH=CH-. In some embodiments, the chain is optionally substituted am. In some embodiments, the chain is optionally substituted am. In some embodiments, the chain is optionally substituted am. In some embodiments, the chain is optionally substituted am. In some embodiments, the chain is optionally substituted am. In some embodiments, the chain is optionally substituted am. In some embodiments, the chain is optionally substituted am. In some embodiments, the chain is optionally substituted am. In some embodiments, the chain is optionally substituted am. In some embodiments, two of R, R′, R ^L , R ^L1 , R ^L2 , etc. on different atoms together form a ring as described herein. For example, in some embodiments, -XR ^L is am. In some embodiments, -XR ^L is am. In some embodiments, -XR ^L is am. In some embodiments, -XR ^L is am. In some embodiments, -XR ^L is am. In some embodiments, -XR ^L is am. In some embodiments, -XR ^L is am. In some embodiments, -XR ^L is am. In some embodiments, -XR ^L is am. In some embodiments, -XR ^L is am. In some embodiments, -N(R') ₂ , -N(R) ₂ , -N(R ^L ) ₂ , -NR'R ^L , -NR'R ^L1 , -NR'R ^L2 , -NR ^L1 R ^L2 and the like are formed rings. In some embodiments, a ring is optionally substituted am. In some embodiments, a ring is optionally substituted am. In some embodiments, a ring is optionally substituted am. In some embodiments, a ring is optionally substituted am. In some embodiments, a ring is optionally substituted am. In some embodiments, a ring is optionally substituted am. In some embodiments, a ring is optionally substituted am. In some embodiments, a ring is optionally substituted am. In some embodiments, a ring is optionally substituted am. In some embodiments, a ring is optionally substituted am. In some embodiments, a ring is optionally substituted am. In some embodiments, a ring is optionally substituted am. In some embodiments, a ring is optionally substituted am. In some embodiments, a ring is optionally substituted am. In some embodiments, a ring is optionally substituted am.

In some embodiments, R ^L1 and R ^L2 are the same. In some embodiments, R ^L1 and R ^L2 are different. In some embodiments, R ^L1 and R ^L2 are each independently R ^L , eg, as described herein below.

In some embodiments, R ^L is an optionally substituted C _1-30 aliphatic. In some embodiments, R ^L is an optionally substituted C _1-30 alkyl. In some embodiments, R ^L is linear. In some embodiments, R ^L is an optionally substituted linear C _1-30 alkyl. In some embodiments, R ^L is an optionally substituted C _1-6 alkyl. In some embodiments, R ^L is methyl. In some embodiments, R ^L is ethyl. In some embodiments, R ^L is n-propyl. In some embodiments, R ^L is isopropyl. In some embodiments, R ^L is n-butyl. In some embodiments, R ^L is tert-butyl. In some embodiments, R ^L is (E)-CH ₂ -CH=CH-CH ₂ -CH ₃ . In some embodiments, R ^L is (Z)-CH ₂ -CH=CH-CH ₂ -CH ₃ . In some embodiments, R ^L is am. In some embodiments, R ^L is am. In some embodiments, R ^L is CH ₃ (CH ₂ ) ₂ C≡CC≡C(CH ₂ ) ₃ —. In some embodiments, R ^L is CH ₃ (CH ₂ ) ₅ C≡C-. In some embodiments, R ^L is optionally substituted aryl. In some embodiments, R ^L is an optionally substituted phenyl. In some embodiments, R ^L is phenyl substituted with one or more halogen. In some embodiments, R ^L is phenyl optionally substituted with halogen, -N(R'), or -N(R')C(O)R'. In some embodiments, R ^L is phenyl optionally substituted with -Cl, -Br, -F, -N(Me) ₂ , or -NHCOCH ₃ . In some embodiments, R ^L is -L ^L -R′, and L ^L is an optionally substituted C _1-20 saturated, partially unsaturated, or unsaturated hydrocarbon chain. In some embodiments, these hydrocarbon chains are linear. In some embodiments, these hydrocarbon chains are unsubstituted. In some embodiments, L ^L is (E)-CH ₂ -CH=CH-. In some embodiments, L ^L is -CH ₂ -C≡C-CH ₂ -. In some embodiments, L ^L is -(CH ₂ ) ₃ -. In some embodiments, L ^L is -(CH ₂ ) ₄ -. In some embodiments, L ^L is -(CH ₂ ) _n -, where n is 1-30 (eg, 1-20, 5-30, 6-30, 1, 2, 3, 4, 5, 6 , 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30, etc. ). In some embodiments, R' is an optionally substituted aryl as described herein. In some embodiments, R' is an optionally substituted phenyl. In some embodiments, R' is phenyl. In some embodiments, R' is an optionally substituted heteroaryl as described herein. In some embodiments, R' is 2'-pyridinyl. In some embodiments, R' is 3'-pyridinyl. In some embodiments, R ^L is am. In some embodiments, R ^L is am. In some embodiments, R ^L is am. In some embodiments, R ^L is -L ^L -N(R′) ₂ , and each variable is independently as described herein. In some embodiments, each R' is independently C _1-6 aliphatic as described herein. In some embodiments, -N(R') ₂ is -N(CH ₃ ) ₂ . In some embodiments, -N(R') ² is -NH ₂ . In some embodiments, R ^L is -(CH ₂ ) _n -N(R′) ₂ , where n is 1-30 (eg, 1-20, 5-30, 6-30, 1, 2, 3 , 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28 , 29, or 30, etc.). In some embodiments, R ^L is -(CH ₂ CH ₂ O) _n -CH ₂ CH ₂ -N(R′) ₂ and n is 1-30 (eg, 1-20, 5-30, 6 ~30, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 , 25, 26, 27, 28, 29, or 30, etc.). In some embodiments, R ^L is am. In some embodiments, R ^L is am. In some embodiments, R ^L is am. In some embodiments, R ^L is -(CH ₂ ) _n -NH ₂ . In some embodiments, R ^L is -(CH ₂ CH ₂ O) _n -CH ₂ CH ₂ -NH ₂ . In some embodiments, R ^L is -(CH ₂ CH ₂ O) _n -CH ₂ CH ₂ -R′, where n is 1-30 (eg, 1-20, 5-30, 6-30, 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26 , 27, 28, 29, or 30, etc.). In some embodiments, R ^L is -(CH ₂ CH ₂ O) _n -CH ₂ CH ₂ CH ₃ , where n is 1-30 (eg, 1-20, 5-30, 6-30, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30, etc.). In some embodiments, R ^L is -(CH ₂ CH ₂ O) _n -CH ₂ CH ₂ OH, where n is 1-30 (eg, 1-20, 5-30, 6-30, 1, 2 , 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27 , 28, 29, or 30, etc.). In some embodiments, R ^L is or comprises a carbohydrate moiety, eg GalNAc. In some embodiments, R ^L is -L ^L -GalNAc. In some embodiments, R ^L is am. In some embodiments, one or more methylene units of L ^L are independently -Cy- (eg, optionally substituted 1,4-phenylene, optionally substituted 3-30 membered divalent monocyclic, bicyclic, or polycyclic cycloaliphatic rings, etc.), -O-, -N(R')- (e.g., -NH), -C(O)-, -C(O)N(R')- (e.g., -C( O)NH-), -C(NR')-(e.g. -C(NH)-), -N(R')C(O)(N(R')-(e.g. -NHC (O)NH-), -N(R')C(NR')(N(R')-(eg -NHC(NH)NH-), -(CH ₂ CH ₂ O) _n - etc. For example, in some embodiments, R ^L is am. In some embodiments, R ^L is am. In some embodiments, R ^L is am. In some embodiments, R ^L is am. In some embodiments, R ^L is And, n is 0 to 20. In some embodiments, R ^L is or comprises one or more additional optionally substituted chemical moieties (eg, carbohydrate moieties, GalNAc moieties, etc.) linked through a linker (which can be divalent or polyvalent). For example, in some embodiments, R ^L is And, n is 0 to 20. In some embodiments, R ^L is And, n is 0 to 20. In some embodiments, R ^L is R' as described herein. As described herein, many variables can independently be R'. In some embodiments, R′ is R as described herein. As described herein, many variables can independently be R. In some embodiments, R is optionally substituted C _1-6 aliphatic. In some embodiments, R is an optionally substituted C _1-6 alkyl. In some embodiments, R is methyl. In some embodiments, R is an optionally substituted cycloaliphatic. In some embodiments, R is an optionally substituted cycloalkyl. In some embodiments, R is an optionally substituted aryl. In some embodiments, R is an optionally substituted phenyl. In some embodiments, R is an optionally substituted heteroaryl. In some embodiments, R is an optionally substituted heterocyclyl. In some embodiments, R is an optionally substituted C _1-20 heterocyclyl having 1-5 heteroatoms (eg one of which is nitrogen). In some embodiments, R is optionally substituted am. In some embodiments, R is optionally substituted am. In some embodiments, R is optionally substituted am. In some embodiments, R is optionally substituted am. In some embodiments, R is optionally substituted am. In some embodiments, R is optionally substituted am. In some embodiments, R is optionally substituted am. In some embodiments, R is optionally substituted am. In some embodiments, R is optionally substituted am. In some embodiments, R is optionally substituted am. In some embodiments, R is optionally substituted am. In some embodiments, R is optionally substituted am. In some embodiments, R is optionally substituted am. In some embodiments, R is optionally substituted am. In some embodiments, R is optionally substituted am.

In some embodiments, -XR ^L is am. In some embodiments, -XR ^L is am. In some embodiments, -XR ^L is am. In some embodiments, -XR ^L is am. In some embodiments, -XR ^L is am. In some embodiments, -XR ^L is am. In some embodiments, -XR ^L is am. In some embodiments, -XR ^L is am. In some embodiments, -XR ^L is am. In some embodiments, -XR ^L is am. In some embodiments, -XR ^L is am. In some embodiments, -XR ^L is am. In some embodiments, -XR ^L is And, n is 1 to 20. In some embodiments, -XR ^L is And, n is 1 to 20. In some embodiments, -XR ^L is

, and is selected from In some embodiments, -XR ^L is am. In some embodiments, -XR ^L is am. In some embodiments, -XR ^L is am.

In some embodiments, R ^L is R” as described herein. In some embodiments, R ^L is R as described herein.

In some embodiments, R” or R ^L is or comprises an additional chemical moiety. In some embodiments, R” or R ^L is or comprises an additional chemical moiety, and the additional chemical moiety is or comprises a carbohydrate moiety. In some embodiments, R” or RL is or comprises GalNAc. In some embodiments, R ^L or R” is replaced by, or used to connect to, additional chemical moieties.

In some embodiments, X is -O-. In some embodiments, X is -S-. In some embodiments, X is -L ^L -N(-L ^L -R ^L )-L ^L -. In some embodiments, X is -N(-L ^L -R ^L )-L ^L -. In some embodiments, X is -L ^L -N(-L ^L -R ^L )-. In some embodiments, X is -N(-L ^L -R ^L )-. In some embodiments, X is -L ^L -N=C(-L ^L -R ^L )-L ^L -. In some embodiments, X is -N=C(-L ^L -R ^L )-L ^L -. In some embodiments, X is -L ^L -N=C(-L ^L -R ^L )-. In some embodiments, X is -N=C(-L ^L -R ^L )-. In some embodiments, X is L ^L . In some embodiments, X is a covalent bond.

In some embodiments, Y is a covalent bond. In some embodiments, Y is -O-. In some embodiments, Y is -N(R')-. In some embodiments, Z is a covalent bond. In some embodiments, Z is -O-. In some embodiments, Z is -N(R')-. In some embodiments, R' is R. In some embodiments, R is -H. In some embodiments, R is an optionally substituted C _1-6 aliphatic. In some embodiments, R is methyl. In some embodiments, R is ethyl. In some embodiments, R is propyl. In some embodiments, R is an optionally substituted phenyl. In some embodiments, R is phenyl.

As described herein, various variables in the structures of the present invention may be or include R. Suitable embodiments for R are described extensively in the present invention. As will be appreciated by those skilled in the art, an embodiment of R described for a variable that can be R can also be applied to another variable that can be R. Likewise, implementations described for a component/moiety for a variable (eg, L) may also apply to other variables that may be or include a component/moiety.

In some embodiments, R” is R'. In some embodiments, R” is —N(R′) ₂ .

In some embodiments, -XR ^L is -SH. In some embodiments, -XR ^L is -OH.

In some embodiments, -XR ^L is -N(R′) ₂ . In some embodiments, each R' is independently an optionally substituted C _1-6 aliphatic. In some embodiments, each R' is independently methyl.

In some embodiments, the non-negatively charged internucleotide linkage has the structure -OP(=0)(-N=C((N(R') ₂ ) ₂ -O-. In some embodiments, one The R' groups of N(R') ₂ are R, the R' groups of the remaining N(R') ₂ are R, and the two R groups, together with their intervening atoms, are optionally substituted rings, such as in n001 In some embodiments, each R' is independently R, and each R is independently optionally substituted C _1-6 aliphatic.

In some embodiments, -XR ^L is -N=C(-L ^L -R') ₂ . In some embodiments, -XR ^L is -N=C(-L ^L1 -L ^L2 -L ^L3 -R') ₂ , each of L ^L1 , L ^L2 , and L ^L3 is independently L”, and each L ” is independently a covalent bond or a divalent optionally substituted linear or branched group selected from C _1-10 aliphatic groups and C _1-10 heteroaliphatic groups having 1 to 10 heteroatoms, wherein one or more methylene units are optionally and independently C _1-6 alkylene, C _1-6 alkenylene, , a divalent C ₁ -C ₆ heteroaliphatic group having 1 to 5 heteroatoms, -C(R') ₂ -, -Cy-, -O-, -S-, -SS-, -N(R' )-, -C(O)-, -C(S)-, -C(NR')-, -C(O)N(R')-, -N(R')C(O)N(R ')-, -N(R')C(O)O-, -S(O)-, -S(O) ₂ -, -S(O) ₂ N(R')-, -C(O) S-, -C(O)O-, -P(O)(OR')-, -P(O)(SR')-, -P(O)(R')-, -P(O)( NR')-, -P(S)(OR')-, -P(S)(SR')-, -P(S)(R')-, -P(S)(NR')-, - P(R')-, -P(OR')-, -P(SR')-, -P(NR')-, -P(OR')[B(R') ₃ ]-, -OP( O)(OR')O-, -OP(O)(SR')O-, -OP(O)(R')O-, -OP(O)(NR')O-, -OP(OR') )O-, -OP(SR')O-, -OP(NR')O-, -OP(R')O-, or -OP(OR')[B(R') ₃ ]O- is replaced with an optionally substituted group, and one or more nitrogen or carbon atoms are optionally and independently replaced with Cy ^L . In some embodiments, L ^L2 is -Cy-. In some embodiments, L ^L1 is a covalent bond. In some embodiments, L ^L3 is a covalent bond. In some embodiments, -XR ^L is -N=C(-L ^L1 -Cy-L ^L3 -R') ₂ . In some embodiments, -XR ^L is am. In some embodiments, -XR ^L is am. In some embodiments, -XR ^L is am. In some embodiments, -XR ^L is am. In some embodiments, -XR ^L is am. In some embodiments, -XR ^L is am.

In some embodiments, as used herein, L is a covalent bond. In some embodiments, L is a divalent optionally substituted linear or branched group selected from C _1-30 aliphatic groups and C _1-30 heteroaliphatic groups having 1-10 heteroatoms, wherein one or more methylene units are optionally and independently C _1-6 alkylene, C _1-6 alkenylene, , a divalent C ₁ -C ₆ heteroaliphatic group having 1 to 5 heteroatoms, -C(R') ₂ -, -Cy-, -O-, -S-, -SS-, -N(R' )-, -C(O)-, -C(S)-, -C(NR')-, -C(O)N(R')-, -N(R')C(O)N(R ')-, -N(R')C(O)O-, -S(O)-, -S(O) ₂ -, -S(O) ₂ N(R')-, -C(O) S-, -C(O)O-, -P(O)(OR')-, -P(O)(SR')-, -P(O)(R')-, -P(O)( NR')-, -P(S)(OR')-, -P(S)(SR')-, -P(S)(R')-, -P(S)(NR')-, - P(R')-, -P(OR')-, -P(SR')-, -P(NR')-, -P(OR')[B(R') ₃ ]-, -OP( O)(OR')O-, -OP(O)(SR')O-, -OP(O)(R')O-, -OP(O)(NR')O-, -OP(OR') )O-, -OP(SR')O-, -OP(NR')O-, -OP(R')O-, or -OP(OR')[B(R') ₃ ]O- is replaced with an optionally substituted group, and one or more nitrogen or carbon atoms are optionally and independently replaced with Cy ^L . In some embodiments, L is a divalent optionally substituted linear or branched group selected from C _1-30 aliphatic groups and C _1-30 heteroaliphatic groups having 1-10 heteroatoms, wherein one or more methylene units are optionally and independently C _1-6 alkylene, C _1-6 alkenylene, , -C(R') ₂ -, -Cy-, -O-, -S-, -SS-, -N(R')-, -C(O)-, -C(S)-, -C (NR')-, -C(O)N(R')-, -N(R')C(O)N(R')-, -N(R')C(O)O-, -S (O)-, -S(O) ₂ -, -S(O) ₂ N(R')-, -C(O)S-, -C(O)O-, -P(O)(OR' )-, -P(O)(SR')-, -P(O)(R')-, -P(O)(NR')-, -P(S)(OR')-, -P( S)(SR')-, -P(S)(R')-, -P(S)(NR')-, -P(R')-, -P(OR')-, -P(SR ')-, -P(NR')-, -P(OR')[B(R') ₃ ]-, -OP(O)(OR')O-, -OP(O)(SR')O -, -OP(O)(R')O-, -OP(O)(NR')O-, -OP(OR')O-, -OP(SR')O-, -OP(NR') O-, -OP(R')O-, or -OP(OR')[B(R') ₃ ]O-, and one or more nitrogen or carbon atoms are optionally and independently replaced by Cy ^L ). In some embodiments, L is a divalent optionally substituted linear or branched group selected from C _1-10 aliphatic groups and C _1-10 heteroaliphatic groups having 1-10 heteroatoms, wherein one or more methylene units are optionally and independently C _1-6 alkylene, C _1-6 alkenylene, , -C(R') ₂ -, -Cy-, -O-, -S-, -SS-, -N(R')-, -C(O)-, -C(S)-, -C (NR')-, -C(O)N(R')-, -N(R')C(O)N(R')-, -N(R')C(O)O-, -S (O)-, -S(O) ₂ -, -S(O) ₂ N(R')-, -C(O)S-, -C(O)O-, -P(O)(OR' )-, -P(O)(SR')-, -P(O)(R')-, -P(O)(NR')-, -P(S)(OR')-, -P( S)(SR')-, -P(S)(R')-, -P(S)(NR')-, -P(R')-, -P(OR')-, -P(SR ')-, -P(NR')-, -P(OR')[B(R') ₃ ]-, -OP(O)(OR')O-, -OP(O)(SR')O -, -OP(O)(R')O-, -OP(O)(NR')O-, -OP(OR')O-, -OP(SR')O-, -OP(NR') O-, -OP(R')O-, or -OP(OR')[B(R') ₃ ]O-, and one or more nitrogen or carbon atoms are optionally and independently replaced by Cy ^L ). In some embodiments, one or more methylene units are optionally and independently , -C(R') ₂ -, -Cy-, -O-, -S-, -SS-, -N(R')-, -C(O)-, -C(S)-, -C (NR')-, -C(O)N(R')-, -N(R')C(O)N(R')-, -N(R')C(O)O-, -S Replaced with an optionally substituted group selected from (O)-, -S(O) ₂ -, -S(O) ₂ N(R')-, -C(O)S-, or -C(O)O- do.

In some embodiments, the internucleotidic linkages are phosphoryl guanidine internucleotidic linkages. In some embodiments, -XR ^L is -N=C[N(R′) ₂ ] ₂ . In some embodiments, each R' is independently R. In some embodiments, R is an optionally substituted C _1-6 aliphatic. In some embodiments, R is methyl. In some embodiments, -XR ^L is am. In some embodiments, one R' on a nitrogen atom together with R' on another nitrogen form a ring as described herein.

In some embodiments, -X-RL is , and R ¹ and R ² are independently R'. In some embodiments, -XR ^L is am. In some embodiments, -XR ^L is am. In some embodiments, two R' on the same nitrogen together form a ring as described herein. In some embodiments, -XR ^L is am. In some embodiments, -XR ^L is am. In some embodiments, -XR ^L is am. In some embodiments, -XR ^L is am. In some embodiments, -XR ^L is am. In some embodiments, -XR ^L is am. In some embodiments, -XR ^L is am. In some embodiments, -XR ^L is am. In some embodiments, -XR ^L is am.

In some embodiments, -XR ^L is R as described herein. In some embodiments, R is not hydrogen. In some embodiments, R is an optionally substituted C _1-6 aliphatic. In some embodiments, R is an optionally substituted C _1-6 alkyl. In some embodiments, R is methyl.

In some embodiments, -XR ^L is selected from the table below. In some embodiments, X is as described herein. In some embodiments, R ^L is as described herein. In some embodiments, the linkage has the structure -YP ^L (-XR ^L )-Z-, where -XR ^L is selected from the table below, and each other variable is independently as described herein. In some embodiments, the linkage has or comprises the structure -P(O)(-XR ^L )-, and -XR ^L is selected from the table below. In some embodiments, the linkage has or comprises the structure -P(S)(-XR ^L )-, and -XR ^L is selected from the table below. In some embodiments, the linkage has or comprises the structure -P(-XR ^L )-, and -XR ^L is selected from the table below. In some embodiments, the linkage has or comprises the structure -P(O)(-XR ^L )-O-, and -XR ^L is selected from the table below. In some embodiments, the linkage has or comprises the structure -P(S)(-XR ^L )-O-, and -XR ^L is selected from the table below. In some embodiments, the linkage has or comprises the structure -P(-XR ^L )-O-, and -XR ^L is selected from the table below. In some embodiments, the linkage has the structure -P(O)(-XR ^L )-O-, and -XR ^L is selected from the table below. In some embodiments, the linkage has the structure -P(S)(-XR ^L )-O-, and -XR ^L is selected from the table below. In some embodiments, the linkage has the structure -P(-XR ^L )-O-, and -XR ^L is selected from the table below. In some embodiments, P is bonded to a nitrogen atom (eg, the nitrogen atom in sm01, sm18, etc.). In some embodiments, the linkage has or comprises the structure -OP(O)(-XR ^L )-O-, and -XR ^L is selected from the table below. In some embodiments, the linkage has or comprises the structure -OP(S)(-XR ^L )-O-, and -XR ^L is selected from the table below. In some embodiments, the linkage has or comprises the structure -OP(-XR ^L )-O-, and -XR ^L is selected from the table below. In some embodiments, the linkage has the structure -OP(O)(-XR ^L )-O-, and -XR ^L is selected from the table below. In some embodiments, the linkage has the structure -OP(S)(-XR ^L )-O-, and -XR ^L is selected from the table below. In some embodiments, the linkage has the structure -OP(-XR ^L )-O-, and -XR ^L is selected from the table below. In some embodiments, in the table below, n is 0-20 or as described herein. As will be appreciated by those skilled in the art, linkages may exist in salt form.

[Table L-1] Useful specific moieties bound to linking phosphorus (eg, -XR ^L ).

(each R ^LS is independently R ^s ). In some embodiments, each R ^LS is independently -Cl, -Br, -F, -N(Me) ₂ , or -NHCOCH ₃ .

[Table L-2] Useful specific moieties bound to linking phosphorus (eg, -XR ^L ).

[Table L-3] Useful specific moieties bound to linking phosphorus (eg, -XR ^L ).

[Table L-4] Useful specific moieties bound to linking phosphorus (eg, -XR ^L ).

[Table L-5] Useful specific moieties bound to linking phosphorus (eg, -XR ^L ).

[Table L-6] Useful specific moieties bound to linking phosphorus (eg, -XR ^L ).

In some embodiments, an internucleotide linkage, eg, a non-negatively charged internucleotide linkage or a neutral internucleotide linkage, has the structure -L ^L1 -Cy ^IL -L ^L2 -. In some embodiments, L ^L1 is bonded to the 3'-carbon of the sugar. In some embodiments, L ^L2 is attached to the 5'-carbon of the sugar. In some embodiments, L ^L1 is -O-CH ₂ -. In some embodiments, L ^L2 is a covalent bond. In some embodiments, L ^L2 is -N(R')-. In some embodiments, L ^L2 is -NH-. In some embodiments, L ^L2 is attached to the 5'-carbon of the sugar and the 5'-carbon is substituted with =0. In some embodiments, Cy ^IL is an optionally substituted 3-10 membered saturated, partially unsaturated, or aromatic ring having 0-5 heteroatoms. In some embodiments, Cy ^IL is an optionally substituted triazole ring. In some embodiments, Cy ^IL is am. In some embodiments, linking is am.

In some embodiments, the non-negatively charged internucleotidic linkage has the structure -OP(=W)(-N(R') ₂ )-O-.

In some embodiments, R' is R. In some embodiments, R' is H. In some embodiments, R' is -C(0)R. In some embodiments, R' is -C(O)OR. In some embodiments, R' is -S(O) ₂ R.

In some embodiments, R” is -NHR'. In some embodiments, -N(R') ₂ is -NHR'.

As described herein, in some embodiments, R is H. In some embodiments, R is an optionally substituted C _1-6 aliphatic. In some embodiments, R is an optionally substituted C _1-6 alkyl. In some embodiments, R is methyl. In some embodiments, R is substituted methyl. In some embodiments, R is ethyl. In some embodiments, R is substituted ethyl.

In some embodiments, as described herein, non-negatively charged internucleotidic linkages are neutral internucleotidic linkages.

In some embodiments, a modified internucleotide linkage (eg, a non-negatively charged internucleotide linkage) comprises an optionally substituted triazolyl. In some embodiments, R is or comprises an optionally substituted triazolyl. In some embodiments, a modified internucleotidic linkage (eg, a non-negatively charged internucleotidic linkage) comprises an optionally substituted alkynyl. In some embodiments, R' is an optionally substituted alkynyl. In some embodiments, R' comprises an optionally substituted triple bond. In some embodiments, the modified internucleotidic linkage comprises a triazole or alkyne moiety. In some embodiments, R' comprises an optionally substituted triazole or alkyne moiety. In some embodiments, a triazole moiety (eg, a triazolyl group) is optionally substituted. In some embodiments, a triazole moiety (eg, a triazolyl group) is substituted. In some embodiments, the triazole moiety is unsubstituted. In some embodiments, the modified internucleotide linkage comprises an optionally substituted guanidine moiety. In some embodiments, the modified internucleotide linkage comprises an optionally substituted cyclic guanidine moiety. In some embodiments, R', R ^L , or -XR ^L is or comprises an optionally substituted guanidine moiety. In some embodiments, R', R ^L , or -XR ^L is or comprises an optionally substituted cyclic guanidine moiety. In some embodiments, R', R ^L , or -XR ^L comprises an optionally substituted cyclic guanidine moiety and the modified internucleotide linkage is , , or has a structure, and W is O or S. In some embodiments, W is O. In some embodiments, W is S. In some embodiments, the non-negatively charged internucleotidic linkages are stereochemically controlled.

In some embodiments, a non-negatively charged internucleotidic linkage or a neutral internucleotidic linkage is an internucleotidic linkage comprising a triazole moiety. In some embodiments, the non-negatively charged internucleotidic linkage or non-negatively charged internucleotidic linkage comprises an optionally substituted triazolyl group. In some embodiments, an internucleotidic linkage comprising a triazole moiety (eg, an optionally substituted triazolyl group) is has the structure of In some embodiments, an internucleotide linkage comprising a triazole moiety is has the structure of In some embodiments, the internucleotide linkage, eg, a non-negatively charged internucleotide linkage, a neutral internucleotide linkage comprises a cyclic guanidine moiety. In some embodiments, an internucleotidic linkage comprising a cyclic guanidine moiety is has the structure of In some embodiments, a non-negatively charged internucleotidic linkage or a neutral internucleotidic linkage is , , , or Is or comprises a structure selected from, and W is O or S.

In some embodiments, the internucleotidic linkage is a Tmg group ( ). In some embodiments, an internucleotidic linkage comprises a Tmg group It has the structure of ("Tmg internucleotidic linkages"). In some embodiments, neutral internucleotide linkages include internucleotide linkages of PNA and PMO, and Tmg internucleotide linkages.

In some embodiments, the non-negatively charged internucleotidic linkage comprises an optionally substituted 3-20 membered heterocyclyl or heteroaryl group having 1-10 heteroatoms. In some embodiments, the non-negatively charged internucleotidic linkage comprises an optionally substituted 3-20 membered heterocyclyl or heteroaryl group having 1-10 heteroatoms, at least one heteroatom being nitrogen. In some embodiments, such heterocyclyl or heteroaryl groups are five-membered ring groups. In some embodiments, such heterocyclyl or heteroaryl groups are six-membered ring groups.

In some embodiments, the non-negatively charged internucleotidic linkage comprises an optionally substituted 5-20 membered heteroaryl group having 1-10 heteroatoms. In some embodiments, the non-negatively charged internucleotidic linkage comprises an optionally substituted 5-20 membered heteroaryl group having 1-10 heteroatoms, at least one heteroatom being nitrogen. In some embodiments, the non-negatively charged internucleotidic linkage comprises an optionally substituted 5-6 membered heteroaryl group having 1-4 heteroatoms, at least one heteroatom being nitrogen. In some embodiments, the non-negatively charged internucleotidic linkage comprises an optionally substituted 5-membered heteroaryl group having 1-4 heteroatoms, at least one heteroatom being nitrogen. In some embodiments, the heteroaryl group is directly bonded to the linking phosphorus. In some embodiments, the non-negatively charged internucleotidic linkage comprises an optionally substituted 5-20 membered heterocyclyl group having 1-10 heteroatoms. In some embodiments, the non-negatively charged internucleotidic linkage comprises an optionally substituted 5-20 membered heterocyclyl group having 1-10 heteroatoms, at least one heteroatom being nitrogen. In some embodiments, the non-negatively charged internucleotidic linkage comprises an optionally substituted 5-6 membered heterocyclyl group having 1-4 heteroatoms, at least one heteroatom being nitrogen. In some embodiments, the non-negatively charged internucleotidic linkage comprises an optionally substituted 5-membered heterocyclyl group having 1-4 heteroatoms, at least one heteroatom being nitrogen. In some embodiments, at least two heteroatoms are nitrogen. In some embodiments, the heterocyclyl group is directly bonded to the linking phosphorus. In some embodiments, a heterocyclyl group is bonded to the linking phosphorus through a linker, for example, =N- when the heterocyclyl group is part of a guanidine moiety derived and bonded to the linking phosphorus through =N-. In some embodiments, non-negatively charged internucleotidic linkages are optionally substituted includes the flag In some embodiments, non-negatively charged internucleotidic linkages are substituted includes the flag In some embodiments, a non-negatively charged internucleotide linkage is includes the flag In some embodiments, each R ¹ is independently an optionally substituted C _1-6 alkyl. In some embodiments, each R ¹ is independently methyl.

In some embodiments, non-negatively charged internucleotidic linkages, eg, neutral internucleotidic linkages, are not chirally controlled. In some embodiments, the non-negatively charged internucleotidic linkages are chirally controlled. In some embodiments, the non-negatively charged internucleotidic linkage is chirally controlled and its linkage phosphorus is Rp. In some embodiments, the non-negatively charged internucleotidic linkage is chirally controlled and its linkage phosphorus is Sp.

In some embodiments, an internucleotidic linkage does not include a linking phosphorus. In some embodiments, the internucleotide linkage has the structure -C(O)-(O)- or -C(O)-N(R')-, where R' is as described herein. In some embodiments, an internucleotidic linkage has the structure -C(O)-(O)-. In some embodiments, an internucleotide linkage has the structure —C(O)-N(R′)-, where R′ is as described herein. In various embodiments, -C(O)- is bonded to nitrogen. In some embodiments, the internucleotidic linkage is or comprises -C(O)-O- that is part of a carbamate moiety. In some embodiments, the internucleotidic linkage is or comprises -C(O)-O- that is part of a urea moiety.

In some embodiments, the oligonucleotide is 1-20, 1-15, 1-10, 1-5, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more negatively charged It contains unlinked internucleotide linkages. In some embodiments, the oligonucleotide has 1-20, 1-15, 1-10, 1-5, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more neutral internucleotide linkages includes In some embodiments, each non-negatively charged internucleotidic linkage and/or neutral internucleotidic linkage is optionally and independently chirally controlled. In some embodiments, each negatively charged internucleotidic linkage in an oligonucleotide is independently a chirally controlled internucleotidic linkage. In some embodiments, each neutral internucleotidic linkage in an oligonucleotide is independently a chirally controlled internucleotidic linkage. In some embodiments, at least one non-negatively charged internucleotide linkage/neutral internucleotide linkage is has the structure of In some embodiments, the oligonucleotide comprises at least one non-negatively charged internucleotidic linkage in which the linking phosphorus is in the Rp configuration, and at least one non-negatively charged internucleotidic linkage in which the linking phosphorus is in the Sp configuration.

In many embodiments, as has been widely demonstrated, oligonucleotides of the present invention comprise two or more different internucleotidic linkages. In some embodiments, the oligonucleotide comprises phosphorothioate internucleotidic linkages and non-negatively charged internucleotidic linkages. In some embodiments, the oligonucleotide comprises phosphorothioate internucleotidic linkages, non-negatively charged internucleotidic linkages, and natural phosphate linkages. In some embodiments, the non-negatively charged internucleotidic linkages are neutral internucleotidic linkages. In some embodiments, a non-negatively charged internucleotide linkage is or n013. In some embodiments, a non-negatively charged internucleotide linkage is am. In some embodiments, the non-negatively charged internucleotide linkage is n001. In some embodiments, each phosphorothioate internucleotidic linkage is independently chirally controlled. In some embodiments, each chiral modified internucleotidic linkage is independently chirally controlled. In some embodiments, one or more non-negatively charged internucleotidic linkages are not chirally controlled.

A common linkage, as in natural DNA and RNA, is that an internucleotidic linkage forms a bond with two sugars, which may or may not be modified as described herein. In many embodiments, as exemplified herein, the internucleotidic linkage forms a bond with one optionally modified ribose or deoxyribose at the 5' carbon and the other optionally modified ribose at the 3' carbon via an oxygen atom or heteroatom. It forms bonds with ribose or deoxyribose. In some embodiments, internucleotidic linkages link sugars that are not ribose sugars, eg, sugars comprising N ring atoms and acyclic sugars described herein.

In some embodiments, each nucleoside unit linked by an internucleotide linkage is independently an optionally substituted A, T, C, G, or U, or an optionally substituted A, T, C, G, or U Independently includes nucleobases that are tautomers.

In some embodiments, oligonucleotides are disclosed in US 9394333, US 9744183, US 9605019, US 9598458, US 9982257, US 10160969, US 10479995, US 2020/0056173, US 2018/0216107, US 2019/01277 33, US 10450568, US 2019/ 0077817, US 2019/0249173, US 2019/0375774, WO 2018/223056, WO 2018/223073, WO 2018/223081, WO 2018/237194, WO 2019/032607, WO 2019/055951 , WO 2019/075357, WO 2019/ 200185 , WO 2019/217784 , and / or WO 2019/032612 ; -4 , II , II-a-1 , II-a-2 , II-b-1 , II-b-2 , II-c-1 , II-c- 2 , II-d-1 , II-d -2 , etc., or a modified internucleotide linkage having a salt form thereof), and an internucleotide linkage of each of the above references (eg, Formula I , Ia , Ib , or Ic , In-1 , In-2 , In-3 , In-4 , II , II-a-1 , II-a-2 , II-b-1 , II-b-2 , II-c-1 , II-c-2 , II-d -1 , II-d-2, etc.) are independently incorporated herein by reference. In some embodiments, the modified internucleotide linkage is a non-negatively charged internucleotide linkage. In some embodiments, provided oligonucleotides include one or more internucleotidic linkages that are not negatively charged. In some embodiments, the non-negatively charged internucleotidic linkages are positively charged internucleotidic linkages. In some embodiments, the non-negatively charged internucleotidic linkages are neutral internucleotidic linkages. In some embodiments, the present invention provides oligonucleotides comprising one or more neutral internucleotide linkages. In some embodiments, a non-negatively charged internucleotidic linkage or a neutral internucleotidic linkage (e.g., formula In-1 , In-2 , In-3 , In-4 , II , II-a-1 , II- a-2 , II-b-1 , II-b-2 , II-c-1 , II-c-2 , II-d-1 , II-d-2 etc.) are US 9394333, US 9744183, US 9605019, US 9598458, US 9982257, US 10160969, US 10479995, US 2020/0056173, US 2018/0216107, US 2019/0127733, US 10450568, US 2019/0077817 , US 2019/0249173, US 2019/0375774, WO 2018 /223056, WO 2018/223073, WO 2018/223081, WO 2018/237194, WO 2019/032607, WO 2019/055951, WO 2019/075357, WO 2019/200185, WO 2019/217784, and/ or WO 2019/032612 as described in In some embodiments, non-negatively charged internucleotide linkages or neutral internucleotide linkages are described in WO 2018/223056, WO 2019/032607, WO 2019/075357, WO 2019/032607, WO 2019/075357, WO 2019/200185, WO Formulas In-1 , In-2 , In-3 , In-4 , II , II -a- 1 , II- a-2 , II-b- as described in 2019/217784, and/or WO 2019/032612 1 , II-b-2 , II-c-1 , II-c-2 , II-d-1 , II-d-2, etc., wherein each such internucleotide linkage of each of the foregoing is independently referenced herein included as

As described herein, various variables can be R, eg R', R ^L , and the like. Various embodiments of R are described herein (eg, when describing a variable that can be R). This implementation is generally useful for any variable that can be R. In some embodiments, R is hydrogen. In some embodiments, R is optionally substituted C _1-30 (eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 , 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29. or 30) is aliphatic. In some embodiments, R is an optionally substituted C _1-20 aliphatic. In some embodiments, R is an optionally substituted C _1-10 aliphatic. In some embodiments, R is an optionally substituted C _1-6 aliphatic. In some embodiments, R is an optionally substituted alkyl. In some embodiments, R is an optionally substituted C _1-6 alkyl. In some embodiments, R is optionally substituted methyl. In some embodiments, R is methyl. In some embodiments, R is optionally substituted ethyl. In some embodiments, R is optionally substituted propyl. In some embodiments, R is isopropyl. In some embodiments, R is optionally substituted butyl. In some embodiments, R is an optionally substituted pentyl. In some embodiments, R is optionally substituted hexyl.

In some embodiments, R is an optionally substituted 3-30 member (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 , 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) are alicyclic. In some embodiments, R is an optionally substituted cycloalkyl. In some embodiments, an alicyclic is monocyclic, bicyclic, or polycyclic, and each monocyclic unit is independently saturated or partially saturated. In some embodiments, R is an optionally substituted cyclopropyl. In some embodiments, R is an optionally substituted cyclobutyl. In some embodiments, R is an optionally substituted cyclopentyl. In some embodiments, R is optionally substituted cyclohexyl. In some embodiments, R is an optionally substituted adamantyl.

In some embodiments, R is an optionally substituted C _1-30 having 1-10 heteroatoms (eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) heteroaliphatic. In some embodiments, R is an optionally substituted C _1-20 aliphatic having 1-10 heteroatoms. In some embodiments, R is an optionally substituted C _1-10 aliphatic having 1-10 heteroatoms. In some embodiments, R is an optionally substituted C _1-6 aliphatic having 1-3 heteroatoms. In some embodiments, R is an optionally substituted heteroalkyl. In some embodiments, R is an optionally substituted C _1-6 heteroalkyl. In some embodiments, R is an optionally substituted 3-30 member having 1-10 heteroatoms (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) heterocycloaliphatic. In some embodiments, R is an optionally substituted heterocycloalkyl. In some embodiments, the heterocycloaliphatic is monocyclic, bicyclic, or polycyclic, and each monocyclic unit is independently saturated or partially saturated.

In some embodiments, R is an optionally substituted C _6-30 aryl. In some embodiments, R is an optionally substituted phenyl. In some embodiments, R is an optionally substituted phenyl. In some embodiments, R is C _6-14 aryl. In some embodiments, R is an optionally substituted bicyclic aryl. In some embodiments, R is an optionally substituted polycyclic aryl. In some embodiments, R is an optionally substituted C _6-30 arylaliphatic. In some embodiments, R is C _6-30 arylheteroaliphatic having 1-10 heteroatoms.

In some embodiments, R is an optionally substituted 5-30 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 , 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) membered heteroaryl. In some embodiments, R is an optionally substituted 5-20 membered heteroaryl having 1-10 heteroatoms. In some embodiments, R is an optionally substituted 5-10 membered heteroaryl having 1-10 heteroatoms. In some embodiments, R is an optionally substituted 5-membered heteroaryl having 1-5 heteroatoms. In some embodiments, R is an optionally substituted 5-membered heteroaryl having 1-4 heteroatoms. In some embodiments, R is an optionally substituted 5-membered heteroaryl having 1-3 heteroatoms. In some embodiments, R is an optionally substituted 5-membered heteroaryl having 1-2 heteroatoms. In some embodiments, R is an optionally substituted 5-membered heteroaryl having 1 heteroatom. In some embodiments, R is an optionally substituted 6-membered heteroaryl having 1-5 heteroatoms. In some embodiments, R is an optionally substituted 6-membered heteroaryl having 1-4 heteroatoms. In some embodiments, R is an optionally substituted 6-membered heteroaryl having 1-3 heteroatoms. In some embodiments, R is an optionally substituted 6-membered heteroaryl having 1-2 heteroatoms. In some embodiments, R is an optionally substituted 6-membered heteroaryl having 1 heteroatom. In some embodiments, R is an optionally substituted monocyclic heteroaryl. In some embodiments, R is an optionally substituted bicyclic heteroaryl. In some embodiments, R is an optionally substituted polycyclic heteroaryl. In some embodiments, the heteroatom is nitrogen.

In some embodiments, R is an optionally substituted 2-pyridinyl. In some embodiments, R is an optionally substituted 3-pyridinyl. In some embodiments, R is an optionally substituted 4-pyridinyl. In some embodiments, R is optionally substituted am.

In some embodiments, R is an optionally substituted 3-30 (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 , 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) membered heterocyclyl. In some embodiments, R is an optionally substituted 3-membered heterocyclyl having 1-2 heteroatoms. In some embodiments, R is an optionally substituted 4-membered heterocyclyl having 1-2 heteroatoms. In some embodiments, R is an optionally substituted 5-20 membered heterocyclyl having 1-10 heteroatoms. In some embodiments, R is an optionally substituted 5-10 membered heterocyclyl having 1-10 heteroatoms. In some embodiments, R is an optionally substituted 5-membered heterocyclyl having 1-5 heteroatoms. In some embodiments, R is an optionally substituted 5-membered heterocyclyl having 1-4 heteroatoms. In some embodiments, R is an optionally substituted 5-membered heterocyclyl having 1-3 heteroatoms. In some embodiments, R is an optionally substituted 5-membered heterocyclyl having 1-2 heteroatoms. In some embodiments, R is an optionally substituted 5-membered heterocyclyl having 1 heteroatom. In some embodiments, R is an optionally substituted 6-membered heterocyclyl having 1-5 heteroatoms. In some embodiments, R is an optionally substituted 6-membered heterocyclyl having 1-4 heteroatoms. In some embodiments, R is an optionally substituted 6-membered heterocyclyl having 1-3 heteroatoms. In some embodiments, R is an optionally substituted 6-membered heterocyclyl having 1-2 heteroatoms. In some embodiments, R is an optionally substituted 6-membered heterocyclyl having 1 heteroatom. In some embodiments, R is an optionally substituted monocyclic heterocyclyl. In some embodiments, R is an optionally substituted bicyclic heterocyclyl. In some embodiments, R is an optionally substituted polycyclic heterocyclyl. In some embodiments, R is an optionally substituted saturated heterocyclyl. In some embodiments, R is an optionally substituted partially unsaturated heterocyclyl. In some embodiments, the heteroatom is nitrogen. In some embodiments, R is optionally substituted am. In some embodiments, R is optionally substituted am. In some embodiments, R is optionally substituted am.

In some embodiments, two R groups optionally and independently form a covalent bond together. In some embodiments, two or more R groups on the same atom optionally and independently form an optionally substituted 3-30 membered monocyclic, bicyclic or polycyclic ring having 0-10 heteroatoms in addition to the corresponding atom together with the corresponding atom. In some embodiments, two or more R groups on two or more atoms, optionally and independently with atoms intervening therebetween, are optionally substituted 3-30 membered monocycles having 0-10 heteroatoms in addition to those intervening atoms. , form bicyclic or polycyclic rings.

The various variables may include optionally substituted rings, or may form rings with intervening atom(s). In some embodiments, the rings are between 3 and 30 (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 , 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30). In some embodiments, a ring is 3 to 20 members. In some embodiments, a ring is 3 to 15 members. In some embodiments, a ring is 3 to 10 members. In some embodiments, a ring is 3-8 members. In some embodiments, a ring is 3-7 members. In some embodiments, a ring is 3-6 membered. In some embodiments, a ring is 4 to 20 members. In some embodiments, a ring is 5 to 20 members. In some embodiments, a ring is monocyclic. In some embodiments, a ring is bicyclic. In some embodiments, a ring is polycyclic. In some embodiments, each monocyclic ring, or each monocyclic ring unit of a polycyclic or polycyclic ring, is independently saturated, partially saturated, or aromatic. In some embodiments, each monocyclic ring, or each monocyclic ring unit of a polycyclic or polycyclic ring, is independently 3 to 10 members and has 0 to 5 heteroatoms.

In some embodiments, each heteroatom is independently selected oxygen, nitrogen, sulfur, silicon, and phosphorus. In some embodiments, each heteroatom is independently selected oxygen, nitrogen, sulfur, and phosphorus. In some embodiments, each heteroatom is independently selected oxygen, nitrogen, and sulfur. In some embodiments, a heteroatom is in oxidized form.

As will be appreciated by those skilled in the art, many other types of internucleotide linkages can be used in accordance with the present invention (see, e.g., U.S. Pat. Nos. 3,687,808; 4,469,863; 4,476,301; 5,177,195; 5,023,243; 5,034,506; 5,166,315; 5,185,444; 5,188,897; 5,214,134; 5,216,141; 5,235,033; 5,264,423; 5,264,564; 5,276,019; 5 ,278,302;5,286,717;5,321,131 5,399,676; 5,405,938; 5,405,939; 5,434,257; 5,453,496; 5,455,233; 5,466,677; 5,466,677; 5,470,967; 5,476,925; 5,489,677;5,519,126; 5,536,821; 5,541,307; 5,541,316; 5,550,111; 5,561,225; 5,563,253; 5,571,799; 5,587,361; 5,596,086; 5,602,240; 5 ,608,046;5,610,289;5,618,704 5,623,070; 5,625,050; 5,633,360; 5,64,562; 5,663,312; 5,677,437; 5,677,439; 6,160,109; 6,239,265; 6,028,188; 6,124,445;6,169,170 6,172,209; 6,277,603; 6,326,199; 6,346,614; 6,444,423; 6,531,590; 6,534,639; 6,608,035; 6,683,167; 6,858,715; 6,867,294;6,878,805; 7,015,315; 7,041,816; 7,273,933; 7,321,029; or as described in RE39464). In some embodiments, the modified internucleotide linkages are described in US 9394333, US 9744183, US 9605019, US 9598458, US 9982257, US 10160969, US 10479995, US 2020/0056173, US 2018/0216107, US 2019/01 27733, US 10450568, US 2019/0077817, US 2019/0249173, US 2019/0375774, WO 2018/223056, WO 2018/223073, WO 2018/223081, WO 2018/237194, WO 2019/032607, WO 2019/0 55951, WO 2019/075357, WO 2019/200185, WO 2019/217784, and/or WO 2019/032612, and techniques for synthesizing nucleobases, sugars, internucleotide linkages, chiral auxiliaries/reagents, and oligonucleotides (reagents, conditions, cycles, etc.) ) are independently incorporated herein by reference.

In some embodiments, each internucleotide linkage of an oligonucleotide is independently a natural phosphate linkage, a phosphorothioate linkage, and a non-negatively charged internucleotide linkage (e.g., n001, n002, n003, n004, n005 , n006, n007, n008, n009, n010, n013, etc.). In some embodiments, each internucleotide linkage of an oligonucleotide is independently a natural phosphate linkage, a phosphorothioate linkage, and a neutral internucleotide linkage (e.g., n001, n002, n003, n004, n005, n006, n007 , n008, n009, n010, n013, etc.).

The oligonucleotide may contain a variable number of natural phosphate linkages, for example 1-50, 1-40, 1-30, 1-25, 1-20, 1-10, 1-5, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more may be included. In some embodiments, one or more of the natural phosphate linkages in the oligonucleotide (e.g., 1-50, 1-40, 1-30, 1-25, 1-20, 1-10, 1-5, or 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more) are consecutive. In some embodiments, provided oligonucleotides do not contain natural phosphate linkages. In some embodiments, a provided oligonucleotide comprises one natural phosphate linkage. In some embodiments, a provided oligonucleotide comprises 1 to 30 or more natural phosphate linkages.

In some embodiments, the modified internucleotide linkage is a chiral internucleotide linkage comprising a chiral linkage phosphorus. In some embodiments, the chiral internucleotidic linkage is a phosphorothioate linkage. In some embodiments, chiral internucleotidic linkages are non-negatively charged internucleotidic linkages. In some embodiments, chiral internucleotidic linkages are neutral internucleotidic linkages. In some embodiments, chiral internucleotidic linkages are chirally controlled with respect to the chiral linkage phosphorus. In some embodiments, a chiral internucleotidic linkage is stereochemically pure with respect to the chiral linking phosphorus. In some embodiments, chiral internucleotidic linkages are not chirally controlled. In some embodiments, the backbone chiral center pattern comprises or consists of positions of chiral control internucleotidic linkages ( R p or Sp ) and linkage phosphorous arrangements and positions of achiral internucleotidic linkages (eg, natural phosphate linkages). do.

In some embodiments, provided oligonucleotides include one or more internucleotidic linkages that are not negatively charged. In some embodiments, provided oligonucleotides include one or more neutral internucleotide linkages. In some embodiments, provided oligonucleotides include one or more phosphorylguanidine internucleotidic linkages. In some embodiments, the neutral internucleotide linkage or the non-negatively charged internucleotide linkage is a phosphoryl guanidine internucleotide linkage. In some embodiments, each neutral internucleotidic linkage or non-negatively charged internucleotidic linkage is independently a phosphorylguanidine internucleotidic linkage. In some embodiments, each neutral internucleotide linkage and each non-negatively charged internucleotide linkage are independently n001.

In some embodiments, each internucleotide linkage within a provided oligonucleotide is independently selected from phosphorothioate internucleotide linkages, phosphoryl guanidine internucleotide linkages, and natural phosphate linkages. In some embodiments, each internucleotide linkage within a provided oligonucleotide is independently selected from phosphorothioate internucleotide linkages, n001 and natural phosphate linkages.

Various types of internucleotidic linkages may be used in combination with other structural elements, such as sugars, to achieve the desired oligonucleotide properties and/or activities. For example, the present invention routinely uses modified internucleotide linkages and modified sugars, optionally along with natural phosphate linkages and natural sugars, in designed oligonucleotides. In some embodiments, the present invention provides oligonucleotides comprising one or more modified sugars. In some embodiments, the present invention provides an oligonucleotide comprising one or more modified sugars and one or more modified internucleotidic linkages, at least one of which is a natural phosphate linkage.

In some embodiments, the internucleotide linkage is a phosphoryl guanidine, phosphoryl amidine, phosphoryl isourea, phosphoryl isothiourea, phosphoryl imidate, or phosphoryl imidothioate internucleotidic linkage, see e.g. US 20170362270 those listed

As will be appreciated by those skilled in the art, many other types of internucleotide linkages can be used in accordance with the present invention (see, e.g., U.S. Pat. Nos. 3,687,808; 4,469,863; 4,476,301; 5,177,195; 5,023,243; 5,034,506; 5,166,315; 5,185,444; 5,188,897; 5,214,134; 5,216,141; 5,235,033; 5,264,423; 5,264,564; 5,276,019; 5 ,278,302;5,286,717;5,321,131 5,399,676; 5,405,938; 5,405,939; 5,434,257; 5,453,496; 5,455,233; 5,466,677; 5,466,677; 5,470,967; 5,476,925; 5,489,677;5,519,126; 5,536,821; 5,541,307; 5,541,316; 5,550,111; 5,561,225; 5,563,253; 5,571,799; 5,587,361; 5,596,086; 5,602,240; 5 ,608,046;5,610,289;5,618,704 5,623,070; 5,625,050; 5,633,360; 5,64,562; 5,663,312; 5,677,437; 5,677,439; 6,160,109; 6,239,265; 6,028,188; 6,124,445;6,169,170 6,172,209; 6,277,603; 6,326,199; 6,346,614; 6,444,423; 6,531,590; 6,534,639; 6,608,035; 6,683,167; 6,858,715; 6,867,294;6,878,805; 7,015,315; 7,041,816; 7,273,933; 7,321,029; or as described in RE39464). In some embodiments, the modified internucleotide linkages are described in US 9982257, US 20170037399, US 20180216108, WO 2017192664, WO 2017015575, WO 2017062862, WO 2018067973, WO 2017160741, WO 201719 2679, WO 2017210647, WO 2018098264, WO 2018223056, WO 2018237194, or WO 2019055951, and the nucleobases, sugars, internucleotidic linkages, chiral auxiliaries/reagents, and techniques (reagents, conditions, cycles, etc.) for each oligonucleotide synthesis are independently incorporated herein by reference. In some embodiments, internucleotide linkages are described in WO 2012/030683, WO 2021/030778, WO 2019112485, US 20170362270, WO 2018156056, WO 2018056871, WO 2020/154344, WO 2020/154343, WO 2 020/154342, WO 2020/165077 , WO 2020/201406, WO 2020/216637, or WO 2020/252376, and may be used according to the present invention.

In some embodiments, each internucleotide linkage in an oligonucleotide is independently selected from natural phosphate linkages, phosphorothioate linkages, and non-negatively charged internucleotide linkages (eg, n001). In some embodiments, each internucleotide linkage in an oligonucleotide is independently selected from natural phosphate linkages, phosphorothioate linkages, and neutral internucleotide linkages (eg, n001).

In some embodiments, an oligonucleotide comprises one or more nucleotides independently comprising phosphorus modifications that are susceptible to "autorelease" under certain conditions. That is, under certain conditions, certain phosphorus modifications are designed to self-cleave from oligonucleotides, for example to provide native phosphate linkages. In some embodiments, such phosphorus modifications have the structure -OLR ¹ , L is ^LB as described herein, and R ¹ is R' as described herein. In some embodiments, the phosphorus variant has the structure -SLR ¹ , and each L and R ¹ are independently as described herein. Specific examples of such phosphorus modifying groups can be found in US 9982257. In some embodiments, the autoreleasing group comprises a morpholino group. In some embodiments, autoreleasing groups are characterized by the ability to deliver an agent to an internucleotidic phosphorus linker, which agent catalyzes further modification of the phosphorus atom, such as desulfurization. In some embodiments, this agent is water and further transformation is hydrolysis to form native phosphate linkages.

In some embodiments, an oligonucleotide comprises one or more internucleotidic linkages that improve one or more pharmacological properties and/or activities of the oligonucleotide. It is well documented in the art that certain oligonucleotides are rapidly degraded by nucleases and exhibit poor cellular uptake through the cytoplasmic cell membrane (Poijarvi-Virta et al., Curr. Med. Chem. (2006), 13(28);3441-65;Wagner et al., Med. Res. Rev. (2000), 20(6):417-51;Peyrottes et al., Mini Rev. Med. Chem. (2004), 4(4):395-408;Gosselin et al., (1996), 43(1):196-208;Bologna et al., (2002), Antisense & Nucleic Acid Drug Development 12:33-41]). Vives et al. (Nucleic Acids Research (1999), 27(20):4071-76) reported that tert-butyl SATE pro-oligonucleotides exhibit significantly increased cell penetration compared to parental oligonucleotides under certain conditions.

An oligonucleotide may contain a variable number of natural phosphate linkages. In some embodiments, 5% or more of the internucleotidic linkages of a provided oligonucleotide are native phosphate linkages. In some embodiments, 10% or more of the internucleotidic linkages of a provided oligonucleotide are natural phosphate linkages. In some embodiments, at least 15% of the internucleotidic linkages of a provided oligonucleotide are natural phosphate linkages. In some embodiments, 20% or more of the internucleotidic linkages of a provided oligonucleotide are natural phosphate linkages. In some embodiments, at least 25% of the internucleotidic linkages of a provided oligonucleotide are natural phosphate linkages. In some embodiments, 30% or more of the internucleotidic linkages of a provided oligonucleotide are natural phosphate linkages. In some embodiments, at least 35% of the internucleotidic linkages of a provided oligonucleotide are natural phosphate linkages. In some embodiments, at least 40% of the internucleotidic linkages of a provided oligonucleotide are natural phosphate linkages. In some embodiments, a provided oligonucleotide comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more natural phosphate linkages. In some embodiments, a provided oligonucleotide comprises 4, 5, 6, 7, 8, 9, 10 or more natural phosphate linkages. In some embodiments, the number of natural phosphate linkages is two. In some embodiments, the number of natural phosphate linkages is three. In some embodiments, the number of natural phosphate linkages is four. In some embodiments, the number of natural phosphate linkages is five. In some embodiments, the number of natural phosphate linkages is 6. In some embodiments, the number of natural phosphate linkages is 7. In some embodiments, the number of natural phosphate linkages is eight. In some embodiments, some or all of the natural phosphate linkages are contiguous. In some embodiments, up to a certain number of internucleotide linkages of a provided oligonucleotide are natural phosphate linkages, e.g., 1 or less, 2 or less, 3 or less, 4 or less, 5 or less, 6 or less, 7 or less Not more than 8, not more than 9, not more than 10, not more than 11, not more than 12, not more than 13, not more than 14, not more than 15, not more than 16, not more than 17, not more than 18, not more than 19 , not more than 20, not more than 21, not more than 22, not more than 23, not more than 24, not more than 25, not more than 26, not more than 27, not more than 28, not more than 29, or not more than 30 natural phosphate linkages . In some embodiments, provided oligonucleotides do not contain natural phosphate linkages.

In some embodiments, the present invention indicates that in at least some cases, particularly 5'-terminal and/or 3'-terminal Sp internucleotide linkages can improve oligonucleotide stability. In some embodiments, the present invention specifically indicates that natural phosphate linkages and/or R p internucleotidic linkages may improve oligonucleotide clearance from the system. As will be appreciated by those skilled in the art, various assays known in the art can be used to evaluate these properties in accordance with the present invention.

In some embodiments, each phosphorothioate internucleotidic linkage in an oligonucleotide or portion thereof (eg, a domain, subdomain, etc.) is independently chirally controlled. In some embodiments, each is independently Sp or R p. In some embodiments, the high level is Sp as described herein. In some embodiments, each phosphorothioate internucleotidic linkage in an oligonucleotide or portion thereof is chirally controlled and is Sp . In some embodiments, one or more, for example about 1-5 (eg, about 1, 2, 3, 4, or 5) is R p .

In some embodiments, as exemplified in certain examples, the oligonucleotides or portions thereof include one or more non-negatively charged internucleotide linkages, each optionally and independently chirally controlled. In some embodiments, each non-negatively charged internucleotide linkage is independently n001. In some embodiments, non-negatively charged chiral internucleotidic linkages are not chirally controlled. In some embodiments, each chiral internucleotidic linkage that is not negatively charged is not chirally controlled. In some embodiments, non-negatively charged chiral internucleotidic linkages are chirally controlled. In some embodiments, non-negatively charged chiral internucleotidic linkages are chirally controlled and R p . In some embodiments, the non-negatively charged chiral internucleotidic linkages are chirally controlled and are Sp . In some embodiments, each non-negatively charged chiral internucleotidic linkage is chirally controlled. In some embodiments, the number of non-negatively charged internucleotidic linkages in an oligonucleotide or portion thereof is about 1-10, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, or It is 10 pieces. In some embodiments, the number is about 1. In some embodiments, the number is about two. In some embodiments, the number is about 3. In some embodiments, the number is about 4. In some embodiments, the number is about 5. In some embodiments, the number is about 6. In some embodiments, the number is about 7. In some embodiments, the number is about 8. In some embodiments, the number is about 9. In some embodiments, the number is about 10. In some embodiments, linkages between two or more nucleotides that are not negatively charged are contiguous. In some embodiments, linkages between two non-negatively charged nucleotides are not contiguous. In some embodiments, all non-negatively charged internucleotidic linkages in an oligonucleotide or portion thereof are contiguous (eg, three consecutive non-negatively charged internucleotidic linkages). In some embodiments, non-negatively charged internucleotidic linkages, or two or more (e.g., about 2, about 3, about 4, etc.) consecutive non-negatively charged internucleotidic linkages are oligonucleotides or at the 3'-end of a part thereof. In some embodiments, the last two or three or four internucleotide linkages of an oligonucleotide or portion thereof comprise at least one internucleotide linkage that is not a negatively charged internucleotide linkage. In some embodiments, the last two or three or four internucleotide linkages of the oligonucleotide or portion thereof comprise at least one internucleotide linkage other than n001. In some embodiments, the internucleoside linkage connecting the first two nucleosides of an oligonucleotide or portion thereof is a non-negatively charged internucleoside linkage. In some embodiments, the internucleoside linkage connecting the last two nucleosides of an oligonucleotide or portion thereof is a non-negatively charged internucleoside linkage. In some embodiments, the internucleoside linkage linking the first two nucleosides of an oligonucleotide or portion thereof is a phosphorothioate internucleoside linkage. In some embodiments, it is Sp . In some embodiments, the internucleoside linkage linking the last two nucleosides of an oligonucleotide or portion thereof is a phosphorothioate internucleoside linkage. In some embodiments, it is Sp .

In some embodiments, one or more chiral internucleotide linkages are chirally controlled and one or more chiral internucleotide linkages are not chirally controlled. In some embodiments, each phosphorothioate internucleotidic linkage is independently chirally controlled and one or more non-negatively charged internucleotidic linkages are not chirally controlled. In some embodiments, each phosphorothioate internucleotide linkage is independently chirally controlled and each non-negatively charged internucleotidic linkage is not chirally controlled. In some embodiments, the internucleoside linkage between the first two nucleosides of an oligonucleotide is a non-negatively charged internucleoside linkage. In some embodiments, the internucleotide linkages between the last two nucleosides are each independently non-negatively charged internucleotidic linkages. In some embodiments, both are independently non-negatively charged internucleotidic linkages. In some embodiments, an oligonucleotide comprises one or more additional internucleotide linkages. For example, one of these is the nucleoside at positions -1 and -2 relative to the nucleoside opposite the target nucleoside (e.g. target adenosine) (3 to the nucleoside opposite the target nucleoside). between two adjacent nucleosides in the ' direction (e.g., ...N ₀ N _-1 N _-2 ..., where N ₀ is the nucleoside opposite the target nucleoside, and N _-1 and N _-2 are positions -1 and -2, respectively). In some embodiments, each non-negatively charged internucleotidic linkage is independently a neutral internucleotidic linkage. In some embodiments, each non-negatively charged internucleotide linkage is independently n001.

As shown herein, in some embodiments, non-negatively charged internucleotidic linkages, such as n001, may provide improved properties and/or activity. In some embodiments, a 5'-terminal internucleotide linkage and/or a 3'-terminal internucleotide linkage in an oligonucleotide, each independently linked to two nucleosides comprising a nucleobase as described herein is a non-negatively charged internucleotidic linkage as described herein. In some embodiments, the first one or more (eg, the first 1, 2, and/or 3), and/or the last one or more (eg, the last 1, 2, 3, 4, 5, 6, or 7) internucleotidic linkages, each independently linked to two nucleosides of the first domain, are independently, non-negatively charged internucleotidic linkages. In some embodiments, the first internucleotide linkage of the first domain is a non-negatively charged internucleotide linkage. In some embodiments, the last internucleotide linkage that binds the two nucleosides of the first domain is a non-negatively charged internucleotide linkage. In some embodiments, the last internucleotide linkage of the second domain is a non-negatively charged internucleotide linkage. In some embodiments, one or more of the internucleotide linkages in the middle of the second domain, e.g., the 4th, 5th, and 6th internucleotide linkages, each independently binding to two nucleosides of the second domain wherein at least one of the nucleotides is independently a non-negatively charged internucleotidic linkage. In some embodiments, the 11th internucleotide linkage that binds the two nucleosides of the second domain is a non-negatively charged internucleotide linkage. In some embodiments, an internucleoside linkage that does not bind to the nucleoside opposite the target nucleoside but to the 3′ adjacent nucleoside is a non-negatively charged internucleoside linkage. In some embodiments, the non-negatively charged internucleotidic linkages are neutral internucleotidic linkages. In some embodiments, the non-negatively charged internucleotide linkage is n001. In some embodiments, each internucleotide linkage that is not negatively charged is n001. In some embodiments, the non-negatively charged internucleotidic linkages are sterically random. In some embodiments, the non-negatively charged internucleotidic linkages are chirally controlled and R p . In some embodiments, the non-negatively charged internucleotidic linkages are chirally controlled and are Sp . In some embodiments, each non-negatively charged internucleotide linkage is independently chirally controlled. In some embodiments, one or more internucleotide linkages of the first domain, e.g., the 4th, 5th, 6th, 7th, and 8th internucleotide linkages (each independently two nucleosides of the first domain) bonded to) is not independently a non-negatively charged internucleotidic linkage. In some embodiments, one or more internucleotide linkages of the second domain, e.g., the 1st, 2nd, 3rd, 7th, 8th, 9th, 12th, and 13th internucleotide linkages (each independently at least one of the two nucleosides of the first domain) is not independently a non-negatively charged internucleotidic linkage. In some embodiments, one or both of the second and third internucleotide linkages of the second domain are not negatively charged internucleotide linkages. In some embodiments, internucleotide linkages that are not negatively charged internucleotidic linkages are phosphorothioate internucleotidic linkages. In some embodiments, it is a sterically random phosphorothioate internucleotidic linkage. In some embodiments, it is an R p chirally controlled phosphorothioate internucleotidic linkage. In some embodiments, it is an S p chirally controlled phosphorothioate internucleotidic linkage.

In some embodiments, one or more or all internucleotide linkages at positions +11, +9, +5, -2, and -5 of the nucleoside opposite the target adenosine are independently non-negatively charged internucleotide linkages. ("+" counts from the nucleoside opposite the target adenosine towards the 5'-end of the oligonucleotide, and the internucleotide linkage at position +1 is between the nucleoside opposite the target adenosine and its 5' adjacent nucleoside. An internucleotide linkage (eg, between N ₁ and N ₀ of 5′-N ₁ N ₀ N _-1 -3′, where N ₀ is the nucleoside opposite the target adenosine, as described herein); , "-" counts from the nucleoside toward the 3'-end of the oligonucleotide, and the internucleotide linkage at position -1 is an internucleotide linkage between the nucleoside opposite the target adenosine and its 3'-side adjacent nucleoside ( between N -1 and N ₀ of eg, 5'-N _{1 N 0} N _-1 -3', where N ₀ is the nucleoside opposite the target adenosine, as described herein). In some embodiments, the first internucleotidic linkage of an oligonucleotide is a non-negatively charged internucleotidic linkage. In some embodiments, the last internucleotidic linkage of an oligonucleotide is a non-negatively charged internucleotidic linkage. In some embodiments, the first and last internucleotidic linkages of an oligonucleotide are each independently non-negatively charged internucleotidic linkages. In some embodiments, +21, +20, +18, +17, +16, +15, +14, +13, +12, +11, +10, +6, +5, +4, and -2 One or more or all internucleotide linkages at position 1 are independently non-negatively charged internucleotide linkages (eg, phosphoryl guanidine internucleotide linkages such as n001). In some embodiments, +24, +23, +22, +19, +16, +15, +14, +13, +12, +11, +10, +6, +5, +4, -2, and one or more or all internucleotide linkages at position -5 are independently non-negatively charged internucleotide linkages (eg, phosphoryl guanidine internucleotide linkages such as n001). In some embodiments, at positions +23, +22, +19, +16, +15, +14, +13, +12, +11, +10, +6, +5, +4, and -2 One or more or all internucleotide linkages present are independently non-negatively charged internucleotide linkages (eg, phosphoryl guanidine internucleotide linkages such as n001). In some embodiments, the first and last internucleotide linkages of an oligonucleotide are independently non-negatively charged internucleotide linkages (eg, phosphoryl guanidine internucleotide linkages such as n001). In some embodiments, the first and last internucleotide linkages and +23, +22, +19, +16, +15, +14, +13, +12, +11, +10, +6, +5, + One or more or all internucleotide linkages at positions 4, and -2 are independently non-negatively charged internucleotide linkages (eg, phosphorylguanidine internucleotide linkages such as n001). In some embodiments, the first and last internucleotide linkages are both R p . In some embodiments, each phosphorothioate internucleotidic linkage is Sp . In some embodiments, the internucleotidic linkage at position -2 is a non-negatively charged internucleotidic linkage. In some embodiments, the internucleotide linkage at position -5 is a non-negatively charged internucleotide linkage. In some embodiments, the internucleotide linkage at position +5 is a non-negatively charged internucleotide linkage. In some embodiments, the internucleotidic linkage at position +9 is a non-negatively charged internucleotidic linkage. In some embodiments, the internucleotidic linkage at position +11 is a non-negatively charged internucleotidic linkage. In some embodiments, each internucleotide linkage at positions -2 and -5 is independently a non-negatively charged internucleotide linkage. In some embodiments, each internucleotide linkage at positions +5, -2 and -5 is independently a non-negatively charged internucleotide linkage. In some embodiments, each internucleotide linkage at positions +11, +9, -2 and -5 is independently a non-negatively charged internucleotide linkage. In some embodiments, each internucleotide linkage at positions +11, +9, +5, -2 and -5 is independently a non-negatively charged internucleotide linkage. In some embodiments, one or more or each of the 1st, 14th, 16th, 20th, 26th and 29th internucleotide linkages (from the 5'-end, unless otherwise specified) are independently negatively charged. It is a linkage between nucleotides. In some embodiments, the oligonucleotide is negatively charged with respect to the 5' side of the nucleoside opposite the target adenosine, except that the first internucleotide linkage of the oligonucleotide may optionally be a non-negatively charged internucleotide linkage. It does not contain unresolved internucleotide linkages. In some embodiments, the oligonucleotide does not contain internal non-negatively charged internucleotidic linkages except at position -2. In some embodiments, one or both of the first and last internucleotide linkages of the first domain are independently non-negatively charged internucleotide linkages. In some embodiments, one or both of the first and last internucleotide linkages of the second domain are independently non-negatively charged internucleotide linkages. In some embodiments, one or both of the first and last internucleotidic linkages of an oligonucleotide are independently non-negatively charged internucleotidic linkages. In some embodiments, both the first and last internucleotide linkages of the first domain are independently non-negatively charged internucleotide linkages. In some embodiments, both the first and last internucleotidic linkages of the second domain are independently non-negatively charged internucleotidic linkages. In some embodiments, both the first and last internucleotidic linkages of an oligonucleotide are independently non-negatively charged internucleotidic linkages. In some embodiments, each non-negatively charged internucleotidic linkage is independently a neutral internucleotidic linkage. In some embodiments, the non-negatively charged internucleotide linkages are phosphoryl guanidine internucleotide linkages. In some embodiments, each non-negatively charged internucleotidic linkage is independently a phosphoryl guanidine internucleotidic linkage. In some embodiments, the non-negatively charged internucleotide linkage is n001. In some embodiments, each non-negatively charged internucleotide linkage is independently n001. In some embodiments, each non-negatively charged internucleotidic linkage is independently R p, Sp , or non-chirally controlled. In some embodiments, one or more non-negatively charged internucleotidic linkages are not independently chirally controlled. In some embodiments, each non-negatively charged internucleotide linkage is independently chirally controlled. In some embodiments, one or more non-negatively charged internucleotidic linkages are independently chirally controlled. In some embodiments, each non-negatively charged internucleotide linkage is independently chirally controlled. In some embodiments, each internucleotidic linkage that is not negatively charged is R p. In some embodiments, each internucleotidic linkage that is not negatively charged is Sp . In some embodiments, a bond at the 3' position to inosine or deoxyinosine or to a 2'-modified inosine (e.g., 2'-OH replaced by a non-H moiety such as -F, -OMe, -MOE, etc.) A modified internucleotide linkage, eg n001, is either not chirally controlled or is chirally controlled and is Sp . In some embodiments, it is chirally controlled and is Sp . In some embodiments, a chirally controlled negatively charged S p internucleotidic linkage (e.g., a phosphoryl guanidine internucleotidic linkage such as n001) linked to the 3'-position of a nucleoside comprising hypoxanthine is Oligonucleotides and compositions thereof comprising provide various advantages over corresponding stereorandom or R p internucleotidic linkages, such as equal or better properties and/or activity, improved manufacturing efficiency and/or lower manufacturing cost, etc. . In some embodiments, a chirally controlled negatively charged S p internucleotidic linkage (e.g., a phosphoryl guanidine internucleotidic linkage such as n001) linked to the 3'-position of a nucleoside comprising hypoxanthine is The constructing process may be performed more easily (eg, higher reagent concentrations, smaller solution volumes, shorter reaction times, etc.) and/or at lower cost (eg, more readily accessible materials). It has been observed that can In some embodiments, oligonucleotides comprising a chirally controlled R p phosphorothioate internucleotidic linkage linked to the 3′-position of a nucleoside comprising hypoxanthine and compositions thereof are composed of the corresponding sterically random or Sp nucleotides It provides various advantages over liver linkages, such as equal or better properties and/or activity, improved manufacturing efficiency and/or lower manufacturing cost. In some embodiments, the process of constructing a chirally controlled R p phosphorothioate internucleotidic linkage linked to the 3′-position of a nucleoside comprising hypoxanthine is easier (e.g., at higher reagent concentrations) , smaller solution volume, shorter reaction time, etc.) and/or lower cost (eg, more readily accessible materials).

In some embodiments, an oligonucleotide comprises one or more (eg, 1-10, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more, etc.) natural phosphate linkages. In some embodiments, both nucleosides linked to a natural phosphate linkage are independently 2'-modified sugars. In some embodiments, both nucleosides linked to a majority (e.g., at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95% or more) of a natural phosphate linkage are independent. as per 2'-strain. In some embodiments, both nucleosides linked to each natural phosphate linkage are independently 2'-modified sugars. In some embodiments, a 2'-modified sugar is a bicyclic sugar or a 2'-OR modified sugar, where R is an optionally substituted C _1-6 aliphatic. In some embodiments, each 2'-modified sugar is independently a bicyclic sugar or a 2'-OR modified sugar, where R is an optionally substituted C _1-6 aliphatic. In some embodiments, each 2'-modified sugar is independently a 2'-OR modified sugar, where R is an optionally substituted C _1-6 aliphatic. In some embodiments, each 2'-modified sugar is independently a 2'-OMe modified sugar or a 2'-MOE modified sugar. In some embodiments, each 2'-modified sugar is independently a 2'-OMe modified sugar. In some embodiments, each 2'-modified sugar is independently a 2'-MOE modified sugar. In some embodiments, natural phosphate linkages are used with non-negatively charged internucleotide linkages (eg, phosphoryl guanidine internucleotide linkages such as n001). In some embodiments, the oligonucleotide comprises alternating natural phosphate linkages and non-negatively charged internucleotidic linkages (eg, phosphoryl guanidine internucleotide linkages such as n001) (see, eg, WV-43047) .

In some embodiments, the one or more internucleotide linkages at positions -1 and -2 are independently R p phosphorothioate internucleotide linkages. In some embodiments, -3, -2, -1, +1, +3, +4, +5, +7, +8, +9, +10, +11, +12, +13, +16, One or more internucleotide linkages at positions +17 and +18 are independently R p phosphorothioate internucleotidic linkages. In some embodiments, the internucleotide linkage at position -3 is an R p phosphorothioate internucleotidic linkage. In some embodiments, the internucleotidic linkage at position -2 is an R p phosphorothioate internucleotidic linkage. In some embodiments, the internucleotide linkage at position -1 is an R p phosphorothioate internucleotidic linkage. In some embodiments, the internucleotidic linkage at position +1 is an R p phosphorothioate internucleotidic linkage. In some embodiments, the internucleotidic linkage at position +3 is an R p phosphorothioate internucleotidic linkage. In some embodiments, the internucleotidic linkage at position +4 is an R p phosphorothioate internucleotidic linkage. In some embodiments, the internucleotidic linkage at position +5 is an R p phosphorothioate internucleotidic linkage. In some embodiments, the internucleotidic linkage at position +7 is an R p phosphorothioate internucleotidic linkage. In some embodiments, the internucleotidic linkage at position +8 is an R p phosphorothioate internucleotidic linkage. In some embodiments, the internucleotidic linkage at position +9 is an R p phosphorothioate internucleotidic linkage. In some embodiments, the internucleotidic linkage at position +10 is an R p phosphorothioate internucleotidic linkage. In some embodiments, the internucleotide linkage at position +11 is an R p phosphorothioate internucleotidic linkage. In some embodiments, the internucleotidic linkage at position +12 is an R p phosphorothioate internucleotidic linkage. In some embodiments, the internucleotidic linkage at position +13 is an R p phosphorothioate internucleotidic linkage. In some embodiments, the internucleotidic linkage at position +16 is an R p phosphorothioate internucleotidic linkage. In some embodiments, the internucleotidic linkage at position +17 is an R p phosphorothioate internucleotidic linkage. In some embodiments, the internucleotidic linkage at position +18 is an R p phosphorothioate internucleotidic linkage. In some embodiments, an oligonucleotide contains only one R p phosphorothioate internucleotidic linkage. In some embodiments, an oligonucleotide contains two or fewer. In some embodiments, an oligonucleotide contains 3 or fewer. In some embodiments, an oligonucleotide contains 4 or fewer. In some embodiments, an oligonucleotide contains 5 or fewer.

In some embodiments, the negatively charged internucleotidic linkage attached to the 3'-carbon of dI is S p. In some embodiments, the negatively charged internucleotidic linkage attached to the 3'-carbon of dI is S p. In some embodiments, the phosphoryl guanidine internucleotide linkage attached to the 3'-carbon of dI is S p. In some embodiments, the n001 internucleotidic linkage attached to the 3'-carbon of dI is Sp . In some embodiments, each non-negatively charged internucleotidic linkage attached to the 3'-carbon of dI is independently Sp . In some embodiments, each neutral internucleotidic linkage attached to the 3'-carbon of dI is independently Sp . In some embodiments, each phosphoryl guanidine internucleotide linkage attached to the 3'-carbon of dI is independently Sp . In some embodiments, each n001 bonded to the 3'-carbon of dI is independently Sp .

In some embodiments, the controlled level of an oligonucleotide in a composition is a desired oligonucleotide. In some embodiments, among all oligonucleotides in a composition that share a common base sequence (eg, a sequence desired for a purpose), or among all oligonucleotides in a composition, the desired oligonucleotide may be present in various forms (eg, salt form). and generally differ only in non-chiral controlled internucleotidic linkages (various forms of the same stereoisomer may be considered identical for this purpose), the level of about 5% to 100%, 10% to 100%, 20% ~100%, 30%~100%, 40%~100%, 50%~100%, 60%~100%, 70%~100%, 80~100%, 90~100%, 95~100%, 50 %~90%, about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75% , 80%, 85%, 90%, 95%, or 100%, or at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55 %, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99%. In some embodiments, the level is at least about 50%. In some embodiments, the level is at least about 60%. In some embodiments, the level is at least about 70%. In some embodiments, the level is at least about 75%. In some embodiments, the level is at least about 80%. In some embodiments, the level is at least about 85%. In some embodiments, the level is at least about 90%. In some embodiments, the level is (DS) ^nc or higher, and the DS is about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5%. and nc is the number of chirally controlled internucleotide linkages as described herein (e.g., 1-50, 1-40, 1-30, 1-25, 1-20, 5-50, 5-40, 5 ~30, 5~25, 5~20, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 , 21, 22, 23, 24, 25 or more). In some embodiments, the level is (DS) ^nc or higher, and the DS is between 95% and 100%.

Various types of internucleotidic linkages may be used in combination with other structural elements, such as sugars, to achieve the desired oligonucleotide properties and/or activities. For example, the present invention routinely uses modified internucleotide linkages and modified sugars, optionally along with natural phosphate linkages and natural sugars, in designing oligonucleotides. In some embodiments, the present invention provides oligonucleotides comprising one or more modified sugars. In some embodiments, the present invention provides an oligonucleotide comprising one or more modified sugars and one or more modified internucleotidic linkages, at least one of which is a natural phosphate linkage.

In some embodiments, a given oligonucleotide is selected from multiple (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 , 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 or more, two or more or all of which are optionally consecutive) of natural RNA sugars. In some embodiments, such oligonucleotides are modified sugars at one or both ends, for example, 2' modifications (eg, 2'-F, etc.) and/or 2'-OR modifications (R is -H). not) (eg, 2-OME, 2-MOE, etc.), and/or various modified internucleotide linkages (eg, phosphorothioate internucleotide linkages, non-negatively charged internucleotide linkages, etc.). In some embodiments, one or more such 2'-OR modifications (R is -H not) exists. In some embodiments, the 3'-end has one or more, for example 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more such 2'-OR variants (R is -H not) exists. In some embodiments, each 2'-modified sugar is independently a 2'-OR modified sugar (R is not -H). In some embodiments, as described herein, 2'-OR is 2'-OMe. In some embodiments, a 2'-OR is a 2'-MOE. In some embodiments, each 2'-OR is independently 2'-OMe or 2'-MOE. In some embodiments, each 2'-OR is 2'-OMe.

In some embodiments, the stability of various internucleotide linkages is assessed. In some embodiments, internucleotidic linkages are formed under various conditions used to prepare oligonucleotides, including, for example, reagents, solvents, temperatures (in some cases above room temperature), cleavage conditions, deprotection conditions, purification conditions, and the like. It is exposed to solid phase oligonucleotide synthesis and stability is assessed. In some embodiments, stable internucleotide linkages (e.g., 10%, 9%, 8%, 7%, 6%, 5%, 4% after exposure to one or more conditions and/or processes or complete oligonucleotide manufacturing process) , 3%, 2%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2% or 0.1%) of various oligonucleotide compositions and applications. selected for use in

Additional Chemical Moieties

In some embodiments, oligonucleotides include one or more additional chemical moieties. A variety of additional chemical moieties are known in the art, such as targeting moieties, carbohydrate moieties, lipid moieties, and the like, and may include properties and/or activities of a given oligonucleotide, such as stability, half-life, activity, delivery, It can be used according to the present invention to modulate pharmacodynamic properties, pharmacokinetic properties and the like. In some embodiments, certain additional chemical moieties facilitate delivery of the oligonucleotide to desired cells, tissues and/or organs, including but not limited to cells of the central nervous system. In some embodiments, certain additional chemical moieties promote internalization of the oligonucleotide. In some embodiments, certain additional chemical moieties increase oligonucleotide stability. In some embodiments, the present invention provides techniques for incorporating various additional chemical moieties into oligonucleotides.

In some embodiments, the additional chemical moiety is or comprises a small molecule moiety. In some embodiments, the small molecule is a ligand of a protein (eg, a receptor). In some embodiments, a small molecule binds a polypeptide. In some embodiments, the small molecule is an inhibitor of a polypeptide. In some embodiments, the additional chemical moiety is or comprises a peptide moiety (eg, an antibody). In some embodiments, the additional chemical moiety is or comprises a nucleic acid moiety. In some embodiments, nucleic acids provide new properties and/or activities. In some embodiments, the nucleic acid moiety forms a duplex or other secondary structure with the original oligonucleotide chain (prior to conjugation) or a portion thereof. In some embodiments, a nucleic acid is or comprises an oligonucleotide that targets the same or a different target and may exert its action through the same or a different mechanism. In some embodiments, the nucleic acid is or comprises an RNAi agent. In some embodiments, a nucleic acid is or comprises a miRNA agent. In some embodiments, the nucleic acid is dependent on or comprises RNase H. In some embodiments, a nucleic acid is or comprises a gRNA. In some embodiments, a nucleic acid is or comprises an aptamer. In some embodiments, the additional chemical moiety is or comprises a carbohydrate moiety as described herein. Many useful agents, such as small molecules, peptides, carbohydrates, nucleic acid agents, and the like, can be conjugated to oligonucleotides herein according to the present invention.

In some embodiments an oligonucleotide comprising an additional chemical moiety has increased delivery to and/or release to a tissue compared to a reference oligonucleotide, e.g., an otherwise identical reference oligonucleotide that does not have the additional chemical moiety. show activity.

In some embodiments, non-limiting examples of additional chemical moieties include carbohydrate moieties, targeting moieties, etc., which can improve one or more properties when incorporated into an oligonucleotide. In some embodiments, the additional chemical moiety is selected from glucose, GluNAc (N-acetyl amine glucosamine) and anisamide moieties. In some embodiments, a provided oligonucleotide can include two or more additional chemical moieties, wherein the additional chemical moieties are of the same category (e.g., carbohydrate moieties, sugar moieties, targeting moieties, etc.) ) or not in the same category.

In some embodiments, the additional chemical moiety is a targeting moiety. In some embodiments, the additional chemical moiety is or comprises a carbohydrate moiety. In some embodiments, the additional chemical moiety is or comprises a lipid moiety. In some embodiments, the additional chemical moiety is or comprises, for example, a ligand moiety for a cellular receptor, such as a sigma receptor, an asialoglycoprotein receptor, and the like. In some embodiments, a ligand moiety is or comprises an anisamide moiety, which can be a ligand moiety for a sigma receptor. In some embodiments, a ligand moiety is or comprises a GalNAc moiety that can be a ligand moiety for an asialoglycoprotein receptor. In some embodiments, additional chemical moieties facilitate delivery to the liver.

In some embodiments, provided oligonucleotides may include one or more linkers and additional chemical moieties (e.g., targeting moieties), may be chirally controlled or non-chirally controlled, and/or described herein and/or one or more variations and/or formats.

A variety of linkers, carbohydrate moieties and targeting moieties can be used in accordance with the present invention, including many known in the art. In some embodiments, the carbohydrate moiety is a targeting moiety. In some embodiments, the targeting moiety is a carbohydrate moiety.

In some embodiments, a provided oligonucleotide comprises an additional chemical moiety suitable for delivery, such as glucose, GluNAc (N-acetyl amine glucosamine), anisamide, or a structure selected from:

. In some embodiments, n is 1. In some embodiments, n is 2. In some embodiments, n is 3. In some embodiments, n is 4. In some embodiments, n is 5. In some embodiments, n is 6. In some embodiments, n is 7. In some embodiments, n is 8.

In some embodiments, the additional chemical moiety is any of those described in the Examples, including examples of various additional chemical moieties incorporated into various oligonucleotides.

In some embodiments, additional chemical moieties conjugated to oligonucleotides can target the oligonucleotides to cells of the central nervous system.

In some embodiments, the additional chemical moiety comprises or is a cell receptor ligand. In some embodiments, the additional chemical moiety comprises or is a protein binder, eg binds to a cell surface protein. In particular, such moieties may be useful for targeted delivery of oligonucleotides to cells expressing the corresponding receptor or protein. In some embodiments, additional chemical moieties of a provided oligonucleotide include anisamide or a derivative or analog thereof, and the oligonucleotide is capable of targeting cells expressing a specific receptor, such as the sigma 1 receptor.

In some embodiments, provided oligonucleotides are formulated for administration to body cells and/or tissues expressing a target. In some embodiments, an additional chemical moiety conjugated to an oligonucleotide can target the oligonucleotide to a cell.

In some embodiments, additional chemical moieties are optionally substituted phenyl;

wherein n' is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, each other variable being as described herein. In some embodiments, R ^s is F. In some embodiments, R ^s is OMe. In some embodiments, R ^s is OH. In some embodiments, R ^s is NHAc. In some embodiments, R ^s is NHCOCF ₃ . In some embodiments, R' is H. In some embodiments, R is H. In some embodiments, R ^2s is NHAc and R ^5s is OH. In some embodiments, R ^2s is p-anisoyl and R ^5s is OH. In some embodiments, R ^2s is NHAc and R ^5s is p-anisoyl. In some embodiments, R ^2s is OH and R ^5s is p-anisoyl. In some embodiments, additional chemical moieties are

is selected from In some embodiments, n' is 1. In some embodiments, n' is 0. In some embodiments, n" is 1. In some embodiments, n" is 2.

In some embodiments, the additional chemical moiety is or comprises an asialoglycoprotein receptor (ASGPR) ligand.

Without wishing to be bound by any particular theory, the present invention notes that ASGPR1 has also been reported to be expressed in the mouse hippocampal region and/or cerebellar Purkinje cell layer. http://mouse.brain-map.org/experiment/show/2048

A variety of other ASGPR ligands are known in the art and can be used in accordance with the present invention. In some embodiments, an ASGPR ligand is a carbohydrate. In some embodiments, the ASGPR ligand is GalNac or a derivative or analog thereof. In some embodiments, ASGPR ligands are described in Sanhueza et al. J. Am. Chem. Soc., 2017, 139 (9), pp 3528-3536]. In some embodiments, ASGPR ligands are described in Mamidyala et al. J. Am. Chem. Soc., 2012, 134, pp 1978-1981. In some embodiments, an ASGPR ligand is described in US 20160207953. In some embodiments, the ASGPR ligand is a substituted-6,8-dioxabicyclo[3.2.1]octane-2,3-diol derivative disclosed, for example, in US 20160207953. In some embodiments, an ASGPR ligand is described, for example, in US 20150329555. In some embodiments, the ASGPR ligand is a substituted-6,8-dioxabicyclo[3.2.1]octane-2,3-diol derivative disclosed, for example, in US 20150329555. In some embodiments, an ASGPR ligand is described in US 8877917, US 20160376585, US 10086081, or US 8106022. ASGPR ligands described in these documents are incorporated herein by reference. Those skilled in the art will understand that a variety of techniques are known in the art and can be used in accordance with the present invention to assess the binding of chemical moieties to ASGPR, including those described in these documents. In some embodiments, provided oligonucleotides are conjugated to ASGPR ligands. In some embodiments, a provided oligonucleotide comprises an ASGPR ligand. In some embodiments, an additional chemical moiety comprising an ASGPR ligand is , and each variable is independently as described herein. In some embodiments, R is -H. In some embodiments, R' is -C(0)R.

In some embodiments, additional chemical moieties are is or includes In some embodiments, additional chemical moieties are is or includes In some embodiments, additional chemical moieties are is or includes In some embodiments, additional chemical moieties are is or includes In some embodiments, the additional chemical moiety is optionally substituted is or includes In some embodiments, additional chemical moieties are is or includes In some embodiments, additional chemical moieties are is or includes In some embodiments, additional chemical moieties are is or includes In some embodiments, additional chemical moieties are is or includes

In some embodiments, the additional chemical moiety comprises one or more moieties capable of binding, for example, an oligonucleotide target cell. For example, in some embodiments, the additional chemical moiety comprises one or more protein ligand moieties, for example, in some embodiments, the additional chemical moiety is multiple moieties, each of which is independently an ASGPR ligand. ). In some embodiments, as in Mod 001 and Mod083, additional chemical moieties include three such ligands.

Mod001:

Mod083:

In some embodiments, oligonucleotides and each variable is independently as described herein. In some embodiments, each -OR' is -OAc and -N(R') ₂ is -NHAc. In some embodiments, oligonucleotides includes In some embodiments, each R' is -H. In some embodiments, each -OR' is -OH and each -N(R') ₂ is -NHC(O)R. In some embodiments, each -OR' is -OH and each -N(R') ₂ is -NHAc. In some embodiments, oligonucleotides (L025). In some embodiments, a -CH ₂ -linkage is used as the C5 linkage in a sugar. In some embodiments, the linkage on the ring is used as the C3 linkage in the sugar. These moieties are Phosphoamidites such as, for example can be introduced using (one skilled in the art knows that protecting groups for one or more other groups such as -OH, -NH ₂ -, -N(i-Pr) ₂ , -OCH ₂ CH ₂ CN, etc. may alternatively be used; It is understood that it may be removed under a variety of suitable conditions, sometimes during oligonucleotide deprotection and/or cleavage steps). In some embodiments, an oligonucleotide is two, three or more (eg, three or less) includes In some embodiments, an oligonucleotide is two, three or more (eg, three or less) includes In some embodiments, copies of such moieties are linked by internucleotide linkages as described herein, eg, natural phosphate linkages. In some embodiments, when at the 5'-end, the -CH ₂ - linkage is bonded to -OH. In some embodiments, oligonucleotides includes In some embodiments, oligonucleotides includes In some embodiments, each -OR' is -OAc and -N(R') ₂ is -NHAc. In some embodiments, oligonucleotides includes especially, has comparable and / or superior activity and / or properties can be used to introduce In some embodiments, it is the same number of (e.g., compared to Mod001) for improved manufacturing efficiency and/or lower cost.

In some embodiments, the additional chemical moiety is a Mod group described herein (eg, Table 1).

In some embodiments, the additional chemical moiety is Mod001. In some embodiments, the additional chemical moiety is Mod083. In some embodiments, an additional chemical moiety, eg, a Mod group, is conjugated directly (eg, without a linker) to the rest of the oligonucleotide. In some embodiments, additional chemical moieties are conjugated to the remainder of the oligonucleotide via a linker. In some embodiments, additional chemical moieties, such as Mod groups, can be linked directly and/or via linkers to nucleobases, sugars, and/or internucleotide linkages of the oligonucleotide. In some embodiments, the Mod group is linked directly or via a linker to the sugar. In some embodiments, the Mod group is linked directly or via a linker to the 5'-end sugar. In some embodiments, the Mod group is linked directly or through a linker to the 5' end sugar through the 5' carbon. See, for example, the various oligonucleotides in Table 1. In some embodiments, the Mod group is linked directly or via a linker to the 3'-end sugar. In some embodiments, the Mod group is linked directly or through a linker to the 3' end sugar through the 3' carbon. In some embodiments, the Mod group is linked directly or through a linker to the nucleobase. In some embodiments, Mod groups are linked directly or via a linker to an internucleotidic linkage. In some embodiments, a provided oligonucleotide comprises Mod001 linked to the 5'-end of the oligonucleotide chain via L001.

As will be appreciated by those skilled in the art, additional chemical moieties may be oligomers at various positions (e.g., sugars, bases, internucleotidic linkages, etc.), e.g., at the 5'-end, 3'-end, or intermediate positions. Can be linked to nucleotide chains. In some embodiments, it is linked to the 5'-end. In some embodiments, it is linked to the 3'-end. In some embodiments, it is linked to a nucleotide in the middle.

Certain additional chemical moieties (e.g., lipid moieties, targeting moieties, carbohydrate moieties), including but not limited to Mod012, Mod039, Mod062, Mod085, Mod086, and Mod094, and additional chemical moieties Various linkers (including but not limited to L001, L003, L004, L008, L009, and L010) for linking oligonucleotide chains are described in WO 2017/062862, WO 2018/067973, WO 2017/160741, WO 2017/ 192679, WO 2017/210647, WO 2018/098264, WO 2018/022473, WO 2018/223056, WO 2018/223073, WO 2018/223081, WO 2018/237194, WO 2019/032607, WO 2019/032612, WO 2019/ 055951, WO 2019/075357, WO 2019/200185, WO 2019/217784, WO 2019/032612, WO 2020/191252, and/or WO 2021/071858, wherein each additional chemical moiety and linker is independently are incorporated herein by reference and may be used in accordance with the present invention. In some embodiments, the additional chemical moiety is digoxigenin or biotin or a derivative thereof.

In some embodiments, the oligonucleotide comprises a linker, eg, L001 L004, L008, and/or an additional chemical moiety, eg, Mod012, Mod039, Mod062, Mod085, Mod086, or Mod094. In some embodiments, a linker, eg, L001, L003, L004, L008, L009, L110, etc., is linked to a Mod, eg, Mod012, Mod039, Mod062, Mod085, Mod086, Mod094, etc.

L001: -CH ₂ - Linked to Mod via -NH-, if present, as shown in linkage site, phosphate linkage (-OP(O)(OH, which can exist in salt form and can be represented by O or PO) )-O-) or a phosphorothioate linkage (which can be in salt form and can be represented by * if the phosphorothioate is not chirally controlled; or the phosphorothioate is chirally controlled and has the Sp configuration -OP(O)(SH)-, which can be denoted by *S, S, or Sp if the phosphorothioate is chirally controlled and has the Rp configuration, or by *R, R, or Rp -NH-(CH ₂ ) ₆ - linker (also known as C6 linker, C6 amine linker or C6 amino linker) linked to the 5'-end or 3'-end of the oligonucleotide chain via O-). If Mod is not present, L001 is connected to -H through -NH-;

L003: Linker. In some embodiments, it is linked to Mod (-H if Mod is absent) via an amino group, if present, for example a linkage (e.g., a phosphate linkage (O or PO) or a phosphorothioate linkage (chiral linked to the 5'-end or the 3'-end of the oligonucleotide chain via an uncontrolled or chirally controlled ( S p or Rp )));

L004: A linker having a structure of -NH(CH ₂ ) ₄ CH(CH ₂ OH)CH ₂ - (where -NH- is linked to Mod(-C(O)-) or -H, and -CH ₂ - The linking site is a linkage, for example a phosphodiester (-OP(O)(OH)-O- which can exist in salt form and can be represented by O or PO), phosphorothioate (which can exist in salt form can be represented by * if the phosphorothioate is not chirally controlled; or *S, S, or Sp if the phosphorothioate is chirally controlled and has the Sp configuration, or by phosphorothioate. -OP(O)(SH)-O-, which may be represented by *R, R, or Rp, if the thioate is chirally controlled and has the Rp configuration, or phosphorodithioate (which may exist in salt form; linked to the oligonucleotide chain (e.g., at the 3'-end) via an -OP(S)(SH)-O-) linkage which may be denoted PS2 or D). For example, an asterisk immediately preceding L004 (eg, *L004) indicates that the linkage is a phosphorothioate linkage, and an absence of an asterisk immediately preceding L004 indicates that the linkage is a phosphodiester linkage. For example, in the oligonucleotide ending in ...mAL004, the linker L004 is phospholipid at the 3' position of the 3'-end sugar (via the -CH ₂ - site) (2'-OMe modified and linked to nucleobase A). linked through a fordiester linkage, and the L004 linker is linked through -NH- to -H. Similarly, in one or more oligonucleotides, the L004 linker is linked via a phosphodiester linkage to the 3' position of the 3'-terminal sugar (via a -CH ₂ - site), and L004 is linked via -NH-, for example Connected to Mod012, Mod085, Mod086, etc;

L008: Linker having the structure of -C(O)-(CH ₂ ) ₉ -, where -C(O)- is connected to Mod (through -NH-) or -OH (when Mod is not indicated) and the -CH ₂ -linkage is a linkage, for example, a phosphodiester (-OP(O)(OH)-O-, which can exist in salt form and can be represented by O or PO), phosphorothioate ( It can exist in salt form and can be represented by * if the phosphorothioate is not chirally controlled; or can be represented by *S, S, or Sp if the phosphorothioate is chirally controlled and has the Sp configuration , or -OP(O)(SH)-O-, which may be represented by *R, R, or Rp, if the phosphorothioate is chirally controlled and has the Rp configuration, or phosphorodithioate (in salt form) may be present and linked to the oligonucleotide chain (e.g., at the 5'-end) via an -OP(S)(SH)-O-) linkage, which may be denoted PS2 or D). For example, having a sequence of 5'-L008mN * mN * mN * mN * N * N * N * N * N * N * N * N * N * N * mN * mN * mN * mN-3', In an exemplary oligonucleotide having the stereochemistry/linkage of OXXXXXXXXX XXXXXXXX (N is a base, O is a natural phosphate internucleotidic linkage, and X is a stereorandom phosphorothioate), L008 is via -C(O)- -OH and linked to the 5'-end of the oligonucleotide chain via a phosphate linkage (denoted by "O" in "Stereochemistry/Linkage");5'-Mod062L008mN * mN * mN * mN * N * N * N * N * N * N * N * N * N * N * mN * mN * mN * mN-3', and the stereo of OXXXXXXXXX XXXXXXXX In another exemplary oligonucleotide with chemistry/linkage (N is a base), L008 is linked to Mod062 via -C(O)- and a phosphate linkage (denoted by "O" in "Stereochemistry/Linkage") linked to the 5'-end of the oligonucleotide chain via;

L009: -CH ₂ CH ₂ CH ₂ -. In some embodiments, when L009 is present at the 5'-end of an oligonucleotide without Mod, one end of L009 is linked to -OH and the other end is linked to, for example, a linkage (e.g., a phosphate linkage (O or PO) or a phosphorothioate linkage (which may be non-chirally controlled or chirally controlled (Sp or Rp)) to the 5'-carbon of the oligonucleotide chain;

L010: . L010 is linked to other moieties, eg L023, L010, oligonucleotide chains, etc., through various linkages (eg n001; typically phosphate if not indicated). L010 is bonded to -OH when the other moiety is absent. For example, in the WV-39202, L010 is used with n001R L010n001R with the structure of , and the configuration of the connecting phosphorus is R p. In some implementations, multiple L010n001R may be used. For example, WV-39202 contains L023L010n001RL010n001RL010n001R having the following structure (bonded to the 5'-carbon at the 5'-end of the oligonucleotide chain, each linking phosphorus being independently R p):

;

Mod012 (in some embodiments, -C(O)- is connected to -NH- of a linker such as L001, L004, L008, etc.):

;

Mod039 (in some embodiments, -C(O)- is connected to -NH- of a linker such as L001, L003, L004, L008, L009, L110, etc.):

Mod062 (in some embodiments, -C(O)- is connected to -NH- of a linker such as L001, L003, L004, L008, L009, L110, etc.):

;

Mod085 (in some embodiments, -C(O)- is linked to -NH- of a linker such as L001, L003, L004, L008, L009, L110, etc.):

;

Mod086 (in some embodiments, -C(O)- is connected to -NH- of a linker such as L001, L003, L004, L008, L009, L110, etc.):

;

Mod094 (in some embodiments, linked to the 5'-end or 3'-end of the oligonucleotide via an internucleotidic linkage or via a linkage, e.g., a phosphate linkage, a phosphorothioate linkage (optionally chirally controlled), etc. For example, the sequence of 5'-mN * mN * mN * mN * N * N * N * N * N * N * N * N * N * N * mN * mN * mN * mNMod094-3' In an exemplary oligonucleotide with a stereochemistry/linkage of XXXXX XXXXX XXXXX XXO (N is a base), Mod094 is a phosphate group (not shown below, and may exist in salt form; in "Stereochemistry/Linkage" linked to the 3'-end (3'-carbon of the 3'-end sugar) of the oligonucleotide chain via the denoted "O" (...XXXX O )):

.

In some embodiments, additional chemical moieties (eg, linkers, lipids, solubilizing groups, conjugate groups, targeting groups and/or targeting ligands) are those described in WO 2012/030683 or WO 2021/030778. In some embodiments, provided oligonucleotides are described in WO 2012/030683, WO 2021/030778, WO 2019112485, US 20170362270, WO 2018156056, or WO 2018056871, WO 2021/030778, WO 2020/154344, WO 2 020/154343, WO 2020/ 154342, WO 2020/165077, WO 2020/201406, WO 2020/216637, or WO 2020/252376 (eg, linkers, lipids, solubilizing groups, and/or targeting ligands).

In some embodiments, a provided oligonucleotide can contain additional chemical moieties (eg, targeting groups, conjugate groups, etc.) and/or modifications (eg, nucleobases, sugars, internucleotidic linkages, etc.) described below. Includes: U.S. Patent Nos. 5,688,941; 6,294,664; 6,320,017; 6,576,752; 5,258,506; 5,591,584; 4,958,013; 5,082,830; 5,118,802; 5,138,045; 6,783,931; 5,254,469; 5,414,077; 5,486,603; 5,112,963; 5,599,928; 6,900,297; 5,214,136; 5,109,124; 5,512,439; 4,667,025; 5,525,465; 5,514,785; 5,565,552; 5,541,313; 5,545,730; 4,835,263; 4,876,335; 5,578,717; 5,580,731; 5,451,463; 5,510,475; 4,904,582; 5,082,830; 4,762,779; 4,789,737; 4,824,941; 4,828,979; 5,595,726; 5,214,136; 5,245,022; 5,317,098; 5,371,241; 5,391,723; 4,948,882; 5,218,105; 5,112,963; 5,567,810; 5,574,142; 5,578,718; 5,608,046; 4,587,044; 4,605,735; 5,585,481; 5,292,873; 5,552,538; 5,512,667; 5,597,696; 5,599,923; 7,037,646; 5,587,371; 5,416,203; 5,262,536; 5,272,250; or 8,106,022.

In some embodiments, additional chemical moieties, such as Mod, are linked via a linker. A variety of linkers are available in the art and can be used in accordance with the present invention (e.g., those used to conjugate various moieties to proteins (e.g., antibodies to form antibody-drug conjugates), nucleic acids, etc.) . Certain useful linkers are described in US 9982257, US 20170037399, US 20180216108, US 20180216107, US 9598458, WO 2017/062862, WO 2018/067973, WO 2017/160741, WO 2017/19267 9, WO 2017/210647, WO 2018/098264, WO 2018/223056, WO 2018/237194, WO 2019/032607, WO 2019/055951, WO 2019/075357, WO 2019/200185, WO 2019/217784, WO 2019/032612, WO 2020/191 252, and/or WO 2021 /071858, each linker moiety independently incorporated herein by reference. In some embodiments, the linker is L001, L004, L009 or L010 as non-limiting examples. In some embodiments, an oligonucleotide comprises a linker, but no additional chemical moiety other than the linker. In some embodiments, the oligonucleotide comprises a linker, but contains no additional chemical moiety other than the linker, and the linker is L001, L004, L009, or L010. In some embodiments, a linker is or comprises a moiety having a structure of internucleotidic linkages as described herein. In some embodiments, such moieties in a linker do not connect two nucleosides. In some embodiments, the linker has the structure of L. In some embodiments, a linker is divalent. In some embodiments, a linker is multivalent. In some embodiments, a linker may connect two or more additional chemical moieties to an oligonucleotide chain as described herein. For example, in some embodiments, one or two or three or more additional chemical moieties, e.g., a GalNAc moiety, are added to the oligonucleotide chain (e.g., at the 5'-end) via a multivalent linker moiety. ) is connected to

In some embodiments, eg, after administration to a system, cell, tissue, organ, subject, etc., additional chemical moieties are cleaved from the remainder of the oligonucleotide, eg, the oligonucleotide chain. In some embodiments, the additional chemical moiety facilitates, increases and/or accelerates delivery to a particular cell, and after delivery of the oligonucleotide to such cell, the additional chemical moiety is cleaved from the oligonucleotide. In some embodiments, a linker moiety comprises one or more cleavable moieties that can be cleaved at a desired location (eg, within a particular type of cell, in a subcellular compartment such as a lysosome, etc.) and/or at a desired time. In some embodiments, the cleavable moiety is selectively cleaved by a polypeptide, eg, an enzyme such as a nuclease. Many useful cleavable moieties and cleavable linkers have been reported and can be used in accordance with the present invention. In some embodiments, the cleavable moiety is or comprises one or more functional groups selected from amides, esters, ethers, phosphodiesters, disulfides, carbamates, and the like. In some embodiments, a linker is described in WO 2012/030683, WO 2021/030778, WO 2020/154344, WO 2020/154343, WO 2020/154342, WO 2020/165077, WO 2020/201406, WO 2020/216637, or WO 2020 As described in /252376.

As indicated herein, in some embodiments, provided technologies are specific structural elements that are required and/or reported to be necessary (e.g., strains, interconnection arrangements and/or patterns, etc., e.g., as reported in WO 2019/219581). (provided that these specific structural elements can be incorporated into oligonucleotides in combination with various other structural elements according to the present invention). For example, in some embodiments, an oligonucleotide of the invention has fewer nucleosides in the 3' direction to the nucleoside opposite the target nucleoside (e.g., target adenosine), contains one or more phosphorothioate internucleotide linkages at one or more positions where phosphorothioate internucleotide linkages are reported to be unfavorable or disallowed, and/or Sp phosphorothioate internucleotide linkages are not favored or tolerated; contains one or more Sp phosphorothioate internucleotidic linkages at one or more positions reported not to be favorable and/or one or more Rp phosphorothioate internucleotidic linkages at one or more positions reported to be unfavorable or unacceptable; Contains porothioate internucleotide linkages and/or other modifications at one or more positions compared to those reported to be beneficial or required for a particular oligonucleotide property and/or activity (e.g., internucleotide linkage modifications, sugar modifications, etc.) and/or stereochemistry (e.g., presence of 2'-MOE, absence of phosphorothioate linkages at specific positions, absence of Sp phosphorothioate linkages at specific positions, and/or Rp at specific positions). The absence of phosphorothioate linkages has been reported to be advantageous or necessary for certain oligonucleotide properties and/or activities; as shown herein, the technology provided is the use of a 2'-MOE, phosphorothioate at one or more of these specific positions. avoidance of 8 linkages, avoidance of Sp phosphorothioate linkages at one or more of these specific positions, and/or avoidance of Rp phosphorothioate linkages at one or more of these specific positions, will provide the desired properties and/or high activity. can). Additionally or alternatively, provided oligonucleotides may have specific modifications (e.g., base modifications, sugar modifications (eg, 2'-F), linkage modifications (eg, non-negatively charged internucleotidic linkages), additional moieties etc.) and previously unrecognized structural elements such as the use of levels, patterns, and combinations thereof.

For example, in some embodiments, as described herein, provided oligonucleotides have up to 5, 6, 7, 8, 9, contains 10, 11 or 12 nucleosides.

Alternatively or additionally, as described herein (eg, exemplified in certain examples), for structural elements in the 3' direction relative to the nucleoside opposite the target nucleoside (eg, target adenosine) , in some embodiments, about 50% to 100% (e.g., about or at least about 50%) of the internucleotide linkages in the 3' direction to the nucleoside opposite the target nucleoside (e.g., target adenosine); 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%) are each independently a modified internucleotide linkage, optionally chirally controlled. In some embodiments, up to 1, 2, or 3 internucleotide linkages in the 3' direction to the nucleoside opposite the target nucleoside are natural phosphate linkages. In some embodiments, these internucleotidic linkages are not natural phosphate linkages. In some embodiments, at most one such internucleotidic linkage is a natural phosphate linkage. In some embodiments, up to two such internucleotidic linkages are natural phosphate linkages. In some embodiments, up to three such internucleotidic linkages are natural phosphate linkages. In some embodiments, each modified internucleotide linkage is independently a phosphorothioate or non-negatively charged internucleotide linkage (eg, n001). In some embodiments, each phosphorothioate internucleotidic linkage is chirally controlled. In some embodiments, up to 1, 2, or 3 internucleotide linkages in the 3' direction to the nucleoside opposite the target nucleoside are R p phosphorothioate internucleotide linkages. In some embodiments, the internucleotide linkage linked to the nucleoside opposite the target nucleoside at the 3'-position of the sugar (considered position -1) is an R p phosphorothioate internucleotide linkage. In some embodiments, it is the only R p phosphorothioate internucleoside linkage in the 3' direction to the nucleoside opposite the target nucleoside. In some embodiments, an internucleotide linkage at position -3 relative to the nucleoside opposite the target nucleoside (e.g., N ₀ is the nucleoside opposite the target nucleoside, ...N ₀ N _-1 N The internucleotidic linkage linking N _-2 and N _-3 for _-2 N _-3 ...) is not an R p phosphorothioate internucleotidic linkage. In some embodiments, the internucleotide linkage at position -6 relative to the nucleoside opposite the target nucleoside is not an R p phosphorothioate internucleotide linkage. In some embodiments, the internucleotide linkage at positions -4 and/or -5 relative to the nucleoside opposite the target nucleoside is independently a modified internucleotide linkage, e.g., a phosphorothioate internucleotide linkage, independently R p phosphorothioate internucleotidic linkages. In some embodiments, one or more or all internucleotide linkages at positions -1, -3, -4, -5, and -6 are each independently an S p internucleotide linkage. In some embodiments, one or more or all internucleotide linkages at positions -1, -3, -4, -5, and -6 are each independently Sp phosphorothioate internucleotide linkages. In some embodiments, the internucleotide linkage(s) at position(s) -4 and/or -5 are each independently an R p internucleotidic linkage. In some embodiments, the internucleotide linkage(s) at position(s) -4 and/or -5 are each independently an Rp phosphorothioate internucleotide linkage. In many embodiments, at most 1, 2, 3, 4, or 5 internucleotide linkages are Rp phosphorothioate internucleotide linkages.

Alternatively or additionally, as described herein (eg, exemplified in certain examples), in some embodiments, in the 5' direction to the nucleoside opposite the target nucleoside (eg, target adenosine). between about 50% and 100% (e.g., about or at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of the internucleotide linkages in ) are each independently a modified internucleotide linkage, optionally chirally controlled. In some embodiments, zero or at most 1, 2, or 3 internucleotide linkages in the 5' direction to the nucleoside opposite the target nucleoside (eg, target adenosine) are not modified internucleotide linkages. In some embodiments, zero or at most 1, 2, or 3 internucleotide linkages in the 5' direction to the nucleoside opposite the target nucleoside (eg, target adenosine) are phosphorothioate internucleotide linkages. no. In some embodiments, 0 or at most 1, 2, or 3 internucleotide linkages in the 5' direction to the nucleoside opposite the target nucleoside (eg, target adenosine) are Sp phosphorothioate internucleotides not a connection In some embodiments, up to 1, 2, or 3 internucleotide linkages in the 5' direction to the nucleoside opposite the target nucleoside (eg, target adenosine) are natural phosphate linkages. In some embodiments, these internucleotidic linkages are not natural phosphate linkages. In some embodiments, at most one such internucleotidic linkage is a natural phosphate linkage. In some embodiments, up to two such internucleotidic linkages are natural phosphate linkages. In some embodiments, up to three such internucleotidic linkages are natural phosphate linkages. In some embodiments, each modified internucleotide linkage is independently a phosphorothioate or non-negatively charged internucleotide linkage (eg, n001). In some embodiments, there are no 2, 3, or 4 consecutive internucleotide linkages in the 5' direction relative to the nucleoside opposite the target nucleoside, each not a phosphorothioate internucleotide linkage. In some embodiments, there are no 2, 3, or 4 consecutive internucleotide linkages in the 5' direction relative to the nucleoside opposite the target nucleoside, each chirally controlled and containing an Sp phosphorothioate internucleotide linkage. no. In some embodiments, 0 or at most 1, 2, 3, 4, or 5 internucleotide linkages in the 5' direction to the opposite nucleoside of the target nucleoside (eg, target adenosine) are R p phosphors It is a thioate internucleotide linkage. In some embodiments, the internucleotide linkage linked to the nucleoside opposite the target nucleoside at the 5'-position of the sugar (considered the +1 position) is an R p phosphorothioate internucleotide linkage. In some embodiments, it is the only R p phosphorothioate internucleoside linkage in the 3' direction to the nucleoside opposite the target nucleoside. In some embodiments, an internucleotide linkage at position +5 relative to the nucleoside opposite the target nucleoside (e.g., N ₀ is the opposite nucleoside of the target nucleoside, ...N ₊₅ N ₊₄ The internucleotide linkage connecting N ₊₄ and N ₊₅ for N ₊₃ N ₊₂ N ₊₁ N ₀ ...) is not an R p phosphorothioate internucleotide linkage. In some embodiments, the internucleotidic linkage at position +11 is not an Sp phosphorothioate internucleotidic linkage. In some embodiments, one or more or all internucleotide linkages at positions +6 to +8 relative to the nucleoside opposite the target nucleoside are each independently, optionally chirally controlled, modified internucleotide linkage. In some embodiments, each of these independently is a phosphorothioate internucleotidic linkage. In some embodiments, each of these is independently an S p phosphorothioate internucleotidic linkage. In some embodiments, one or more or all internucleotide linkages at positions +6 to +8 relative to the nucleoside opposite the target nucleoside are each independently, optionally chirally controlled, phosphorothioate internucleotide linkage. In some embodiments, one or more or all internucleotide linkages at positions +6, +7, +8, +9, and +11 are each independently R p internucleotidic linkages. In some embodiments, one or more or all internucleotide linkages at positions +6, +7, +8, +9, and +11 are each independently R p phosphorothioate internucleotide linkages. In some embodiments, one or more or all internucleotide linkages at positions +5, +6, +7, +8, and +9 relative to the nucleoside opposite the target adenosine are each independently an Sp internucleotide linkage. In some embodiments, one or more or all internucleotide linkages at positions +5, +6, +7, +8, and +9 relative to the nucleoside opposite the target adenosine are each independently an Sp phosphorothioate nucleotide is a connection between In some embodiments, the internucleotide linkage at position +5 is an S p internucleotidic linkage. In some embodiments, the internucleotidic linkage at position +5 is an Sp phosphorothioate internucleotidic linkage. In some embodiments, the internucleotide linkage at position +6 is an S p internucleotidic linkage. In some embodiments, the internucleotidic linkage at position +6 is an Sp phosphorothioate internucleotidic linkage. In some embodiments, the internucleotide linkage at position +7 is an S p internucleotidic linkage. In some embodiments, the internucleotidic linkage at position +7 is an Sp phosphorothioate internucleotidic linkage. In some embodiments, the internucleotidic linkage at position +8 is an S p internucleotidic linkage. In some embodiments, the internucleotidic linkage at position +8 is an Sp phosphorothioate internucleotidic linkage. In some embodiments, the internucleotidic linkage at position +9 is an S p internucleotidic linkage. In some embodiments, the internucleotidic linkage at position +9 is an Sp phosphorothioate internucleotidic linkage. In some embodiments, at least about 1, 2, 3, 4, 5, 6, 7, 8, 9 of the internucleotide linkages in the 5' direction to the nucleoside opposite the target nucleoside (eg, target adenosine) , or 10, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 18, 19, 20, 21, 22, 23 , 24, 25, 26, 27, 28, 29, 30, 31, or 32, or from about 50% to about 100% (e.g., about or at least about 50%, 55%, 60%, 65%, 70 %, 75%, 80%, 85%, 90%, or 95%) are each independently chirally controlled and are Sp internucleotidic linkages. In some embodiments, at least about 1, 2, 3, 4, 5, 6, 7 of phosphorothioate internucleotide linkages in the 5' direction to the nucleoside opposite the target nucleoside (eg, target adenosine) , 8, 9, or 10, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, or 32, or about 50% to 100% (e.g., about or at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%) are each independently chirally controlled and are Sp . In some embodiments, each phosphorothioate internucleoside linkage in the 5' direction to the nucleoside opposite the target nucleoside (eg, target adenosine) is chirally controlled. In some embodiments, each phosphorothioate internucleoside linkage in the 5' direction to the nucleoside opposite the target nucleoside (eg, target adenosine) is Sp .

Alternatively or additionally, as described herein (e.g., exemplified in certain examples), in some embodiments, about 5% to 90%, about 10 to 80% of all internucleotide linkages of an oligonucleotide , about 10-75%, about 10-70%, 10%-60%, 10-50%, 10-40%, 10-30%, 15-40%, 20-30%, 25-30%, or About or at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45% or 50% independently are natural phosphate linkages. In some embodiments, about 5%-90%, about 10-80%, about 10-80%, of all internucleotide linkages in the 5' direction to the nucleoside opposite the target nucleoside (e.g., target adenosine) 75%, about 10-70%, 10%-60%, 10-50%, 10-40%, 10-30%, 15-40%, 20-30%, 25-30%, or about or at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45% or 50% independently are natural phosphate linkages. In some embodiments, one or more in the oligonucleotide, for example about 1-15, 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 5-6, 5-7 , 5-8, 5-9, 5-10, or about or at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 internucleotide linkages are independently natural phosphate linkages. In some embodiments, one or more, e.g., about 1-15, 1-10, 1-9, 1-, 5' to the nucleoside opposite the target nucleoside (e.g., target adenosine). 8, 1-7, 1-6, 1-5, 5-6, 5-7, 5-8, 5-9, 5-10, or about or at least about 1, 2, 3, 4, 5, Internucleotide linkages of 6, 7, 8, 9 or 10 are independently natural phosphate linkages. In some embodiments, one or more of positions +3 (between N ₊₄ N ₊₃ ), +4, +6, +8, +9, +12, +14, +15, +17, and +18. The above internucleotide linkages are independently natural phosphate linkages. In some embodiments, there are four natural phosphate linkages in the 5' direction to the nucleoside opposite the target nucleoside. In some embodiments, there are five natural phosphate linkages in the 5' direction to the nucleoside opposite the target nucleoside. In some embodiments, there are six natural phosphate linkages in the 5' direction to the nucleoside opposite the target nucleoside. In some embodiments, there are seven natural phosphate linkages in the 5' direction to the nucleoside opposite the target nucleoside. In some embodiments, there are eight natural phosphate linkages in the 5' direction to the nucleoside opposite the target nucleoside. In some embodiments, there are 9 natural phosphate linkages in the 5' direction to the nucleoside opposite the target nucleoside. In some embodiments, there are 10 natural phosphate linkages in the 5' direction to the nucleoside opposite the target nucleoside. In some embodiments, each of the one or more internucleoside linkages in the 3' direction to the nucleoside opposite the target nucleoside (eg, the target adenosine) is a native phosphate linkage. In some embodiments, there is one natural phosphate linkage in the 3' direction to the nucleoside opposite the target nucleoside. In some embodiments, the internucleotide linkage at position -3 is a natural phosphate linkage.

Alternatively or additionally, as described herein (e.g., exemplified in certain examples), in some embodiments, about 5% to 90%, about 10 to 80% of all internucleotide linkages of an oligonucleotide , about 10-75%, about 10-70%, 10%-60%, 10-50%, 10-40%, 10-30%, 15-40%, 20-30%, 25-30%, 30 % to 70%, 40 to 70%, 40% to 65%, 40% to 60%, or about or at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40% , 45%, 50%, 55%, 60% or 65% are independently phosphorothioate internucleotidic linkages. In some embodiments, about 5%-90%, about 10-80%, about 10-80%, of all internucleotide linkages in the 5' direction to the nucleoside opposite the target nucleoside (e.g., target adenosine) 75%, approximately 10-70%, 10%-60%, 10-50%, 10-40%, 10-30%, 15-40%, 20-30%, 25-30%, 30%-70% , 40-70%, 40%-65%, 40%-60%, or about or at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, or 65% are independently natural phosphate linkages. In some embodiments, one or more in the oligonucleotide, for example about 1-30, 1-25, 1-20, 1-15, 5-30, 5-25, 5-20, 5-15, 10-30 , 10-25, 10-20, 10-15, or about or at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 , 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 internucleotide linkages are independently phosphorothioate internucleotide linkages. In some embodiments, one or more, e.g., about 1-30, 1-25, 1-20, 1-20, 1-25, 1-20, 1-25 in the 5' direction to the nucleoside opposite the target nucleoside (e.g., target adenosine). 15, 5-30, 5-25, 5-20, 5-15, 10-30, 10-25, 10-20, 10-15, or about or at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 The internucleotidic linkages are independently phosphorothioate internucleotidic linkages. In some embodiments, +1 (between N ₊₁ N ₀ ), +2, +5, +6, +7, +8, +11, +14, +15, +16, +17, +19, + The one or more internucleotide linkages at one or more of positions 20, +21 and +22 are independently phosphorothioate internucleotide linkages. In some embodiments, there are 5 or more phosphorothioate internucleoside linkages in the 5' direction to the nucleoside opposite the target nucleoside. In some embodiments, there are 10 or more phosphorothioate internucleoside linkages in the 5' direction to the nucleoside opposite the target nucleoside. In some embodiments, there are 11 or more phosphorothioate internucleoside linkages in the 5' direction to the nucleoside opposite the target nucleoside. In some embodiments, there are 12 or more phosphorothioate internucleoside linkages in the 5' direction to the nucleoside opposite the target nucleoside. In some embodiments, there are 13 or more phosphorothioate internucleoside linkages in the 5' direction to the nucleoside opposite the target nucleoside. In some embodiments, there are 14 or more phosphorothioate internucleoside linkages in the 5' direction to the nucleoside opposite the target nucleoside. In some embodiments, there are 15 or more phosphorothioate internucleoside linkages in the 5' direction to the nucleoside opposite the target nucleoside. In some embodiments, each of the one or more internucleotide linkages in the 3' direction to the nucleoside opposite the target nucleoside (eg, target adenosine) is independently a phosphorothioate internucleotide linkage. In some embodiments, there is one phosphorothioate internucleoside linkage in the 3' direction to the nucleoside opposite the target nucleoside. In some embodiments, there are two phosphorothioate internucleoside linkages in the 3' direction to the nucleoside opposite the target nucleoside. In some embodiments, there are three phosphorothioate internucleoside linkages in the 3' direction to the nucleoside opposite the target nucleoside. In some embodiments, one or more or all internucleotide linkages at positions -1, -4, and -5 are independently phosphorothioate internucleotide linkages. In some embodiments, each phosphorothioate internucleotidic linkage is independently chirally controlled. In some embodiments, about or at least about 80%, 85%, 90% or 95% of all phosphorothioate internucleotidic linkages are independently Sp . In some embodiments, each phosphorothioate internucleotidic linkage is independently Sp .

Alternatively or additionally, as described herein (e.g., exemplified in certain examples), in some embodiments, about 5% to 90%, about 10 to 80% of all internucleotide linkages of an oligonucleotide , about 10-75%, about 10-70%, 10%-60%, 10-50%, 10-40%, 10-30%, 10%-20%, 10-15%, 15-40%, 15%-35%, 15%-30%, 15-25%, 15-20%, or about or at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40% , 45% or 50% are independently non-negatively charged internucleotidic linkages. In some embodiments, about 5%-90%, about 10-80%, about 10-80%, of all internucleotide linkages in the 5' direction to the nucleoside opposite the target nucleoside (e.g., target adenosine) 75%, approximately 10-70%, 10%-60%, 10-50%, 10-40%, 10-30%, 10%-20%, 10-15%, 15-40%, 15%-35 %, 15%-30%, 15-25%, 15-20%, or about or at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45% or 50% are internucleotidic linkages that are not independently negatively charged. In some embodiments, one or more in the oligonucleotide, for example about 1-15, 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 5-6, 5-7 , 5 to 8, 5 to 9, 5 to 10, or about or at least about 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 internucleotide linkages are independently negatively charged It is an internucleotide linkage. In some embodiments, one or more, e.g., about 1-15, 1-10, 1-9, 1-, 5' to the nucleoside opposite the target nucleoside (e.g., target adenosine). 8, 1-7, 1-6, 1-5, 5-6, 5-7, 5-8, 5-9, 5-10, or about or at least about 1, 2, 3, 4, 5, The 6, 7, 8, 9 or 10 internucleotide linkages are independently non-negatively charged internucleotide linkages. In some embodiments, the one or more internucleotidic linkages at one or more or all of positions +5 (between N ₊₅ N ₊₄ ), +10, +13, or +23 are independently non-negatively charged internucleotidic linkages. . In some embodiments, there are two or more non-negatively charged internucleotide linkages in the 5' direction to the nucleoside opposite the target nucleoside. In some embodiments, there are three or more non-negatively charged internucleotide linkages in the 5' direction to the nucleoside opposite the target nucleoside. In some embodiments, there are 4 or more non-negatively charged internucleotide linkages in the 5' direction to the nucleoside opposite the target nucleoside. In some embodiments, there are 5 or more non-negatively charged internucleotide linkages in the 5' direction to the nucleoside opposite the target nucleoside. In some embodiments, the one or more internucleoside linkages in the 3' direction to the opposite nucleoside of the target nucleoside (eg, target adenosine) are each independently non-negatively charged internucleotidic linkages. In some embodiments, there is one non-negatively charged internucleotide linkage in the 3' direction to the nucleoside opposite the target nucleoside. In some embodiments, there are two or more non-negatively charged internucleotide linkages in the 3' direction to the nucleoside opposite the target nucleoside. In some embodiments, there are two non-negatively charged internucleotide linkages in the 3' direction to the nucleoside opposite the target nucleoside. In some embodiments, the internucleotide linkage at one or both positions -2 and -6 is independently a non-negatively charged internucleotide linkage. In some embodiments, the non-negatively charged internucleotidic linkages are neutral internucleotidic linkages. In some embodiments, the non-negatively charged internucleotidic linkages are independently neutral internucleotidic linkages. In some embodiments, the non-negatively charged internucleotide linkages are phosphoryl guanidine internucleotide linkages. In some embodiments, each non-negatively charged internucleotidic linkage is independently a phosphoryl guanidine internucleotidic linkage. In some embodiments, the non-negatively charged internucleotide linkage is n001. In some embodiments, each non-negatively charged internucleotide linkage is independently n001. In some embodiments, the non-negatively charged internucleotidic linkages are chirally controlled. In some embodiments, each non-negatively charged internucleotide linkage is independently chirally controlled. In some embodiments, the non-negatively charged internucleotidic linkage is R p. In some embodiments, the non-negatively charged internucleotidic linkage is Sp . In some embodiments, each non-negatively charged internucleotide linkage is independently Sp . In some embodiments, each n001 is independently Sp , except that each n001 bonded to the 3'-carbon of dI is independently R p.

ADAR

In particular, the provided technology can provide modification/editing of the target adenosine by converting A to I. In some embodiments, an oligonucleotide and/or a duplex formed by an oligonucleotide and a target nucleic acid interacts with a protein, eg an ADAR protein. In some embodiments, such proteins contain adenosine modifying activity and are capable of modifying target adenosines in target nucleic acids (eg, converting them to inosines).

ADAR proteins are proteins that are naturally expressed in a variety of cells, tissues, organs and/or organisms. Reportedly, some ADAR proteins, such as ADAR1 and ADAR2, can edit adenosine through deamination, which converts adenosine to inosine, which has several functions including being read as G or G-like during translation. can provide The mechanism of ADAR-mediated mRNA editing (eg deamination) has been reported. For example, ADAR proteins are reported to catalyze the conversion of adenosine to inosine in mismatched double-stranded RNA substrates. As will be appreciated by those skilled in the art, inosine can be recognized as guanosine by cellular translation and/or splicing machinery. Thus, ADARs can be used for functional adenosine to guanosine editing of nucleic acids, such as pre-mRNA and mRNA substrates.

In some embodiments, the present invention provides oligonucleotides and compositions thereof for ADAR-mediated editing of a target adenosine in a target nucleic acid (eg, RNA). ADAR-mediated RNA editing may offer several advantages over DNA editing. Delivery is simplified because expression of a recombinant protein, eg Cas9, is not required. Since both ADAR1 and ADAR2 are endogenous enzymes, cell delivery of oligonucleotides may be sufficient for editing. Even off-target effects are transient and do not result in changes to genomic DNA. Additionally, ADAR-mediated editing can be used in post-mitotic cells and does not require HDR templates for restoration. Three vertebrate ADAR genes have been reported with common functional domains (Nishikura Nat Rev Mol Cell Biol. 2016 Feb; 17(2): 83-96.; Nishikura Annu Rev Biochem. 2010; 79: 321-349.; Thomas and Beal Bioessays.2017 Apr;39(4)). All three ADARs contain a dsRNA-binding domain (dsRBD) capable of contacting a dsRNA substrate. Some ADAR1s also contain a Z-DNA-binding domain. ADAR1 has been reported to be significantly expressed in the brain, lung, kidney, liver, and heart, and may appear in two isoforms. In some embodiments, isoform p150 may be induced by interferon, whereas isoform p110 may be constitutively expressed. In some embodiments, it may be advantageous to use p110 as it is reported to be constitutively expressed in all tissues. ADAR2 can be highly expressed, for example in the brain and lung, and is reported to be localized to the nucleus. ADAR3 is reported to be catalytically inactive and expressed only in the brain. Potential differences in tissue expression can be considered when selecting therapeutic targets.

The use of oligonucleotides for RNA editing by ADAR has been reported. In particular, the present invention provides that previously reported technologies generally have low stability (e.g., oligonucleotides with native RNA sugars), low editing efficiency, and low editing specificity (e.g., target nucleic acids substantially complementary to oligonucleotides). multiple A's are edited in part of), specific structures of oligonucleotides for ADAR recognition/replenishment, exogenous proteins (eg, oligonucleotides with specific structures for editing and/or duplexes thereof (eg, targets) engineered to recognize duplexes with nucleic acids) and the like. Also, previously reported technologies generally use stereorandom oligonucleotide compositions when the oligonucleotide contains one or more chiral linkages of modified internucleotidic linkages.

For example, various reported oligonucleotides contain ADAR-complementary domains. See Merkle et al., Nat Biotechnol. 2019 Feb;37(2):133-138] disclosed an oligonucleotide comprising an incomplete 20bp hairpin ADAR-complementary domain, an intramolecular stem loop that recruits endogenous human ADAR2 to edit the endogenous transcript. See Mali et al., Nat Methods. 2019 Mar;16(3):239-242] contains the ADAR substrate GluR2 pre-messenger RNA sequence or MS2 hairpin in addition to the specificity domain that hybridizes to the target mRNA.

Certain editing approaches reported utilize exogenous or engineered proteins (e.g. proteins using the CRISPR/Cas9 system). For example, Nature 2016 volume 533, pages 420-424 disclosed a deaminase coupled with CRISPR-Cas9 to create a programmable DNA base editor. Because it involves exogenous editing proteins, it requires delivery of both the CRISPR/Cas9 system and guide RNA.

In particular, the present invention addresses one or more or all disadvantages of conventional adenosine editing techniques by, for example, providing a chirally controlled oligonucleotide composition of the designed oligonucleotides described herein, such as sugar modification, base modification, internucleotidic linkage modification, Techniques are provided that include one or more features, such as control of stereochemistry, various patterns thereof, and the like. For example, as shown herein, ADAR-supplemental loops are optional and not required in the provided technology.

As will be appreciated by those skilled in the art, oligonucleotides of the prior art using one or more of these useful features (e.g., those described in WO 2016097212, WO 2017220751, WO 2018041973, WO 2018134301, oligonucleotides and oligonucleotide compositions, respectively) are independently incorporated by reference) may be improved. In some embodiments, the present invention provides an improvement over the prior art by applying one or more useful features described herein to previously reported oligonucleotide sequences. In some embodiments, the present invention provides chirally controlled oligonucleotide compositions of previously reported oligonucleotides that may be useful for adenosine editing. In some embodiments, the present invention provides an improvement over previously reported adenosine editing using stereorandom oligonucleotide compositions by performing adenosine editing using chirally controlled oligonucleotide compositions.

As reported, ADAR proteins can have various isoforms. For example, ADAR1 in particular has a reported p110 isoform and a reported p150 isoform. In some embodiments, certain chirally controlled oligonucleotide compositions can use multiple isoforms (in some embodiments, both p110 and p150 isoforms) to provide high levels of adenosine modification (e.g., A to I conversion). In contrast, sterically random compositions have been observed to provide low levels of adenosine modification for more than one isoform (eg, p110). In some embodiments, the chirally controlled oligonucleotide composition expresses a high level of the p110 isoform of ADAR1 compared to a system (eg, cell, tissue, organ, organism, subject, etc.) expressing or comprising the p110 isoform of ADAR1, in particular the p110 isoform of ADAR1. It is particularly useful for adenosine modification in those that do, contain, or do not express or express low levels of ADAR1 p150.

In some embodiments, the present invention provides Cis-acting (CisA) oligonucleotides that do not require stem loops in their structure. In some embodiments, a provided oligonucleotide is capable of forming a dsRNA structure with a target mRNA through base pairing. In some embodiments, the formed dsRNA structure (optionally including secondary mismatches) may include bulges that promote ADAR binding and thus facilitate ADAR-mediated editing (eg, deamination of a target adenosine). In some embodiments, oligonucleotides of the invention are shorter than LSL oligonucleotides or CSL oligonucleotides (e.g., about 32 nt or less, about 31 nt or less, about 30 nt or less, about 29 nt or less, about 28 nt or less, length of about 27 nt or less, or about 26 nt or less), high editing efficiency can be provided.

Duplexing and targeting regions

In some embodiments, the present invention

duplexing area; and

Provides an oligonucleotide comprising a targeting region,

At this time,

The duplexing region can form a duplex with a nucleic acid;

The targeting region can form a duplex with a target nucleic acid comprising a target adenosine.

In some embodiments, the duplexing region is or comprises a first domain as described herein. In some embodiments, the targeting region is or comprises a second domain as described herein.

In some embodiments, the duplexing region is capable of forming a duplex with a nucleic acid, wherein the nucleic acid is not a target nucleic acid. In some embodiments, the duplexing region forms a duplex with a target nucleic acid. In some embodiments, the duplexing region forms a duplex with a nucleic acid expressed in a system, eg, a cell. In some embodiments, the duplexing region forms a duplex with an exogenous nucleic acid, eg, an oligonucleotide. In some embodiments, the duplexing region forms a duplex with a nucleic acid that is or comprises an RNA portion. In some embodiments, the formed duplex can be recognized by an ADAR polypeptide, eg, a polypeptide such as ADAR1 (p110 or p150 or both), ADAR2, and the like. In some embodiments, the formed duplex may be supplemented with an ADAR polypeptide, eg, a polypeptide such as ADAR1 (p110 or p150 or both), ADAR2, and the like. In some embodiments, the formed duplex recruits ADAR1. In some embodiments, the duplex formed recruits ADAR1 p110. In some embodiments, the duplex formed recruits ADAR1 p150. In some embodiments, the duplex supplements ADAR2. In some embodiments, the formed duplex complements ADAR1 p110 and p150. In some embodiments, the formed duplex complements ADAR1 and ADAR2. In some embodiments, the duplex formed complements ADAR1 p110, ADAR p150 and/or ADAR2. In some embodiments, the duplex formed complements ADAR1 p110 and p150 and ADAR2.

In some embodiments, the duplexed region forms a duplex with an oligonucleotide (the oligonucleotide may be referred to as a "duplexed oligonucleotide"). In some embodiments, a duplexed oligonucleotide comprises one or more modified nucleobases, modified sugars, and/or modified internucleotidic linkages. In some embodiments, the duplexed oligonucleotide comprises a duplex forming region complementary to the duplexed region. As will be appreciated by those skilled in the art, in many cases perfect complementarity is not required and one or more wobbles, bulges, inconsistencies, etc. may well be tolerated. For example, ADAR proteins have been reported to bind to and/or utilize both fully and incompletely complementary duplexes as substrates.

The duplexed region and/or duplexed forming region can be of various lengths. In some embodiments, they are at least 10 in length (e.g., about or at least about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 or more, approximately 10-20, 10-25, 10-30, 10-40, 10-50, 10-100, 14-20, 14-25, 14-30 , 14~40, 14~50, 14~100, 15~20, 15~25, 15~30, 15~40, 15~50, 15~100, 16~20, 16~25, 16~30, 16 ~40, 16~50, 16~100, 17~20, 17~25, 17~30, 17~40, 17~50, 17~100, 18~20, 18~25, 18~30, 18~40 , 18~50, 18~100, 19~20, 19~25, 19~30, 19~40, 19~50, 19~100, 20~25, 20~30, 20~40, 20~50, 20 ~100, etc.) nucleosides. In some embodiments, the length is about or at least about 10 nucleosides in length. In some embodiments, the length is about or at least about 11 nucleosides in length. In some embodiments, the length is about or at least about 12 nucleosides in length. In some embodiments, the length is about or at least about 13 nucleosides in length. In some embodiments, the length is about or at least about 14 nucleosides in length. In some embodiments, the length is about or at least about 15 nucleosides in length. In some embodiments, the length is about or at least about 16 nucleosides in length. In some embodiments, the length is about or at least about 17 nucleosides in length. In some embodiments, the length is about or at least about 18 nucleosides in length. In some embodiments, the length is about or at least about 19 nucleosides in length. In some embodiments, the length is about or at least about 20 nucleosides in length.

In some embodiments, a duplexed oligonucleotide consists of or consists essentially of a duplex forming region. In some embodiments, the duplexed oligonucleotide further comprises one or more additional regions in addition to the duplex forming region. In some embodiments, the duplexed oligonucleotide comprises a stem-loop region (eg, as described in FIG. 35 ). In some embodiments, a duplexed oligonucleotide comprises or consists of a duplex forming region and a stem-loop region. In some embodiments, the stem region is about or at least about 2, 3, 4, 5, 6, 7, 8, 9, or 10 (e.g., about or at least about 2, 3, 4, 5, 6) in length. , 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 or more , about 4~10, 4~15, 4~20, 4~25, 4~30, 4~40, 4~50, 5~10, 5~15, 5~20, 5~25, 5~30, 5~40, 5~50, 6~10, 6~15, 6~20, 6~25, 6~30, 6~40, 6~50, 7~10, 7~15, 7~20, 7~ 25, 7~30, 7~40, 7~50, 8~10, 8~15, 8~20, 8~25, 8~30, 8~40, 8~50, 9~10, 9~15, 9-20, 9-25, 9-30, 9-40, 9-50, 10-15, 10-25, 10-30, 10-40, 10-50, 10-100, etc.) are nucleobases. In some embodiments, it is about or at least about 5 nucleobases in length. In some embodiments, it is about or at least about 6 nucleobases in length. In some embodiments, it is about or at least about 7 nucleobases in length. In some embodiments, it is about or at least about 8 nucleobases in length. In some embodiments, it is about or at least about 9 nucleobases in length. In some embodiments, it is about or at least about 10 nucleobases in length.

In some embodiments, the one or more additional regions may promote, encourage, facilitate and/or contribute to the recruitment and/or recognition and/or interaction by a polypeptide, e.g., ADAR1 (p110 and/or p150) and/or ADAR2. can In some embodiments, to duplex an oligonucleotide comprising one or more additional regions, a shorter duplex forming region can be used compared to the absence of such additional regions.

In some embodiments, the duplex structure formed by the duplex region and the duplexed oligonucleotide can supplement a polypeptide, eg, ADAR1 (p110 and/or p150) and/or ADAR2. In some embodiments, the duplex structure is or comprises a supplemental moiety as described in WO 2016/097212.

In some embodiments, the duplexed oligonucleotide comprises one or more sugar, nucleobase and/or internucleobase linkage modifications as described herein. In some embodiments, a duplexed oligonucleotide contains one or more sugar modifications. In some embodiments, most or all of the sugars in the duplexed oligonucleotide, as described herein, are modified sugars. In some embodiments, a modified sugar is a 2'-modified sugar. In some embodiments, each modified sugar is independently a 2'-modified sugar. In some embodiments, each modified sugar is independently selected from a 2'-F modified sugar, a bicyclic sugar, or a 2'-OR modified sugar where R is not hydrogen. In some embodiments, each modified sugar is independently selected from a 2'-F modified sugar, a bicyclic sugar, or a 2'-OR modified sugar, where R is an optionally substituted C _1-6 aliphatic. In some embodiments, each modified sugar is independently selected from a 2'-F modified sugar or a 2'-OR modified sugar, where R is an optionally substituted C _1-6 aliphatic. In some embodiments, each 2'-OR modified sugar is independently a 2'-OMe or 2'-MOE modified sugar. In some embodiments, each 2'-OR modified sugar is independently a 2'-OMe modified sugar. In some embodiments, each 2'-OR modified sugar is independently a 2'-MOE modified sugar. In some embodiments, each 2'-OR modified sugar is independently a 2'-F modified sugar. In some embodiments, the duplexed oligonucleotide comprises one or more modified internucleotide linkages, such as phosphorothioate internucleotide linkages. In some embodiments, most or all of the internucleotide linkages of the duplexed oligonucleotide, as described herein, are independently modified internucleotide linkages. In some embodiments, each internucleotidic linkage of the duplexed oligonucleotide is independently a modified internucleotidic linkage. In some embodiments, the modified internucleotidic linkage is a phosphorothioate internucleotidic linkage. In some embodiments, the modified internucleotide linkage is a non-negatively charged internucleotide linkage. In some embodiments, a modified internucleotide linkage is a neutral internucleotide linkage. In some embodiments, the modified internucleotide linkage is a phosphoryl guanidine internucleotide linkage. In some embodiments, the modified internucleotide linkage is n001. In some embodiments, each modified internucleotide linkage is a phosphorothioate internucleotide linkage. In some embodiments, each internucleotidic linkage is a phosphorothioate internucleotidic linkage. In some embodiments, the phosphorothioate internucleotidic linkages are chirally controlled. In some embodiments, the phosphorothioate internucleotidic linkages are not chirally controlled. In some embodiments, most or all of the chirally controlled phosphorothioate internucleotidic linkages, as described herein, are independently Sp . In some embodiments, all phosphorothioate internucleotidic linkages are Sp . In some embodiments, the duplexed oligonucleotide is one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 or more) natural phosphate linkages. In some embodiments, where the oligonucleotide comprises one or more native phosphate linkages, one or several internucleotide linkages at the 5' and/or 3' ends are independently modified internucleotide linkages, as described herein. In some embodiments, several internucleotide linkages at both the 5' and 3' ends are independently modified internucleotide linkages. In some embodiments, one or more at the 5' end (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 2-20, 2-10, 2-5, 3 ~5, etc.) internucleotide linkages are modified internucleotide linkages, such as phosphorothioate internucleotide linkages, as described herein. In some embodiments, one or more at the 3' end (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 2-20, 2-10, 2-5, 3 ~5, etc.) internucleotide linkages are modified internucleotide linkages, such as phosphorothioate internucleotide linkages, as described herein. In some embodiments, increasing the number of modified internucleotide linkages, e.g., phosphorothioate internucleotide linkages, etc., e.g., per more natural DNA/RNA, per 2'-F modification, etc. When coupled to modified internucleotide linkages, such as porothioate internucleotide linkages, it can increase editing efficiency.

In some embodiments, the duplexing region comprises one or more sugar, nucleobase and/or internucleobase linkage modifications as described herein. In some embodiments, the duplexed region is one or more (e.g., 1-30, 1-20, 1-10, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more) modified sugars as described herein. In some embodiments, most or all of the sugars in the duplexing region, as described herein, are each independently a modified sugar as described herein. In some embodiments, a modified sugar is a 2'-modified sugar. In some embodiments, each modified sugar is independently a 2'-modified sugar. In some embodiments, each modified sugar is independently selected from a 2'-F modified sugar, a bicyclic sugar, or a 2'-OR modified sugar where R is not hydrogen. In some embodiments, each modified sugar is independently selected from a 2'-F modified sugar, a bicyclic sugar, or a 2'-OR modified sugar, where R is an optionally substituted C _1-6 aliphatic. In some embodiments, each modified sugar is independently selected from a 2'-F modified sugar or a 2'-OR modified sugar, where R is an optionally substituted C _1-6 aliphatic. In some embodiments, each 2'-OR modified sugar is independently a 2'-OMe or 2'-MOE modified sugar. In some embodiments, each 2'-OR modified sugar is independently a 2'-OMe modified sugar. In some embodiments, each 2'-OR modified sugar is independently a 2'-MOE modified sugar. In some embodiments, each 2'-OR modified sugar is independently a 2'-F modified sugar. In some embodiments, between about 50%-100%, 60%-100%, 70%-100%, 50%-90%, 50%-80%, 60%-90%, 60%- 80%, 70% to 90%, 70% to 80%, or about or at least about 60%, 70%, 75%, 80%, 85%, 90%, 95% or more, each independently a 2'-F modification It is party. In some embodiments, as described herein, one or more sugars at the terminal of an oligonucleotide are independently modified sugars. In some embodiments, as described herein, one or more sugars at the terminal of an oligonucleotide are independently a bicyclic sugar or a 2′-OR modified sugar (R is C _1-6 aliphatic). In some embodiments, as described herein, one or more of the oligonucleotide termini (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 2-20, 2-10, 2-5, 3-5, etc.) sugars are each independently a 2'-OR modified sugar (where R is C _1-6 aliphatic). In some embodiments, one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 2-20, 2-10, 2-5, 3 to 5, etc.) sugars are each independently a modified sugar; For example, in some oligonucleotides, three or more sugars at the 5' end are 2'-OMe modified sugars, and four or more sugars at the 3' end are 2'-OMe modified sugars. In some embodiments, the duplexed region is one or more (e.g., 1-30, 1-20, 1-10, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more) of modified internucleotide linkages as described herein. In some embodiments, one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 2-20, 2- 10, 2-5, 3-5, etc.) of the internucleotide linkages are each independently a modified internucleotide linkage, e.g., in some embodiments each independently a non-negatively charged internucleotide linkage, a neutral internucleotide linkage , phosphoryl guanidine internucleotide linkages, n001 and phosphorothioate internucleotide linkages. In some embodiments, one or more of the 5' ends of the oligonucleotide (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 2-20, 2-10, 2-5 , 3-5, etc.) are each independently a modified internucleotide linkage, and one or more (eg, 1, 2, 3, 4, 5, 6, 7, 8 , 9, 10, 2-20, 2-10, 2-5, 3-5, etc.) internucleotide linkages are each independently modified internucleotide linkages. In some embodiments, the modified internucleotidic linkage is a phosphorothioate internucleotidic linkage. In some embodiments, the modified internucleotide linkage is a non-negatively charged internucleotide linkage. In some embodiments, a modified internucleotide linkage is a neutral internucleotide linkage. In some embodiments, the modified internucleotide linkage is a phosphoryl guanidine internucleotide linkage. In some embodiments, the modified internucleotide linkage is n001. In some embodiments, each modified internucleotide linkage is a phosphorothioate internucleotide linkage. In some embodiments, each internucleotidic linkage is a phosphorothioate internucleotidic linkage. In some embodiments, the phosphorothioate internucleotidic linkages are chirally controlled. In some embodiments, the phosphorothioate internucleotidic linkages are not chirally controlled. In some embodiments, most or all of the chirally controlled phosphorothioate internucleotidic linkages, as described herein, are independently Sp . In some embodiments, all phosphorothioate internucleotidic linkages are Sp . In some embodiments, chirally modified internucleotide linkages, eg, phosphorothioate internucleotide linkages, are not chirally controlled. In some embodiments, one or more duplexed regions (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 , 19 or 20 or more) natural phosphate linkages. In some embodiments, where the oligonucleotide comprises one or more native phosphate linkages, one or several internucleotide linkages at the 5' and/or 3' ends are independently modified internucleotide linkages, as described herein. In some embodiments, several internucleotide linkages at both the 5' and 3' ends are independently modified internucleotide linkages. In some embodiments, one or more at the 5' end (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 2-20, 2-10, 2-5, 3 ~5, etc.) internucleotide linkages are modified internucleotide linkages, such as phosphorothioate internucleotide linkages, as described herein. In some embodiments, one or more at the 3' end (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 2-20, 2-10, 2-5, 3 ~5, etc.) internucleotide linkages are modified internucleotide linkages, such as phosphorothioate internucleotide linkages, as described herein. In some implementations, one or more (eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 2-20, 2-10, 2-5, 3 Incorporation of ~5, etc.) natural phosphate linkages increases editing efficiency. In some embodiments, most internucleotide linkages in the duplexed region (e.g., 50%-100%, 60%-100%, 70%-100%, 50%-90%, 50%-80%, 60%-80%) % to 90%, 60% to 80%, 70% to 90%, 70% to 80%, or about or at least about 60%, 70%, 75%, 80%, 85%, 90%, 95% or more) is independently a natural phosphate linkage. In some embodiments, the different internucleotidic linkages in the duplexed region are independently native phosphate linkages, except for one or more native phosphate linkages at the ends of the oligonucleotide (if present).

In some embodiments, a targeting region is or comprises an editing region as described herein. In some embodiments, the targeting region comprises 5'-N ₁ N ₀ N _-1 -3' as described herein.

In some embodiments, the targeting region comprises one or more sugar, nucleobase and/or internucleobase linkage modifications as described herein. In some embodiments, the targeting region is one or more (e.g., 1-30, 1-20, 1-10, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 , 13, 14, 15, 16, 17, 18, 19, 20 or more, etc.) modified sugars as described herein. In some embodiments, most or all of the sugars in the targeting region, as described herein, are each independently modified sugars as described herein. In some embodiments, a modified sugar is a 2'-modified sugar. In some embodiments, each modified sugar is independently a 2'-modified sugar. In some embodiments, each modified sugar is independently selected from a 2'-F modified sugar, a bicyclic sugar, or a 2'-OR modified sugar where R is not hydrogen. In some embodiments, each modified sugar is independently selected from a 2'-F modified sugar, a bicyclic sugar, or a 2'-OR modified sugar, where R is an optionally substituted C _1-6 aliphatic. In some embodiments, each modified sugar is independently selected from a 2'-F modified sugar or a 2'-OR modified sugar, where R is an optionally substituted C _1-6 aliphatic. In some embodiments, each 2'-OR modified sugar is independently a 2'-OMe or 2'-MOE modified sugar. In some embodiments, each 2'-OR modified sugar is independently a 2'-OMe modified sugar. In some embodiments, each 2'-OR modified sugar is independently a 2'-MOE modified sugar. In some embodiments, each 2'-OR modified sugar is independently a 2'-F modified sugar. In some embodiments, from about 50% to 100%, 60% to 100%, 70% to 100%, 50% to 90%, 50% to 80%, 60% to 90%, 60% to 80% of the sugars in the targeting region. %, 70% to 90%, 70% to 80%, or about or at least about 60%, 70%, 75%, 80%, 85%, 90%, 95% or more, each independently, a morbidity or 2'- OR per variant (R is not hydrogen). In some embodiments, from about 50% to 100%, 60% to 100%, 70% to 100%, 50% to 90%, 50% to 80%, 60% to 90%, 60% to 80% of the sugars in the targeting region. %, 70% to 90%, 70% to 80%, or about or at least about 60%, 70%, 75%, 80%, 85%, 90%, 95% or more, each independently, a morbidity or 2'- OR is a modified sugar (R is an optionally substituted C _1-6 aliphatic). In some embodiments, from about 50% to 100%, 60% to 100%, 70% to 100%, 50% to 90%, 50% to 80%, 60% to 90%, 60% to 80% of the sugars in the targeting region. %, 70% to 90%, 70% to 80%, or about or at least about 60%, 70%, 75%, 80%, 85%, 90%, 95% or more each independently 2'-OMe or 2 '- is per MOE strain. In some embodiments, each sugar in the targeting region other than the sugar in the editing region is independently a modified sugar as described herein. In some embodiments, each sugar in the targeting region, excluding sugars in the editing region, is independently a bicyclic sugar or a 2'-OR modified sugar, where R is an optionally substituted C _1-6 aliphatic. In some embodiments, each sugar in the targeting region, excluding sugars in the editing region, is independently a 2'-OR modified sugar (R is an optionally substituted C _1-6 aliphatic). In some embodiments, each sugar in the targeting region other than the sugar in the editing region is independently a 2'-OMe or 2'-MOE modified sugar. In some embodiments, each sugar in the targeting region other than the sugar in the editing region is independently a 2'-OMe modified sugar. In some embodiments, the editing region comprises or consists of 3 nucleosides, wherein the nucleoside opposite the target adenosine is in the middle of the 3. In some embodiments, the editing region consists of 3 nucleosides, wherein the nucleoside opposite the target adenosine is in the middle of the 3. In some embodiments, an editing region comprising or consisting of 5'-N ₁ N ₀ N _-1 -3'. In some embodiments, as described herein, one or more of the oligonucleotide termini (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 2-20, 2-10, 2-5, 3-5, etc.) are independently modified sugars. In some embodiments, as described herein, one or more of the oligonucleotide termini (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 2-20, 2-10, 2-5, 3-5, etc.) sugars are independently bicyclic sugars or 2'-OR modified sugars where R is C _1-6 aliphatic. In some embodiments, as described herein, one or more of the oligonucleotide termini (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 2-20, 2-10, 2-5, 3-5, etc.) sugars are each independently a 2'-OR modified sugar (where R is C _1-6 aliphatic). In some embodiments, one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 2-20, 2-10, 2-5, 3 to 5, etc.) sugars are each independently a modified sugar; For example, in some oligonucleotides, three or more sugars at the 5' end are 2'-OMe modified sugars, and four or more sugars at the 3' end are 2'-OMe modified sugars. In some embodiments, the targeting region is one or more (e.g., 1-30, 1-20, 1-10, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 , 13, 14, 15, 16, 17, 18, 19, 20 or more, etc.) of modified internucleotide linkages as described herein. In some embodiments, one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 2-20, 2- 10, 2-5, 3-5, etc.) of the internucleotide linkages are each independently a modified internucleotide linkage, e.g., in some embodiments each independently a non-negatively charged internucleotide linkage, a neutral internucleotide linkage , phosphoryl guanidine internucleotide linkages, n001 and phosphorothioate internucleotide linkages. In some embodiments, one or more of the 5' ends of the oligonucleotide (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 2-20, 2-10, 2-5 , 3-5, etc.) are each independently a modified internucleotide linkage, and one or more (eg, 1, 2, 3, 4, 5, 6, 7, 8 , 9, 10, 2-20, 2-10, 2-5, 3-5, etc.) internucleotide linkages are each independently modified internucleotide linkages. In some embodiments, the modified internucleotidic linkage is a phosphorothioate internucleotidic linkage. In some embodiments, the modified internucleotide linkage is a non-negatively charged internucleotide linkage. In some embodiments, a modified internucleotide linkage is a neutral internucleotide linkage. In some embodiments, the modified internucleotide linkage is a phosphoryl guanidine internucleotide linkage. In some embodiments, the modified internucleotide linkage is n001. In some embodiments, each modified internucleotide linkage is a phosphorothioate internucleotide linkage. In some embodiments, each internucleotidic linkage is a phosphorothioate internucleotidic linkage. In some embodiments, the phosphorothioate internucleotidic linkages are chirally controlled. In some embodiments, the phosphorothioate internucleotidic linkages are not chirally controlled. In some embodiments, most or all of the chirally controlled phosphorothioate internucleotidic linkages, as described herein, are independently Sp . In some embodiments, all phosphorothioate internucleotidic linkages are Sp . In some embodiments, chirally modified internucleotide linkages, eg, phosphorothioate internucleotide linkages, are not chirally controlled. In some embodiments, one or more targeting regions (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 or more) natural phosphate linkages. In some embodiments, where the oligonucleotide comprises one or more native phosphate linkages, one or several internucleotide linkages at the 5' and/or 3' ends are independently modified internucleotide linkages, as described herein. In some embodiments, several internucleotide linkages at both the 5' and 3' ends are independently modified internucleotide linkages. In some embodiments, one or more at the 5' end (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 2-20, 2-10, 2-5, 3 ~5, etc.) internucleotide linkages are modified internucleotide linkages, such as phosphorothioate internucleotide linkages, as described herein. In some embodiments, one or more at the 3' end (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 2-20, 2-10, 2-5, 3 ~5, etc.) internucleotide linkages are modified internucleotide linkages, such as phosphorothioate internucleotide linkages, as described herein. In some embodiments, one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 2-20, 2-10, 2-5, 3-10) in the targeting region 5, etc.) of natural phosphate linkages increases editing efficiency. In some embodiments, a majority of internucleotide linkages within a targeting region (e.g., 50%-100%, 60%-100%, 70%-100%, 50%-90%, 50%-80%, 60% ~90%, 60%~80%, 70%~90%, 70%~80%, or about or at least about 60%, 70%, 75%, 80%, 85%, 90%, 95% or more) It is independently a natural phosphate linkage. In some embodiments, the different internucleotidic linkages in the targeting region are independently native phosphate linkages, except for one or more native phosphate linkages at the ends of the oligonucleotide (if present).

In some embodiments, a targeting region is complementary to a sequence within a target nucleic acid. In some embodiments, a nucleic acid is or comprises RNA. In some embodiments, a nucleic acid is RNA. In some embodiments, the sequence in the target nucleic acid to which the target region is complementary comprises a target adenosine. As will be appreciated by those skilled in the art, in many cases complete complementarity is not required, and one or more wobbles, bulges, inconsistencies, etc. may be present.

The length of the targeting region can vary. In some embodiments, the targeting region is at least 10 in length (e.g., about or at least about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 or more, approximately 10-20, 10-25, 10-30, 10-40, 10-50, 10-100, 14-20, 14-25, 14 ~30, 14~40, 14~50, 14~100, 15~20, 15~25, 15~30, 15~40, 15~50, 15~100, 16~20, 16~25, 16~30 , 16~40, 16~50, 16~100, 17~20, 17~25, 17~30, 17~40, 17~50, 17~100, 18~20, 18~25, 18~30, 18 ~40, 18~50, 18~100, 19~20, 19~25, 19~30, 19~40, 19~50, 19~100, 20~25, 20~30, 20~40, 20~50 , 20 to 100, etc.) is a nucleoside. In some embodiments, the length is about or at least about 10 nucleosides in length. In some embodiments, the length is about or at least about 11 nucleosides in length. In some embodiments, the length is about or at least about 12 nucleosides in length. In some embodiments, the length is about or at least about 13 nucleosides in length. In some embodiments, the length is about or at least about 14 nucleosides in length. In some embodiments, the length is about or at least about 15 nucleosides in length. In some embodiments, the length is about or at least about 16 nucleosides in length. In some embodiments, the length is about or at least about 17 nucleosides in length. In some embodiments, the length is about or at least about 18 nucleosides in length. In some embodiments, the length is about or at least about 19 nucleosides in length. In some embodiments, the length is about or at least about 20 nucleosides in length. In some embodiments, the length is about or at least about 21 nucleosides in length. In some embodiments, the length is about or at least about 22 nucleosides in length. In some embodiments, the length is about or at least about 23 nucleosides in length. In some embodiments, the length is about or at least about 24 nucleosides in length. In some embodiments, the length is about or at least about 25 nucleosides in length.

In some embodiments, an oligonucleotide comprises a targeting region and a duplexing region, and the targeting region is on the 3' side of the duplexing region. In some embodiments, an oligonucleotide comprises a targeting region and a duplexing region, and the targeting region is on the 5' side of the duplexing region. In some embodiments, an oligonucleotide consists of a targeting region and a duplexing region, the targeting region being on the 3' side of the duplexing region. In some embodiments, an oligonucleotide consists of a targeting region and a duplexing region, the targeting region being on the 5' side of the duplexing region. In some embodiments, an oligonucleotide comprises a targeting region, a duplexed region and a linker region between the targeting region and the duplexed region. In some embodiments, a linker region is or comprises an oligonucleotide moiety.

In some embodiments, an oligonucleotide comprising a duplexed and targeting region forms a duplex with another nucleic acid, eg, a complex comprising the duplexed oligonucleotide. In some embodiments, the invention provides a duplex comprising an oligonucleotide comprising a duplexed and targeting region and a nucleic acid forming a duplex with the duplexed region. In some embodiments, the present invention provides oligonucleotides comprising duplexed and targeting regions and duplexes comprising duplexed oligonucleotides. In some embodiments, a chirally controlled oligonucleotide composition of oligonucleotides comprising duplexing and targeting regions is used (eg, WV-42707). In some embodiments, non-chirally controlled oligonucleotide compositions of oligonucleotides comprising duplexing and targeting regions are used. In some embodiments, a chirally controlled oligonucleotide composition of duplexed oligonucleotides is used (eg, WV-42724). In some embodiments, non-chirally controlled oligonucleotide compositions of duplexed oligonucleotides are used (eg, WV-42721).

In some embodiments, duplexes are formed prior to administration. In some embodiments, the oligonucleotide comprising the duplexed and targeting regions and the nucleic acid that forms the duplex therewith (which may be referred to as a "duplexed nucleic acid") are administered separately. In some embodiments, oligonucleotides comprising duplexed and targeting regions are administered before, simultaneously (in single or multiple compositions) or after duplexed nucleic acids (eg, various duplexed oligonucleotides described herein). In some embodiments, the duplexed nucleic acid may not need to be administered directly as it may be present in and/or expressed in a cell.

Particular oligonucleotides and/or uses comprising duplexed and targeting regions and/or duplexed nucleic acids (eg, duplexed oligonucleotides) are illustrated in FIGS. 33 , 34 and 35 , etc., by way of example.

In some embodiments, a target nucleic acid is or comprises RNA. In some embodiments, a target nucleic acid is or comprises mRNA. In some embodiments, the target adenosine in the target nucleic acid is edited with I.

Generation of oligonucleotides and compositions

A variety of methods can be used for the production of oligonucleotides and compositions and can be used in accordance with the present invention. For example, stereorandom oligonucleotides and compositions can be prepared using conventional phosphoramidite chemistry (eg phosphoramidites comprising -CH ₂ CH ₂ CN and -N(i-Pr) ₂ ). can be prepared, and chirally controlled oligonucleotide compositions can be prepared using specific reagents and chiral control techniques, which are described in, for example, US 9982257, US 20170037399, US 20180216108, US 20180216107, US 9598458, WO 2017/062862, WO 2018/067973, WO 2017/160741, WO 2017/192679, WO 2017/210647, WO 2018/098264, WO 2018/223056, WO 2018/237194, WO 2019/032607, WO 2019/0559 51, WO 2019/075357, WO 2019/200185, WO 2019/217784, WO 2019/032612, WO 2020/191252, and/or WO 2021/071858, each reagent and method being incorporated herein by reference.

In some embodiments, chirally controlled/stereoselective preparation of oligonucleotides and compositions thereof is performed using, for example, monomers, dimers (eg, chiral pure dimers from isolation), monomeric phosphoramidites, dimer phosphors As part of formamidites (eg chiral pure dimers from isolation) and the like, this includes the use of chiral auxiliaries. Examples of such chiral auxiliary reagents, monomers, dimers and phosphoramidites are described in US 9982257, US 20170037399, US 20180216108, US 20180216107, US 9598458, WO 2017/062862, WO 2018/067973, WO 2017/16 0741, WO 2017 /192679, WO 2017/210647, WO 2018/098264, WO 2018/223056, WO 2018/237194, WO 2019/032607, WO 2019/055951, WO 2019/075357, WO 2019/200185, WO 2019/217784, WO 2019 /032612, WO 2020/191252 and/or WO 2021/071858, each of which chiral auxiliary reagents, monomers, dimers and phosphoramidites are independently incorporated herein by reference. In some embodiments, chiral auxiliaries are described in WO 2018/022473, WO 2018/098264, WO 2018/223056, WO 2018/223073, WO 2018/223081, WO 2018/237194, WO 2019/032607, WO 2019/055951, WO 2019/075357, WO 2019/200185, WO 2019/217784, WO 2019/032612, WO 2020/191252, and/or WO 2021/071858, each chiral auxiliary described independently herein. is incorporated by reference in

In some embodiments, chiral controlled manufacturing techniques including oligonucleotide synthesis cycles, reagents and conditions are described in US 9982257, US 20170037399, US 20180216108, US 20180216107, US 9598458, WO 2017/062862, WO 2018/067973, WO 2017/160 741; WO 2017/192679, WO 2017/210647, and/WO 2018/098264, WO 2018/022473, WO 2018/223056, WO 2018/223073, WO 2018/223081, WO 2018/237194, WO 2019/ 032607, WO 2019/ 032612, WO 2019/055951, WO 2019/075357, WO 2019/200185, WO 2019/217784, WO 2019/032612, WO 2018/223056, WO 2018/223073, WO 2018/223081, WO 2018/237194, WO 2019/ 032607, WO 2019/055951, WO 2019/075357, WO 2019/200185, WO 2019/217784, WO 2019/032612, WO 2020/191252, and/or WO 2021/071858, respectively oligonucleotide synthesis methods , cycles, reagents and conditions are independently incorporated herein by reference.

Once synthesized, provided oligonucleotides and compositions are generally further purified. Suitable purification techniques are well known and practiced by the person skilled in the art and include US 9982257, US 20170037399, US 20180216108, US 20180216107, US 9598458, WO 2017/062862, WO 2018/067973, WO 2017/160741, WO 2 017/192679; WO 2017/210647, WO 2018/098264, WO 2018/223056, WO 2018/237194, WO 2019/032607, WO 2019/055951, WO 2019/075357, WO 2019/200185, WO 2019/217 784, WO 2019/032612; Each purification technique is independently incorporated herein by reference, including but not limited to those described in WO 2020/191252, and/or WO 2021/071858.

In some embodiments, a cycle comprises or consists of coupling, capping, transforming, and unblocking. In some embodiments, a cycle comprises or consists of coupling, capping, modifying, capping, and unblocking. These steps are generally performed in the order listed, but in some implementations the order of certain steps, such as capping and transforming, may be changed as will be appreciated by those skilled in the art. If desired, one or more steps may be repeated to improve conversion, yield and/or purity, as often done by those skilled in the art in synthesis. For example, in some embodiments coupling can be repeated; In some implementations, the transformation (eg, oxidation to install =O, sulfide to install =S, etc.) can be repeated; In some embodiments, coupling is repeated after transformation that can transform a P(III) linkage into a P(V) linkage that may be more stable in certain circumstances, and coupling typically results in a newly formed P(III) linkage. A transformation to convert to a P(V) connection follows. In some embodiments, different conditions (eg, concentrations, temperatures, reagents, times, etc.) may be used when steps are repeated.

Techniques for formulating provided oligonucleotides and/or preparing pharmaceutical compositions, eg, for administration to a subject via a variety of routes, are readily available in the art and can be used in accordance with the present invention (eg ; US 9982257; US 20170037399; 2017/210647, WO 2018/098264, WO 2018/223056 , or as described in WO 2018/237194 and references cited therein).

Techniques for formulating provided oligonucleotides and/or preparing pharmaceutical compositions, eg, for administration to a subject via a variety of routes, are readily available in the art and can be used in accordance with the present invention (eg ; US 9982257; US 20170037399; 2017/210647, WO 2018/098264, WO 2018/223056 , or as described in WO 2018/237194 and references cited therein).

In some embodiments, useful chiral auxiliaries are , , or a salt thereof, R ^C11 is -L ^C1 -R ^C1 , L ^C1 is an optionally substituted -CH ₂ -, R ^C1 is R, -Si(R) ₃ , -SO ₂ R or an electron withdrawal is a male group, and R ^C2 and R ^C3 together with the atoms intervening therebetween form an optionally substituted 3-10 membered saturated ring having 0-2 heteroatoms in addition to nitrogen atoms. In some embodiments, useful chiral auxiliaries are Has a structure of, R ^C1 is R, -Si(R) ₃ , or -SO ₂ R, and R ^C2 and R ^C3 together with the atoms intervened therebetween, 0 to 2 heteroatoms in addition to the nitrogen atom Forms an optionally substituted 3-7 membered saturated ring with The ring formed is an optionally substituted 5-membered ring. In some embodiments, useful chiral auxiliaries are , , or has a salt structure thereof. In some embodiments, useful chiral auxiliaries are has the structure of In some embodiments, a useful chiral auxiliary is a DPSE chiral auxiliary. In some embodiments, the purity or stereochemical purity of a chiral auxiliary is at least 85%, 90%, 95%, 96%, 97%, 98%, or 99%. In some embodiments, the percentage is at least 85%. In some embodiments, the percentage is at least 90%. In some embodiments, the percentage is at least 95%. In some embodiments, the percentage is at least 96%. In some embodiments, the percentage is at least 97%. In some embodiments, the percentage is at least 98%. In some embodiments, the percentage is at least 99%.

In some embodiments, L ^C1 is -CH ₂ -. In some embodiments, L ^C1 is substituted -CH ₂ -. In some embodiments, L ^C1 is monosubstituted -CH ₂ -.

In some embodiments, R ^C1 is R. In some embodiments, R ^C1 is optionally substituted phenyl. In some embodiments, R ^C1 is -SiR ₃ . In some embodiments, R ^C1 is -SiPh ₂ Me. In some embodiments, R ^C1 is -SO ₂ R. In some embodiments, R is not hydrogen. In some embodiments, R is an optionally substituted phenyl. In some embodiments, R is phenyl. In some embodiments, R is an optionally substituted C _1-6 aliphatic. In some embodiments, R is C _1-6 alkyl. In some embodiments, R is methyl. In some embodiments, R is t-butyl.

In some embodiments, R ^C1 is -C(O)R, -OP(O)(OR) ₂ , -OP(O)(R) ₂ , -P(O)(R) ₂ , -S(O) electron withdrawing groups such as R, -S(O) ₂ R and the like. In some embodiments, chiral auxiliaries comprising an electron withdrawing group R ^C1 group are particularly useful for making chiral controlled internucleotidic linkages and/or non-negatively charged chiral controlled internucleotidic linkages linked to native RNA sugars.

In some embodiments, R ^C2 and R ^C3 are optionally substituted 3-10 membered (eg, 3, 4, 5, 6, 7, 3, 4, 5, 6, 7 , 8, 9, or 10 members) form a saturated ring. In some embodiments, R ^C2 and R ^C3 together with the intervening atoms form an optionally substituted 5-membered saturated ring having no heteroatoms other than nitrogen atoms.

In some embodiments, the compound has the structure HX ^C -C(R ^C5 ) ₂ -C(R ^C6 ) ₂ -SH or a salt thereof, wherein X ^C is O or S, and each R ^C5 and R ^C6 are independently R as described herein. In some embodiments, these compounds are useful for making monomers. In some embodiments, these compounds are useful as chiral auxiliaries. In some embodiments, these compounds are particularly useful for preparing monomers (eg, monomers including sm01, sm18, etc.) that form bonds between nitrogen atoms with linking phosphorus when used in oligonucleotide synthesis. In some embodiments, X ^C is O. In some embodiments, X ^C is S. In some embodiments, one R ^C5 is -H. In some embodiments, one R ^C6 is -H. In some embodiments, the compound has the structure HX ^C -CHR ^C5 -CHR ^C6 -SH or a salt thereof. In some embodiments, R ^C5 is an optionally substituted C _1-6 aliphatic. In some embodiments, R ^C5 is an optionally substituted C1-6 alkyl. In some embodiments, R ^C5 is methyl. In some embodiments, R ^C6 is an optionally substituted C _1-6 aliphatic. In some embodiments, R ^C6 is an optionally substituted C1-6 alkyl. In some embodiments, R ^C6 is methyl. In some embodiments, the compound is HOCH(CH ₃ )CH(CH ₃ )SH. In some embodiments, the compound is HSCH(CH ₃ )CH(CH ₃ )SH. In some embodiments, one R ^C5 is not hydrogen. In some embodiments, one R ^C6 is not hydrogen. In some embodiments, one R ^C5 and one R ^C6 are optionally substituted 3-20 having 0-5 heteroatoms together with intervening atoms (eg, 3-15, 3-10, 5-10, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) membered monocyclic, bicyclic, or polycyclic rings. In some embodiments, the formed ring is monocyclic. In some embodiments, one R ^C5 and one R ^C6 together with intervening atoms are optionally substituted 4-8, 4-7, 5-8, 5-7, 4, 5, 6, 7 or 8 membered monocyclic rings. form In some embodiments, the ring formed is a saturated cycloalkyl ring. In some embodiments, the ring formed is a cyclohexyl ring. In some embodiments, the rings formed are bicyclic. In some embodiments, the rings formed do not contain heteroatomic ring atoms. In some embodiments, each monocyclic ring unit is independently 3-10 membered and/or independently saturated, partially unsaturated or aromatic and has 0-5 heteroatoms. In some embodiments, the compound or a salt thereof, wherein the cyclohexyl ring is optionally substituted. In some embodiments, the compound or , or a salt thereof, wherein the cyclohexyl ring is optionally substituted. In some embodiments, the substituent is C _1-6 aliphatic, eg -C(CH ₃ )=CH ₂ . For example, in some embodiments, the compound am. In some embodiments, the compound or a salt thereof, wherein the cyclohexyl ring is optionally substituted.

In some embodiments, methods of making oligonucleotides and/or compositions include using chiral auxiliaries described herein to construct, for example, one or more chirally controlled internucleotide linkages. In some embodiments, one or more chirally controlled internucleotidic linkages are independently constructed using a DPSE chiral auxiliary. In some embodiments, each chirally controlled phosphorothioate internucleotidic linkage is independently constructed using a DPSE chiral auxiliary. In some embodiments, one or more chiral controlled internucleotidic linkages are independently , or a salt thereof, and R ^AU is as described herein. In some embodiments, each non-negatively charged chiral control internucleotidic linkage (eg, n001) is independently , or a salt thereof. In some embodiments, each chiral control internucleotidic linkage is independently , or a salt thereof. In some embodiments, R ^AU is optionally substituted C _1-20 , C _1-10 , C _1-6 , C _1-5 , or C _1-4 aliphatic. In some embodiments, R ^AU is an optionally substituted C _1-20 , C _1-10 , C _1-6 , C _1-5 , or C _1-4 alkyl. In some embodiments, R ^AU is optionally substituted aryl. In some embodiments, R ^AU is phenyl. In some embodiments, one or more chirally controlled internucleotidic linkages are constructed using a PSM chiral auxiliary. In some embodiments, each non-negatively charged chiral control internucleotidic linkage (eg, n001) is independently constructed using a PSM chiral auxiliary. In some embodiments, each chiral control internucleotidic linkage is independently constructed using a PSM chiral auxiliary. As will be appreciated by those of ordinary skill in the art, chiral auxiliaries are phosphoramidites (e.g., (DPSE phosphoramidite), (R ^AU is independently as described herein; when R ^AU is -Ph, a PSM phosphoramidite), and R ^NS is an optionally substituted/protected nucleoside (e.g., for oligonucleotide synthesis optionally protected), or salts thereof, etc.). In some embodiments, the phosphoramidite is , or a salt thereof, wherein each variable is independently as described herein. In some embodiments, R ^AU is optionally substituted phenyl. In some embodiments, R ^AU is phenyl. In some embodiments, R ^NS is an optionally substituted or protected nucleoside comprising hypoxanthine. In some embodiments, R ^NS comprises an optionally substituted or protected hypoxanthine. In some embodiments, R ^NS is an optionally substituted or protected inosine. In some embodiments, R ^NS is an optionally substituted or protected deoxyinosine. In some embodiments, R ^NS is an optionally substituted or protected 2'-F inosine (2'-OH replaced with 2'-F). In some embodiments, R ^NS is an optionally substituted or protected 2'-OR modified inosine (2'-OR modified (eg, 2'-OMe, 2'-MOE, etc.) replaced with a 2'-OR modified as described herein). '-OH). In some embodiments, hypoxanthine is protected O ⁶ . In some embodiments, hypoxanthine is O ⁶ protected with -L-Si(R) ₃ , where L is optionally substituted -CH ₂ -CH ₂ -, and each R is independently as described herein and - not H. In some embodiments, each R is independently an optionally substituted group selected from C _1-6 aliphatic and phenyl. In some embodiments, each R is independently an optionally substituted C _1-6 alkyl. In some embodiments, -L-Si(R) ₃ is -CH ₂ CH ₂ Si(Me) ₃ . In some embodiments, a compound comprising an O ⁶ protected hypoxanthine (eg, having -CH ₂ CH ₂ Si(Me) ₃ ) has a higher solubility than the corresponding unprotected O ⁶ compound and is suitable for use according to the present invention. When used in oligonucleotide synthesis, it can provide various benefits and advantages. In some embodiments, , or a salt structure thereof, R ^NS includes O ⁶ protected hypoxanthine (eg, with -CH ₂ CH ₂ Si(Me) ₃ ). In some embodiments, R ^NS is O ⁶ -protected inosine. In some embodiments, R ^NS is O ⁶ -protected deoxyinosine. In some embodiments, R ^NS is O ⁶ -protected 2′-F inosine. In some embodiments, R ^NS is an O ⁶ -protected 2′-OR modified inosine, wherein the 2′-OR variant is as described herein (eg, 2′-OMe, 2′-MOE, etc.). Among other things, the present invention encompasses the recognition that such compounds have sufficient solubility for oligonucleotide synthesis and can be used for oligonucleotide synthesis, whereas corresponding compounds lacking O ⁶ protection may not have sufficient solubility for efficient oligonucleotide synthesis. do. In some embodiments, the phosphoramidite is (1S,3S,3aS)-1-(((2R,3S,5R)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl) -5-(6-(2-(trimethylsilyl)ethoxy)-9H-purin-9-yl)tetrahydrofuran-3-yl)oxy)-3-((methyldiphenylsilyl)methyl)tetrahydro- 1H,3H-pyrrolo[1,2-c][1,3,2]oxazaphosphole. In some embodiments, the phosphoramidite is (1S,3S,3aS)-1-(((2R,3S,5R)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl) -5-(6-(2-(trimethylsilyl)ethoxy)-9H-purin-9-yl)tetrahydrofuran-3-yl)oxy)-3-((phenylsulfonyl)methyl)tetrahydro-1H ,3H-pyrrolo[1,2-c][1,3,2]oxazaphosphole. In some embodiments, , or a compound having a salt structure thereof, R ^NS includes O ⁶ unprotected hypoxanthine. In some embodiments, R ^NS is an optionally substituted or protected inosine, wherein hypoxanthine is unprotected. In some embodiments, R ^NS is an optionally substituted or protected deoxyinosine, wherein hypoxanthine is unprotected. In some embodiments, R ^NS is an optionally substituted or protected 2′-F inosine, wherein hypoxanthine is unprotected. In some embodiments, R ^NS is an optionally substituted or protected 2′-OR modified inosine, wherein hypoxanthine is unprotected and its 2′-OR variants are as described herein (eg, 2′-OMe, 2′-MOE, etc.). Among other things, the present invention encompasses the recognition that such compounds have sufficient solubility for oligonucleotide synthesis and can be used for oligonucleotide synthesis without O ⁶ protection.

In some embodiments, the method comprises providing a DPSE and/or PSM phosphoramidite or salt thereof. In some embodiments, provided methods include contacting a DPSE and/or PSM phosphoramidite or salt thereof with -OH (eg, the 5'-OH of a nucleoside or oligonucleotide chain). As will be appreciated by those skilled in the art, contacting can be performed under a variety of suitable conditions to form a phosphorus bond. In some embodiments, preparation of each chirally controlled internucleotidic linkage involves contacting a DPSE or PSM phosphoramidite or salt thereof with -OH (e.g., the 5'-OH of a nucleoside or oligonucleotide chain). Include steps. In some embodiments, preparation of each chirally controlled phosphorothioate internucleotidic linkage independently converts the DPSE phosphoramidite or salt thereof to -OH (e.g., the 5'-OH of the nucleoside or oligonucleotide chain). ). In some embodiments, preparation of each non-negatively charged chiral control internucleotidic linkage (eg, n001) independently converts the PSM phosphoramidite or salt thereof to -OH (eg, a nucleoside or 5'-OH) of the oligonucleotide chain. In some embodiments, preparation of each chirally controlled internucleotidic linkage comprises contacting a PSM phosphoramidite or salt thereof with -OH (e.g., the 5'-OH of a nucleoside or oligonucleotide chain). include In some embodiments, the contact is a phosphorus atom bonded to two sugars and a chiral auxiliary moiety (e.g., , or a salt form thereof (eg, derived from DPSE phosphoramidite or a salt thereof), , or a salt form thereof (R ^AU is independently as described herein; when R ^AU is -Ph, eg derived from PSM phosphoramidite or a salt thereof), etc.) (III ) form a connection. In some embodiments, the oligonucleotide comprises a P(III) linkage comprising a chiral auxiliary moiety, for example derived from a DPSE or PSM phosphoramidite. In some embodiments, a P(III) linkage comprising a chiral auxiliary moiety is chirally controlled. In some embodiments, a chiral auxiliary moiety can be protected, eg, before converting a P(III) linkage to a P(V) linkage (eg, prior to sulfurization, reaction with an azide, etc.). In some embodiments, the protected chiral auxiliary is , or a salt form thereof (eg, R′ is independently as described herein; eg, derived from DPSE phosphoramidite or a salt thereof), or , or a salt form thereof (each R' and R ^AU is independently as described herein; when R ^AU is -Ph, for example derived from PSM phosphoramidite or a salt thereof). and each R' is independently as described herein. In some embodiments, R' is -C(0)R, and R is as described herein. In some embodiments, R is -CH ₃ . In some embodiments, the oligonucleotide comprises a protected chiral auxiliary. In some embodiments, each chiral control internucleotidic linkage in an oligonucleotide is independently , or a salt form thereof, or , or a salt form thereof. In some embodiments, each chiral control internucleotidic linkage in an oligonucleotide is independently , or a salt form thereof. In some embodiments, R' is -C(0)R. In some embodiments, R' is -C(O)CH ₃ . In some embodiments, R ^AU is Ph. In some embodiments, an oligonucleotide is one or more or a salt form thereof (PIII-1), wherein each variable is independently as described herein. In some embodiments, an oligonucleotide is one or more or a salt form thereof (PIII-2), wherein each variable is independently as described herein. In some embodiments, an oligonucleotide is one or more or a salt form thereof (PIII-5), wherein each variable is independently as described herein. In some embodiments, an oligonucleotide is one or more or a salt form thereof (PIII-6), wherein each variable is independently as described herein. In some embodiments, the 5'-terminal internucleotide linkage is PIII-1, PIII-2, PIII-5, or PIII-6. In some embodiments, the 5'-terminal internucleotide linkage is PIII-1 or PIII-2. In some embodiments, R' is -H. In some embodiments, R' is -C(0)R. In some embodiments, R' is -C(O)CH ₃ . In some embodiments, R ^AU is -Ph. In some embodiments, a P(III) linkage is converted to a P(V) linkage. In some embodiments, a P(V) linkage is a phosphorus atom bonded to two sugars, a chiral auxiliary moiety (e.g., or , or a salt form thereof (R′ is as described herein; eg derived from DPSE phosphoramidite or a salt thereof); , or a salt form thereof (each of R' and R ^AU is independently as described herein; when R ^AU is -Ph, eg derived from PSM phosphoramidite or a salt thereof), etc.), and S or includes In some embodiments, a P(V) linkage is a phosphorus atom bonded to two sugars; , or a salt form thereof (each R' and RAU is independently as described herein; when R ^AU is -Ph, eg derived from PSM phosphoramidite or a salt thereof), etc.), and S or includes In some embodiments, a P(V) linkage is a phosphorus atom bonded to two sugars; , or a salt form thereof (each R' and R ^AU is independently as described herein; when R ^AU is -Ph, eg derived from PSM phosphoramidite or a salt thereof), etc.), and S. In some embodiments, a P(V) linkage is a phosphorus atom bonded to two sugars; , or a salt form thereof (each R' and R ^AU is independently as described herein; when R ^AU is -Ph, eg derived from PSM phosphoramidite or a salt thereof), etc.), and includes Those skilled in the art It will be appreciated that this counterion (eg, in some embodiments, PF ₆ ^- ) may be present. In some embodiments, an oligonucleotide is one or more or a salt form thereof (PV-1), wherein each variable is independently as described herein. In some embodiments, an oligonucleotide is one or more or a salt form thereof (PV-2), wherein each variable is independently as described herein. In some embodiments, an oligonucleotide is one or more or a salt form thereof (PV-3), wherein each variable is independently as described herein. In some embodiments, an oligonucleotide is one or more or a salt form thereof (PV-4), wherein each variable is independently as described herein. In some embodiments, an oligonucleotide is one or more or a salt form thereof (PV-5), wherein each variable is independently as described herein. In some embodiments, an oligonucleotide is one or more or a salt form thereof (PV-6), wherein each variable is independently as described herein. In some embodiments, each chiral internucleotidic linkage, or each chiral control internucleotidic linkage, of an oligonucleotide is independently PIII-1, PIII-2, PIII-5, PIII-6, PV-1, PV-2, is selected from PV-3, PV-4, PV-5, and PV-6. In some embodiments, each chiral internucleotidic linkage, or each chiral control internucleotidic linkage, of an oligonucleotide is independently PIII-1, PIII-2, PV-1, PV-2, PV-3, and PV-4 is selected from In some embodiments, the linkage of PIII-1, PIII-2, PIII-5, or PIII-6 is generally the 5'-end of the internucleotide linkage. In some embodiments, each chiral internucleotidic linkage, or each chiral control internucleotidic linkage, of an oligonucleotide is independently PV-1, PV-2, PV-3, PV-4, PV-5, and PV-6 is selected from In some embodiments, each chiral internucleotidic linkage, or each chiral control internucleotidic linkage, of an oligonucleotide is independently selected from PV-1, PV-2, PV-3, or PV-4. In some embodiments, a provided oligonucleotide is an oligonucleotide as described herein, e.g., of Table 1, wherein each *S is independently replaced with PV-3 or PV-5, and each *R is independently Each n001R is independently replaced with PV-1, and each n001S is independently replaced with PV-2. In some embodiments, a provided oligonucleotide is an oligonucleotide as described herein, e.g., of Table 1, wherein each *S is independently replaced with PV-3, and each *R is independently replaced with PV-4. , each n001R is independently replaced with PV-1, and each n001S is independently replaced with PV-2. In some embodiments, each native phosphate linkage is independently a precursor, e.g. is replaced by In some embodiments, R' is -H. In some embodiments, R' is -C(0)R. In some embodiments, R' is -C(O)CH ₃ . In some embodiments, R ^AU is -Ph. In some embodiments, the method comprises removing one or more chiral auxiliary moieties such that a phosphorothioate and/or negatively charged internucleotidic linkage (eg, n001) is formed (eg, V-1 , from PV-2, PV-3, PV-4, PV-5, PV-6, etc.). In some embodiments, removal of the chiral auxiliary (eg, PSM) comprises contacting the oligonucleotide with a base (eg, N(R) ₃ such as DEA) under anhydrous conditions.

In some embodiments, as will be appreciated by those skilled in the art, for the preparation of chirally controlled internucleotidic linkages, monomers or phosphoramidates (e.g., DPSE or PSM phosphoramidates) are generally chirally enriched or In pure form (e.g., about or at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or about 100%)).

In some embodiments, the present invention provides useful reagents for the preparation of oligonucleotides and compositions thereof. In some embodiments, the monomers and phosphoramidites include nucleosides, nucleobases, and sugars as described herein. In some embodiments, nucleobases and sugars are suitably protected for oligonucleotide synthesis, as will be appreciated by those skilled in the art. In some embodiments, the phosphoramidite has the structure R ^NS -P(OR)N(R) ₂ , where R ^NS is an optionally protected nucleoside moiety. In some embodiments, the phosphoramidite has the structure R ^NS -P(OCH ₂ CH ₂ CN)N(i-Pr) ₂ . In some embodiments, a monomer comprises a nucleobase that is or comprises ring BA or a tautomer of ring BA, wherein ring BA is BA-I, BA-Ia, BA-Ib, BA- II, BA-II-a, BA-II-b, BA-III, BA-III-a, BA-III-b, BA-IV, BA-IV-a, BA-IV-b, BA-V, It has the structure BA-Va, BA-Vb, or BA-VI, and the nucleobases are optionally substituted or protected. In some embodiments, the phosphoramidite comprises a nucleobase that is or comprises Ring BA or a tautomer of Ring BA, or comprising Ring BA or a tautomer of Ring BA, wherein Ring BA is BA-I, BA-Ia, BA-Ib , BA-II, BA-II-a, BA-II-b, BA-III, BA-III-a, BA-III-b, BA-IV, BA-IV-a, BA-IV-b, BA -V, BA-Va, BA-Vb, or BA-VI, wherein the nucleobases are optionally substituted or protected. In some embodiments, the phosphoramidite includes a chiral auxiliary moiety, and phosphorus is bonded to the oxygen and nitrogen atoms of the chiral auxiliary moiety. In some embodiments, the phosphoramidite is , or a salt thereof, where R ^NS is a protected nucleoside moiety (eg, a 5'-OH and/or nucleobase suitably protected for oligonucleotide synthesis), and each other variable is independently as described herein. In some embodiments, the phosphoramidite is where R ^NS is a protected nucleoside moiety (eg, a 5'-OH and/or nucleobase suitably protected for oligonucleotide synthesis), and R ^C1 is R, -Si(R ) ₃ , or -SO ₂ R, and R ^C2 and R ^C3 together with the atoms intervening therebetween form an optionally substituted 3-7 membered saturated ring having 0-2 heteroatoms in addition to nitrogen atoms, Coupling forms internucleotide linkages. In some embodiments, the 5'-OH of R ^NS is protected. In some embodiments, the 5'-OH of R ^NS is protected as -ODMTr. In some embodiments, R ^NS is linked to phosphorus through 3'-0-. In some embodiments, the ring formed by R ^C2 and R ^C3 is an optionally substituted 5-membered ring. In some embodiments, the phosphoramidite is , or has a salt structure thereof. In some embodiments, the phosphoramidite is has the structure of In some embodiments, as described herein, an R ^NS comprises a modified nucleobase that is optionally protected for oligonucleotide synthesis (eg, b001A, b002A, b003A, b008U, b001C, etc.). In some embodiments, the monomer is or a salt structure thereof, wherein R ^NS is an optionally substituted/protected nucleoside (eg, optionally protected for oligonucleotide synthesis) as described herein, and each other variable is independently as described herein. same. In some embodiments, -X ^C -C(R ^C5 ) ₂ -C(R ^C6 ) ₂ -S- is HX ^C -C(R ^C5 ) ₂ -C(R ^C6 ) ₂ -SH as described herein compounds such as HOCH(CH ₃ )CH(CH ₃ )SH, HSCH(CH ₃ )CH(CH ₃ )SH, It is of a back-and-forth structure. In some embodiments, the 5'-OH of R ^NS is protected. In some embodiments, the 5'-OH of R ^NS is protected as -ODMTr.

In some embodiments, R ^NS is , or salts thereof, wherein BA ^s are as described herein. In some embodiments, R ^NS is , or a salt thereof, and BA ^s are as described herein. In some embodiments, each -OH is optionally and independently substituted or protected. In some embodiments, BA ^s is an optionally substituted or protected nucleobase, and each -OH of the nucleoside is independently protected, wherein at least one -OH is protected as DMTrO-. In some embodiments, -OH, for example for coupling with other monomers or phosphoramidites, is protected as DMTrO-. In some embodiments, -OH groups, for example for coupling with other monomers or phosphoramidites, are protected differently from -OH groups not for coupling. In some embodiments, uncoupling -OH is protected so that protection is maintained when DMTrO- is deprotected. In some embodiments, uncoupling -OH is protected such that protection is maintained during oligonucleotide synthesis cycles. In some embodiments, BA ^s is an optionally protected nucleobase selected from A, T, C, G, U, and tautomers thereof.

In some embodiments, the purity or stereochemical purity of the monomer or phosphoramidite is at least 85%, 90%, 95%, 96%, 97%, 98%, or 99%. In some embodiments, the percentage is at least 85%. In some embodiments, the percentage is at least 90%. In some embodiments, the percentage is at least 95%.

In some embodiments, the invention provides a method of making an oligonucleotide or composition comprising coupling a free -OH, e.g., free 5'-OH, of an oligonucleotide or nucleoside with a monomer as described herein provides a way to In some embodiments, the invention provides a method of making an oligonucleotide or composition, comprising coupling a free -OH, e.g., free 5'-OH, of an oligonucleotide or nucleoside with a phosphoramidite as described herein. It provides a method comprising the step of doing.

In some embodiments, the invention provides an oligonucleotide, wherein the oligonucleotide comprises one or more modified internucleotide linkages, each independently of the structure -O ⁵ -P ^L (W)(R ^CA )-O ³ - has,

(Wherein, P ^L is P or P(=W);

W is O, S, or W ^N ;

W ^N is =NC(-N(R ¹ ) ₂ =N ⁺ (R ¹ ) ₂ Q ^-

Q ^- is an anion,

R ^CA is or contains an optionally capped chiral auxiliary moiety,

O ⁵ is an oxygen bonded to the 5'-carbon of the sugar;

O ³ is the oxygen attached to the 3'-carbon of the sugar.

In some embodiments, the modified internucleotidic linkages are optionally chirally controlled. In some embodiments, the modified internucleotidic linkages are optionally chirally controlled.

In some embodiments, provided methods include removing R ^CA from such modified internucleotide linkages. In some embodiments, after removal, the bond to R ^CA is replaced with -OH. In some embodiments, after removal, the bond to R ^CA is replaced with =O and the bond to W ^N is replaced with -N=C(N(R ¹ ) ₂ ) ₂ .

In some embodiments, P ^L is P=S and when R ^CA is removed, these internucleotidic linkages are converted to phosphorothioate internucleotidic linkages.

In some embodiments, P ^L is P=W ^N and when R ^CA is removed, such an internucleotidic linkage is It is converted into an internucleotide linkage having the structure of In some embodiments, The internucleotide linkage having the structure of has the structure of In some embodiments, The internucleotide linkage having the structure of has the structure of

In some embodiments, P ^L is P (eg, in an internucleotide linkage newly formed from coupling a phosphoramidite with a 5'-OH). In some embodiments, W is O or S. In some embodiments, W is S (eg, after sulfurization). In some embodiments, W is O (eg, after oxidation). In some embodiments, certain non-negatively charged internucleotidic linkages or neutral internucleotidic linkages can form P(III) phosphite triester internucleotidic linkages under suitable conditions with an azido imidazolinium salt (e.g., It can be prepared by reacting with a compound containing In some embodiments, the azido imidazolinium salt is a salt of PF ₆ ⁻ . In some embodiments, the azido imidazolinium salt is is the salt of In some embodiments, the azido imidazolinium salt is 2-azido-1,3-dimethylimidazolinium hexafluorophosphate.

As understood by those skilled in the art, Q ⁻ can be a variety of suitable anions present in the system (eg, in oligonucleotide synthesis), and can vary during the oligonucleotide manufacturing process depending on cycles, process steps, reagents, solvents, etc. . In some embodiments, Q ⁻ is PF ₆ ⁻ .

In some embodiments, R ^CA is , R ^C4 is -H or -C(O)R', and each other variable is independently as described herein. In some embodiments, R ^CA is , R ^C1 is R, -Si(R) ₃ , or -SO ₂ R, and R ^C2 and R ^C3 together with the atoms intervening therebetween have 0 to 2 heteroatoms in addition to the nitrogen atom, optionally substituted. form a 3-7 membered saturated ring, and R ^C4 is -H or -C(O)R'. In some embodiments, R ^C4 is -H. In some embodiments, R ^C4 is -C(O)CH ₃ . In some embodiments, R ^C2 and R ^C3 together form an optionally substituted 5-membered ring.

In some embodiments, R ^C4 is -H (eg, in an internucleotidic linkage newly formed from coupling a phosphoramidite with a 5'-OH). In some embodiments, R ^C4 is -C(O)R (eg, after capping the amine). In some embodiments, R is methyl.

In some embodiments, each chirally controlled phosphorothioate internucleotidic linkage is independently converted from -O ⁵ -P ^L (W)(R ^CA )-O ³ -.

Evaluation/characterization of provided technology

As will be appreciated by those skilled in the art, a variety of techniques can be used to evaluate/characterize techniques provided in accordance with the present invention. Certain useful techniques are described in the Examples; As indicated, in particular, the present invention describes a variety of in vivo and in vitro techniques suitable for evaluating and characterizing the provided techniques. In some embodiments, provided techniques are assessed/characterized in the presence or absence of an exogenous ADAR polypeptide, eg, in cells. Additionally or alternatively, in some embodiments, provided techniques are evaluated/characterized in, for example, animals (eg, non-human primates and mice).

In particular, the present invention provides various agents (e.g., oligonucleotides) and compositions thereof capable of conferring editing in a variety of human systems, e.g., cells, in certain cells that do not contain or express a human ADAR (e.g., human ADAR1). eg mouse cells) and certain animals such as rodents (eg mice) may exhibit no or much lower levels of editing. In particular, the mouse commonly used in animal models is that various agents that are active in human cells do not provide or very little activity in mouse cells and animals that have not been engineered to contain or express an appropriate ADAR1 (eg, human ADAR1) polypeptide or feature thereof. Because they provide low levels of activity, their use in evaluating various agents (eg oligonucleotides) for editing in humans may be limited. In some embodiments, the present invention provides engineered cells and non-human animals expressing human ADAR1 polypeptides or features thereof. In some embodiments, such cells and humans are useful for evaluating and characterizing provided technologies. In some embodiments, a human ADAR1 polypeptide or feature thereof is or comprises a human ADAR1 polypeptide or feature thereof. In some embodiments, the human ADAR1 polypeptide or feature thereof is or comprises a human ADAR1 p110 polypeptide or feature thereof. In some embodiments, the human ADAR1 polypeptide or feature thereof is or comprises a human ADAR1 p150 polypeptide or feature thereof. In some embodiments, a human ADAR1 polypeptide or feature thereof is or comprises a human ADAR1. In some embodiments, a human ADAR1 polypeptide or feature thereof is or comprises a human ADAR1 p110 peptide. In some embodiments, a human ADAR1 polypeptide or feature thereof is or comprises a human ADAR1 p150 peptide. In some embodiments, a human ADAR1 polypeptide or feature thereof is or comprises one or more or all of the following domains of human ADAR1: a Z-DNA binding domain, a dsRNA binding domain, and a deaminase domain. In some embodiments, a human ADAR1 polypeptide or feature thereof is or comprises one or both human ADAR1 Z-DNA binding domains; Alternatively or additionally, in some embodiments, a human ADAR1 polypeptide or feature thereof is or comprises one, two, or all of a human ADAR1 dsRNA binding domain; Alternatively or additionally, in some embodiments, a human ADAR1 polypeptide or feature thereof is or comprises a human deaminase domain. In some embodiments, a human ADAR1 polypeptide or feature thereof can be co-expressed with a mouse ADAR1 polypeptide or feature thereof. For example, one or more human dsRNA binding domains can be engineered to be expressed together with a mouse deaminase domain to form a human-mouse hybrid ADAR1 polypeptide. In some embodiments, the cell and/or non-human animal is engineered to contain and/or express a polynucleotide encoding a human ADAR1 polypeptide or features thereof as described herein. In some embodiments, the genome of the cell and/or non-human animal is engineered to include a polynucleotide encoding a human ADAR1 polypeptide or features thereof as described herein. In some embodiments, the cell and/or germline genome of a non-human animal is engineered to include a polynucleotide encoding a human ADAR1 polypeptide or feature thereof as described herein. In some embodiments, the cells and non-human animals have one or more mutations from G to A, each independently associated with a condition, disorder or disease (e.g., substitution of lysine for glutamate at amino acid position 342 (E342K) of the A1AT protein). mutations in the SERPINA1 gene (eg, c. 1024G>A)), eg, in a genome (in some embodiments, a germline genome). As shown herein, such cells and animals in particular are susceptible to provided technology, e.g., various oligonucleotides and compositions thereof, e.g., editing properties and/or activities, including use for one or more conditions, disorders or diseases. useful for evaluating/characterizing In some embodiments, the cell is a rodent cell. In some embodiments, the cell is a mouse cell. In some embodiments, the animal is a rodent. In some embodiments, the animal is a mouse.

Among other things, the present invention relates to sugar modifications, base modifications, internucleotidic linkage modifications, Oligonucleotide designs comprising linkage phosphorus stereochemistry and/or patterns thereof are provided. For example, provided oligonucleotides of various designs and compositions thereof provide high levels of editing in mice that do not express human ADAR protein (eg, mice expressing only mouse ADAR protein), and in some embodiments human ADAR While providing a level of editing comparable to or not lower than in mice engineered to express the protein, comparable oligonucleotides of the reference design and compositions thereof can be used in mice that do not express human ADAR proteins (e.g., mouse ADAR mice expressing only the protein), and in some embodiments significantly lower levels of editing than in mice engineered to express human ADAR proteins. In some embodiments, the reference design is described in WO 2016/097212, WO 2017/220751, WO 2018/041973, WO 2018/134301A1, WO 2019/158475, WO 2019/219581, WO 2020/157008, WO 2020/165077, WO 2020 /201406 or WO 2020/252376. In some embodiments, the reference design is the design of WO 2021/071858.

In some embodiments, the present invention provides evaluation/characterization for evaluating cells and/or non-human animals (including those comprising or engineered to express an ADAR1 polypeptide or a feature thereof, or a polypeptide encoding an ADAR1 polypeptide or feature thereof). The technology is provided wherein the ADAR1 polypeptide or features thereof and/or polynucleotides are not present and/or expressed in cells and/or non-human animals prior to manipulation. In some embodiments, a provided method comprises administering one or more oligonucleotides or compositions to a cell or population thereof, wherein the one or more oligonucleotides or compositions are each independently capable of editing adenosine in a comparable human cell or population thereof. can In some embodiments, provided methods include administering one or more oligonucleotides or compositions to an animal or population thereof, wherein the one or more oligonucleotides or compositions are each independently capable of editing adenosine in a human cell or population thereof. In some embodiments, the level of editing in a cell to be evaluated/characterized, or in a cell of an animal, is compared to that observed in a comparable human cell. In some embodiments, the comparable human cell is of the same type as the cell or animal cell being evaluated/characterized. In some embodiments, the cell is a rodent cell. In some embodiments, the cell is a mouse cell. In some embodiments, the animal is a rodent. In some embodiments, the animal is a mouse. In some embodiments, one or more oligonucleotides or compositions are administered individually to individual cells and/or animals. In some embodiments, one or more oligonucleotides or compositions may be administered to the same collection of cells and/or animals, optionally simultaneously. A variety of oligonucleotides and compositions capable of editing a variety of target adenosines are as described herein and can be used accordingly.

As will be appreciated by those skilled in the art, in some embodiments, provided techniques, e.g., oligonucleotides, compositions, etc., can be evaluated in one or more models, e.g., cells, tissues, organs, animals, etc. In some embodiments, as will be appreciated by those of ordinary skill in the art, a cell, tissue, organ, animal, etc. is of, associated with, or comprises one or more features (e.g., a nucleotide sequence such as a mutation) of a condition, disorder or disease. is or contains such a cell. For example, in some embodiments, a cell, tissue, organ, animal, etc. comprises a G to A mutation associated with a condition, disorder or disease, eg, 1024G>A(E342K) in human SERPINA1. In some embodiments, the animal is a NOD.Cg-Prkdcscid Il2rgtm1Wjl Tg(SERPINA1*E342K)#Slcw/SzJ mouse (eg, The Jackson Laboratory Stock No: 028842; NSG-PiZ, also Borel F; Tang Q; Gernoux G; Greer C; Wang Z; Barzel A; Kay MA; Shultz LD; Greiner DL; Flotte TR; Brehm MA; Mueller C. 2017. Survival Advantage of Both Human Hepatocyte Xenografts and Genome-Edited Hepatocytes for Treatment of alpha-1 Antitrypsin Deficiency.Mol Ther 25(11):2477-2489PubMed:29032169MGI:J:243726] and Li S;Ling C;Zhong L;Li M;Su Q;He R;Tang Q;Greiner DL; Shultz LD; Brehm MA; Flotte TR; Mueller C; Srivastava A; Gao G. 2015). Efficient and Targeted Transduction of Nonhuman Primate Liver with Systemically Delivered Optimized AAV3B Vectors. Mol Ther 23(12):1867-76 PubMed: 26403887MGI: J:230567). In some embodiments, the cell, tissue, organ, animal, etc. comprises one or more cancer cells. In some embodiments, a non-human cell, tissue, organ, animal, etc. is an ADAR1 or its, e.g., through integration (optionally into its genome or germline genome) of a polynucleotide whose sequence encodes an ADAR1 polypeptide or feature thereof. Engineered to contain or express a feature. In some embodiments, ADAR1 is a primate ADAR1. In some embodiments, ADAR1 is human ADAR1. In some embodiments, human ADAR1 is human ADAR1 p110. In some embodiments, human ADAR1 is human ADAR1 p150. As will be appreciated by those skilled in the art, a variety of techniques are available in the art and can be used in accordance with the present invention to produce useful cells, tissues, organs, animals, and the like. For example, for an animal model of a condition, disorder or disease expressing human ADAR1 or a feature thereof, the animal model can be crossed with a huADAR1 mouse described herein to provide an engineered animal model expressing human ADAR1 or feature thereof. there is. In some embodiments, a mouse comprising a G to A mutation, e.g., a NOD.Cg-Prkdcscid Il2rgtm1Wjl Tg(SERPINA1*E342K)#Slcw/SzJ mouse (e.g., The Jackson Laboratory Stock No: 028842 ];NSG-PiZ, also Borel F; Tang Q; Gernoux G; Greer C; Wang Z; Barzel A; Kay MA; Shultz LD; Greiner DL; Flotte TR; Brehm MA; Mueller C. 2017. Survival Advantage of Both Human Hepatocyte Xenografts and Genome-Edited Hepatocytes for Treatment of alpha-1 Antitrypsin Deficiency.Mol Ther 25(11):2477-2489PubMed: 29032169MGI: J:243726, and Li S;Ling C;Zhong L;Li M;Su Q; He R; Tang Q; Greiner DL; Shultz LD; Brehm MA; Flotte TR; Mueller C; Srivastava A; Gao G. 2015]) was crossed with the huADAR1 mouse described herein to produce a G to A mutation (e.g. For example, a mouse comprising 024G>A(E342K) in human SERPINA1 and expressing human ADAR1 or a feature thereof is provided.

As will be appreciated by those skilled in the art, in some embodiments, an animal may be heterozygous for one or more or all sequences. In some embodiments, the animal is homozygous for one or more or all sequences. In some embodiments, the animal is hemizygous for one or more or all engineered sequences. In some embodiments, the animal is homozygous for one or more sequences and heterozygous for one or more sequences. In some embodiments, the animal is heterozygous for a polynucleotide whose sequence encodes an ADAR1 polypeptide or feature thereof. In some embodiments, the animal is homozygous for a polynucleotide whose sequence encodes an ADAR1 polypeptide or feature thereof. In some embodiments, a particular animal is heterozygous for one or more polynucleotide sequences associated with various conditions, disorders or diseases, and whose sequences are heterozygous for a polynucleotide encoding an ADAR1 polypeptide or feature thereof. In some embodiments, a particular animal is homozygous for one or more polynucleotide sequences associated with various conditions, disorders or diseases, and its sequences are heterozygous for a polynucleotide encoding an ADAR1 polypeptide or feature thereof. In some embodiments, an animal is heterozygous for one or more polynucleotide sequences associated with various conditions, disorders or diseases, and its sequences are homozygous for a polynucleotide encoding an ADAR1 polypeptide or feature thereof. In some embodiments, a particular animal is homozygous for one or more polynucleotide sequences associated with various conditions, disorders or diseases, and whose sequences are homozygous for a polynucleotide encoding an ADAR1 polypeptide or feature thereof. A cell or tissue may similarly be heterozygous, hemizygous and/or homozygous for a variety of sequences.

In some embodiments, the present invention provides a method for evaluating an agent, eg, an oligonucleotide or composition thereof, comprising administering the agent or composition to an animal, cell or tissue described herein. In some embodiments, an agent or composition is evaluated to prevent or treat a condition, disorder or disease. In some embodiments, an animal, cell, tissue, e.g., as described in various embodiments herein, whose sequences have been engineered to comprise and/or express a polynucleotide encoding an ADAR1 polypeptide or feature thereof (e.g., For example, mutations associated with various conditions, disorders or diseases, and/or animal models, or cells or tissues, for various conditions, disorders or diseases, including cells, tissues, organs, etc., associated with or associated with various conditions, disorders or diseases. . In some embodiments, the animal is a model animal for various conditions, disorders or diseases, but whose sequence has not been engineered to comprise and/or express a polynucleotide encoding an ADAR1 polypeptide or feature thereof, whose sequence is an ADAR1 polypeptide or It can be provided by mating (eg, IVF, natural mating, etc.) with an animal engineered to contain and/or express a polynucleotide encoding a feature thereof. In some embodiments, a cell or tissue can be provided by introducing into the cell or tissue a polynucleotide whose sequence encodes an ADAR1 polypeptide or feature thereof. In some embodiments, the present invention provides a method of preventing or treating a condition, disorder or disease comprising administering to a subject an effective amount of an agent or composition thereof, wherein the agent or composition is evaluated in an animal provided herein. (e.g., an animal engineered to comprise an ADAR1 polypeptide or feature thereof, an animal whose sequence is engineered to comprise and/or express a polynucleotide encoding an ADAR1 polypeptide or feature thereof, an ADAR1 polypeptide or feature thereof A model animal for a condition, disorder or disease that has been engineered, the sequence of which has been engineered to contain and/or express a polynucleotide encoding an ADAR1 polypeptide or feature thereof). In some embodiments, the present invention provides a method of preventing or treating a condition, disorder or disease comprising administering to a subject an effective amount of an agent or composition thereof, wherein the agent or composition is a cell or tissue provided herein is evaluated in In some embodiments, an animal, cell, or tissue comprises a SERPINA1 mutation (eg, 1024 G>A (E342K)) and its sequence is engineered to contain and/or express a polynucleotide encoding an ADAR1 polypeptide or feature thereof. do. In some embodiments, the animal is a non-human animal. In some embodiments, the cell is a non-human animal cell. In some embodiments, the tissue is a non-human animal tissue. In some embodiments, the non-human animal is a rodent. In some embodiments, the non-human animal is a mouse. In some embodiments, the non-human animal is a rat. In some embodiments, the non-human animal is a non-human primate.

In some embodiments, the present invention relates to 1) evaluating an agent or composition thereof comprising contacting the agent or composition thereof with a provided cell or tissue associated with or of the condition, disorder or disease; and 2) administering an effective amount of an agent or composition thereof to a subject suffering from or susceptible to the condition, disorder or disease. In some embodiments, the present invention provides steps for 1) evaluating an agent or composition thereof comprising administering the agent or composition thereof to a given animal that is an animal model of the condition, disorder or disease, and 2) evaluating the condition, disorder or disease. A method comprising administering an effective amount of an agent or composition thereof to a subject suffering from or susceptible to it is provided. In some embodiments, as described herein, a cell, tissue or animal is engineered to include an ADAR1 polypeptide or feature thereof. In some embodiments, a cell, tissue or animal is engineered such that its sequence comprises and/or expresses a polynucleotide encoding an ADAR1 polypeptide or feature thereof. In some embodiments, the cell, tissue or animal further comprises a nucleotide sequence (eg, a mutation) associated with a condition, disorder or disease. In some embodiments, the animal is a rodent, eg, mouse, rat, etc. In some embodiments, the cell or tissue is rodent, eg, mouse, rat, etc. In some embodiments, the cell is a germline cell. In some embodiments, some but not all cells, e.g., cell populations, tissues, or cells of a particular cell type or tissue or location of an animal, have a nucleotide sequence (e.g., a mutation) associated with a condition, disorder, or disease. , and some of these cells are engineered to contain an ADAR1 polypeptide or feature thereof, or their sequences are engineered to contain and/or express a polynucleotide encoding an ADAR1 polypeptide or feature thereof. In some embodiments, the population of liver cells comprises a SERPINA1 mutation, eg 1024 G>A (E342K) and a polynucleotide whose sequence encodes an ADAR1 polypeptide or feature thereof. One skilled in the art understands that a variety of techniques are available and can be used in accordance with the present invention for the optionally controlled introduction and/or expression of nucleotide sequences in a variety of cells, tissues or organs. In some embodiments, as described herein, a cell, tissue, or animal comprises a polynucleotide whose sequence encodes an ADAR1 polypeptide or feature thereof in a genome, in some embodiments in a germline genome. In some embodiments, as described herein, a cell, tissue, or animal comprises a nucleotide sequence (eg, a mutation) associated with a condition, disorder, or disease in a genome, in some embodiments a germline genome.

As described herein, in some embodiments, the polynucleotide encodes human ADAR1 p110 or features thereof. In some embodiments, the polynucleotide encodes human ADAR1 p110. In some embodiments, the polynucleotide encodes human ADAR1 p150 or features thereof. In some embodiments, the polynucleotide encodes human ADAR1 p150. In some embodiments, a cell, tissue or animal (eg, a huADAR mouse or a cell or tissue therefrom) is engineered to contain and/or express a polynucleotide whose sequence encodes a human ADAR1 p110 polypeptide or features thereof. . In some embodiments, a cell, tissue or animal (eg, a huADAR mouse or a cell or tissue therefrom) is engineered to contain and/or express a polynucleotide whose sequence encodes a human ADAR1 p110 polypeptide. In some embodiments, a cell, tissue or animal (e.g., a huADAR mouse or a cell or tissue therefrom) is engineered to contain and/or express a polynucleotide whose sequence encodes a human ADAR1 p150 polypeptide or feature thereof. . In some embodiments, a cell, tissue or animal (eg, a huADAR mouse or a cell or tissue therefrom) is engineered to contain and/or express a polynucleotide whose sequence encodes a human ADAR1 p150 polypeptide. As described herein, in some embodiments, the animal is a rodent, eg a mouse or rat.

In some embodiments, an ADAR (eg, human ADAR1) transgene is established on or on a zygote, eg, a SERPINA1 mouse zygote comprising a mutation (eg, 1024 G>A(E342K) in human SERPINA1). Opposite. In some embodiments, a zygote is homozygous. In some embodiments, a zygote is heterozygous.

use and purpose

As will be appreciated by those skilled in the art, oligonucleotides are useful for several purposes. In some embodiments, provided technologies (eg, oligonucleotides, compositions, methods, etc.) can be used to measure the level and/or It can be useful for regulating activity. In some embodiments, provided techniques can reduce the level and/or activity of undesirable (eg, comprising undesirable adenosine) target nucleic acids and/or products thereof. In some embodiments, provided techniques can increase the level and/or activity of a target nucleic acid and/or product thereof of interest (eg, comprising an I in place of an undesirable adenosine at one or more positions).

For example, in some embodiments, provided technologies can be used as single-stranded oligonucleotides for site-directed editing of a target adenosine within a target RNA sequence. In some embodiments, provided technologies can modulate the level of expression and activity. In particular, the present invention provides for the improvement of various desired biological functions, including but not limited to treatment and/or prevention of various conditions, disorders or diseases (eg, those associated with G to A mutations). Provide improvement by technology.

In some embodiments, provided technologies can modulate the activity and/or function of a target gene. In some embodiments, a target gene is a gene for which the expression and/or activity of one or more gene products (eg, RNA and/or protein products) is intended to be altered. In many embodiments, the target gene has target adenosine residues to be altered and can benefit from conversion of these residues to inosine residues. In some embodiments, when an oligonucleotide as described herein acts on a particular target gene, the level and/or activity of one or more gene products of that gene is altered in the presence of the oligonucleotide compared to in the absence of the oligonucleotide. can

In some embodiments, provided oligonucleotides and compositions are used by reducing the level and/or activity of a target transcript and/or the product encoded thereby, and optionally less associated with or associated with a condition, disorder or disease. various conditions, disorders or diseases by providing unrelated transcripts and/or products encoded thereby (eg, G to A mutation correction by conversion of target adenosine to inosine, splicing alteration, etc.) useful for treatment In some embodiments, the present invention provides a method for preventing or treating a condition, disorder or disease comprising administering to a subject predisposed to or suffering from the condition, disorder or disease an effective amount of a provided oligonucleotide or composition. provides a way In some embodiments, the present invention provides a method for preventing or treating a condition, disorder or disease, wherein a provided single-stranded oligonucleotide or composition thereof for site-directed editing of nucleotides (eg, targeting adenosine) in a target RNA sequence is used to treat the condition, disorder, or disease; Provided are methods comprising administering to a subject predisposed to or suffering from a disorder or disease. In some embodiments, provided single-stranded oligonucleotides for site-directed editing of nucleotides within a target RNA sequence have a base sequence that is partially or completely complementary to a portion of a transcript associated with a condition, disorder or disease. In some embodiments, the base sequence is such that it preferentially binds a transcript associated with the condition, disorder, or disease over other transcripts not associated with the condition, disorder, or disease. In some embodiments, the condition, disorder or disease is associated with a G to A mutation. In some embodiments, the condition, disorder or disease is associated with a G to A mutation in SERPINA1. In some embodiments, the condition, disorder or disease is associated with the 1024 G>A(E342K) mutation in human SERPINA1. In some embodiments, the condition, disorder or disease is alpha-1 antitrypsin deficiency. In some embodiments, provided techniques provide a desired product (eg, as a percentage of total protein or total A1AT protein) in an absolute amount (eg, ng/mL in serum) and/or relatively (eg, in serum). Properly folded wild-type A1AT protein) and / or undesirable product (eg, mutant (eg, E342K) A1AT protein in serum) or decrease activity. In some embodiments, the present invention provides a method of increasing the level and/or activity of an alpha-1 antitrypsin (A1AT) polypeptide in the serum or blood of a subject comprising administering to the subject an effective amount of an oligonucleotide or composition. to provide. In some embodiments, an A1AT polypeptide provides one or more higher activities compared to a reference A1AT polypeptide. In some embodiments, the A1AT polypeptide is a wild-type A1AT polypeptide. In some embodiments, the method increases the amount of A1AT polypeptide in serum. In some embodiments, the method reduces the amount of a reference A1AT polypeptide in serum. In some embodiments, the method increases the ratio of A1AT polypeptide to reference A1AT polypeptide in serum or blood. In some embodiments, a reference A1AT polypeptide is mutated. In some embodiments, a reference A1AT polypeptide is not folded properly. In some embodiments, a reference A1AT polypeptide is an E342K A1AT polypeptide. In some embodiments, the present invention provides a method for reducing the level and/or activity of a mutant alpha-1 antitrypsin (A1AT) polypeptide in the serum or blood of a subject, comprising administering to the subject an effective amount of an oligonucleotide or composition. provides a way In some embodiments, the subject is predisposed to or suffers from a condition, disorder or disease. In some embodiments, the condition, disorder or disease is alpha-1 antitrypsin deficiency. In some embodiments, the subject is a human. In some embodiments, the subject comprises a mutation in human SERPINA1. In some embodiments, the subject comprises a 1024 G>A(E342K) mutation in human SERPINA1. In some embodiments, the subject is homozygous for the mutation. In some embodiments, the subject is heterozygous for the mutation.

In some embodiments, the condition, disorder or disease is not associated with a G to A mutation. In some embodiments, the condition, disorder or disease is associated with increased levels and/or activity of a transcript and/or encoded product, and provided techniques can be used to treat the transcript, for example by introducing Iylation of one or more A's into the transcript. , reducing the level and/or activity of the transcript and/or encoded product. In some embodiments, the condition, disorder or disease is associated with reduced levels and/or activity of a transcript and/or encoded product, and provided techniques can be used to treat the transcript, e.g., by introducing Iylation of one or more A's into the transcript. , increasing the level and/or activity of the transcript and/or encoded product. In some embodiments, the condition, disorder or disease is associated with splicing, and provided technologies provide regulation of splicing by introducing Iization of one or more A's into a transcript (eg, pre-mRNA).

In some embodiments, an oligonucleotide composition of a provided method is a chirally controlled oligonucleotide composition. In some embodiments, a method of treating a condition, disorder or disease may include administering a composition comprising a plurality of oligonucleotides that share a common base sequence complementary to a target sequence of a target transcript. In particular, the present invention is characterized in that, when contacted with a target transcript in a system, adenosine editing of the transcript is improved compared to that observed under reference conditions selected from the group consisting of the absence of the composition, the presence of the reference composition, and combinations thereof. Provides an improvement comprising administering as an oligonucleotide composition a chirally controlled oligonucleotide composition as described herein. In some embodiments, a reference composition is a racemic preparation of oligonucleotides of the same sequence or composition. In some embodiments, a target transcript is an oligonucleotide transcript.

As will be appreciated by those skilled in the art, in particular, the provided technology can be used for a variety of applications that can involve and/or benefit from the conversion of adenosine to inosine. Specific uses are described below.

One skilled in the art upon reading this disclosure will understand that various G to A mutations, e.g. mutations in the C to T transcript, the most common type of mutation occurring in human genes, can be corrected and thus benefit from the techniques provided. will understand In some embodiments, provided technology can be used to select various polar or charged amino acids (e.g. Ser, Tyr, Asp, Glu, His, Asn, Gln, Lys, etc.), stop codons (Opal, Ocher and Amber), transcription start sites, splices It can be used to target mutations related to sing signals, microRNA recognition sites, repeat elements, microRNAs (miRNAs), protein-encoding transcripts, and the like. In particular, provided techniques can lead to various functional outcomes, such as alteration of splicing, restoration/improvement of protein expression and/or function, and the like.

In some embodiments, provided techniques can, through editing, restore protein function (e.g., correct nonsense and missense mutations that cannot be corrected for splices, remove stop mutations, and reduce protein misfolding and aggregation). can be used for the prevention and/or treatment of various conditions, disorders or diseases, such as recessive or dominant genetically defined diseases, etc., can modify protein function (e.g., protein processing (e.g., protease cleavage) sites), alter protein-protein interactions, modulate signaling pathways, etc., can be used for the prevention and/or treatment of various conditions, disorders or diseases, such as those associated with ion channel permeability), to upregulate proteins. (e.g., miRNA target site modification, upstream ORF modification, modification of ubiquitination site, etc., and can be used for prevention and/or treatment of various conditions, disorders or diseases, such as haplodeletion mutation diseases) . In some embodiments, provided techniques restore or improve the expression, level, function and/or activity of a protein. In some embodiments, provided techniques are useful for preventing or treating genetically defined recessive or dominant conditions, disorders or diseases, such as those associated with G to A mutations. In some embodiments, the condition, disorder or disease is a liver condition, disorder or disease. In some embodiments, the condition, disorder or disease is a metabolic liver condition, disorder or disease. In some embodiments, the condition, disorder or disease is a neurodevelopmental condition, disorder or disease. In some embodiments, provided technologies modify the expression, level, function and/or activity of a protein. In some embodiments, provided technologies reduce the expression, level, function and/or activity of a protein. In some embodiments, provided technologies increase the expression, level, function and/or activity of a protein. In some embodiments, provided technologies modulate ion channel permeability. In some embodiments, provided techniques are useful for preventing or treating conditions, disorders or diseases associated with ion channel permeability. In some embodiments, the condition, disorder or disease is familial epilepsy. In some embodiments, the condition, disorder or disease is neuropathic pain. In some embodiments, the condition, disorder or disease is AATD. In some embodiments, the condition, disorder or disease is Rett Syndrome. In some embodiments, the condition, disorder or disease is a genetically defined recessive or dominant disease. In some embodiments, provided technologies modify nucleic acid (eg, miRNA) target sites. In some embodiments, provided techniques modify, reduce the function or activity of, eliminate or inhibit an upstream ORF (e.g., in some embodiments, (e.g., the ATG start codon of a uORF) ) transform A). In some embodiments, provided techniques modify a modification site, eg, an ubiquitination site, of a protein. In some embodiments, provided techniques are useful for preventing or treating conditions, disorders, or diseases associated with haplodeletion mutations. In some embodiments, provided techniques are useful for preventing or treating a neurological condition, disorder or disease. In some embodiments, provided techniques are useful for preventing or treating a neuromuscular condition, disorder or disease. In some embodiments, provided techniques are useful for preventing or treating dementia. In some embodiments, provided techniques are useful for preventing or treating dementia. In some embodiments, provided techniques are useful for preventing or treating haplotype mutation conditions, disorders or diseases. In some embodiments, provided techniques are methods of preventing or treating a condition, disorder or disease, comprising administering to a subject susceptible to or suffering from the condition, an effective amount of an oligonucleotide or composition thereof as described herein. provides One skilled in the art understands that the protein encoded by it can be edited, for example, through editing of a nucleobase such as A in RNA. In some embodiments, an amino acid residue is replaced with another amino acid residue. In some embodiments, the protein is elongated. In some embodiments, a protein is shortened. In some embodiments, expression, level, function, stability, property and/or activity is modulated. In some embodiments, some properties and/or activities are enhanced while other properties and/or activities are reduced or remain the same. In some embodiments, some properties and/or activities are reduced while other properties and/or activities are enhanced or remain the same.

In some embodiments, provided techniques edit nucleic acids or codons that contain mutations. In some embodiments, the mutation is a nonsense mutation. In some embodiments, the mutation is a missense mutation. In some embodiments, the mutation is a silent mutation. In some embodiments, provided techniques correct nonsense mutations. In some embodiments, provided technologies correct missense mutations. In some embodiments, provided techniques remove stop mutations. In some embodiments, provided techniques prevent or reduce misfolding and/or aggregation. In some embodiments, provided techniques edit codons that contain mutations. In some embodiments, an edited nucleobase is a mutation. In some embodiments, the edited nucleobase is not a mutation but another nucleobase of a codon. In some embodiments, a codon after editing becomes its corresponding wild-type codon. In some embodiments, post-edited codons encode the same amino acids as wild-type codons. In some embodiments, post-edited codons encode different amino acids than wild-type codons. In some embodiments, proteins comprising such different amino acid residues share one or more properties and/or perform one or more functions of their corresponding wild-type proteins. In some embodiments, proteins comprising these different amino acid residues share more similarity with the wild-type protein and/or provide a higher level of desired activity compared to the corresponding mutated, unedited protein. In some embodiments, nonsense or missense mutations cannot be splice corrected. In some embodiments, provided techniques generate silent mutations. In some embodiments, the silent mutation modulates the level of the encoded protein. In some embodiments, protein levels are increased. In some embodiments, protein levels are reduced.

In some embodiments, provided technologies modify protein function. In some embodiments, provided techniques alter one or more properties and/or functions of nucleic acids (eg, transcripts) and/or proteins. In some embodiments, provided technologies increase, promote, or enhance one or more properties and/or functions of nucleic acids (eg, transcripts) and/or proteins. In some embodiments, provided technologies provide one or more novel properties and/or activities, eg, of nucleic acids (eg, transcripts) and/or proteins. In some embodiments, provided technologies reduce, inhibit, or eliminate one or more properties and/or functions of nucleic acids (eg, transcripts) and/or proteins. In some embodiments, provided technologies alter protein processing. For example, in some embodiments, protease cleavage sites are edited. In some embodiments, provided techniques edit one or more residues involved in protein-protein interactions. In some embodiments, provided technologies edit amino acid residues in protein-protein interaction domains. In some embodiments, by editing the mRNA encoding the protein, various regions of the polypeptide, e.g., protease cleavage sites, various domains (eg, protein-protein interaction domains), modification sites, miRNA targeting sites, ubiquitin Residues in the site of fire and the like can be edited. In some embodiments, provided technologies modulate signaling pathways.

In some embodiments, provided technologies restore, increase, or enhance levels of functional proteins. In some embodiments, provided techniques reduce the level and/or activity of mutant or undesirable nucleic acids (eg, RNA transcripts) and proteins. In some embodiments, provided techniques restore or correct expression of one or more polypeptides. In some embodiments, provided technologies are capable of upregulating expression. In some embodiments, provided technologies can upregulate translation. In some embodiments, provided technologies are capable of up-regulating the activity level of a polypeptide. In some embodiments, provided technologies modify the function of a target nucleic acid (eg, an RNA transcript) and/or a product encoded thereby (eg, a polypeptide). In some embodiments, provided technologies modulate post-translational modification of target nucleic acids (eg, RNA transcripts) and/or products encoded thereby (eg, polypeptides). In some embodiments, provided technologies are capable of up-regulating the level of a polypeptide. In some embodiments, provided technologies edit codons that encode amino acid residues involved in protein-protein interactions or protein interactions with other agents, and in some embodiments change an amino acid residue to a different amino acid residue to break the interaction. Including enhancing or reducing In some embodiments, provided technologies modify one or more functions of nucleic acids and/or proteins. In some embodiments, provided technologies can modulate protein-protein interactions. In some embodiments, provided technologies edit coding transcripts to remove, alter or incorporate amino acid residues for post-translational modification. In some embodiments, provided technologies control post-translational modifications. In some embodiments, provided technologies modulate nucleic acid folding. In some embodiments, provided technologies modulate protein folding. In some embodiments, provided technologies modulate the stability of transcripts and/or products thereof. In some embodiments, provided technologies modulate protein stability. In some embodiments, provided technologies modulate processing of transcripts and/or products thereof. In some embodiments, provided technologies modulate nucleic acid (eg, transcript) processing. In some embodiments, provided technologies alter protein processing. In some embodiments, provided technologies modulate post-translational processes. For example, in some embodiments, provided technologies modulate PCSK9 post-translational processing. Among other things, the provided technology can be applied to a wide range of therapeutic applications in large patient populations.

For example, as demonstrated herein, in some embodiments, one or more amino acid residues of one or more proteins may be altered through editing of the encoding mRNA to modulate protein-protein interactions. Amino acid residues suitable for editing include various reported amino acid residues involved in protein-protein interactions, or can be identified through techniques available in the art, such as mutagenesis techniques, structural biology techniques, and the like. In some embodiments, the present invention relates to the level, properties, and/or quality of proteins and/or proteins that interact with them through editing of nucleic acids (eg, transcripts) and/or nucleic acids (eg, transcriptomes). Provides techniques for modulating activity. In some embodiments, the present invention provides techniques for modulating the level and/or activity of a protein (eg, transcription factor) and/or the transcription and/or expression regulated thereby. In some embodiments, provided techniques include editing amino acid residues of a protein (eg, a transcription factor) or a partner protein with which it interacts, wherein the interaction between the protein and the partner protein is reduced or enhanced. In some embodiments, provided techniques include editing amino acid residues of a protein (eg, transcription factor) or a partner protein with which it interacts, wherein interactions between the protein and the partner protein are reduced. In some embodiments, such editing stabilizes a protein such that its level and/or activity (eg, transcriptional activation of a particular nucleic acid) is increased. In some embodiments, the invention provides a protein encoding a protein that regulates expression of a nucleic acid, or a protein that interacts with a protein that regulates expression of a nucleic acid, or a protein that is a member of a pathway that includes a protein that regulates expression of a nucleic acid. Techniques are provided for modulating (eg, activating, increasing, decreasing, inhibiting, etc.) expression of a nucleic acid comprising editing adenosine in a transcript, wherein the editing modulates the level and/or regulate activity. In some embodiments, the level and/or activity of a transcript of a nucleic acid is modulated. In some embodiments, the level and/or activity of a protein encoded by such transcript is modulated. Among other things, the present invention confirms that many of the functions, activities, pathways, etc., that involve protein-protein interactions can be modulated through editing of the interacting amino acid residues of one or more interacting proteins. For example, editing of one or more amino acid residues in NRF2 (e.g. Glu82 (e.g. to Gly), Glu79 (e.g. to Gly), Glu78 (e.g. to Gly), Asp76 (e.g. to Gly), Ile28 (e.g. to Val) Asp27 (e.g. to Gly), Gln26 (e.g. to Arg), etc.) or editing of one or more amino acid residues in Keap1 (e.g. Ser603 (e.g. to Gly), Tyr572 (e.g. to Cys), Tyr525 ( e.g. to Cys), Ser508 (e.g. to Gly), His436 (e.g. to Arg), Asn382 (e.g. to Asp), Arg380 (e.g. to Gly), Tyr334 (e.g. to Cys) etc.) of NRF2 level and/or activity, and/or expression of various nucleic acids (eg, various genes) regulated by NRF2. In some embodiments, the invention provides a method of modulating, e.g., reducing, NRF2-Keap1 interaction in a system comprising administering an oligonucleotide or composition thereof to a system comprising NRF2 or Keap1 mRNA, , wherein the oligonucleotide edits the adenosine of the mRNA such that an amino acid residue in the protein encoded by the mRNA is edited to another residue. In some embodiments, the invention provides a method of increasing the level and/or activity of NRF2 in a system comprising administering an oligonucleotide or composition thereof to a system comprising NRF2 or Keap1 mRNA, wherein the oligonucleotide Edits the adenosine of an mRNA so that an amino acid residue in the protein encoded by the mRNA is edited to a different residue. In some embodiments, the invention provides a method of increasing transcription or expression of a NRF2-regulated nucleic acid (eg, gene) comprising administering an oligonucleotide or composition thereof to a system comprising NRF2 or Keap1 mRNA. , wherein the oligonucleotide edits the adenosine of the mRNA such that an amino acid residue in the protein encoded by the mRNA is edited to a different residue. In some embodiments, the level and/or activity of an NRF2 regulatory nucleic acid, e.g., a transcript from a gene such as SRGN, HMOX1, SLC7a11, NQO1, etc., and/or a product (e.g., protein) encoded thereby is increased. . In some embodiments, systems comprising NRF2 and Keap1 mRNAs and NRF2 and Keap1 proteins are translated from such mRNAs. In some embodiments, the target adenosine of NRF2 and/or Keap1 mRNA is post-translationally edited such that an amino acid residue is replaced with another amino acid residue. In some embodiments, the administered oligonucleotide or composition thereof targets NRF2 mRNA. In some embodiments, the oligonucleotide or composition thereof administered targets Keap1 mRNA. In some embodiments, an amino acid residue of NRF2 (eg, Glu82 (eg, as Gly), Glu79 (eg, as Gly), Glu78 (eg, as Gly), Asp76 (eg, as Gly), Ile28 (eg, as Gly). to Val), Asp27 (eg to Gly), Gln26 (eg to Arg), etc.) are edited. In some embodiments, an amino acid residue of Keap1 (eg, Ser603 (eg, as Gly), Tyr572 (eg, as Cys), Tyr525 (eg, as Cys), Ser508 (eg, as Gly), His436 (eg, as Gly). to Arg), Asn382 (eg to Asp), Arg380 (eg to Gly), Tyr334 (eg to Cys), etc.) are edited. In some embodiments, two or more amino acid residues are edited. In some embodiments, each edited amino acid residue is independently a NRF2 residue. In some embodiments, each edited amino acid residue is independently a Keap1 residue. In some embodiments, the edited amino acid residue is a Keap1 residue and the edited amino acid residue is a NRF2 residue. In some embodiments, a system is or comprises a cell. In some embodiments, a system is or comprises a tissue. In some embodiments, a system is or comprises an organ. In some embodiments, the system is an organism. In some embodiments, the system is an in vitro system. Specific NRF2 targeting and Keap1 targeting oligonucleotides and/or oligonucleotide compositions are shown in the table(s) as examples. In some embodiments, provided techniques are useful for treating conditions, disorders or diseases associated with NRF2. In some embodiments, provided techniques are useful for treating conditions, disorders or diseases associated with Keap1. In some embodiments, provided techniques are useful for treating conditions, disorders or diseases associated with NRF2-Keap1 interactions.

In some embodiments, provided technologies modulate enzymatic activity. In some embodiments, provided techniques increase enzymatic activity by editing codons, for example, with codons encoding amino acid residues capable of increasing enzymatic activity. In some embodiments, provided techniques reduce enzymatic activity, eg, those associated with conditions, disorders or diseases, by editing codons with codons encoding amino acid residues capable of reducing enzymatic activity. In many cases with amino acid residues involved in these activities, various enzymatic activities have been reported or can be identified and characterized and modulated according to the present invention. In some embodiments, the activity is a kinase activity.

In some embodiments, editing of a protein (eg, through editing of its encoding mRNA to alter one or more amino acid residues) reduces degradation of the protein or the protein that interacts with it. In some embodiments, editing of a protein upregulates its level. In some embodiments, editing of a protein modulates protein processing. In some embodiments, editing of a protein modulates its folding. In some embodiments, editing of a protein modulates its stability. In some embodiments, editing of a protein modulates protein modification (eg, increase, decrease, elimination or introduction of modification sites, etc.). In some embodiments, editing of a protein modulates post-translational modifications (eg, increase, decrease, elimination or introduction of modified sites, etc.). In some embodiments, the provided techniques are useful for treating related conditions, disorders or diseases, such as dementia, familial epilepsy, neuropathic pain, neuromuscular disorders, dementia, hemiplegic mutation diseases, loss-of-function conditions, disorders or diseases, and the like.

The techniques of the present invention can provide efficient editing in various types of cells, tissues, organs and/or organisms. In some embodiments, provided technologies can provide efficient editing in a variety of immune cells. As demonstrated herein, the provided technology can provide high-level editing in human peripheral blood mononuclear cells (PBMCs). Among other things, the provided technology can provide high-level editing in various cell populations such as CD4+ T cells, CD8+ T cells, CD14 monocytes, CD19 B cells, NK cells, Treg T cells, and the like. In some embodiments, the immune cell is activated prior to contacting the oligonucleotide (eg, by PHA). In some embodiments, the cell is not activated. In some embodiments, similar levels of editing are observed in activated and inactivated cells. In some embodiments, higher levels of editing are observed in activated cells. In some embodiments, after cell editing, eg, PBMCs can be sorted into various cell types. In some embodiments, cells may be first sorted prior to being contacted with oligonucleotides. As will be appreciated by those skilled in the art, immune cells have multiple functions and can be used for multiple purposes, including treatment of a variety of conditions, disorders or diseases. In some embodiments, immune cells are used in immunotherapy, for example for various types of cancer. Among other things, the present invention provides techniques for editing one or more transcripts expressed in immune cells to improve properties and/or activity for immunotherapy. In some embodiments, provided technologies can reduce the expression and/or activity of one or more genes in immune cells, such as FAS, BID, CTLA4, PDCD1, CBLB, PTPN6, TRAC, TRBC, and the like. In some embodiments, transcripts from these genes are edited. In some embodiments, the target cell is a T cell, eg, a CD8+ T cell (eg, a CD8+ naive T cell, a central memory T cell, or an effector memory T cell), a CD4+ T cell, a natural killer T cell (NK T cells), regulatory T cells (Treg), stem cell memory T cells, lymphoid progenitor cells, hematopoietic stem cells, natural killer cells (NK cells) or dendritic cells. In some embodiments, the cell is a CD4+ cell, eg a CD4+ T cell. In some embodiments, the cell is a CD8+ cell, eg a CD8+ T cell. In some embodiments, the cell is a CD14+ cell, eg a CD14+ monocyte. In some embodiments, the cell is a CD19+ cell, eg a CD19+ B cell. In some embodiments, the cell is a NC cell. In some embodiments, the cell is a T regulatory cell. In some embodiments, the target cell is an induced pluripotent stem (iPS) cell or a cell derived from an iPS cell, e.g., generated from a subject and containing one or more genes, e.g., FAS, BID, CTLA4, PDCD1, CBLB, engineered to alter expression of (eg, induce mutations in) the PTPN6, TRAC or TRBC genes, eg, T cells, eg, CD8+ T cells (eg, CD8+ naive T cells, central memory) T cells, or effector memory T cells), CD4+ T cells, stem cell memory T cells, lymphoid progenitor cells or iPS cells differentiated into hematopoietic stem cells.

Among other things, the provided techniques are useful for increasing, enhancing, improving or up-regulating the level, properties, activity, etc., of various polypeptides, including various proteins. In some embodiments, provided technologies modify a binding or target site, eg, a miRNA target site. In some embodiments, provided technologies modify regulatory elements in transcripts. In some embodiments, provided techniques modify an upstream ORF (eg, A of ATG). In some embodiments, provided techniques modify amino acid residues that can be modified, eg, ubiquitination sites. One skilled in the art understands that the techniques provided may also be useful for reducing or down-regulating the level, properties, activity, etc. of various polypeptides, including various proteins, through RNA modification.

In some embodiments, the editing site, eg, the target adenosine, is in a coding region. In some implementations, it is in the non-encrypted area. In some embodiments, a target nucleic acid is a non-coding RNA.

Specific applications are for example WO 2016/097212, WO 2017/220751, WO 2018/041973, WO 2018/134301A1, WO 2020/154344, WO 2020/154343, WO 2020/154342, WO 2020/165077, WO 2020/201406 , WO 2020/216637, or WO 2020/252376.

Many adenosines associated with a variety of conditions, disorders or diseases have been reported or can be identified and can be targeted using the provided technologies, for example to prevent or treat a related condition, disorder or disease. For example, SNCA (eg Parkinson's disease), APP (eg Alzheimer's disease), Tau (eg Alzheimer's disease), Nav1.7 (eg chronic pain), C9orf72 (eg Alzheimer's disease), C9orf72 (eg Alzheimer's disease) eg, amyotrophic lateral sclerosis), SOD1 (eg, amyotrophic lateral sclerosis), DYRK1A (eg, Down syndrome), IT15 (eg, Huntington's disease), HEXA (eg, Tay-Sachs disease) , RAI1 (e.g. Protocki-Lupski syndrome), ABCA4 (e.g. Stargardt's disease), USH2A (e.g. Usher syndrome), NRP1 (e.g. wet AMD, dry AMD, etc.), PCSK9 (e.g. cardiovascular conditions, disorders or diseases), LIPA (e.g. cholesteryl ester storage disease), HFE (e.g. hemochromatosis), ALAS1 (e.g. porphyria/acute hepatic porphyria), ATP7B (e.g. Wilson's disease), COL4A5 (eg, Alport syndrome), LDHA (eg, primary hyperoxia), HAO1 (eg, primary hyperoxia type 2), DUX4 (eg, facial scapulohumeral dystonia), DMPK (eg dystonia), BCL11A (eg sickle cell disease), Mex3B (eg asthma), CIDEC (eg obesity), SCD1 (eg obesity) ), GNB3 (eg obesity), FGFR3 (eg achondroplasia), CLCN7 (eg osteopetrosis), PMP22 (eg Charcot-Marie-Tooth disease), ENAC (eg For example, cystic fibrosis), GHR (eg acromegaly), TTR (eg transthyretin amyloidosis (familial)), etc. In some embodiments, the present invention provides oligonucleotides and compositions that target such adenosine, and methods of preventing or treating such conditions, disorders or diseases.

In some embodiments, the condition, disorder or disease that can be treated is, for example, alpha-1 antitrypsin deficiency, Alzheimer's disease, amyloid disease, Becker's muscular dystrophy, breast cancer predisposition mutation, Canavan's disease, Charcot-Marie-Tooth disease, cystic Fibrosis, factor V Leiden deficiency, type 1 diabetes, type 2 diabetes, Duchenne muscular dystrophy, Fabry disease, hereditary tyrosinemia type 1 (HTI), familial adenomatous polyposis, familial amyloid cardiomyopathy, familial amyloidosis Polyneuropathy, familial dysautonomia, familial hypercholesterolemia, Friedreich's ataxia, Gaucher disease type I, Gaucher disease type II, glycogen storage disease type II, GM2 gangliosidosis, hemochromatosis, hemophilia A, B Hemophilia C, hexosaminidase A deficiency, ovarian cancer predisposition mutation, obesity, phenylketonuria, polycystic kidney disease, prion disease, senile systemic amyloidosis, sickle cell disease, Smith-Lemley-Opitz syndrome, spinal muscular atrophy, Wilson's disease, Parkinson's disease and hereditary blindness. In some embodiments, diseases/targets include: Cystic Fibrosis Transmembrane Conductance Regulatory Gene (CFTR); Albinism, amyotrophic lateral sclerosis, asthma, β-thalassemia, Cardasil syndrome, chronic obstructive pulmonary disease (COPD), distal spinal muscular atrophy (DSMA), Duchenne/Becker muscular dystrophy, dystrophic epidermolysis bullae, epidermolysis bullosa, dystrophin gene (DMD); amyloid beta (A4) precursor protein gene (APP); Factor V Leiden-related disorder, glucose-6-phosphate dehydrogenase, hemophilia, hereditary hemochromatosis, Hunter syndrome, Huntington's disease, Hurler syndrome, inflammatory bowel disease (IBD), hereditary polyaggregation syndrome, Leber congenital amaurosis, Lesch-Nyhan syndrome, Lynch syndrome, Marfan syndrome, mucopolysaccharidosis, myotonic dystrophy types I and II, neurofibromatosis, Niemann-Pick disease types A, B, and C, NY-eso1-associated cancer, Rett syndrome, NY-ESO-1-associated cancer, 11- thalassemia, galactosemia, Gaucher disease, factor XII gene; factor IX gene; factor XI gene; HgbS; insulin receptor gene; adenosine deaminase gene; alpha-1 antitrypsin gene; breast cancer 1 gene (BRCA1); breast cancer 2 gene (BRCA2); aspartocyclase gene (ASPA); galactosidase alpha gene (GLA); adenomatous polyposis coli gene (APC); inhibitor of kappa light chain polypeptide gene enhancer in B cells, kinase complex-associated protein (IKBKAP); glucosidase beta acid gene (GBA); glucosidase alpha acid gene (GAA); hemochromatosis gene (HFE); apolipoprotein B gene (APOB); low-density lipoprotein receptor gene (LDLR), low-density lipoprotein receptor adapter protein 1 gene (LDLRAP1); proprotein convertase subtilisin/kexin type 9 gene (PCSK9); polycystic kidney disease 1 (autosomal dominant) gene (PKD-1); prion protein gene (PRNP); PTP-1B; 7-dehydrocholesterol reductase gene (DHCR7); survival of motor neuron 1, telomeric gene (SMN1); biquitin-like modifier activating enzyme 1 gene (UBA1); dynein, cytoplasmic 1, heavy chain 1 gene (DYNC1H1), survival of motor neuron 2, centrosome gene (SMN2); (vesicle-associated membrane proteins) associated proteins B and C (VAPB); hexosaminidase A (alpha polypeptide) gene (HEXA); transthyretin gene (TTR); ATPase, Cu++ transport, beta polypeptide gene (ATP7B); phenylalanine hydroxylase gene (PAH); rhodopsin gene; retinitis pigmentosa 1 (autosomal dominant) gene (RP1); Retinitis pigmentosa 2 (X-linked recessive) gene (RP2), Sturge-Weber syndrome, Parkinson's disease, Peutz-Jaegers syndrome, Pompe disease, primary ciliary disease, disorders associated with prothrombin mutations such as prothrombin G20210A mutation, pulmonary hypertension, Sandhoff disease, severe combined immunodeficiency syndrome (SCID), Stargardt disease, Tay-Sachs disease, Usher syndrome, X-linked immunodeficiency syndrome, various forms of cancer (eg BRCA1 and 2-associated breast and ovarian cancer), and other notices genetic target. Other disorders include point mutations or small deletions or insertions or, for example, updated on 24 September 2021, http://www.omim.org/ Online Mendelian Inheritance in Man® An Online Catalog of Human Genes and Genetic Disorders diseases that can be corrected by point mutations or small deletions or insertions listed in .

In some embodiments, the present invention provides techniques for targeting IDUA. In some embodiments, the invention provides a method for preventing or treating a condition, disorder or disease associated with IDUA, comprising administering to a subject predisposed to or suffering from the condition, disorder or disease an effective amount of an oligonucleotide or composition. Provides a way to include In some embodiments, the subject benefits from a G to A edit in IDUA. In some embodiments, the condition, disorder or disease is Hurler syndrome. In some embodiments, the present invention provides techniques for targeting PINK1. In some embodiments, the invention provides a method of preventing or treating a PINK1-associated condition, disorder or disease comprising administering to a subject predisposed to or suffering from the condition, disorder or disease an effective amount of an oligonucleotide or composition. provides a way to In some embodiments, the subject benefits from G to A editing in PINK1. In some embodiments, the condition, disorder or disease is Parkinson's disease. In some embodiments, the present invention provides techniques for targeting Factor V Leiden. In some embodiments, the present invention provides a method for preventing or treating a Factor V Leiden-related condition, disorder or disease, comprising administering to a subject predisposed to or suffering from the condition, disorder or disease an effective amount of an oligonucleotide or composition. Provides a method including. In some embodiments, the subject benefits from G to A editing in Factor V Leiden. In some embodiments, the condition, disorder or disease is Factor V Leiden deficiency. In some embodiments, the present invention provides technologies for targeting CFTR. In some embodiments, the invention provides a method of preventing or treating a CFTR-related condition, disorder or disease comprising administering to a subject predisposed to or suffering from the condition, disorder or disease an effective amount of an oligonucleotide or composition. provides a way to In some embodiments, the subject benefits from G to A editing in CFTR. In some embodiments, the condition, disorder or disease is cystic fibrosis.

It has been reported that there are over 32,000 pathogenic human SNPs and nearly half of them are G to A mutations that can be corrected by the techniques provided. Indeed, tens of thousands of diseases have been reported to be associated with G to A mutations and can be prevented or treated by the techniques provided. Among other things, the provided technology can be used to prevent or treat many conditions, disorders or diseases associated with premature stop codons; Approximately 12% of all reported disease-causing mutations are reported to be single point mutations resulting in premature stop codons. In some embodiments, provided technologies correct premature stop codons. For example, the ClinVar database; See Gaudelli NM et al., Nature. 2017 Nov 23; 551(7681): 464-471]; Keeling KM et al., Madame Curie Bioscience Database 2000-2013; See etc.

In some embodiments, when an oligonucleotide or oligonucleotide composition is contacted with a target nucleic acid comprising a target adenosine in the system, the target adenosine within the target nucleic acid is modified. In some embodiments, when the oligonucleotide or oligonucleotide composition is contacted with a target nucleic acid comprising a target adenosine in the system, the level of the target nucleic acid is reduced relative to the absence of the product or the presence of a reference oligonucleotide. In some embodiments, when an oligonucleotide or oligonucleotide composition is contacted with a target nucleic acid comprising a target adenosine in the system, splicing of the target nucleic acid or product thereof is altered relative to the absence of the oligonucleotide or the presence of a reference oligonucleotide. . In some embodiments, when an oligonucleotide or oligonucleotide composition is contacted with a target nucleic acid comprising a target adenosine in the system, the level of the product of the target nucleic acid is altered relative to the absence of the product or the presence of a reference oligonucleotide. In some embodiments, the level of a product is increased and the product is a nucleic acid that is identical to or encoded by the target nucleic acid except that the target adenosine is modified. In some embodiments, the level of a product is increased and the product is or is encoded by the same nucleic acid as the target nucleic acid except that the target adenosine is replaced with inosine. In some embodiments, the product level is increased and the product is a nucleic acid that is identical to or encoded by the target nucleic acid except that the adenine of the target adenosine is replaced with guanine. In some embodiments, the product is a protein. In some embodiments, the target adenosine is a mutation from guanine. In some embodiments, the target adenosine is more associated with the condition, disorder or disease than the guanine at the same position. In some embodiments, an oligonucleotide is capable of forming a double-stranded complex with a target nucleic acid. In some embodiments, the target nucleic acid or portion thereof is or comprises RNA. In some embodiments, the target is of adenosine or RNA. In some embodiments, a target adenosine is modified, and the modification is or comprises deamination of the target adenosine. In some embodiments, a target adenosine is modified, and the modification is or comprises conversion of the target adenosine to inosine. In some embodiments, modification is catalyzed by an ADAR protein. In some embodiments, the system is an in vitro or ex vivo system comprising an ADAR protein. In some embodiments, a system is or comprises a cell comprising or expressing an ADAR protein. In some embodiments, the system is a subject comprising a cell comprising or expressing an ADAR protein. In some embodiments, the ADAR protein is ADAR1. In some embodiments, the ADAR1 protein is or comprises a p110 isotype. In some embodiments, the ADAR1 protein is or comprises a p150 isotype. In some embodiments, the ADAR1 protein is or comprises p110 and p150 isoforms. In some embodiments, the ADAR protein is ADAR2. As set forth herein, the present invention relates specifically to techniques for recruiting enzymes to a target site (e.g., a site comprising target A) by contacting a provided oligonucleotide or composition thereof with such a target site, or Provided are techniques comprising administering to a system comprising or expressing a polynucleotide (eg, RNA) comprising a target site. In some embodiments, the enzyme is an RNA-editing enzyme as described herein, such as ADAR1, ADAR2, etc.

In some embodiments, an oligonucleotide composition comprising a plurality of oligonucleotides provides a greater level (eg, a target adenosine is modified to a greater degree) than that observed in a comparable reference oligonucleotide composition. In some embodiments, the reference oligonucleotide composition does not include or includes a lower level of the plurality of oligonucleotides. In some embodiments, the reference composition does not contain oligonucleotides having the same composition as the plurality of oligonucleotides. In some embodiments, the reference composition does not contain oligonucleotides having the same structure as the plurality of oligonucleotides. In some embodiments, a reference oligonucleotide composition is a composition wherein an oligonucleotide having the same base sequence as the plurality of oligonucleotides comprises a lower level of 2'-F modification than the plurality of oligonucleotides. In some embodiments, a reference oligonucleotide composition is a composition wherein an oligonucleotide having the same base sequence as the plurality of oligonucleotides comprises a lower level of 2'-OMe modification than the plurality of oligonucleotides. In some embodiments, a reference oligonucleotide composition is a composition in which an oligonucleotide having the same base sequence as a plurality of oligonucleotides has a different sugar modification pattern compared to the plurality of oligonucleotides. In some embodiments, a reference oligonucleotide composition is a composition wherein an oligonucleotide having the same base sequence as the plurality of oligonucleotides comprises a lower level of modified internucleotide linkages compared to the plurality of oligonucleotides. In some embodiments, a reference oligonucleotide composition is a composition wherein an oligonucleotide having the same base sequence as the plurality of oligonucleotides comprises a lower level of phosphorothioate internucleotidic linkages than the plurality of oligonucleotides. In some embodiments, the composition is a stereorandom oligonucleotide composition. In some embodiments, a reference composition is a stereorandomized oligonucleotide composition of a plurality of oligonucleotides and oligonucleotides of identical composition.

In some embodiments, the invention provides techniques for modifying a target adenosine in a target nucleic acid comprising contacting the target nucleic acid with a provided oligonucleotide or oligonucleotide composition as described herein. In some embodiments, the invention provides a method of deamination of a target adenosine in a target nucleic acid comprising contacting the target nucleic acid with an oligonucleotide or composition as described herein. In some embodiments, the invention provides a method of producing a product of a specific nucleic acid, or restoring or increasing the level of a product of a specific nucleic acid, comprising contacting a target nucleic acid with a provided oligonucleotide or composition, wherein the target nucleic acid is a target adenosine wherein the specific nucleic acid differs from the target nucleic acid in that it has I or G instead of the target adenosine. In some embodiments, the invention provides a method of reducing product levels of a target nucleic acid comprising contacting the target nucleic acid with an oligonucleotide or composition of the invention, wherein the target nucleic acid comprises a target adenosine. . In some embodiments, the product is a protein. In some embodiments, the product is mRNA.

In some embodiments, the present invention

A method comprising contacting an oligonucleotide or composition with a sample comprising a target nucleic acid and an adenosine deaminase,

The base sequence of the oligonucleotide in the oligonucleotide or oligonucleotide composition is substantially complementary to the base sequence of the target nucleic acid,

the target nucleic acid comprises target adenosine;

The target adenosine is modified.

In some embodiments, the present invention

1) obtaining a modification of a first level of target adenosine in a target nucleic acid (the level observed when the first oligonucleotide composition is contacted with a sample comprising the target nucleic acid and adenosine deaminase), wherein the first oligonucleotide composition comprises: Comprising a first plurality of oligonucleotides sharing the same base sequence that is substantially complementary to the base sequence of the target nucleic acid, and

2) obtaining a modification of the reference level of target adenosine in the target nucleic acid (the level observed when the reference oligonucleotide composition is contacted with a sample comprising the target nucleic acid and adenosine deaminase), wherein the reference oligonucleotide composition is Comprising a plurality of reference oligonucleotides sharing the same base sequence that is substantially complementary to the base sequence,

At this time,

the first plurality of oligonucleotides comprises more sugars with 2'-F modifications, sugars with 2'-OR modifications (R is not -H), and/or chiral internucleotidic linkages than the reference plurality of oligonucleotides; do,

The first oligonucleotide composition provides a higher level of modification compared to the oligonucleotides of the reference oligonucleotide composition.

In some embodiments, the present invention

A method comprising obtaining a modification of a first level of target adenosine in a target nucleic acid (the level observed when the first oligonucleotide composition is contacted with a sample comprising the target nucleic acid and adenosine deaminase), wherein the first oligonucleotide The composition comprises a first plurality of oligonucleotides sharing the same base sequence that is substantially complementary to the base sequence of the target nucleic acid,

The first level of modification of the target adenosine is higher than the reference level of modification of the target adenosine, the reference level observed when the reference oligonucleotide composition is contacted with a sample comprising a target nucleic acid and an adenosine deaminase, wherein the reference oligonucleotide composition is It includes a plurality of reference oligonucleotides that share the same base sequence that is substantially complementary to the base sequence of the target nucleic acid,

At this time,

the first plurality of oligonucleotides comprises more sugars with 2'-F modifications, sugars with 2'-OR modifications (R is not -H), and/or chiral internucleotidic linkages than the reference plurality of oligonucleotides; It provides a way to do it.

In some embodiments, the present invention

1) obtaining a modification of a first level of target adenosine in a target nucleic acid (the level observed when the first oligonucleotide composition is contacted with a sample comprising the target nucleic acid and adenosine deaminase), wherein the first oligonucleotide composition comprises: Comprising a first plurality of oligonucleotides sharing the same base sequence that is substantially complementary to the base sequence of the target nucleic acid, and

2) obtaining a modification of the reference level of target adenosine in the target nucleic acid (the level observed when the reference oligonucleotide composition is contacted with a sample comprising the target nucleic acid and adenosine deaminase), wherein the reference oligonucleotide composition is Comprising a plurality of reference oligonucleotides sharing the same base sequence that is substantially complementary to the base sequence,

At this time,

The first plurality of oligonucleotides has more sugars with 2'-F modifications, sugars with 2'-OR modifications (R is not -H), and/or chiral controlled chiral internucleotidic linkages than the reference plurality of oligonucleotides. contains a lot,

The first oligonucleotide composition provides a higher level of modification compared to the oligonucleotides of the reference oligonucleotide composition.

In some embodiments, the present invention

A method comprising obtaining a modification of a first level of target adenosine in a target nucleic acid (the level observed when the first oligonucleotide composition is contacted with a sample comprising the target nucleic acid and adenosine deaminase), wherein the first oligonucleotide The composition comprises a first plurality of oligonucleotides sharing the same base sequence that is substantially complementary to the base sequence of the target nucleic acid,

The first level of modification of the target adenosine is higher than the reference level of modification of the target adenosine, the reference level observed when the reference oligonucleotide composition is contacted with a sample comprising a target nucleic acid and an adenosine deaminase, wherein the reference oligonucleotide composition is It includes a plurality of reference oligonucleotides that share the same base sequence that is substantially complementary to the base sequence of the target nucleic acid,

At this time,

The first plurality of oligonucleotides has more sugars with 2'-F modifications, sugars with 2'-OR modifications (R is not -H), and/or chiral controlled chiral internucleotidic linkages than the reference plurality of oligonucleotides. Provides a method, including many.

In some embodiments, the present invention

1) obtaining a modification of a first level of target adenosine in a target nucleic acid (the level observed when the first oligonucleotide composition is contacted with a sample comprising the target nucleic acid and adenosine deaminase), wherein the first oligonucleotide composition comprises: Comprising a first plurality of oligonucleotides sharing the same base sequence that is substantially complementary to the base sequence of the target nucleic acid, and

2) obtaining a modification of the reference level of target adenosine in the target nucleic acid (the level observed when the reference oligonucleotide composition is contacted with a sample comprising the target nucleic acid and adenosine deaminase), wherein the reference oligonucleotide composition is Comprising a plurality of reference oligonucleotides sharing the same base sequence that is substantially complementary to the base sequence,

At this time,

the first plurality of oligonucleotides comprises one or more chirally controlled chiral internucleotidic linkages;

the reference plurality of oligonucleotides does not contain chiral controlled chiral internucleotide linkages (the reference oligonucleotide composition is a stereorandom composition);

The first oligonucleotide composition provides a higher level of modification compared to the oligonucleotides of the reference oligonucleotide composition.

In some embodiments, the present invention

A method comprising obtaining a modification of a first level of target adenosine in a target nucleic acid (the level observed when the first oligonucleotide composition is contacted with a sample comprising the target nucleic acid and adenosine deaminase), wherein the first oligonucleotide The composition comprises a first plurality of oligonucleotides sharing the same base sequence that is substantially complementary to the base sequence of the target nucleic acid,

The first level of modification of the target adenosine is higher than the reference level of modification of the target adenosine, the reference level observed when the reference oligonucleotide composition is contacted with a sample comprising a target nucleic acid and an adenosine deaminase, wherein the reference oligonucleotide composition is It includes a plurality of reference oligonucleotides that share the same base sequence that is substantially complementary to the base sequence of the target nucleic acid,

At this time,

the first plurality of oligonucleotides comprises one or more chirally controlled chiral internucleotidic linkages;

The reference plurality of oligonucleotides does not contain chiral controlled chiral internucleotide linkages (the reference oligonucleotide composition is a stereorandom composition).

In some embodiments, the first oligonucleotide composition is an oligonucleotide composition as described herein. In some embodiments, the first oligonucleotide composition is a chirally controlled oligonucleotide composition. In some embodiments, the deaminase is an ADAR enzyme. In some embodiments, the deaminase is ADAR1. In some embodiments, the deaminase is ADAR2. In some embodiments, a sample is or comprises a cell. In some embodiments, the target nucleic acid has an I or G at the position of the target adenosine instead of the target adenosine, compared to a nucleic acid that differs from the target nucleic acid in that it is more related to a condition, disorder or disease, or reduces a desired property or function. is more related to, or more related to, an increase in an undesirable property or function. In some embodiments, the target adenosine is a G to A mutation.

In particular, the oligonucleotide design of the present invention, eg, nucleobase, sugar, internucleotide linkage modifications, control of linkage phosphorus stereochemistry, and/or patterns thereof, can be applied to improve upon the prior art. In some embodiments, the present invention is achieved by incorporating one or more structural features of the present invention, e.g., nucleobases, sugars, internucleotidic linkage modifications, control of linkage phosphorus stereochemistry, and/or patterns thereof, into prior art oligonucleotides. It provides improvements over the prior art. In some embodiments, the improvement is or includes an improvement from control of linkage phosphorus stereochemistry.

In some embodiments, the invention provides adenosine editing by a polypeptide, e.g., ADAR1, ADAR2, etc., comprising incorporating a design (e.g., one or more modifications and/or patterns thereof) as described herein into an oligonucleotide. provides techniques to improve In some embodiments, the design is or comprises a modified base as described herein, eg, at a position opposite a target adenosine and/or one or both adjacent positions thereof. In some embodiments, the design comprises one or more sugar modifications and/or patterns thereof, one or more base modifications and/or patterns thereof, one or more modified internucleotide linkages and/or patterns thereof, and/or controlled conformation at one or more positions. is or includes a chemistry and/or pattern thereof. In some embodiments, provided techniques improve editing by ADAR1 more than ADAR2. In some embodiments, provided techniques improve editing by ADAR2 more than ADAR1. In some embodiments, provided technologies improve editing by ADAR1 p110 more than p150 (eg, in some embodiments, R p at one or more positions (eg, of a phosphorothioate internucleotidic linkage)) . In some embodiments, provided techniques improve editing by ADAR1 p150 more than p110.

In some embodiments, provided techniques include increasing the level of an adenosine editing polypeptide, eg, ADAR1 (p110 or p150) or ADAR2, or portion thereof. In some embodiments, the increase is through expression of an exogenous polypeptide.

In some embodiments, provided oligonucleotides or oligonucleotide compositions do not cause significant degradation of nucleic acids (e.g., at most about 5%-100% (e.g., at most about 10%-100%, 20-100%, 30%~100%, 40%~100%, 50%~80%, 50%~85%, 50%~90%, 50%~95%, 60%~80%, 60%~85%, 60% ~90%, 60%~95%, 60%~100%, 65%~80%, 65%~85%, 65%~90%, 65%~95%, 65%~100%, 70%~80 %, 70%~85%, 70%~90%, 70%~95%, 70%~100%, 75%~80%, 75%~85%, 75%~90%, 75%~95%, 75% to 100%, 80% to 85%, 80% to 90%, 80% to 95%, 80% to 100%, 85% to 90%, 85% to 95%, 85% to 100%, 90% ~95%, 90%~100%, 10%, 20%, 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% etc.)). In some embodiments, the composition does not cause significant undesirable exon skipping or altered exon inclusion in the target nucleic acid (e.g., at most about 5%-100% (e.g., at most about 10%-100%, 20-100%)). 100%, 30%~100%, 40%~100%, 50%~80%, 50%~85%, 50%~90%, 50%~95%, 60%~80%, 60%~85% , 60%~90%, 60%~95%, 60%~100%, 65%~80%, 65%~85%, 65%~90%, 65%~95%, 65%~100%, 70 %~80%, 70%~85%, 70%~90%, 70%~95%, 70%~100%, 75%~80%, 75%~85%, 75%~90%, 75%~ 95%, 75% to 100%, 80% to 85%, 80% to 90%, 80% to 95%, 80% to 100%, 85% to 90%, 85% to 95%, 85% to 100% , 90%~95%, 90%~100%, 10%, 20%, 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95 %, or 100%, etc.)).

In some embodiments, provided technologies can provide high-level adenosine editing (eg, conversion to inosine). In some embodiments, the percentage of target adenosine editing is between about 10% and 100%, for example at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, or 95%. In some embodiments, the percentage is at least 10%. In some embodiments, the percentage is at least 15%. In some embodiments, the percentage is at least 20%. In some embodiments, the percentage is at least 25%. In some embodiments, the percentage is at least 30%. In some embodiments, the percentage is at least 35%. In some embodiments, the percentage is at least 40%. In some embodiments, the percentage is at least 45%. In some embodiments, the percentage is at least 50%. In some embodiments, the percentage is at least 60%. In some embodiments, the percentage is at least 70%. In some embodiments, the percentage is at least 75%. In some embodiments, the percentage is at least 80%. In some embodiments, the percentage is at least 85%. In some embodiments, the percentage is at least 90%. In some embodiments, the percentage is at least 95%. In some embodiments, the percentage is at least about 100%.

In some embodiments, an oligonucleotide or composition thereof can mediate a decrease in the expression or level of a target nucleic acid or its product (eg, by modifying a target adenosine to inosine). In some embodiments, an oligonucleotide or composition thereof can mediate a decrease in the expression or level of a target gene or gene product thereof (eg, by modifying a target adenosine to inosine) in a cell in vitro. In some embodiments, the expression or level is at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 75%, 80%, It may be reduced by 85%, 90%, or 95%. In some embodiments, the expression or level of a target gene or gene product thereof is reduced by at least about 10%, e.g., by ADAR-mediated deamination directed by an oligonucleotide or composition thereof at a concentration of 10 uM or less in the cell(s) in vitro. %, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, or 95%. can In some embodiments, oligonucleotides or compositions thereof are capable of providing suitable levels of activity at concentrations of 1 nM, 5 nM, 10 nM or less (eg, when assayed in vitro or in vivo cells).

In some embodiments, the activity of provided oligonucleotides and compositions can be assessed by IC50, which is an inhibitory concentration that reduces the level of a target nucleic acid or its product by 50% in suitable conditions, eg, a cell-based in vitro assay. In some embodiments, a provided oligonucleotide or composition is at most 0.001, 0.01, 0.1, 0.5, 1, 2, 5, 10, 50, 100, 200, 500, or 1000 nM, as assessed, for example, in a cell-based assay. It has an IC50. In some embodiments, the IC50 is at most about 500 nM. In some embodiments, the IC50 is up to about 200 nM. In some embodiments, the IC50 is at most about 100 nM. In some embodiments, the IC50 is at most about 50 nM. In some embodiments, the IC50 is at most about 25 nM. In some embodiments, the IC50 is at most about 10 nM. In some embodiments, the IC50 is at most about 5 nM. In some embodiments, the IC50 is at most about 2 nM. In some embodiments, the IC50 is at most about 1 nM. In some embodiments, the IC50 is at most about 0.5 nM.

In some embodiments, provided technologies can provide selective editing of a target adenosine over other adenosine residues on the target adenosine. In some embodiments, the selectivity of a target adenosine over a non-target adenosine is at least 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 60 , 70, 80, 90, 100-fold or more (e.g., by the level of editing of target adenosine relative to non-target adenosine under suitable conditions, or at a specific level of editing (e.g., 0.5%, 1%, 2%) , 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, etc.). In some embodiments, the selectivity is at least about 2-fold. In some embodiments, the selectivity is at least about 3-fold. In some embodiments, the selectivity is at least about 4-fold. In some embodiments, the selectivity is at least about 5-fold. In some embodiments, the selectivity is at least about 10 fold. In some embodiments, the selectivity is at least about 25 fold. In some embodiments, the selectivity is at least about 50 fold. In some embodiments, the selectivity is at least about 100 fold.

In some embodiments, the present invention provides a method of inhibiting transcripts from a target nucleic acid sequence in which there is one or more similar nucleic acid sequences in a population, wherein the target and similar sequences are each a specific sequence defining the target sequence relative to the similar sequences. characterized sequence elements, wherein the method comprises contacting a transcript of a target nucleic acid sequence with an oligonucleotide or a composition comprising a plurality of oligonucleotides that share a common base sequence, wherein the base sequence of the oligonucleotide, or The common base sequence of the plurality of oligonucleotides is or includes a sequence complementary to a characteristic sequence element defining a target nucleic acid sequence. In some embodiments, when an oligonucleotide or oligonucleotide composition is contacted with a system comprising transcripts of both a target nucleic acid sequence and a similar nucleic acid sequence, the transcript of the target nucleic acid sequence is less than the level of inhibition observed for the similar nucleic acid sequence. suppressed to a greater degree. In some embodiments, inhibition of a transcript of a target nucleic acid sequence is 1.1-100, 2-100, 1.5, 2, 2.5, 3, 4, 5, 6, 7, 8, 9, or 10 times larger. In some embodiments, a target nucleic acid sequence is associated with (or more associated with, a similar nucleic acid sequence) a condition, disorder or disease. As will be appreciated by those skilled in the art, selectively reducing transcripts (and/or products thereof) associated with a condition, disorder, or disease while retaining transcripts not or less associated with the condition, disorder, or disease can result in one or more desired It can provide many benefits, such as providing disease treatment and/or prevention while retaining its biological function (in particular, it can provide fewer or less severe side effects).

In some embodiments, as shown herein, the selectivity is at least 10-fold, or 20, 30, 40, or 50-fold or more in a system, eg, a reporter assay described herein. In some embodiments, the oligonucleotide or composition maintains the level of wild-type protein in the system (e.g., at least 70%, 75%, 80%, 85%, 90%, 95% or more of wild-type protein remains), The level of the mutant protein can be effectively reduced (eg, a reduction of at least 50%, 60%, 70% or more of the mutant protein). In some embodiments, provided oligonucleotides are stable (e.g., at least 70% after 1, 2, 3, 4, 5, 6, 7, or 8 days) in a variety of biological systems, e.g., mouse brain homogenate; 75%, 80%, 85%, 90%, 95% or more remaining). In some embodiments, provided oligonucleotides have low toxicity. In some embodiments, provided oligonucleotides and compositions thereof, e.g., chirally controlled oligonucleotides and compositions thereof, do not significantly activate TLR9 (e.g., a reference oligonucleotide and composition thereof (e.g., a corresponding sterically randomized oligonucleotides and compositions thereof)). In some embodiments, provided oligonucleotides and compositions thereof, e.g., chirally controlled oligonucleotides and compositions thereof, do not significantly induce complement activation (e.g., a reference oligonucleotide and composition thereof (e.g., a corresponding steric when compared to random oligonucleotides and compositions thereof).

For various uses, provided oligonucleotides and/or compositions may be provided as pharmaceutical compositions. In some embodiments, the present invention provides pharmaceutical compositions comprising or delivering an effective amount of an oligonucleotide or pharmaceutically acceptable salt thereof. In some embodiments, the pharmaceutical composition may include various forms of oligonucleotides, such as acids, bases, and various pharmaceutically acceptable salt forms. In some embodiments, the pharmaceutically acceptable salt is the sodium salt. In some embodiments, the pharmaceutically acceptable salt is a potassium salt. In some embodiments, a pharmaceutically acceptable salt is an amine salt (eg, a salt of an amine having the structure N(R) ₃ ). In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition is or comprises a liquid solution. In some embodiments, the liquid composition has a controlled pH range, for example at or near physiological pH. In some embodiments, the pharmaceutical composition comprises or is formulated with a solution in a physiologically compatible buffer such as Hanks' solution, Ringer's solution, cerebrospinal fluid, artificial cerebrospinal fluid (aCSF), or physiological saline buffer. In some embodiments, the pharmaceutical composition comprises or is formulated with a solution in artificial cerebral spinal fluid (aCSF). In some embodiments, the pharmaceutical composition is an injectable suspension or solution. In certain embodiments, injectable suspensions or solutions are prepared using appropriate liquid carriers, suspending agents, and the like. A pharmaceutical composition can be administered by a variety of suitable routes. In some embodiments, the pharmaceutical composition is, for example, drench (aqueous or non-aqueous solution or suspension), tablet, e.g., targeted for buccal, sublingual, and systemic absorption, bolus, powder, granule, lingual for oral administration as a paste for application to the skin; In a physiologically compatible buffer such as Hanks' solution, Ringer's solution, artificial cerebrospinal fluid (aCSF) or physiological saline buffer, for example as a sterile solution or suspension, or as a sustained-release formulation, for example subcutaneously, intramuscularly. , for parenteral administration by intravenous, intrathecal, intraventricular or epidural injection; for topical application, for example as a cream, ointment or controlled release patch or spray applied to the skin, lungs or oral cavity; for vaginal or intrarectal administration, eg as a pessary, cream or foam; for sublingual administration; for ocular administration; for transdermal administration; or formulated for nasal, pulmonary and other mucosal surface administration.

In particular, the present invention provides techniques for preventing or treating conditions, disorders or diseases. In some embodiments, the invention provides a method of preventing or treating a condition, disorder or disease comprising administering or delivering to a subject predisposed to or suffering from it an effective amount of an oligonucleotide or composition as described herein. provides a way In some embodiments, the condition, disorder or disease is amenable to (eg, may benefit from) conversion from A to I. In some embodiments, the invention provides a method for preventing or treating a condition, disorder, or disease associated with a G to A mutation, wherein a subject predisposed to or suffering from such condition, disorder, or disease is treated with an oligonucleotide as described herein. A method comprising administering an effective amount of a nucleotide or composition is provided. In some embodiments, the present invention provides a method for preventing or treating a condition, disorder or disease prone to G to A mutation, comprising administering to a subject predisposed to or suffering from it an effective amount of an oligonucleotide or composition as described herein. It provides a method comprising the step of administering. In some embodiments, the invention provides a method for preventing or treating a condition, disorder, or disease associated with a G to A mutation, wherein a subject predisposed to or suffering from such condition, disorder, or disease is treated with an oligonucleotide as described herein. A method comprising administering an effective amount of a nucleotide or composition is provided. In some embodiments, the base sequence of an oligonucleotide in an oligonucleotide or oligonucleotide composition is substantially complementary to the base sequence of a target nucleic acid comprising a target adenosine. In some embodiments, the cell, tissue or organ associated with the condition, disorder or disease comprises or expresses an ADAR protein. In some embodiments, a cell, tissue, or organ associated with a condition, disorder, or disease comprises or expresses ADAR1 (eg, in the form of p110 and/or p150). In some embodiments, the cell, tissue or organ associated with the condition, disorder or disease comprises or expresses ADAR2. In some embodiments, the condition, disorder or disease is as described herein. In some embodiments, the condition, disorder or disease is alpha-1 antitrypsin deficiency. In some embodiments, the method comprises converting the target adenosine to I.

In some embodiments, the present invention provides an oligonucleotide comprising a sequence complementary to a target sequence. In some embodiments, the present invention provides oligonucleotides that direct site-specific (also referred to as site-directed) editing (eg, deamination). In some embodiments, the present invention provides oligonucleotides that direct site-specific adenosine editing mediated by ADARs (eg, endogenous ADARs). A variety of provided oligonucleotides can be used as single-stranded oligonucleotides for site-directed editing of nucleotides within a target RNA sequence. In some embodiments, the present invention uses provided single-stranded oligonucleotides for site-directed editing of nucleotides in a target RNA sequence and compositions thereof to prevent and/or prevent conditions, disorders or diseases associated with G to A mutations in a target sequence. Or provide a method for treatment. In some embodiments, the present invention provides oligonucleotides and compositions thereof for use as a medicament, for example for a condition, disorder or disease associated with a G to A mutation in a target sequence. In some embodiments, the present invention provides oligonucleotides and compositions thereof for use in the treatment of a condition, disorder or disease associated with a G to A mutation in a target sequence. In some embodiments, the present invention provides oligonucleotides and compositions thereof for the manufacture of a medicament for the treatment of a related condition, disorder or disease associated with a G to A mutation in a target sequence.

In some embodiments, the present invention provides a method for preventing, treating, or ameliorating in a subject susceptible to or suffering from a condition, disorder, or disease associated with a G to A mutation in a target sequence, comprising an oligonucleotide or pharmaceutical composition thereof A method comprising administering to a subject a therapeutically effective amount is provided.

In some embodiments, the invention provides a method of deamination of a target adenosine in a target sequence in a cell comprising contacting the cell with an oligonucleotide or composition thereof. In some embodiments, the present invention provides a method of deamination of a target adenosine in a target sequence (eg, transcript) of a cell comprising contacting the cell with an oligonucleotide or composition thereof. In some embodiments, the present invention provides a method of reducing the level of a protein associated with a G to A mutation in a cell, comprising contacting the cell with an oligonucleotide or composition thereof. In some embodiments, provided methods are capable of selectively reducing the level of a transcript and/or product encoded thereby associated with a condition, disorder or disease associated with a G to A mutation. In some embodiments, provided methods selectively select a target nucleic acid, e.g., a transcript, (e.g., a G to A mutation) comprising an undesirable A relative to the same nucleic acid but having a G at the position of the target A. can be edited with

In some embodiments, the present invention provides a method for reducing expression of a mutant gene (e.g., a G to A mutation) in a mammal in need thereof, provided for site-directed editing of nucleotides in a target RNA sequence. A method comprising administering a nucleic acid-lipid particle comprising a single-stranded oligonucleotide or composition thereof to a mammal is provided.

In some embodiments, the present invention provides a method for in vivo delivery of an oligonucleotide, comprising administering an oligonucleotide or composition thereof to a mammal.

In some embodiments, a subject or patient suitable for treatment of a condition, disorder or disease associated with a G to A mutation can be identified or diagnosed by a health care professional.

In some embodiments, the symptoms of a condition, disorder or disease associated with a G to A mutation can be any condition, disorder or disease that could benefit from an A to I transformation.

In some embodiments, a provided single-stranded oligonucleotide or composition thereof for site-directed editing of nucleotides in a target RNA sequence is a condition, disorder or disease associated with a G to A mutation, or a condition, disorder associated with a G to A mutation. or to prevent, treat, ameliorate or slow the progression of at least one symptom of a disease.

In some embodiments, a method of the invention may be for treatment of a condition, disorder or disease associated with a G to A mutation in a subject, comprising administering to the subject a therapeutically effective amount of an oligonucleotide or pharmaceutical composition thereof. do.

In some embodiments, provided methods are capable of reducing at least one symptom of a condition, disorder or disease associated with a G to A mutation, comprising administering to a subject a therapeutically effective amount of an oligonucleotide or pharmaceutical composition thereof.

In some embodiments, administration of an oligonucleotide to a patient or subject can mediate any one or more of the following: slowing the progression of a condition, disorder or disease associated with a G to A mutation; delaying the onset of a condition, disorder or disease associated with a G to A mutation or at least one symptom thereof; improving one or more indicators of a condition, disorder or disease associated with a G to A mutation; and/or increasing the survival or longevity of a patient or subject.

In some embodiments, slowing disease progression is a clinically undesirable change in one or more clinical parameters in an individual predisposed to or suffering from a condition, disorder, or disease associated with a G to A mutation, as described herein. may be related to the prevention or delay of Using one or more of the disease assessment tests described herein, it is within the competence of a physician to identify a slowing of disease progression in an individual predisposed to or suffering from a condition, disorder or disease associated with a G to A mutation. It is further understood that a physician may subject an individual to diagnostic tests other than those described herein to assess the rate of disease progression in an individual predisposed to or suffering from a condition, disorder, or disease associated with a G to A mutation. do.

A physician may use a family history of a condition, disorder, or disease associated with the G to A mutation, or comparison with other patients with a similar genetic profile.

In some embodiments, indicators of conditions, disorders or diseases associated with G to A mutations include parameters used by medical professionals, such as physicians, to diagnose or measure the progression of a condition, disorder or disease.

In some embodiments, the subject is administered an oligonucleotide or composition thereof and an additional agent and/or method, eg, an additional therapeutic agent and/or method. In some embodiments, an oligonucleotide or composition thereof may be administered alone or in combination with one or more additional therapeutic agents and/or treatments. When administered in combination, each component may be administered simultaneously or sequentially in any order at different times. In some embodiments, each component may be administered separately but sufficiently close in time to provide the desired therapeutic effect. In some embodiments, a provided oligonucleotide and an additional therapeutic component are administered concurrently. In some embodiments, provided oligonucleotides and additional therapeutic components can be administered as one composition. In some embodiments, the administered subject may be simultaneously exposed at a given point in time to both the provided oligonucleotide and the additional component.

In some embodiments, an additional therapeutic agent can be physically conjugated to an oligonucleotide. In some embodiments, the additional agent is GalNAc. In some embodiments, provided single-stranded oligonucleotides for site-directed editing of nucleotides within a target RNA sequence may be physically conjugated with additional agents. In some embodiments, an oligonucleotide that is an additional agent is a base sequence, sugar, nucleobase, internucleotide linkage, sugar, nucleobase, and/or internucleotide linkage modification pattern, backbone chiral center pattern, etc., as described herein, or can have any combination of these, and each T can be independently replaced with a U and vice versa. In some embodiments, the oligonucleotide is capable of reducing (directly or indirectly) the expression, activity and/or level of a target sequence, or a second oligonucleotide useful for treating a condition, disorder or disease associated with a G to A mutation. It is physically conjugated to a nucleotide.

In some embodiments, a provided single-stranded oligonucleotide for site-directed editing of nucleotides in a target RNA sequence is administered with one or more additional (or second) therapeutic agents for a condition, disorder or disease associated with a G to A mutation. It can be.

In some embodiments, a subject may be administered an oligonucleotide and an additional therapeutic agent, wherein the additional therapeutic agent is an agent described herein or known in the art useful for the treatment of the condition, disorder or disease being treated.

In some embodiments, provided single-stranded oligonucleotides for site-directed editing of nucleotides within a target RNA sequence are useful for one or more treatments (aptamers, lncRNAs, lncRNA inhibitors, antibodies, peptides, small molecules, other oligonucleotides directed against other targets), or used as part of a treatment regimen.

In some embodiments, the additional therapeutic treatment is a gene editing method, as a non-limiting example.

In some embodiments, the additional therapeutic agent is an oligonucleotide by way of non-limiting example.

In some embodiments, the second or additional therapeutic agent can be administered to the subject before, concurrently with, or after the oligonucleotide. In some embodiments, the second or additional therapeutic agent can be administered to the subject multiple times, and the oligonucleotide is also administered to the subject multiple times, and the administrations are in any order.

In some embodiments, the amelioration is by reducing the expression, activity and/or level of a gene or gene product that is too high in a diseased state; increasing the expression, activity and/or level of a gene or gene product that is too low in a disease state; and/or reducing the expression, activity and/or level of mutations and/or disease-associated variants of a gene or gene product.

In some embodiments, an oligonucleotide or composition useful for the treatment, amelioration and/or prevention of a condition, disorder or disease associated with a G to A mutation can be administered (eg, to a subject) via a variety of suitable available techniques. there is.

In some embodiments, provided oligonucleotides, e.g., single-stranded oligonucleotides for site-directed editing of nucleotides within a target RNA sequence, are pharmaceutically acceptable, e.g., for the treatment, amelioration and/or prevention of a condition, disorder or disease. It can be administered as a composition. In some embodiments, provided oligonucleotides include one or more chirally controlled internucleotide linkages. In some embodiments, provided oligonucleotide compositions are chirally controlled.

In particular, the technology of the present invention, e.g., oligonucleotides and compositions thereof, can be compared with reference technology (e.g., in the absence or low level of chiral control (e.g., oligonucleotides of the same sequence or configuration, etc.) ) various improvements and advantages compared to sterically random oligonucleotide compositions), and/or absence or low levels of certain modifications and patterns thereof (eg, 2'-F, non-negatively charged internucleotidic linkages, etc.) , such as improved stability, delivery, editing efficiency, pharmacokinetics, and/or pharmacodynamics. In some embodiments, the reference oligonucleotide composition is a stereorandomized oligonucleotide composition of oligonucleotides having the same base sequence. In some embodiments, the reference oligonucleotide composition is a stereorandom oligonucleotide composition of oligonucleotides having the same configuration (as will be appreciated by those skilled in the art, in some embodiments, various salt forms may be properly considered to have the same configuration). can). In some embodiments, a reference oligonucleotide is an oligonucleotide that does not contain non-negatively charged internucleotidic linkages. In some embodiments, the reference oligonucleotide does not include n001. In some embodiments, a reference oligonucleotide composition is a composition of oligonucleotides that do not include non-negatively charged internucleotide linkages. In some embodiments, a reference oligonucleotide composition is a composition of oligonucleotides that does not include n001. In some embodiments, a provided technology can be used in lower unit or total doses and/or in smaller doses and/or longer dose intervals (eg, to achieve a comparable or superior effect) as compared to a reference technology. can be administered. In some implementations, provided techniques may provide longer editing durability. In some embodiments, a provided technique, once administered, is administered over a period of time, e.g., about or at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30 after the last administration. , active for more than 35, 40, 45, 50, 60 days, or 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 months, e.g. target editing to a certain level (e.g. , at a level useful and/or sufficient to provide a particular biological and/or therapeutic effect). In some embodiments, provided technologies provide low toxicity. In some embodiments, a provided technology can be used in higher unit or total doses and/or administered in higher doses and/or shorter dose intervals (eg, to achieve a greater effect) than a reference technology. can In some embodiments, the total dose can be administered as a single dose. In some embodiments, the total dose can be administered as two or more single doses. In some embodiments, a total dose administered in a single dose may provide a higher maximal level of editing compared to administration in two or more single doses.

In some cases, patients administered oligonucleotides as medications suffer from thrombocytopenia, renal toxicity, glomerulonephritis, and/or coagulopathy; genotoxicity, repeated dose toxicity in target organs and pathological effects; dose response and exposure relationships; chronic toxicity; developmental toxicity; reproductive and developmental toxicity; cardiovascular safety; injection site reaction; cytokine response complement effect; immunogenicity; and/or may experience certain side effects or adverse effects, including carcinogenicity. In some embodiments, an additional therapeutic agent is administered to overcome side effects or adverse effects of oligonucleotide administration. In some embodiments, a particular single-stranded oligonucleotide for site-directed editing of nucleotides within a target RNA sequence has a reduced ability to induce side effects or adverse effects compared to a different single-stranded oligonucleotide for site-directed editing of nucleotides within a target RNA sequence. can have

In some embodiments, an additional therapeutic agent may be administered to the patient to control or alleviate one or more side effects or adverse effects associated with administration of the oligonucleotide.

In some embodiments, the oligonucleotide and one or more additional therapeutic agents can be administered to the patient (in any order), and the additional therapeutic agent can be administered to the patient to control or alleviate one or more side effects or adverse effects associated with administration of the oligonucleotide. can

In some embodiments, the oligonucleotide and one or more additional therapeutic agents can be administered to the patient (in any order), and the additional therapeutic agent can be administered to the patient to control or alleviate one or more side effects or adverse effects associated with administration of the oligonucleotide. can

In some embodiments, an oligonucleotide and one or more additional therapeutic agents may be administered (in any order) to a patient, wherein the additional therapeutic agent is administered to the patient to control or alleviate one or more side effects or adverse effects associated with administration of the oligonucleotide. When administered, the oligonucleotides act through any biochemical mechanism, including but not limited to: reduction of the level, expression and/or activity of the target gene or its gene product, one or more in the target gene mRNA. increased or decreased exon skipping, ADAR-mediated deamination, RNaseH-mediated mechanisms, steric hindrance-mediated mechanisms, and/or RNA interference-mediated mechanisms, wherein the oligonucleotides are single or double stranded.

In some embodiments, an oligonucleotide composition and one or more additional therapeutic agents may be administered (in any order) to a patient, and the additional therapeutic agent may be administered to the patient to control or alleviate one or more side effects or adverse effects associated with administration of the oligonucleotide composition. The oligonucleotide composition may be chirally controlled or contain at least one chirally controlled internucleotidic linkage (including but not limited to a chirally controlled phosphorothioate).

Those associated with G to A mutations, e.g., cystic fibrosis, Hurler syndrome, alpha-1 antitrypsin (A1AT) deficiency, Parkinson's disease, Alzheimer's disease, albinism, amyotrophic lateral sclerosis, asthma, beta thalassemia, cardasil Syndrome, Charcomaritus Disease, Chronic Obstructive Pulmonary Disease (COPD), Distal Spinal Muscular Atrophy (DSMA), Duchenne/Becker Muscular Dystrophy, Dystrophic Epidermal Desquamation Blister, Epidermolysis bullosa, Fabry Disease, Factor V Leiden-related Disorder, Familial Adenoma, Polyposis, galactosemia, Gaucher disease, glucose-6-phosphate dehydrogenase, hemophilia, hereditary hemochromatosis, Hunter syndrome, Huntington's disease, inflammatory bowel disease (IBD), hereditary polyaggregation syndrome, Leber congenital amaurosis, Lesch-Nyhan syndrome, Lynch syndrome, Marfan syndrome, mucopolysaccharidosis, muscular dystrophy, myotonic dystrophy types I and II, neurofibromatosis, Niemann-Pick disease types A, B, and C, NY-eso1-associated cancer, Peutz-Jaeger syndrome, phenylketonuria, Pompe disease, primary Ciliary disease, disorders associated with prothrombin mutations such as prothrombin G20210A mutation, pulmonary hypertension, retinitis pigmentosa, Sandhoff disease, severe combined immunodeficiency syndrome (SCID), sickle cell anemia, spinal muscular atrophy, Stargardt disease, Tay-Sachs disease, Usher A variety of conditions, disorders or diseases can benefit from adenosine editing, including syndrome, X-linked immunodeficiency, Sturge-Weber syndrome, and various cancers. Certain conditions, disorders or diseases are described in WO 2020/154344, WO 2020/154343, WO 2020/154342, WO 2020/165077, WO 2020/201406, WO 2020/216637, or WO 2020/252376.

In some embodiments, the condition, disorder or disease is alpha-1 antitrypsin (A1AT) deficiency (AATD).

Alpha-1 antitrypsin (A1AT) deficiency (AATD) is a genetic disorder reported to result from defects in the SERPINA1 gene (PI; AIA; AAT; PI1; A1AT; PR02275; also known as alpha1AT). Severe A1AT deficiency is associated with a variety of phenotypes including lung and liver phenotypes.

A1AT deficiency is reported to be one of the most common genetic disorders in subjects of Nordic ancestry. The prevalence of severe A1AT deficiency is 80,000 to 100,000 in the United States alone. Similar figures are assumed to be found in the EU. The global estimate for severe A1AT deficiency is fixed at 3 million. A1AT deficiency causes emphysema, and subjects develop emphysema in their 30s or 40s. A1AT deficiency can also lead to liver failure and hepatocellular carcinoma, and up to 30% of subjects with severe A1AT deficiency develop severe liver disease, including cirrhosis, fulminant liver failure, and hepatocellular carcinoma.

A mutation in the SERPINA1 gene (i.e. c. 1024G>A) results in a glutamate to lysine substitution at amino acid position 342 (E342K, "Z mutation") of the mature A1AT protein. These missense mutations reduce circulating A1AT levels by affecting protein conformation and secretion. Alleles carrying the Z mutation are identified as PiZ alleles. Subjects who are homozygous for the PiZ allele are referred to as PiZZ carriers, and they exhibit 10-15% of normal levels of serum A1AT. Approximately 95% of subjects with symptoms of A1AT deficiency carry the PiZZ genotype. Subjects heterozygous for the Z mutation are referred to as PiMZ mutants, and they exhibit 60% of normal levels of serum A1AT. Among diagnosed patients, 90% of patients with severe A1AT deficiency have ZZ mutations. About 30,000 to 50,000 individuals in the United States have the PiZZ genotype.

The pathophysiology of A1AT deficiency may differ depending on the organ affected. Liver disease is reported to occur due to gain-of-function mechanisms. Aberrantly folded A1ATs, especially Z-type A1ATs (Z-ATs), aggregate and polymerize within hepatocytes. A1AT inclusion bodies are found in PiZZ subjects and are believed to cause cirrhosis and, in some cases, hepatocellular carcinoma. Evidence for a gain-of-function mechanism in liver disease is supported by null homozygotes. These subjects do not produce A1AT and do not develop hepatocellular inclusion bodies or liver disease.

A1AT deficiency is reported to cause liver disease in up to about 50% of A1AT subjects and lead to severe liver disease in up to about 30% of subjects. Liver disease can present as (a) self-limiting childhood cirrhosis, (b) severe childhood or adult cirrhosis requiring liver transplantation or leading to death, and (c) often fatal hepatocellular carcinoma. The incidence of liver disease is reported to be bimodal, primarily affecting children or adults. Childhood disease is in many cases self-limiting but can lead to late-stage fatal cirrhosis. It is reported that up to about 18% of subjects with the PiZZ genotype may develop clinically significant liver abnormalities in childhood. Approximately 2% of PiZZ subjects are reported to develop severe cirrhosis leading to death in childhood (Sveger 1988; Volpert 2000). Adult-onset liver disease can affect subjects with any genotype, but appears earlier in subjects with the PiZZ genotype. Approximately 2-10% of A1AT-deficient subjects are reported to develop adult-onset liver disease.

Pulmonary diseases associated with A1AT deficiency are currently treated with intravenous administration of human-derived replacement A1AT proteins, but besides being expensive and requiring frequent injections throughout the subject's lifetime, this approach is only partially effective. AlAT-deficient subjects with hepatocellular carcinoma are currently being treated with chemotherapy and surgery, but there is no satisfactory approach to prevent the potentially fatal liver manifestations of A1AT deficiency.

In particular, the present invention recognizes the need for improved treatment of A1AT deficiencies, including hepatic and pulmonary manifestations. In some embodiments, the present invention provides oligonucleotides and/or compositions capable of converting, for example, an A mutation to an I that can be read as G during protein translation, by correcting a G to A mutation for protein translation. Provided are techniques for preventing or treating conditions, disorders or diseases associated with alpha-1 antitrypsin (A1AT) deficiency. In particular, alteration of SERPINA1 in one or more hepatocytes can prevent the progression of liver disease in subjects with A1AT deficiency by reducing or eliminating the production of toxic Z protein (Z-AAT). In certain embodiments, Z protein production is eliminated or reduced by using provided techniques. In certain embodiments, the disease is cured, does not progress, or progress is delayed compared to a subject not receiving treatment.

In some embodiments, AATD dual pathology has been reported in liver and lung. In some embodiments, the inability to secrete polymerized Z-ATT has been reported to cause liver damage/cirrhosis, for example. In some embodiments, one or both lungs are open to an unidentified protease that causes inflammation and lung damage in some embodiments. A large number of patients (eg, approximately 200,000 reported in the US and EU) carry a homozygous ZZ genotype that has been reported to be associated with the most common form of severe AATD. Approved therapies have been reported to modestly increase circulating levels of extensive AAT in people with lung pathology, and there are no treatments addressing liver pathology. In some embodiments, provided technologies increase or restore the expression, level, properties, and/or activity of wild-type AAT in the liver. In some embodiments, provided technologies target the liver, for example, by incorporating a liver-targeting moiety (eg, a ligand such as GalNAc that targets a receptor expressed in the liver) into an oligonucleotide. In some embodiments, provided technologies restore, increase, or enhance wild-type AAT physiological regulation in the liver. In some embodiments, provided technologies reduce Z-AAT protein aggregation. In some embodiments, provided technologies restore, increase or enhance wild-type AAT physiological regulation in the liver and reduce Z-AAT protein aggregation. In some embodiments, provided techniques increase secretion into the bloodstream. In some embodiments, provided technologies increase circulating wild-type AAT. In some embodiments, provided technologies increase circulating lung-bound wild-type AAT. In some embodiments, provided technologies increase or restore the expression, level, properties and/or activity of wild-type AAT in the lung. In some embodiments, provided technologies protect the lungs from undesirable proteases. In some embodiments, provided technologies reduce or prevent inflammation and/or lung damage. In some embodiments, provided technologies provide benefit in both liver and lung. In some embodiments, provided technologies reduce or prevent liver damage or cirrhosis, and reduce or prevent inflammation and/or lung damage. In some embodiments, provided oligonucleotides, eg, oligonucleotides comprising specific moieties such as ligands (eg, GalNAc) that target receptors expressed in the liver provide benefits in the liver and lung. In some embodiments, provided technologies provide benefit in the liver and lungs simultaneously. In some embodiments, provided technologies address pulmonary and/or hepatic manifestations of AATD. In some embodiments, provided technologies simultaneously address pulmonary and hepatic manifestations of AATD. In some embodiments, provided techniques include using GalNAc conjugated oligonucleotides and compositions thereof to correct RNA base mutations in mRNA encoded by the SERPINA1 Z allele that cause AATD. In some embodiments, provided technologies reduce aggregation of mutated and misfolded alpha-1 protein while simultaneously increasing circulating levels of wild-type alpha-1 antitrypsin protein, and in some embodiments address both hepatic and pulmonary symptoms of AATD. In some embodiments, provided techniques avoid the risk of permanent off-target changes to DNA.

In certain embodiments, techniques as described herein may provide a selective advantage for the survival of one or more of the treated hepatocytes. In certain embodiments, target cells are modified. In some embodiments, cells treated with the techniques herein may not produce toxic Z proteins. In some embodiments, diseased cells that are not transformed produce toxic Z proteins and can undergo apoptosis secondary to endoplasmic reticulum (ER) stress induced by Z protein misfolding. In certain embodiments, after treatment using a provided technique, treated cells will survive and untreated cells will die. This selective advantage can lead to eventual colonization of hepatocytes, most of which are SERPINA1 proofreading cells.

In some embodiments, the oligonucleotide, when administered to a patient suffering from or susceptible to a condition, disorder or disease associated with a G to A mutation, is capable of reducing at least one symptom of the condition, disorder or disease; delay or prevent onset, deterioration, and/or reduce the rate and/or extent of deterioration of at least one symptom of a condition, disorder or disease due to a G to A mutation in a gene or gene product.

In some embodiments, provided techniques may provide for editing of two or more sites in a system (eg, a cell, tissue, organ, animal, etc.) (“multiple editing”). In some embodiments, provided technologies are capable of targeting and editing two or more sites of the same transcript. In some embodiments, provided technologies can target and edit two or more different transcripts of the same nucleic acid or different nucleic acids. In some embodiments, provided technologies are capable of targeting and editing the transcripts of two or more different nucleic acids. In some embodiments, provided technologies are capable of targeting and editing the transcripts of two or more different genes. In some embodiments, each of the simultaneously edited targets is independently biologically and/or therapeutically relevant. In some embodiments, one or more or all targets in multiple edits are edited independently, to a similar degree to edits performed individually under similar conditions. In some embodiments, multiple editing is performed using two or more separate compositions, each independently targeting one or more targets. In some embodiments, the compositions are administered concurrently. In some embodiments, the compositions are administered at appropriate intervals. In some embodiments, one or more compositions are administered before or after one or more other compositions. In some embodiments, multiple editing is performed using a single composition, eg, a composition comprising two or more pluralities of oligonucleotides, the pluralities targeting different targets. In some embodiments, each plurality independently targets a different adenosine. In some embodiments, each plurality independently targets a different transcript. In some embodiments, each plurality independently targets a different gene. In some embodiments, two or more pluralities may target the same target, but together the pluralities target a desired target.

As described herein, the provided technology can provide many advantages. For example, in some embodiments, provided techniques are safer than techniques that act on DNA, because provided techniques can provide reversible and tunable RNA editing (eg, via modulating doses). Additionally and alternatively, as shown herein, provided technologies can provide for high-level editing in systems that express endogenous ADAR proteins, avoiding the requirement for introduction of exogenous proteins in many cases. In addition, the provided technology does not require complex oligonucleotides that rely on auxiliary delivery vehicles such as viral vectors or lipid nanoparticles as used in many other technologies, particularly for applications other than cell culture. In some embodiments, provided technologies can provide sequence-specific A-I RNA editing with high efficiency using endogenous ADAR enzymes and can be delivered to a variety of systems, eg, cells, in the absence of artificial delivery agents.

Those skilled in the art upon reading this disclosure will understand that provided oligonucleotides and compositions thereof can be delivered using several techniques in accordance with the present invention. In some embodiments, provided oligonucleotides and compositions thereof can be delivered via transfection or lipofection. In some embodiments, provided oligonucleotides and compositions thereof can be delivered without delivery adjuvants such as those used for transfection or lipofection. In some embodiments, provided oligonucleotides and compositions thereof can be delivered via transfection or lipofection. In some embodiments, provided oligonucleotides and compositions thereof are delivered by zymotic delivery. In some embodiments, provided oligonucleotides include additional chemical moieties capable of facilitating delivery. For example, in some embodiments, the additional chemical moiety is or comprises a ligand moiety (eg, N -acetylgalactosamine (GalNAc)) for a receptor (eg, an asialoglycoprotein receptor). In some embodiments, provided oligonucleotides and compositions thereof can be delivered via GalNAc mediated delivery.

In particular, the present invention provides the following exemplary embodiments:

1. As an oligonucleotide,

first domain; and

Including a second domain;

At this time,

the first domain comprises one or more 2'-F modifications;

The oligonucleotide of claim 1 , wherein the second domain comprises one or more sugars without a 2'-F modification.

2. An oligonucleotide comprising a modified nucleobase, nucleoside, sugar or internucleotide linkage as described herein.

3. An oligonucleotide wherein about or at least about 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% of all sugars are 2'-F modified sugars.

4. An oligonucleotide comprising a second subdomain as described herein.

5. An oligonucleotide comprising one or more modified sugars and/or one or more modified internucleotidic linkages, wherein the oligonucleotide comprises a first domain and a second domain, each independently comprising one or more nucleobases.

6. The oligonucleotide of any one of embodiments 1-5, wherein the target adenosine in the target nucleic acid is modified when the oligonucleotide is contacted with a target nucleic acid comprising the target adenosine in the system.

7. The method according to any one of embodiments 1 to 5, wherein when the oligonucleotide is contacted with a target nucleic acid comprising a target adenosine in the system, the level of the target nucleic acid is reduced relative to the absence of the product or the presence of the reference oligonucleotide. , oligonucleotides.

8. The method according to any one of embodiments 1 to 5, wherein when the oligonucleotide is contacted with a target nucleic acid comprising a target adenosine in the system, splicing of the target nucleic acid or product thereof occurs in the absence of the oligonucleotide or the reference oligonucleotide Oligonucleotides, which are altered relative to the presence of.

9. The method according to any one of embodiments 1 to 5, wherein when the oligonucleotide is contacted with a target nucleic acid comprising a target adenosine in the system, the product level of the target nucleic acid is altered relative to the absence of the product or the presence of the reference oligonucleotide. which is, an oligonucleotide.

10. The oligonucleotide according to any one of embodiments 7-9, wherein the target nucleic acid is modified.

11. The oligonucleotide of any one of embodiments 6-10, wherein the product level is increased and the product is or is encoded by the same nucleic acid as the target nucleic acid except that the target adenosine is modified.

12. The oligonucleotide of any one of embodiments 6-10, wherein the product level is increased and the product is or is encoded by the same nucleic acid as the target nucleic acid except that the target adenosine is replaced by inosine.

13. The oligonucleotide of any one of embodiments 6-10, wherein the product level is increased and the product is or is encoded by the same nucleic acid as the target nucleic acid except that the adenine of the target adenosine is replaced by guanine.

14. The oligonucleotide of any one of embodiments 11-13, wherein the product is a protein.

15. The oligonucleotide of any one of the preceding embodiments, wherein the target adenosine is a mutation from guanine.

16. The oligonucleotide of any one of the above embodiments, wherein the target adenosine is more associated with the condition, disorder or disease than the guanine at the same position.

17. The oligonucleotide of any one of the preceding embodiments, wherein the target adenosine is associated with alpha-1 antitrypsin (A1AT) deficiency.

18. The oligonucleotide of any one of the above embodiments, wherein the target adenosine is in the human SERPINA1 gene.

19. The oligonucleotide according to any one of the above embodiments, wherein the target adenosine is the 1024 G>A(E342K) mutation in the human SERPINA1 gene.

20. An oligonucleotide according to any one of the preceding embodiments, capable of forming a double-stranded complex with a target nucleic acid.

21. The oligonucleotide of any one of embodiments 6-20, wherein the target nucleic acid or portion thereof is or comprises RNA.

22. The oligonucleotide of any one of embodiments 6-21, wherein the target adenosine is of RNA.

23. The oligonucleotide according to any one of embodiments 6-22, wherein the target adenosine is modified and the modification comprises or is deamination of the target adenosine.

24. The oligonucleotide according to any one of embodiments 6 to 23, wherein the target adenosine is modified and the modification is or comprises conversion of the target adenosine to inosine.

25. The oligonucleotide of any one of embodiments 6-24, wherein the modification is catalyzed by an ADAR protein.

26. The oligonucleotide of any one of embodiments 6-25, wherein the system is an in vitro or ex vivo system comprising an ADAR protein.

27. The oligonucleotide of any one of embodiments 6-25, wherein the system is or comprises a cell comprising or expressing an ADAR protein.

28. The oligonucleotide of any one of embodiments 6-25, wherein the system is a subject comprising a cell comprising or expressing an ADAR protein.

29. The oligonucleotide of any one of embodiments 25-28, wherein the ADAR protein is ADAR1.

30. The oligonucleotide of any one of embodiments 25-28, wherein the ADAR protein is ADAR2.

31. The method of any one of the above embodiments, about 10-200 (e.g., about 10-20, 10-30, 10-40, 10-50, 10-60, 10-70, 10- 80, 10~90, 10~100, 10~120, 10~150, 20~30, 20~40, 20~50, 20~60, 20~70, 20~80, 20~90, 20~100, 20~120, 20~150, 20~200, 25~30, 25~40, 25~50, 25~60, 25~70, 25~80, 25~90, 25~100, 25~120, 25~ 150, 25~200, 30~40, 30~50, 30~60, 30~70, 30~80, 30~90, 30~100, 30~120, 30~150, 30~200, 10, 20, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 45, 50, 60, etc.) nucleobases in length.

32. The oligonucleotide of any one of the preceding embodiments, having a length of about 26-35 nucleobases.

33. The oligonucleotide of any one of the preceding embodiments, having a length of about 26 nucleobases.

34. The oligonucleotide of any one of the preceding embodiments, having a length of about 27 nucleobases.

35. The oligonucleotide of any one of the preceding embodiments, having a length of about 28 nucleobases.

36. The oligonucleotide of any one of the preceding embodiments, having a length of about 29 nucleobases.

37. The oligonucleotide of any one of the preceding embodiments, having a length of about 30 nucleobases.

38. The oligonucleotide of any one of the preceding embodiments, having a length of about 31 nucleobases.

39. The oligonucleotide of any one of the preceding embodiments, having a length of about 32 nucleobases.

40. The oligonucleotide of any one of the preceding embodiments, having a length of about 33 nucleobases.

41. The oligonucleotide of any one of the preceding embodiments, having a length of about 34 nucleobases.

42. The oligonucleotide of any one of the preceding embodiments, having a length of about 35 nucleobases.

43. In any one of the above embodiments, the base sequence of the oligonucleotide is 0 to 10 non-Watson-Crick base pairs (eg, 0 to 1, 0 to 2, 0 to 3, 0 to 4, 0~5, 0~6, 0~7, 0~8, 0~9, 0~10, 1~2, 1~3, 1~4, 1~5, 1~6, 1~7, 1~ 8, 1~9, 1~10, 2~3, 2~4, 2~5, 2~6, 2~7, 2~8, 2~9, 2~10, 3~4, 3~5, 3-6, 3-7, 3-8, 3-9, 3-10, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 targets with discrepancies An oligonucleotide complementary to the nucleotide sequence of a target nucleic acid portion containing adenosine.

44. The oligonucleotide of embodiment 43, wherein the one or more mismatches are independently wobble base pairs.

45. The method of embodiment 43 or 44, wherein the complementarity is between about 50% and 100% (e.g., between about 50% and 80%, 50% and 85%, 50% and 90%, 50% and 95%, 60% ~80%, 60%~85%, 60%~90%, 60%~95%, 60%~100%, 65%~80%, 65%~85%, 65%~90%, 65%~95 %, 65%~100%, 70%~80%, 70%~85%, 70%~90%, 70%~95%, 70%~100%, 75%~80%, 75%~85%, 75%~90%, 75%~95%, 75%~100%, 80%~85%, 80%~90%, 80%~95%, 80%~100%, 85%~90%, 85% ~95%, 85%~100%, 90%~95%, 90%~100%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%, etc.), oligonucleotides.

46. The oligonucleotide of embodiment 43 or 44, wherein the complementarity is between about 90% and 100% or between about 95% and 100%.

47. The oligonucleotide of embodiment 43 or 44, wherein the complementarity is 100%.

48. The oligonucleotide of embodiment 43 or 44, wherein the complementarity is 100% except for the nucleoside opposite to the target nucleoside (eg, adenosine).

49. The oligonucleotide according to any one of the preceding embodiments, consisting of a first domain and a second domain.

50. The method of any one of the preceding embodiments, wherein the first domain is from about 2 to about 50 (e.g., about 5, 6, 7, 8, 9, or 10 to about 10, 11, 12, 13 , 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 40 or 50, or about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 40 or 50, etc.) nucleobases in length.

51. The oligonucleotide of any one of the preceding embodiments, wherein the first domain is about 10-25 nucleobases in length.

52. The oligonucleotide of any one of the preceding embodiments, wherein the first domain is about 15 nucleobases in length.

53. The method of any one of the preceding embodiments, wherein when the oligonucleotide is aligned with a target nucleic acid for complementarity, the first domain can be one or more (e.g., 1-10, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, etc.) mismatches.

54. The oligonucleotide of any one of the preceding embodiments, wherein the first domain comprises two or more mismatches when the oligonucleotide is aligned with a target nucleic acid for complementarity.

55. The oligonucleotide of any one of embodiments 1-50, wherein the first domain comprises no more than one mismatch when the oligonucleotide is aligned with a target nucleic acid for complementarity.

56. The oligonucleotide of any one of embodiments 1-50, wherein the first domain comprises no more than two mismatches when the oligonucleotide is aligned with a target nucleic acid for complementarity.

57. The method of any one of the preceding embodiments, wherein when the oligonucleotide is aligned with a target nucleic acid for complementarity, the first domain can be one or more (e.g., 1-10, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, etc.) bulges.

58. The oligonucleotide of embodiment 57, wherein each bulge independently comprises one or more base pairs that are not Watson-Crick or wobble pairs.

59. The method of any one of the preceding embodiments, wherein when the oligonucleotide is aligned with a target nucleic acid for complementarity, the first domain can be one or more (e.g., 1-10, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, etc.) wobble pairs.

60. The oligonucleotide of any one of the preceding embodiments, wherein the first domain comprises two or more wobble pairs when the oligonucleotide is aligned with a target nucleic acid for complementarity.

61. The oligonucleotide of any one of the preceding embodiments, wherein the first domain comprises no more than two pairs of wobbles when the oligonucleotide is aligned with a target nucleic acid for complementarity.

62. The oligonucleotide of any one of embodiments 1-50, wherein the first domain is completely complementary to the target nucleic acid.

63. The method of any one of the preceding embodiments, wherein the first domain has from about 1 to about 50 (e.g., about 5, 6, 7, 8, 9, or 10 to about 2'-F modifications). 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 40 or 50, or about 5, 6, 7, 8, 9 , 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 40 or 50, etc., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, etc.) sugars.

64. according to any one of the above embodiments, about 5%-100% (e.g., about 10%-100%, 20-100%, 30%-100%, 40%) of the sugars in the first domain ~100%, 50%~80%, 50%~85%, 50%~90%, 50%~95%, 60%~80%, 60%~85%, 60%~90%, 60%~95 %, 60%~100%, 65%~80%, 65%~85%, 65%~90%, 65%~95%, 65%~100%, 70%~80%, 70%~85%, 70% to 90%, 70% to 95%, 70% to 100%, 75% to 80%, 75% to 85%, 75% to 90%, 75% to 95%, 75% to 100%, 80% ~85%, 80%~90%, 80%~95%, 80%~100%, 85%~90%, 85%~95%, 85%~100%, 90%~95%, 90%~100 %, 10%, 20%, 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%) independently of 2 An oligonucleotide comprising a '-F modification.

65. The method of any one of the above embodiments, wherein about 50% to 100% (e.g., about 50% to 80%, 50% to 85%, 50% to 90%, 50% to about 50%) of the sugars in the first domain. %~95%, 60%~80%, 60%~85%, 60%~90%, 60%~95%, 60%~100%, 65%~80%, 65%~85%, 65%~ 90%, 65% to 95%, 65% to 100%, 70% to 80%, 70% to 85%, 70% to 90%, 70% to 95%, 70% to 100%, 75% to 80% , 75%~85%, 75%~90%, 75%~95%, 75%~100%, 80%~85%, 80%~90%, 80%~95%, 80%~100%, 85 %~90%, 85%~95%, 85%~100%, 90%~95%, 90%~100%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%, etc.) independently contain a 2'-F modification.

66. The method of any one of the preceding embodiments, wherein about 30% to 70% (e.g., about 30% to 60%, 30% to 50%, or about 30%, 40%) of the sugars in the first domain , 50%, 60% or 70%) independently comprises a 2'-F modification.

67. The method of any one of the preceding embodiments, wherein at most about 1% to 95% (e.g., at most about 1%, 5%, 10%, 15%, 20%, 25%) of the sugars in the first domain. , 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, etc.) Including, oligonucleotides.

68. The method of any one of the preceding embodiments, wherein about 30% to 70% (e.g., about 30% to 60%, 30% to 50%, or about 30%, 40%) of the sugars in the first domain , 50%, 60% or 70%) comprises 2'-OMe.

69. The oligonucleotide of any one of the preceding embodiments, wherein up to about 50% of the sugars in the first domain comprise 2'-OMe.

70. Any one of the preceding embodiments, wherein from up to about 1% to about 95% (e.g., up to about 1%, 5%, 10%, 15%, 20%, 25%) of the sugars in the first domain. , 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, etc.) is 2'-OR ( R is an optionally substituted C _1-6 aliphatic.

71. The method of any one of the preceding embodiments, wherein about 30% to 70% (e.g., about 30% to 60%, 30% to 50%, or about 30%, 40%) of the sugars in the first domain , 50%, 60% or 70%) comprises a 2'-OR, wherein R is an optionally substituted C _1-6 aliphatic.

72. The oligonucleotide of any one of the preceding embodiments, wherein up to about 50% of the sugars in the first domain comprise a 2'-OR, wherein R is an optionally substituted C _1-6 aliphatic.

73. Any one of the preceding embodiments, wherein from up to about 1% to about 95% (e.g., up to about 1%, 5%, 10%, 15%, 20%, 25%) of the sugars in the first domain. , 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, etc.) Including, oligonucleotides.

74. The method of any one of the preceding embodiments, wherein about 30% to 70% (e.g., about 30% to 60%, 30% to 50%, or about 30%, 40%) of the sugars in the first domain , 50%, 60% or 70%) comprises a 2'-OR (R is not -H).

75. The oligonucleotide of any one of the preceding embodiments, wherein up to about 50% of the sugars in the first domain comprise a 2'-OR.

76. The method of any one of _the preceding embodiments, wherein the first domain comprises one or more (eg, about 1-20; 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, etc.) nucleotide.

77. The method of any one of the preceding embodiments, wherein the first domain comprises one or more (e.g., about 1-20, 1, 2, 3, 4, 5, 6, 7 , 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, etc.) modified sugars.

78. The method of any one of the preceding embodiments, wherein the first domain comprises one or more (e.g., about 1-20, 1, 2, 3, 4, 5, 6, 7 , 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, etc.) modified sugars.

79. The method of any one of the preceding embodiments, wherein the first about 1-5 sugars from the 5'-end of the first domain, for example 1, 2, 3, 4, or 5 sugars, are independently 2'- an OR modified sugar, wherein R is independently an optionally substituted C _1-6 aliphatic.

80. The method of any one of the preceding embodiments, wherein the first about 1-5 sugars from the 5'-end of the first domain, for example 1, 2, 3, 4, or 5 sugars, are independently 2'- MOE modified sugars, oligonucleotides.

81. The method of any one of the preceding embodiments, wherein the first domain comprises one or more (eg, 2′-N(R) ₂ variants comprising a 2′-N(R) 2 variant, each R being an optionally substituted C _1-6 aliphatic). , about 1 to 20, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, etc.) An oligonucleotide containing a sugar.

82. The method of _any one of the preceding embodiments, wherein the first domain comprises one or more (eg, about 1-20, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, etc.) modified sugars.

83. The method of any one of the preceding embodiments, wherein the first domain is one or more (e.g., about 1-20, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, etc.) LNA sugars.

84. The method of any one of the preceding embodiments, wherein the first domain is one or more (e.g., about 1-20, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, etc.) of acyclic sugars (eg, UNA sugars).

85. The method of any one of the preceding embodiments, wherein the first domain comprises one or more (e.g., about 1-20, 1, 2, 3, 4, 5, 6, 7 , 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, etc.) modified sugars.

86. The method of any one of the preceding embodiments, wherein the first domain comprises one or more (e.g., about 1-20, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, etc.) sugars.

87. The method of any one of the above embodiments, wherein the first domain comprises one or more (e.g., about 1-20, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, etc.) sugars.

88. The oligonucleotide according to any one of embodiments 1 to 75, wherein the sugar of the first domain does not comprise a 2'-OR.

89. The oligonucleotide according to any one of embodiments 1-75, wherein the sugar of the first domain does not comprise 2'-OMe.

90. The oligonucleotide of any one of embodiments 1-75, wherein the sugar of the first domain does not comprise a 2'-OR, where R is an optionally substituted C _1-6 aliphatic.

91. The oligonucleotide of any one of embodiments 1-75, wherein each sugar in the first domain comprises a 2'-F.

92. The method of any one of the preceding embodiments, wherein the first domain is from about 1 to about 50 (e.g., about 5, 6, 7, 8, 9, or 10 to about 10, 11, 12, 13 , 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 40 or 50, or about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 40 or 50 etc, about 1, 2, 3, 4, 5, 6, 7, 8 , 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, etc.) modified internucleotide linkages.

93. The method of any one of the preceding embodiments, wherein about 5% to 100% (e.g., about 10% to 100%, 20 to 100%, 30% to 100%) of the internucleotide linkages in the first domain , 40%~100%, 50%~80%, 50%~85%, 50%~90%, 50%~95%, 60%~80%, 60%~85%, 60%~90%, 60 %~95%, 60%~100%, 65%~80%, 65%~85%, 65%~90%, 65%~95%, 65%~100%, 70%~80%, 70%~ 85%, 70% to 90%, 70% to 95%, 70% to 100%, 75% to 80%, 75% to 85%, 75% to 90%, 75% to 95%, 75% to 100% , 80%~85%, 80%~90%, 80%~95%, 80%~100%, 85%~90%, 85%~95%, 85%~100%, 90%~95%, 90 %~100%, 10%, 20%, 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%, etc.) Oligonucleotides, linkages between modified nucleotides.

94. The method of any one of the preceding embodiments, wherein about 50% to 100% (e.g., about 50% to 80%, 50% to 85%, 50% to 90%) of the internucleotide linkages in the first domain %, 50%~95%, 60%~80%, 60%~85%, 60%~90%, 60%~95%, 60%~100%, 65%~80%, 65%~85%, 65%~90%, 65%~95%, 65%~100%, 70%~80%, 70%~85%, 70%~90%, 70%~95%, 70%~100%, 75% ~80%, 75%~85%, 75%~90%, 75%~95%, 75%~100%, 80%~85%, 80%~90%, 80%~95%, 80%~100 %, 85%~90%, 85%~95%, 85%~100%, 90%~95%, 90%~100%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%, etc.) are linkages between modified nucleotides.

95. The oligonucleotide of any one of the preceding embodiments, wherein each modified internucleotidic linkage is independently a chiral internucleotidic linkage.

96. The oligonucleotide of any one of the preceding embodiments, wherein each modified internucleotide linkage is independently a phosphorothioate internucleotide linkage or a non-negatively charged internucleotide linkage.

97. The oligonucleotide of any one of the preceding embodiments, wherein each modified internucleotide linkage is independently a phosphorothioate internucleotide linkage or a neutral internucleotide linkage.

98. The oligonucleotide of any one of the preceding embodiments, wherein the first domain comprises one or more phosphorothioate internucleotidic linkages.

99. The oligonucleotide of any one of the preceding embodiments, wherein the first domain comprises 1, 2, 3, 4, or 5 internucleotide linkages that are not negatively charged.

100. The oligonucleotide of any one of the preceding embodiments, wherein the internucleoside linkage between the first nucleoside and the second nucleoside of the first domain is a non-negatively charged internucleoside linkage.

101. The oligonucleotide of any one of the above embodiments, wherein the internucleotide linkage between the last nucleoside and the penultimate nucleoside of the first domain is a non-negatively charged internucleotide linkage.

102. The method of any one of the preceding embodiments, wherein at least about 1-50 (e.g., about 5, 6, 7, 8, 9, or 10 to about 10, 11, 12 , 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 40 or 50, or about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 40 or 50 etc, about 1, 2, 3, 4, 5, 6, 7 , 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, etc.) chiral internucleotide linkages are chirally controlled.

103. The method of any one of the preceding embodiments, wherein about 5%-100% (e.g., about 10%-100%, 20-100%, 30%-100%) of the chiral internucleotide linkages in the first domain %, 40%~100%, 50%~80%, 50%~85%, 50%~90%, 50%~95%, 60%~80%, 60%~85%, 60%~90%, 60%~95%, 60%~100%, 65%~80%, 65%~85%, 65%~90%, 65%~95%, 65%~100%, 70%~80%, 70% ~85%, 70%~90%, 70%~95%, 70%~100%, 75%~80%, 75%~85%, 75%~90%, 75%~95%, 75%~100 %, 80%~85%, 80%~90%, 80%~95%, 80%~100%, 85%~90%, 85%~95%, 85%~100%, 90%~95%, 90% to 100%, 10%, 20%, 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%, etc.) is a chirally controlled oligonucleotide.

104. The oligonucleotide of any one of the preceding embodiments, wherein the internucleoside linkage between the first nucleoside and the second nucleoside of the first domain is chirally controlled.

105. The oligonucleotide of any one of the above embodiments, wherein the internucleotide linkage between the last nucleoside and the penultimate nucleoside of the first domain is chirally controlled.

106. The oligonucleotide of any one of the preceding embodiments, wherein each chiral internucleotidic linkage is independently a chiral control internucleotidic linkage.

107. The method of any one of the preceding embodiments, wherein at least about 1-50 (e.g., about 5, 6, 7, 8, 9, or 10 to about 10, 11, 12 , 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 40 or 50, or about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 40 or 50 etc, about 1, 2, 3, 4, 5, 6, 7 , 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, etc.) chiral internucleotide linkages are Sp .

108. The method of any one of the preceding embodiments, wherein at least 5%-100% (e.g., about 10%-100%, 20-100%, 30%-100%) of the chiral internucleotide linkages in the first domain %, 40%~100%, 50%~80%, 50%~85%, 50%~90%, 50%~95%, 60%~80%, 60%~85%, 60%~90%, 60%~95%, 60%~100%, 65%~80%, 65%~85%, 65%~90%, 65%~95%, 65%~100%, 70%~80%, 70% ~85%, 70%~90%, 70%~95%, 70%~100%, 75%~80%, 75%~85%, 75%~90%, 75%~95%, 75%~100 %, 80%~85%, 80%~90%, 80%~95%, 80%~100%, 85%~90%, 85%~95%, 85%~100%, 90%~95%, 90% to 100%, 10%, 20%, 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%, etc.) is S p, an oligonucleotide.

109. The oligonucleotide according to any one of the preceding embodiments, wherein each chiral internucleotide linkage in the first domain is S p.

110. The oligonucleotide according to any one of embodiments 1 to 108, wherein the internucleotide linkage between the first nucleoside and the second nucleoside of the first domain is R p.

111. The oligonucleotide according to any one of embodiments 1 to 108 and 110, wherein the internucleotide linkage between the last nucleoside and the penultimate nucleoside of the first domain is R p.

112. The oligonucleotide of any one of the preceding embodiments, wherein each internucleotide linkage in the first domain is independently a modified internucleotide linkage.

113. The oligonucleotide according to any one of embodiments 1 to 111, wherein the first domain comprises one or more native phosphate linkages.

114. The oligonucleotide according to any one of the preceding embodiments, wherein the first domain enables, facilitates, or contributes to recruitment of the ADAR protein to the target nucleic acid.

115. The oligonucleotide of any one of the preceding embodiments, wherein the first domain enables, promotes, or contributes to interaction of the ADAR with the target nucleic acid.

116. The oligonucleotide of any one of the preceding embodiments, wherein the first domain contacts the RNA binding domain (RBD) of an ADAR.

117. The oligonucleotide of any one of the preceding embodiments, wherein the first domain is not substantially in contact with the second RBD domain of an ADAR.

118. The oligonucleotide of any one of the preceding embodiments, wherein the first domain is not substantially in contact with the catalytic domain having deaminase activity of the ADAR.

119. The method of any one of the preceding embodiments, wherein the second domain is from about 2 to about 50 (e.g., about 5, 6, 7, 8, 9, or 10 to about 10, 11, 12, 13 , 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 40 or 50, or about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 40 or 50, etc.) nucleobases in length.

120. The oligonucleotide of any one of the preceding embodiments, wherein the second domain is about 1-7 nucleobases in length.

121. The oligonucleotide of any one of the preceding embodiments, wherein the second domain is about 5-15 nucleobases in length.

122. The oligonucleotide of any one of the preceding embodiments, wherein the second domain has a length of about 10-25 nucleobases.

123. The oligonucleotide of any one of the preceding embodiments, wherein the second domain is about 15 nucleobases in length.

124. The method of any one of the preceding embodiments, wherein the second domain is one or more (eg, 1-10, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, etc.) mismatches.

125. The oligonucleotide of any one of the preceding embodiments, wherein the second domain comprises two or more mismatches when the oligonucleotide is aligned with a target nucleic acid for complementarity.

126. The oligonucleotide of any one of embodiments 1-119, wherein the second domain comprises no more than one mismatch when the oligonucleotide is aligned with a target nucleic acid for complementarity.

127. The oligonucleotide according to any one of embodiments 1 to 119, wherein the second domain comprises no more than two mismatches when the oligonucleotide is aligned with the target nucleic acid for complementarity.

128. The method according to any one of the preceding embodiments, wherein when the oligonucleotide is aligned with a target nucleic acid for complementarity, the second domain is one or more (e.g., 1-10, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, etc.) bulges.

129. The oligonucleotide of embodiment 128, wherein each bulge independently comprises one or more base pairs that are not Watson-Crick or wobble pairs.

130. The method according to any one of the preceding embodiments, wherein when the oligonucleotide is aligned with a target nucleic acid for complementarity, the second domain is one or more (e.g., 1-10, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, etc.) wobble pairs.

131. The oligonucleotide of any one of the preceding embodiments, wherein the second domain comprises two or more wobble pairs when the oligonucleotide is aligned with a target nucleic acid for complementarity.

132. The oligonucleotide of any one of the preceding embodiments, wherein the second domain comprises no more than two pairs of wobbles when the oligonucleotide is aligned with a target nucleic acid for complementarity.

133. The oligonucleotide according to any one of embodiments 1 to 119, wherein the second domain is completely complementary to the target nucleic acid.

134. The oligonucleotide of any one of the preceding embodiments, wherein the second domain comprises a nucleoside opposite to the target adenosine when the oligonucleotide is aligned with the target nucleic acid for complementarity.

135. The oligonucleotide according to embodiment 134, wherein the opposite nucleobase is an optionally substituted or protected U or an optionally substituted or protected tautomer of U.

136. The oligonucleotide of embodiment 134, wherein the opposite nucleobases are U.

137. The oligonucleotide of embodiment 134, wherein the opposite nucleobase is an optionally substituted or protected C, or an optionally substituted or protected tautomer of C.

138. The oligonucleotide of embodiment 134, wherein the opposite nucleobases are C.

139. The oligonucleotide according to embodiment 134, wherein the opposite nucleobase is an optionally substituted or protected A or an optionally substituted or protected tautomer of A.

140. The oligonucleotide of embodiment 134, wherein the opposite nucleobases are A.

141. The oligonucleotide according to embodiment 134, wherein the opposite nucleobase is an optionally substituted or protected nucleobase of pseudoisocytosine or an optionally substituted or protected tautomer of a nucleobase of pseudoisocytosine.

142. The oligonucleotide of embodiment 134, wherein the opposite nucleobase is a nucleobase of pseudoisocytosine.

143. The oligonucleotide according to any one of the preceding embodiments, wherein the oligonucleotide comprises nucleobase BA, wherein BA is or comprises ring BA or a tautomer thereof, wherein ring BA comprises 0 to 10 heterocyclic groups. An oligonucleotide that is an optionally substituted 5-20 membered monocyclic, bicyclic or polycyclic ring having atoms.

144. An oligonucleotide comprising the nucleobase BA, wherein BA is or comprises ring BA or a tautomer thereof, wherein ring BA is an optionally substituted 5-20 membered monocyclic ring having 0-10 heteroatoms , an oligonucleotide that is bicyclic or polycyclic.

145. The method according to embodiment 134, wherein the nucleobase is BA, BA is or comprises ring BA or a tautomer thereof, and ring BA has 0 to 10 heteroatoms, optionally substituted 5-20 Oligonucleotides that are monocyclic, bicyclic or polycyclic.

146. The oligonucleotide according to any one of embodiments 143 to 145, wherein BA has weaker hydrogen bonds with adenine, the target of adenosine, than U.

147. The oligonucleotide according to any one of embodiments 143 to 146, wherein BA forms fewer hydrogen bonds with the target adenine of adenosine than U.

148. The oligonucleotide according to any one of embodiments 143 to 147, wherein BA forms one or more hydrogen bonds with one or more amino acid residues of ADAR and the residues form one or more hydrogen bonds with U opposite to the target adenosine.

149. The oligonucleotide according to any one of embodiments 143 to 148, wherein BA forms one or more hydrogen bonds with each amino acid residue of ADAR that forms one or more hydrogen bonds with U opposite to the target adenosine.

150. The method according to any one of embodiments 143 to 149, wherein ring BA is X ² X ³ Including, oligonucleotide.

151. The method according to any one of embodiments 143 to 149, wherein ring BA is X ² X ³ X ⁴ Including, oligonucleotide.

152. The method of any one of embodiments 143 to 149, wherein ring BA is -X ¹ ( ) X ² X ³ Including, oligonucleotide.

153. The method of any one of embodiments 143 to 149, wherein ring BA is -X ¹ ( ) X ² X ³ X ⁴ Including, oligonucleotide.

154. The oligonucleotide according to any one of embodiments 143 to 153, wherein ring BA has the structure of formula BA-I.

155. The oligonucleotide according to any one of embodiments 143 to 153, wherein ring BA has the structure of Formula BA-I-a.

156. The oligonucleotide according to any one of embodiments 143 to 153, wherein ring BA has the structure of Formula BA-I-b.

157. The oligonucleotide according to any one of embodiments 143 to 153, wherein ring BA has the structure of formula BA-II.

158. The oligonucleotide according to any one of embodiments 143 to 153, wherein ring BA has the structure of formula BA-II-a.

159. The oligonucleotide according to any one of embodiments 143 to 153, wherein ring BA has the structure of formula BA-II-b.

160. The oligonucleotide according to any one of embodiments 143 to 153, wherein ring BA has the structure of formula BA-III.

161. The oligonucleotide according to any one of embodiments 143 to 153, wherein ring BA has the structure of Formula BA-III-a.

162. The oligonucleotide according to any one of embodiments 143 to 153, wherein ring BA has the structure of Formula BA-III-b.

163. according to any one of embodiments 143-162, X ¹ , X ² , X ³ , X ⁴ , X ⁵ , X ⁶ , X 1′ , X ^2′ , X ^{3 ′} , X ^4′ , X ^5' , X ^6' , and X ^7' are each -CH=, -C(OH)=, -C(-NH ₂ )=, -CH ₂ -, -C(=NH)-, or -NH- oligonucleotides, which are independently and optionally substituted, if any.

164. The oligonucleotide according to any one of embodiments 150 to 163, wherein X ¹ is -N(-)-.

165. The oligonucleotide according to any one of embodiments 150 to 163, wherein X ¹ is -C(-)=.

166. The oligonucleotide according to any one of embodiments 150 to 165, wherein X ² is -C(O)-.

167. The oligonucleotide according to any one of embodiments 150 to 166, wherein X ³ is -NR'-.

168. The oligonucleotide according to any one of embodiments 150 to 167, wherein X ³ is optionally substituted -NH-.

169. The oligonucleotide according to any one of embodiments 150 to 167, wherein X ³ is -NH-.

170. The method of any one of embodiments 150-169, wherein X ⁴ is -C(R ^B4 )=, -C(-N(R ^B4 ) ₂ )=, -C(R ^B4 ) ₂ -, or - C(=NR ^B4 )-in, oligonucleotide.

171. The oligonucleotide according to any one of embodiments 150-169, wherein X ⁴ is -C(R ^B4 )=.

172. The oligonucleotide according to any one of embodiments 150 to 169, wherein X ⁴ is optionally substituted -CH=.

173. The oligonucleotide according to any one of embodiments 150 to 169, wherein X ⁴ is -CH=.

174. The oligonucleotide according to any one of embodiments 150-169, wherein X ⁴ is -C(-N(R ^B4 ) ₂ )=.

175. The oligonucleotide according to any one of embodiments 150 to 169, wherein X ⁴ is optionally substituted -C(-NH ₂ )=.

176. The oligonucleotide according to any one of embodiments 150-169, wherein X ⁴ is -C(-NH ₂ )=.

177. The oligonucleotide according to any one of embodiments 150 to 169, wherein X ⁴ is -C(-N=CHNR ₂ )=.

178. The oligonucleotide according to any one of embodiments 150-169, wherein X ⁴ is -C(-N=CHN(CH ₃ ) ₂ )=.

179. The oligonucleotide according to any one of embodiments 150 to 169, wherein X ⁴ is -C(-NHR')=.

180. The oligonucleotide according to any one of embodiments 150 to 169, wherein X ⁴ is -C(R ^B4 ) ₂ -.

181. The oligonucleotide according to any one of embodiments 150 to 169, wherein X ⁴ is optionally substituted —CH ₂ —.

182. The oligonucleotide according to any one of embodiments 150 to 169, wherein X ⁴ is -CH ₂ -.

183. The oligonucleotide according to any one of embodiments 150 to 169, wherein X ⁴ is optionally substituted -C(=NH)-.

184. The oligonucleotide according to any one of embodiments 150 to 169, wherein X ⁴ is -C(=NR ^B4 )-.

185. according to any one of embodiments 150 to 169, wherein X ⁴ is -C(O)= and the oxygen atom has a weaker hydrogen bond acceptor than the corresponding -C(O)- in U; nucleotide.

186. The oligonucleotide according to any one of embodiments 150 to 169, wherein X ⁴ is -C(O)= and the oxygen atom forms an intramolecular hydrogen bond.

187. The oligonucleotide according to any one of embodiments 150 to 169, wherein X ⁴ is -C(O)= and the oxygen atom forms a hydrogen bond with a hydrogen in the same nucleobase.

188. The oligonucleotide according to any one of embodiments 157 to 187, wherein X ⁵ is -C(R ^B5 ) ₂ -.

189. The oligonucleotide according to any one of embodiments 157 to 187, wherein X ⁵ is optionally substituted —CH ₂ —.

190. The oligonucleotide according to any one of embodiments 157-187, wherein X ⁵ is -CH ₂ -.

191. The oligonucleotide according to any one of embodiments 157-187, wherein X ⁵ is -C(R ^B5 )=.

192. The oligonucleotide according to any one of embodiments 157-187, wherein X ⁵ is optionally substituted -C(-NO ₂ )=.

193. The oligonucleotide according to any one of embodiments 157 to 187, wherein X ⁵ is optionally substituted -CH=.

194. The oligonucleotide according to any one of embodiments 157 to 187, wherein X ⁵ is -CH=.

195. The method of any one of embodiments 157 to 187, wherein X ⁵ is -C(-L ^B5 -R ^B51 )=, and R ^B51 is -R', -N(R') ₂ , -OR', or -SR', an oligonucleotide.

196. The method of any one of embodiments 157-187, wherein X ⁵ is -C(-L ^B5 -R ^B51 )= and R ^B51 is -N(R') ₂ , -OR', or -SR' Phosphorus, oligonucleotide.

197. The oligonucleotide according to any one of embodiments 157-187, wherein X ⁵ is C(-L ^B5 -R ^B51 )= and R ^B51 is -NHR'.

198. The oligonucleotide according to any one of embodiments 195 to 197, wherein L ^B5 is or comprises -C(O).

199. The oligonucleotide according to any one of embodiments 157 to 187, wherein X ⁵ is -N=.

200. The oligonucleotide according to embodiment 197 or 198, wherein X ⁴ is -C(O)= and the oxygen atom forms a hydrogen bond with a hydrogen of -NHR', -OH or -SH in R ^B51 .

201. The oligonucleotide according to any one of embodiments 143 to 153, wherein ring BA has the structure of Formula BA-IV.

202. The oligonucleotide according to any one of embodiments 143 to 153, wherein ring BA has the structure of Formula BA-IV-a.

203. The oligonucleotide according to any one of embodiments 143 to 153, wherein ring BA has the structure of Formula BA-IV-b.

204. The oligonucleotide according to any one of embodiments 143 to 153, wherein ring BA has the structure of Formula BA-V.

205. The oligonucleotide according to any one of embodiments 143 to 153, wherein ring BA has the structure of Formula BA-V-a.

206. The oligonucleotide according to any one of embodiments 143 to 153, wherein ring BA has the structure of formula BA-V-b.

207. The oligonucleotide according to any one of embodiments 143 to 153, wherein ring BA has the structure of formula BA-VI.

208. The method of any one of embodiments 201-207, wherein X ¹ , X ² , X ³ , X ⁴ , X ⁵ , X ⁶ , X 1′ , X ^2′ , X ^{3 ′} , X ^4′ , X ^5' , X ^6' , and X ^7' are each -CH=, -C(OH)=, -C(-NH ₂ )=, -CH ₂ -, -C(=NH)-, or -NH- oligonucleotides, which are independently and optionally substituted, if any.

209. The oligonucleotide according to any one of embodiments 201 to 208, wherein X ¹ is -N(-)-.

210. The oligonucleotide according to any one of embodiments 201 to 208, wherein X ¹ is -C(-)=.

211. The oligonucleotide of any one of embodiments 201-210, wherein X ² is optionally substituted -CH=.

212. The oligonucleotide according to any one of embodiments 201 to 210, wherein X ² is -CH=.

213. The oligonucleotide according to any one of embodiments 201 to 210, wherein X ² is -C(O)-.

214. The oligonucleotide according to any one of embodiments 201 to 213, wherein X ³ is -NR'-.

215. The oligonucleotide according to any one of embodiments 201 to 213, wherein X ³ is optionally substituted -NH-.

216. The oligonucleotide according to any one of embodiments 201 to 213, wherein X ³ is -NH-.

217. The oligonucleotide according to any one of embodiments 201 to 216, wherein ring BA ^A is 5 membered.

218. The oligonucleotide according to any one of embodiments 201 to 216, wherein ring BA ^A is 6 membered.

219. The oligonucleotide according to any one of embodiments 201 to 218, wherein ring BA ^A is an optionally substituted ring having 1-3 heteroatoms.

220. The oligonucleotide of embodiment 219, wherein the heteroatom is nitrogen.

221. The oligonucleotide of embodiment 219 or 220, wherein ring BA ^A contains two nitrogens.

222. The oligonucleotide of embodiment 219 or 220, wherein the heteroatom is oxygen.

223. The method of any one of embodiments 160-222, wherein X ⁶ is -C(R ^B6 )=, -C(OR ^B6 )=, -C(R ^B6 ) ₂ -, or -C(O)- Phosphorus, oligonucleotide.

224. The oligonucleotide of any one of embodiments 160-222, wherein X ⁶ is -C(R)=, -C(R) ₂ -, or -C(O)-.

225. The oligonucleotide according to any one of embodiments 160 to 222, wherein X ⁶ is optionally substituted -CH=.

226. The oligonucleotide according to any one of embodiments 160 to 222, wherein X ⁶ is -CH=.

227. The oligonucleotide according to any one of embodiments 160 to 222, wherein X ⁶ is optionally substituted —CH ₂ —.

228. The oligonucleotide according to any one of embodiments 160 to 222, wherein X ⁶ is -CH ₂ -.

229. The oligonucleotide according to any one of embodiments 160-222, wherein X ⁶ is -C(O)-.

230. The method according to any one of embodiments 134 to 149, wherein ring BA is X ^4' X ^5' Including, oligonucleotide.

231. The oligonucleotide according to any one of embodiments 143 to 149 or 230, wherein ring BA has the structure of formula BA-VI.

232. The oligonucleotide of embodiment 230, wherein X ^1' is -N(-)-.

233. The oligonucleotide of embodiment 230, wherein X ^1' is -C(-)=.

234. The oligonucleotide according to any one of embodiments 230 to 233, wherein X ^2' is -C(O)-.

235. The oligonucleotide according to any one of embodiments 230 to 233, wherein X ^2' is optionally substituted -CH=.

236. The oligonucleotide according to any one of embodiments 230 to 233, wherein X ^2' is -CH=.

237. The oligonucleotide according to any one of embodiments 230 to 233, wherein X ^2' is -C(-)=.

238. The oligonucleotide according to any one of embodiments 230 to 236, wherein X ^3' is -NR'-.

239. The oligonucleotide according to any one of embodiments 230 to 236, wherein X ^3' is optionally substituted -NH-.

240. The oligonucleotide according to any one of embodiments 230 to 236, wherein X ³ 'is -NH-.

241. The oligonucleotide according to any one of embodiments 230 to 236, wherein X ³ 'is -N=.

242. The oligonucleotide according to any one of embodiments 230 to 241, wherein X ^4' is -C(O)=.

243. The oligonucleotide according to any one of embodiments 230 to 241, wherein X ^4' is -C(OR ^B4' )=.

244. The oligonucleotide according to any one of embodiments 230 to 241, wherein X ^4' is -C(R ^B4' )=.

245. The oligonucleotide according to any one of embodiments 230 to 241, wherein X ^4' is optionally substituted -CH=.

246. The oligonucleotide according to any one of embodiments 230 to 241, wherein X ^4' is -CH=.

247. The oligonucleotide according to any one of embodiments 230 to 241, wherein X ^4' is -C(-N(R ^B4' ) ₂ )=.

248. The oligonucleotide according to any one of embodiments 230 to 241, wherein X ^4' is optionally substituted -C(-NH ₂ )=.

249. The oligonucleotide according to any one of embodiments 230 to 241, wherein X ^4' is -C(-NH ₂ )=.

250. The oligonucleotide according to any one of embodiments 230-241, wherein X ^4' is -C(-N=CHN(CH ₃ ) ₂ )=.

251. The oligonucleotide according to any one of embodiments 230 to 241, wherein X ^4' is -C(-NC(O)R')=.

252. The oligonucleotide according to any one of embodiments 230 to 251, wherein X ^5' is optionally substituted -NH-.

253. The oligonucleotide according to any one of embodiments 230 to 251, wherein X ⁵ 'is -NH-.

254. The oligonucleotide according to any one of embodiments 230 to 251, wherein X ⁵ 'is -N=.

255. The oligonucleotide according to any one of embodiments 230 to 251, wherein X ^5' is -C(R ^B5' )=.

256. The oligonucleotide according to any one of embodiments 230 to 251, wherein X ^5' is optionally substituted -CH=.

257. The oligonucleotide according to any one of embodiments 230 to 251, wherein X ^5' is -CH=.

258. The oligonucleotide according to any one of embodiments 230 to 257, wherein X ^6' is -C(R ^B6' )=.

259. The oligonucleotide according to any one of embodiments 230 to 257, wherein X ^6' is optionally substituted -CH=.

260. The oligonucleotide according to any one of embodiments 230 to 257, wherein X ^6' is -CH=.

261. The oligonucleotide according to any one of embodiments 230 to 257, wherein X ^6' is -C(O)=.

262. The oligonucleotide according to any one of embodiments 230 to 257, wherein X ^6' is -C(OR ^B6' )=.

263. The oligonucleotide according to any one of embodiments 230 to 257, wherein X ^6' is -C(OR')=.

264. The oligonucleotide according to any one of embodiments 230 to 263, wherein X ^7' is -C(R ^B7' )=.

265. The oligonucleotide according to any one of embodiments 230 to 263, wherein X ^7' is optionally substituted -CH=.

266. The oligonucleotide according to any one of embodiments 230 to 263, wherein X ^7' is -CH=.

267. The oligonucleotide according to any one of embodiments 230 to 263, wherein X ^7' is optionally substituted -NH-.

268. The oligonucleotide according to any one of embodiments 230 to 263, wherein X ^7' is -NH-.

269. The oligonucleotide according to any one of embodiments 230 to 263, wherein X ^7' is -N=.

270. The method of any one of embodiments 143 to 149, wherein ring BA is Phosphorus, oligonucleotide.

271. The method of any one of embodiments 143 to 149, wherein ring BA is Phosphorus, oligonucleotide.

272. The method of any one of embodiments 143 to 149, wherein ring BA is Phosphorus, oligonucleotide.

273. The method of any one of embodiments 143 to 149, wherein ring BA is and R' is -C(O)R.

274. The method of any one of embodiments 143 to 149, wherein ring BA is and R' is -C(O)Ph.

275. The method of any one of embodiments 143 to 149, wherein ring BA is Phosphorus, oligonucleotide.

276. The method of any one of embodiments 143 to 149, wherein ring BA is Phosphorus, oligonucleotide.

277. The method of any one of embodiments 143 to 149, wherein ring BA is Phosphorus, oligonucleotide.

278. The method of any one of embodiments 143 to 149, wherein ring BA is Phosphorus, oligonucleotide.

279. The method according to any one of embodiments 143 to 149, wherein ring BA is Phosphorus, oligonucleotide.

280. The method of any one of embodiments 143 to 149, wherein ring BA is Phosphorus, oligonucleotide.

281. The method of any one of embodiments 143 to 149, wherein ring BA is Phosphorus, oligonucleotide.

282. The method of any one of embodiments 143 to 149, wherein ring BA is Phosphorus, oligonucleotide.

283. The method of any one of embodiments 143 to 149, wherein ring BA is Phosphorus, oligonucleotide.

284. The method of any one of embodiments 143 to 149, wherein ring BA is Phosphorus, oligonucleotide.

285. The method of any one of embodiments 143 to 149, wherein ring BA is Phosphorus, oligonucleotide.

286. The method of any one of embodiments 143 to 149, wherein ring BA is Phosphorus, oligonucleotide.

287. The method of any one of embodiments 143 to 149, wherein ring BA is Phosphorus, oligonucleotide.

288. The method of any one of embodiments 143 to 149, wherein ring BA is Phosphorus, oligonucleotide.

289. The method of any one of embodiments 143 to 149, wherein ring BA is Phosphorus, oligonucleotide.

290. The method of any one of embodiments 143 to 149, wherein ring BA is Phosphorus, oligonucleotide.

291. The method of any one of embodiments 143 to 149, wherein ring BA is Phosphorus, oligonucleotide.

292. The method of any one of embodiments 143 to 149, wherein ring BA is Phosphorus, oligonucleotide.

293. The method of any one of embodiments 143 to 149, wherein ring BA is Phosphorus, oligonucleotide.

294. The oligonucleotide according to any one of embodiments 143 to 293, wherein the nucleobase is ring BA or a tautomer thereof.

295. The oligonucleotide according to any one of embodiments 143 to 293, wherein the nucleobase is a substituted ring BA or a tautomer thereof.

296. The method of any one of embodiments 143 to 293, wherein the nucleobase is an optionally substituted ring BA or a tautomer thereof, and each ring -CH=, -CH ₂ -, and -NH- is optionally and independently substituted oligonucleotides.

297. The oligonucleotide according to any one of embodiments 143 to 293, wherein the nucleobase is an optionally substituted ring BA or a tautomer thereof, and each ring -CH= and -CH ₂ - is optionally and independently substituted .

298. The oligonucleotide according to any one of embodiments 143 to 293, wherein the nucleobase is an optionally substituted ring BA or a tautomer thereof, and each ring -CH= is optionally and independently substituted.

299. The method of any one of the preceding embodiments, wherein the second domain has about 1-50 (e.g., about 5, 6, 7, 8, 9, or 10 to about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 40 or 50, or about 5, 6, 7 , 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 40 or 50, etc., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, etc.) modified sugars.

300. The method of any one of the preceding embodiments, from about 5% to about 100% (e.g., about 10% to about 100%, about 20 to about 100%, about 30% to about 100%, about 40% of the sugars in the second domain). ~100%, 50%~80%, 50%~85%, 50%~90%, 50%~95%, 60%~80%, 60%~85%, 60%~90%, 60%~95 %, 60%~100%, 65%~80%, 65%~85%, 65%~90%, 65%~95%, 65%~100%, 70%~80%, 70%~85%, 70% to 90%, 70% to 95%, 70% to 100%, 75% to 80%, 75% to 85%, 75% to 90%, 75% to 95%, 75% to 100%, 80% ~85%, 80%~90%, 80%~95%, 80%~100%, 85%~90%, 85%~95%, 85%~100%, 90%~95%, 90%~100 %, 10%, 20%, 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%) independently of 2 An oligonucleotide, which is a modified sugar with a modification other than '-F.

301. The method of any one of the preceding embodiments, wherein about 50% to 100% (e.g., about 50% to 80%, 50% to 85%, 50% to 90%, 50% to about 50%) of the sugars in the second domain. %~95%, 60%~80%, 60%~85%, 60%~90%, 60%~95%, 60%~100%, 65%~80%, 65%~85%, 65%~ 90%, 65% to 95%, 65% to 100%, 70% to 80%, 70% to 85%, 70% to 90%, 70% to 95%, 70% to 100%, 75% to 80% , 75%~85%, 75%~90%, 75%~95%, 75%~100%, 80%~85%, 80%~90%, 80%~95%, 80%~100%, 85 %~90%, 85%~95%, 85%~100%, 90%~95%, 90%~100%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%, etc.) are independently modified sugars with modifications other than 2'-F.

302. The method according to any one of embodiments 139 to 301, wherein the modified sugars are independently bicyclic sugars (eg LNA sugars), acyclic sugars (eg UNA sugars), sugars with 2'-OR modifications, or 2 An oligonucleotide selected from sugars with the '-N(R) ₂ variant, wherein each R is independently optionally substituted C _1-6 aliphatic.

303. The method of any one of the preceding embodiments, wherein the second domain comprises one or more (e.g., about 1-20, 1, 2, 3, 4, 5, 6, 7 , 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, etc.) modified sugars.

304. The method of any one of the preceding embodiments, wherein _the second domain comprises one or more (eg, about 1-20, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, etc.) nucleotide.

305. The method of any one of the preceding embodiments, wherein the second domain comprises one or more (e.g., about 1-20, 1, 2, 3, 4, 5, 6, 7 , 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, etc.) modified sugars.

306. The method according to any one of the preceding embodiments, wherein the second domain comprises one or more (eg, 2′-N(R) ₂ variants comprising a 2′-N(R) 2 variant, each R being an optionally substituted C _1-6 aliphatic). , about 1 to 20, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, etc.) An oligonucleotide containing a sugar.

307. The method of any one of _the preceding embodiments, wherein the second domain comprises one or more (eg, about 1-20, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, etc.) modified sugars.

308. The method of any one of the preceding embodiments, wherein the second domain is one or more (eg, about 1-20, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, etc.) LNA sugars.

309. The method of any one of the preceding embodiments, wherein the second domain is one or more (eg, about 1-20, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, etc.) of acyclic sugars (eg, UNA sugars).

310. The method of any one of the preceding embodiments, wherein the second domain comprises one or more (e.g., about 1-20, 1, 2, 3, 4, 5, 6, 7 , 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, etc.) modified sugars.

311. The method of any one of the preceding embodiments, wherein the second domain comprises one or more (e.g., about 1-20, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, etc.) sugars.

312. The method of any one of the preceding embodiments, wherein the second domain comprises one or more (e.g., about 1-20, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, etc.) sugars.

313. The method of any one of the preceding embodiments, wherein the second domain is from about 1 to about 50 (e.g., about 5, 6, 7, 8, 9, or 10 to about 10, 11, 12, 13 , 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 40 or 50, or about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 40 or 50 etc, about 1, 2, 3, 4, 5, 6, 7, 8 , 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, etc.) modified internucleotide linkages.

314. The method of any one of the preceding embodiments, wherein about 5%-100% (e.g., about 10%-100%, 20-100%, 30%-100%) of the internucleotide linkages in the second domain , 40%~100%, 50%~80%, 50%~85%, 50%~90%, 50%~95%, 60%~80%, 60%~85%, 60%~90%, 60 %~95%, 60%~100%, 65%~80%, 65%~85%, 65%~90%, 65%~95%, 65%~100%, 70%~80%, 70%~ 85%, 70% to 90%, 70% to 95%, 70% to 100%, 75% to 80%, 75% to 85%, 75% to 90%, 75% to 95%, 75% to 100% , 80%~85%, 80%~90%, 80%~95%, 80%~100%, 85%~90%, 85%~95%, 85%~100%, 90%~95%, 90 %~100%, 10%, 20%, 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%, etc.) Oligonucleotides, linkages between modified nucleotides.

315. The method of any one of the preceding embodiments, wherein about 50% to 100% (e.g., about 50% to 80%, 50% to 85%, 50% to 90%) of the internucleotide linkages in the second domain %, 50%~95%, 60%~80%, 60%~85%, 60%~90%, 60%~95%, 60%~100%, 65%~80%, 65%~85%, 65%~90%, 65%~95%, 65%~100%, 70%~80%, 70%~85%, 70%~90%, 70%~95%, 70%~100%, 75% ~80%, 75%~85%, 75%~90%, 75%~95%, 75%~100%, 80%~85%, 80%~90%, 80%~95%, 80%~100 %, 85%~90%, 85%~95%, 85%~100%, 90%~95%, 90%~100%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%, etc.) are linkages between modified nucleotides.

316. The oligonucleotide of any one of the preceding embodiments, wherein each modified internucleotide linkage is independently a chiral internucleotide linkage.

317. The oligonucleotide of any one of the preceding embodiments, wherein each modified internucleotide linkage is independently a phosphorothioate internucleotide linkage or a non-negatively charged internucleotide linkage.

318. The oligonucleotide of any one of the preceding embodiments, wherein each modified internucleotide linkage is independently a phosphorothioate internucleotide linkage or a neutral internucleotide linkage.

319. The oligonucleotide of any one of the preceding embodiments, wherein the second domain comprises one or more phosphorothioate internucleotidic linkages.

320. The oligonucleotide of any one of the preceding embodiments, wherein the second domain comprises 1, 2, 3, 4, or 5 internucleotide linkages that are not negatively charged.

321. The oligonucleotide of any one of the preceding embodiments, wherein the internucleotide linkage between the last nucleoside and the penultimate nucleoside of the second domain is a non-negatively charged internucleotide linkage.

322. The oligonucleotide of any one of the preceding embodiments, wherein the internucleoside linkage between the first nucleoside and the second nucleoside of the second domain is a non-negatively charged internucleoside linkage.

323. The method of any one of the preceding embodiments, wherein at least about 1-50 (e.g., about 5, 6, 7, 8, 9, or 10 to about 10, 11, 12 in the second domain) , 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 40 or 50, or about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 40 or 50 etc, about 1, 2, 3, 4, 5, 6, 7 , 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, etc.) chiral internucleotide linkages are chirally controlled.

324. The method of any one of the preceding embodiments, wherein at least 5%-100% (e.g., about 10%-100%, 20-100%, 30%-100%) of the chiral internucleotide linkages in the second domain %, 40%~100%, 50%~80%, 50%~85%, 50%~90%, 50%~95%, 60%~80%, 60%~85%, 60%~90%, 60%~95%, 60%~100%, 65%~80%, 65%~85%, 65%~90%, 65%~95%, 65%~100%, 70%~80%, 70% ~85%, 70%~90%, 70%~95%, 70%~100%, 75%~80%, 75%~85%, 75%~90%, 75%~95%, 75%~100 %, 80%~85%, 80%~90%, 80%~95%, 80%~100%, 85%~90%, 85%~95%, 85%~100%, 90%~95%, 90% to 100%, 10%, 20%, 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%, etc.) is a chirally controlled oligonucleotide.

325. The oligonucleotide of any one of the above embodiments, wherein the internucleotide linkage between the last nucleoside and the penultimate nucleoside of the second domain is chirally controlled.

326. The oligonucleotide of any one of the preceding embodiments, wherein the internucleotide linkage between the first nucleoside and the second nucleoside of the second domain is chirally controlled.

327. The oligonucleotide of any one of the preceding embodiments, wherein each chiral internucleotidic linkage is independently a chiral control internucleotidic linkage.

328. The method of any one of the preceding embodiments, wherein at least about 1-50 (e.g., about 5, 6, 7, 8, 9, or 10 to about 10, 11, 12 , 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 40 or 50, or about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 40 or 50 etc, about 1, 2, 3, 4, 5, 6, 7 , 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, etc.) chiral internucleotide linkages are Sp .

329. The method of any one of the preceding embodiments, wherein at least 5%-100% (e.g., about 10%-100%, 20-100%, 30%-100%) of the chiral internucleotide linkages in the second domain %, 40%~100%, 50%~80%, 50%~85%, 50%~90%, 50%~95%, 60%~80%, 60%~85%, 60%~90%, 60%~95%, 60%~100%, 65%~80%, 65%~85%, 65%~90%, 65%~95%, 65%~100%, 70%~80%, 70% ~85%, 70%~90%, 70%~95%, 70%~100%, 75%~80%, 75%~85%, 75%~90%, 75%~95%, 75%~100 %, 80%~85%, 80%~90%, 80%~95%, 80%~100%, 85%~90%, 85%~95%, 85%~100%, 90%~95%, 90% to 100%, 10%, 20%, 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%, etc.) is Sp , or each chiral internucleotidic linkage in the second domain is Sp .

330. The oligonucleotide according to any one of the above embodiments, wherein the internucleotide linkage between the first nucleoside and the second nucleoside of the second domain is R p.

331. The oligonucleotide according to any one of the above embodiments, wherein the internucleotide linkage between the last nucleoside and the penultimate nucleoside of the second domain is R p.

332. The oligonucleotide of any one of the preceding embodiments, wherein each internucleotide linkage in the second domain is independently a modified internucleotide linkage.

333. The oligonucleotide of any one of embodiments 1-331, wherein the second domain comprises one or more native phosphate linkages.

334. The oligonucleotide of any one of the preceding embodiments, wherein the second domain enables, promotes, or contributes to recruitment of the ADAR protein to the target nucleic acid.

335. The oligonucleotide of any one of the preceding embodiments, wherein the second domain enables, promotes, or contributes to interaction of the ADAR with the target nucleic acid.

336. The oligonucleotide according to any one of the preceding embodiments, wherein the second domain is in contact with a domain having enzymatic activity.

337. The oligonucleotide according to any one of the above embodiments, wherein the second domain is in contact with a domain having deaminase activity of ADAR1.

338. The oligonucleotide according to any one of the preceding embodiments, wherein the second domain is in contact with a domain having deaminase activity of ADAR2.

339. The oligonucleotide of any one of the preceding embodiments, wherein the second domain comprises or consists of a first subdomain, a second subdomain, and a third subdomain in a 5' to 3' direction.

340. The oligonucleotide according to any one of the above embodiments, wherein the second domain consists of a first subdomain, a second subdomain, and a third subdomain in a 5' to 3' direction.

341. The method of any one of the preceding embodiments, wherein the first subdomain is from about 1 to about 50 (e.g., about 5, 6, 7, 8, 9, or 10 to about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 40 or 50, or about 5, 6, 7, 8, 9, 10, 11, 12 , 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 40 or 50, etc.) nucleobases in length.

342. The first subdomains of any one of the preceding embodiments can be about 10-20 (e.g., about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) nucleobases in length.

343. The method of any one of the preceding embodiments, wherein the first subdomain is one or more (e.g., 1-10, 1, 2, 3, 4, 5 , 6, 7, 8, 9, or 10, etc.) mismatches.

344. The oligonucleotide of any one of the preceding embodiments, wherein the first subdomain comprises two or more mismatches when the oligonucleotide is aligned with a target nucleic acid for complementarity.

345. The oligonucleotide of any one of embodiments 1-343, wherein the first subdomain comprises no more than one mismatch when the oligonucleotide is aligned with a target nucleic acid for complementarity.

346. The oligonucleotide of any one of embodiments 1-343, wherein the first subdomain comprises no more than two mismatches when the oligonucleotide is aligned with the target nucleic acid for complementarity.

347. The method of any one of the preceding embodiments, wherein the first subdomain is one or more (e.g., 1-10, 1, 2, 3, 4, 5 , 6, 7, 8, 9, or 10 bulges, etc.).

348. The oligonucleotide of embodiment 347, wherein each bulge independently comprises one or more base pairs that are not Watson-Crick or wobble pairs.

349. The method of any one of the preceding embodiments, wherein the first subdomain is one or more (e.g., 1-10, 1, 2, 3, 4, 5 , 6, 7, 8, 9, or 10, etc.) wobble pairs.

350. The oligonucleotide of any one of the preceding embodiments, wherein the first subdomain comprises two or more wobble pairs when the oligonucleotide is aligned with a target nucleic acid for complementarity.

351. The oligonucleotide of any one of the preceding embodiments, wherein the first subdomain comprises no more than two wobble pairs when the oligonucleotide is aligned with a target nucleic acid for complementarity.

352. The oligonucleotide according to any one of embodiments 1-341, wherein the first subdomain is completely complementary to the target nucleic acid.

353. The method of any one of the above embodiments, wherein the first subdomain has about 1-50 (e.g., about 5, 6, 7, 8, 9, or 10 to about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 40 or 50, or about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 40 or 50, etc., about 1, 2 , 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 modified sugars, etc.).

354. The method of any one of the preceding embodiments, wherein about 5%-100% (e.g., about 10%-100%, 20-100%, 30%-100%, 40%) of the sugars in the first subdomain %~100%, 50%~80%, 50%~85%, 50%~90%, 50%~95%, 60%~80%, 60%~85%, 60%~90%, 60%~ 95%, 60% to 100%, 65% to 80%, 65% to 85%, 65% to 90%, 65% to 95%, 65% to 100%, 70% to 80%, 70% to 85% , 70%~90%, 70%~95%, 70%~100%, 75%~80%, 75%~85%, 75%~90%, 75%~95%, 75%~100%, 80 %~85%, 80%~90%, 80%~95%, 80%~100%, 85%~90%, 85%~95%, 85%~100%, 90%~95%, 90%~ 100%, 10%, 20%, 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%) independently. Oligonucleotides, which are modified sugars with modifications other than 2'-F.

355. The method of any one of the preceding embodiments, wherein about 50% to 100% (e.g., about 50% to 80%, 50% to 85%, 50% to 90%, 50%~95%, 60%~80%, 60%~85%, 60%~90%, 60%~95%, 60%~100%, 65%~80%, 65%~85%, 65% ~90%, 65%~95%, 65%~100%, 70%~80%, 70%~85%, 70%~90%, 70%~95%, 70%~100%, 75%~80 %, 75%~85%, 75%~90%, 75%~95%, 75%~100%, 80%~85%, 80%~90%, 80%~95%, 80%~100%, 85% to 90%, 85% to 95%, 85% to 100%, 90% to 95%, 90% to 100%, 50%, 60%, 65%, 70%, 75%, 80%, 85% , 90%, 95%, or 100%, etc.) are independently modified sugars with modifications other than 2'-F.

356. The method according to any one of embodiments 353 to 355, wherein the first subdomain is a bicyclic sugar (eg LNA sugar), an acyclic sugar (eg UNA sugar), a sugar with a 2'-OR variant, or a 2 from about ₁ to about 50 ₍ e.g., about 5, 6, 7, 8, 9, or 10 to about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 40, or 50, or about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 40 or 50 etc., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, etc.) , oligonucleotides.

357. The method of any one of embodiments 353-355, wherein about 5% to 100% (e.g., about 10% to 100%, 20 to 100%, 30% to 100%) of the sugars in the first subdomain , 40%~100%, 50%~80%, 50%~85%, 50%~90%, 50%~95%, 60%~80%, 60%~85%, 60%~90%, 60 %~95%, 60%~100%, 65%~80%, 65%~85%, 65%~90%, 65%~95%, 65%~100%, 70%~80%, 70%~ 85%, 70% to 90%, 70% to 95%, 70% to 100%, 75% to 80%, 75% to 85%, 75% to 90%, 75% to 95%, 75% to 100% , 80%~85%, 80%~90%, 80%~95%, 80%~100%, 85%~90%, 85%~95%, 85%~100%, 90%~95%, 90 %~100%, 10%, 20%, 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%, etc.) independently a bicyclic sugar (e.g. LNA sugar), an acyclic sugar (e.g. UNA sugar), a sugar with a 2'-OR strain, or a sugar with a 2'-N(R) ₂ strain (each R is independently optionally substituted C _1-6 aliphatic).

358. The method of any one of embodiments 353-355, wherein about 50% to 100% (e.g., about 50% to 80%, 50% to 85%, 50% to 90%) of the sugars in the first subdomain %, 50%~95%, 60%~80%, 60%~85%, 60%~90%, 60%~95%, 60%~100%, 65%~80%, 65%~85%, 65%~90%, 65%~95%, 65%~100%, 70%~80%, 70%~85%, 70%~90%, 70%~95%, 70%~100%, 75% ~80%, 75%~85%, 75%~90%, 75%~95%, 75%~100%, 80%~85%, 80%~90%, 80%~95%, 80%~100 %, 85%~90%, 85%~95%, 85%~100%, 90%~95%, 90%~100%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%, etc.) are independently bicyclic sugars (e.g., LNA sugars), acyclic sugars (e.g., UNA sugars), sugars with 2'-OR modifications, or 2'-N (R) is a modified sugar selected from sugars with ₂ modifications, each R independently being an optionally substituted C _1-6 aliphatic oligonucleotide.

359. The method of any one _of the preceding embodiments, wherein the first subdomain comprises one _or more (eg, For example, about 1 to 20, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, etc.) An oligonucleotide containing a modified sugar.

360. The method of any one of _the preceding embodiments, wherein the first subdomain contains one or more (eg, about 1-20, 1, 2, 3, 4, 5, 6 , 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, etc.) modified sugars.

361. The method of any one of the preceding embodiments, wherein the first subdomain is one or more (e.g., about 1-20, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, etc.) LNA sugars.

362. The method of any one of the preceding embodiments, wherein the first subdomain is one or more (e.g., about 1-20, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, etc.) of acyclic sugars (eg, UNA sugars).

363. The method of any one of the preceding embodiments, wherein the first subdomain comprises one or more (e.g., about 1-20, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, etc.) modified sugars.

364. The method of any one of the preceding embodiments, wherein the first subdomain contains one or more (e.g., about 1-20, 1, 2, 3, 4, 5, 6, 7 , 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, etc.) sugars.

365. The method of any one of the preceding embodiments, wherein the first subdomain comprises one or more (e.g., about 1-20, 1, 2, 3, 4, 5, 6 , 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, etc.) sugars.

366. The method of any one of the preceding embodiments, wherein the first subdomain comprises one or more (eg, about 1-20) comprising a 2'-OR variant (R is an optionally substituted C _1-6 aliphatic). , 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, etc.) oligonucleotide.

367. The method of any one of the preceding embodiments, wherein the first subdomain contains one or more (e.g., about 1-20, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, etc.) modified sugars.

368. The method of any one of embodiments 339 to 358, wherein each sugar in the first subdomain is independently a 2'-OR variant (R is an optionally substituted C _1-6 aliphatic) or 2'-OL An oligonucleotide comprising ^{a B} -4' modification.

369. is according to embodiment 368, wherein each sugar in the first subdomain is independently a 2'-OR variant (R is an optionally substituted C _1-6 aliphatic) or a 2'-OL ^B -4' variant (L ^B is an optionally substituted -CH ₂ -.

370. The oligonucleotide of embodiment 368, wherein each sugar in the first subdomain independently comprises 2'-OMe.

371. The oligonucleotide of any one of the preceding embodiments, wherein the first subdomain comprises a 5'-end portion having a length of about 3-8 nucleobases.

372. The oligonucleotide of embodiment 371, wherein the 5'-end has a length of about 3-6 nucleobases.

373. The oligonucleotide of embodiment 371 or 372, wherein the 5'-terminal portion comprises the 5'-terminal nucleobase of the first subdomain.

374. The oligonucleotide according to any one of embodiments 371 to 373, wherein at least one of the sugars at the 5'-terminus is independently a modified sugar.

375. The method of embodiment 374, wherein the modified sugars are independently bicyclic sugars (eg LNA sugars), acyclic sugars (eg UNA sugars), sugars with 2'-OR modifications, or 2'-N(R) ₂ An oligonucleotide selected from modified sugars, wherein each R is independently an optionally substituted C _1-6 aliphatic.

376. The oligonucleotide of embodiment 374, wherein at least one of the modified sugars independently comprises 2'-F or 2'-OR, wherein R is independently optionally substituted C _1-6 aliphatic.

377. The oligonucleotide of embodiment 374, wherein at least one of the modified sugars is independently 2'-F or 2'-OMe.

378. The oligonucleotide according to any one of embodiments 371 to 377, wherein the 5'-end comprises one or more mismatches.

379. The oligonucleotide according to any one of embodiments 371 to 378, wherein the 5'-end comprises one or more wobbles.

380. The method according to any one of embodiments 371 to 379, wherein the 5'-end is about 60-100% (e.g., 66%, 70%, 75%, 80%, 85%, 90%) relative to the target nucleic acid %, 95% or more) complementary, oligonucleotides.

381. The oligonucleotide of any one of the preceding embodiments, wherein the first subdomain comprises a 3'-end portion having a length of about 3-8 nucleobases.

382. The oligonucleotide of embodiment 381, wherein the 3'-terminus has a length of about 1-3 nucleobases.

383. The oligonucleotide of embodiment 381 or 382, wherein the 3'-terminal portion comprises the 3'-terminal nucleobase of the first subdomain.

384. The oligonucleotide according to any one of embodiments 381 to 383, wherein at least one of the sugars at the 3'-terminus is independently a modified sugar.

385. The method of embodiment 384, wherein the modified sugars are independently bicyclic sugars (eg LNA sugars), acyclic sugars (eg UNA sugars), sugars with 2'-OR modifications, or 2'-N(R) ₂ An oligonucleotide selected from modified sugars, wherein each R is independently an optionally substituted C _1-6 aliphatic.

386. The oligonucleotide of embodiment 384, wherein at least one of the modified sugars independently comprises 2'-F.

387. The oligonucleotide according to any one of embodiments 384 to 386, wherein the modified sugar does not comprise 2'-OMe.

388. The oligonucleotide according to any one of embodiments 381 to 387, wherein each sugar at the 3'-terminus independently comprises two 2'-H or 2'-F modifications.

389. The oligonucleotide according to any one of embodiments 371 to 377, wherein the 3'-end comprises one or more mismatches.

390. The oligonucleotide according to any one of embodiments 371 to 378, wherein the 3'-end comprises one or more wobbles.

391. The method of any one of embodiments 371 to 379, wherein the 3'-end is about 60-100% (e.g., 66%, 70%, 75%, 80%, 85%, 90%) relative to the target nucleic acid %, 95% or more) complementary, oligonucleotides.

392. The method of any one of the preceding embodiments, wherein the first subdomain is from about 1 to about 50 (e.g., about 5, 6, 7, 8, 9, or 10 to about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 40 or 50, or about 5, 6, 7, 8, 9, 10, 11, 12 , 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 40 or 50, etc., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, etc.) modified internucleotide linkages.

393. The method of any one of the preceding embodiments, wherein about 5%-100% (e.g., about 10%-100%, 20-100%, 30%-100%) of the internucleotide linkages in the first subdomain %, 40%~100%, 50%~80%, 50%~85%, 50%~90%, 50%~95%, 60%~80%, 60%~85%, 60%~90%, 60%~95%, 60%~100%, 65%~80%, 65%~85%, 65%~90%, 65%~95%, 65%~100%, 70%~80%, 70% ~85%, 70%~90%, 70%~95%, 70%~100%, 75%~80%, 75%~85%, 75%~90%, 75%~95%, 75%~100 %, 80%~85%, 80%~90%, 80%~95%, 80%~100%, 85%~90%, 85%~95%, 85%~100%, 90%~95%, 90% to 100%, 10%, 20%, 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%, etc.) is an oligonucleotide that is a linkage between modified nucleotides.

394. The method of any one of the preceding embodiments, wherein between about 50% and 100% (e.g., between about 50% and 80%, 50% and 85%, 50% and 90%, 50%~95%, 60%~80%, 60%~85%, 60%~90%, 60%~95%, 60%~100%, 65%~80%, 65%~85% , 65%~90%, 65%~95%, 65%~100%, 70%~80%, 70%~85%, 70%~90%, 70%~95%, 70%~100%, 75 %~80%, 75%~85%, 75%~90%, 75%~95%, 75%~100%, 80%~85%, 80%~90%, 80%~95%, 80%~ 100%, 85% to 90%, 85% to 95%, 85% to 100%, 90% to 95%, 90% to 100%, 50%, 60%, 65%, 70%, 75%, 80% , 85%, 90%, 95%, or 100%, etc.) are modified internucleotide linkages.

395. The oligonucleotide according to any one of the preceding embodiments, wherein each modified internucleotide linkage is independently a chiral internucleotide linkage.

396. The oligonucleotide of any one of the preceding embodiments, wherein the internucleotide linkage between the first nucleoside and the second nucleoside of the first subdomain is a non-negatively charged internucleotide linkage.

397. The oligonucleotide of any one of the preceding embodiments, wherein each modified internucleotide linkage is independently a phosphorothioate internucleotide linkage or a non-negatively charged internucleotide linkage.

398. The oligonucleotide of any one of the preceding embodiments, wherein each modified internucleotide linkage is independently a phosphorothioate internucleotide linkage or a neutral internucleotide linkage.

399. The method of any one of the preceding embodiments, wherein at least about 1-50 (e.g., about 5, 6, 7, 8, 9, or 10 to about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 40 or 50, or about 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 40 or 50, etc., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, etc.) chiral internucleotide linkages are chirally controlled.

400. The method of any one of the preceding embodiments, wherein at least 5%-100% (e.g., about 10%-100%, 20-100%, 30%- 100%, 40% to 100%, 50% to 80%, 50% to 85%, 50% to 90%, 50% to 95%, 60% to 80%, 60% to 85%, 60% to 90% , 60%~95%, 60%~100%, 65%~80%, 65%~85%, 65%~90%, 65%~95%, 65%~100%, 70%~80%, 70% %~85%, 70%~90%, 70%~95%, 70%~100%, 75%~80%, 75%~85%, 75%~90%, 75%~95%, 75%~ 100%, 80% to 85%, 80% to 90%, 80% to 95%, 80% to 100%, 85% to 90%, 85% to 95%, 85% to 100%, 90% to 95% , 90%~100%, 10%, 20%, 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%, etc. ) is a chirally controlled oligonucleotide.

401. The oligonucleotide of any one of the preceding embodiments, wherein the internucleotide linkage between the first nucleoside and the second nucleoside of the first subdomain is chirally controlled.

402. The oligonucleotide of any one of the preceding embodiments, wherein each chiral internucleotidic linkage is independently a chiral control internucleotidic linkage.

403. The method of any one of the preceding embodiments, wherein at least about 1-50 (e.g., about 5, 6, 7, 8, 9, or 10 to about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 40 or 50, or about 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 40 or 50, etc., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, etc.) chiral internucleotide linkages are Sp .

404. The method of any one of the preceding embodiments, wherein at least 5% to 100% (e.g., about 10% to 100%, 20 to 100%, 30% to 100%, 40% to 100%, 50% to 80%, 50% to 85%, 50% to 90%, 50% to 95%, 60% to 80%, 60% to 85%, 60% to 90% , 60%~95%, 60%~100%, 65%~80%, 65%~85%, 65%~90%, 65%~95%, 65%~100%, 70%~80%, 70% %~85%, 70%~90%, 70%~95%, 70%~100%, 75%~80%, 75%~85%, 75%~90%, 75%~95%, 75%~ 100%, 80% to 85%, 80% to 90%, 80% to 95%, 80% to 100%, 85% to 90%, 85% to 95%, 85% to 100%, 90% to 95% , 90%~100%, 10%, 20%, 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%, etc. ) is S p, an oligonucleotide.

405. The oligonucleotide according to any one of the preceding embodiments, wherein each chiral internucleotide linkage in the first subdomain is Sp .

406. The oligonucleotide according to any one of embodiments 1 to 405, wherein the internucleotide linkage between the first nucleoside and the second nucleoside of the first subdomain is R p.

407. The oligonucleotide of any one of the above embodiments, wherein each internucleotide linkage in the first subdomain is independently a modified internucleotide linkage.

408. The oligonucleotide according to any one of embodiments 1 to 406, wherein the first subdomain comprises one or more native phosphate linkages.

409. The oligonucleotide according to any one of the preceding embodiments, wherein the first subdomain enables, facilitates, or contributes to recruitment of the ADAR protein to the target nucleic acid.

410. The oligonucleotide of any one of the preceding embodiments, wherein the first subdomain enables, promotes, or contributes to interaction of the ADAR with the target nucleic acid.

411. The oligonucleotide according to any one of the preceding embodiments, wherein the first subdomain is in contact with a domain having enzymatic activity.

412. The oligonucleotide according to any one of the above embodiments, wherein the first subdomain is in contact with a domain having a deaminase activity of ADAR1.

413. The oligonucleotide according to any one of the above embodiments, wherein the first subdomain is in contact with a domain having a deaminase activity of ADAR2.

414. The second subdomain of any one of the preceding embodiments, from about 1 to about 10 (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 subdomains). ) oligonucleotides with a length of nucleobases.

415. The oligonucleotide of any one of the preceding embodiments, wherein the second subdomain is about 1-5 (e.g., about 1, 2, 3, 4, or 5) nucleobases in length. .

416. The oligonucleotide of any one of the preceding embodiments, wherein the second subdomain is about 1, 2, or 3 nucleobases in length.

417. The oligonucleotide of any one of the preceding embodiments, wherein the second subdomain is three nucleobases in length.

418. The oligonucleotide according to any one of the preceding embodiments, wherein the second subdomain comprises a nucleoside opposite to the target adenosine.

419. The oligonucleotide of any one of the preceding embodiments, wherein the second domain comprises no more than one nucleoside opposite the target adenosine.

420. The method of any one of the preceding embodiments, wherein the second subdomain is one or more (e.g., 1-10, 1, 2, 3, 4, 5 , 6, 7, 8, 9, or 10, etc.) mismatches.

421. The oligonucleotide of any one of the preceding embodiments, wherein the second subdomain comprises two or more mismatches when the oligonucleotide is aligned with a target nucleic acid for complementarity.

422. The oligonucleotide of any one of embodiments 1-420, wherein the second subdomain comprises no more than one mismatch when the oligonucleotide is aligned with a target nucleic acid for complementarity.

423. The oligonucleotide of any one of embodiments 1-420, wherein the second subdomain comprises no more than two mismatches when the oligonucleotide is aligned with the target nucleic acid for complementarity.

424. The method according to any one of the preceding embodiments, wherein the second subdomain is one or more (e.g., 1-10, 1, 2, 3, 4, 5 , 6, 7, 8, 9, or 10 bulges, etc.).

425. The oligonucleotide of embodiment 424, wherein each bulge independently comprises one or more base pairs that are not Watson-Crick or wobble pairs.

426. The method of any one of the preceding embodiments, wherein the second subdomain is one or more (e.g., 1-10, 1, 2, 3, 4, 5 , 6, 7, 8, 9, or 10, etc.) wobble pairs.

427. The oligonucleotide of any one of the preceding embodiments, wherein the second subdomain comprises two or more wobble pairs when the oligonucleotide is aligned with a target nucleic acid for complementarity.

428. The oligonucleotide of any one of the preceding embodiments, wherein the second subdomain comprises no more than two wobble pairs when the oligonucleotide is aligned with a target nucleic acid for complementarity.

429. The oligonucleotide according to any one of embodiments 1 to 419, wherein the second subdomain is completely complementary to the target nucleic acid.

430. The oligonucleotide of any one of the preceding embodiments, wherein the second subdomain comprises one or more sugars (eg, natural DNA sugars) comprising two 2'-Hs.

431. The oligonucleotide of any one of the preceding embodiments, wherein the second subdomain comprises one or more sugars (eg, native RNA sugars) comprising 2'-OH.

432. The number of second subdomains according to any one of the preceding embodiments, from about 1 to about 10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) An oligonucleotide comprising a modified sugar of

433. The method of embodiment 432, wherein each modified sugar is independently a bicyclic sugar (eg, LNA sugar), an acyclic sugar (eg, UNA sugar), a sugar with a 2'-OR variant, or a 2'-N (R ) sugars with ₂ modifications, wherein each R is independently an optionally substituted C _1-6 aliphatic oligonucleotide.

434. The oligonucleotide according to any one of the preceding embodiments, wherein the second subdomain does not contain a modified sugar comprising a 2'-OMe modification.

435. The oligonucleotide of any one of the preceding embodiments, wherein the second subdomain does not contain a modified sugar comprising a 2'-OR variant (R is an optionally substituted C _1-6 aliphatic).

436. The oligonucleotide of embodiment 432, wherein each 2'-modified sugar is a sugar comprising a 2'-F modification.

437. The oligonucleotide according to any one of embodiments 1 to 435, wherein the sugar of the opposite nucleoside is an acyclic sugar (eg, UNA sugar).

438. The oligonucleotide according to any one of embodiments 1 to 435, wherein the sugar of the opposite nucleoside comprises two 2'-Hs.

439. The oligonucleotide according to any one of embodiments 1 to 435, wherein the sugar of the opposite nucleoside comprises a 2'-OH.

440. The oligonucleotide according to any one of embodiments 1 to 435, wherein the sugar of the opposing nucleoside is a natural DNA sugar.

441. The oligonucleotide according to any one of embodiments 1 to 435, wherein the sugar of the opposite nucleoside is a modified sugar.

442. The oligonucleotide according to any one of embodiments 1 to 435, wherein the sugar of the opposite nucleoside comprises a 2'-F.

443. The sugar of the adjacent nucleoside in the 5' direction to the opposite nucleoside (5'-...N ₁ N where N ₀ is opposite to the target adenosine when aligned with the target) according to any one of the above embodiments. oligonucleotide, wherein the sugar of N ₁ at ₀ …3') contains two 2'-Hs.

444. The sugar of any one of the preceding nucleosides in the 5' direction to the opposite nucleoside (when aligned with the target, N ₀ is opposite to the target adenosine, 5'-...N ₁ N ₀ …3' to the N ₁ sugar) comprises a 2'-OH.

445. The method according to any one of the above embodiments, wherein the sugar of the adjacent nucleoside in the 5' direction to the opposite nucleoside (when aligned with the target, N ₀ is opposite to the target adenosine, 5'-...N ₁ N oligonucleotide, wherein the sugar of N ₁ at ₀ …3') is a natural DNA sugar.

446. The sugar of any one of the preceding nucleosides in the 5' direction to the opposite nucleoside (when aligned with the target, N ₀ is opposite to the target adenosine, 5'-...N ₁ N ₀ …3' to the sugar of N ₁ ) comprises a 2'-F.

447. The sugar of any one of the preceding nucleosides in the 3' direction to the opposite nucleoside (when aligned with the target, N ₀ is opposite to the target adenosine, 5'-...N ₀ N _-1 ... 3' to N _-1 sugars) contain two 2'-H, an oligonucleotide.

448. The sugar of the adjacent nucleoside in the 3' direction to the opposite nucleoside (5'-...N ₀ N, where N ₀ is opposite to the target adenosine when aligned with the target) according to any one of the above embodiments. _-1 ... 3' to N _-1 sugars) contain 2'-OH.

449. The sugar of the adjacent nucleoside in the 3' direction to the opposite nucleoside (5'-...N ₀ N where N ₀ is opposite to the target adenosine when aligned with the target) according to any one of the above embodiments. _-1 ... 3 'to N _-1 sugar) is a natural DNA sugar, an oligonucleotide.

450. The sugar of any one of the preceding nucleosides in the 3' direction to the opposite nucleoside (when aligned with the target, N ₀ is opposite to the target adenosine, 5'-...N ₀ N _-1 ... 3' to N _-1 sugar) comprises 2'-F, an oligonucleotide.

451. The method according to any one of embodiments 1 to 435, wherein each sugar of the opposite nucleoside, i.e., the sugar of the 5' adjacent nucleoside to the opposite nucleoside (when aligned with the target, N0 is the target adenosine of N 1 at 5′-…N ₁ N ₀ …3′ _, opposite to , and the sugar of the adjacent nucleoside 3′ to the opposite nucleoside (when aligned with the target, N ₀ is the target adenosine’s sugar). Opposite, 5'-...N ₀ N _-1 ... 3' to N _-1 sugars) are independently natural DNA sugars, oligonucleotides.

452. is according to any one of embodiments 1 to 435, wherein the sugar of the opposite nucleoside is a natural DNA sugar, and the sugar of the 5' adjacent nucleoside to the opposite nucleoside (N ₀ when aligned with the target is the sugar of the N 1 in 5′-…N ₁ N ₀ …3 _′ , opposite the target adenosine) is the 2′-F modified sugar, and the sugar of the adjacent nucleoside in the 3′ direction to the opposite nucleoside (the target 5′-… _{N 0 N −1} . . 3′ to N ₋₁ , where N 0 is opposite to the target adenosine when aligned with .

453. The oligonucleotide according to any one of the preceding embodiments, wherein the second subdomain comprises a 5'-terminus linked to the 5' side of the opposite nucleoside.

454. The oligonucleotide of embodiment 450, wherein the 5'-end comprises one or more mismatches or wobbles when aligned with the target nucleic acid for complementarity.

455. The oligonucleotide of embodiment 450 or 454, wherein the 5'-end has a length of 1, 2 or 3 nucleobases.

456. The oligonucleotide according to any one of embodiments 450 to 455, wherein the sugar at the 5'-terminus is selected from sugars having two 2'-H (eg, natural DNA sugars) and 2'-F modified sugars. nucleotide.

457. The oligonucleotide according to any one of the preceding embodiments, wherein the second subdomain comprises a 3'-terminus linked to the 3' side of the opposite nucleoside.

458. The oligonucleotide of embodiment 457, wherein the 3'-end comprises one or more mismatches or wobbles when aligned with the target nucleic acid for complementarity.

459. The oligonucleotide of embodiment 457, wherein the 3'-end when aligned with the target nucleic acid for complementarity comprises one or more mismatches and/or wobbles.

460. The oligonucleotide of embodiment 457, wherein the 3'-end comprises one or more wobbles when aligned with the target nucleic acid for complementarity.

461. The oligonucleotide of embodiment 457, wherein the 3'-terminus comprises I or a derivative thereof.

462. The oligonucleotide of embodiment 457, wherein the 3'-end comprises I and I-C wobbles when aligned with the target nucleic acid for complementarity.

463. The oligonucleotide according to any one of embodiments 457 to 462, wherein the 3'-end has a length of 1, 2 or 3 nucleobases.

464. The oligonucleotide according to any one of embodiments 457 to 463, wherein the sugar at the 3'-terminus is selected from sugars having two 2'-H (eg, natural DNA sugars) and 2'-F modified sugars. nucleotide.

465. The oligonucleotide according to any one of embodiments 457 to 463, wherein the sugar at the 3'-terminus is a sugar having two 2'-Hs (eg, a natural DNA sugar).

466. The method of any one of the preceding embodiments, wherein the second subdomain is about 1-10 (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, or 20, etc.) modified internucleotide linkages.

467. The method of any one of the preceding embodiments, wherein about 5%-100% (e.g., about 10%-100%, 20-100%, 30%-100%) of the internucleotide linkages in the second subdomain %, 40%~100%, 50%~80%, 50%~85%, 50%~90%, 50%~95%, 60%~80%, 60%~85%, 60%~90%, 60%~95%, 60%~100%, 65%~80%, 65%~85%, 65%~90%, 65%~95%, 65%~100%, 70%~80%, 70% ~85%, 70%~90%, 70%~95%, 70%~100%, 75%~80%, 75%~85%, 75%~90%, 75%~95%, 75%~100 %, 80%~85%, 80%~90%, 80%~95%, 80%~100%, 85%~90%, 85%~95%, 85%~100%, 90%~95%, 90% to 100%, 10%, 20%, 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%, etc.) is an oligonucleotide that is a linkage between modified nucleotides.

468. The method of any one of the preceding embodiments, wherein about 50%-100% (e.g., about 50%-80%, 50%-85%, 50%- 90%, 50%~95%, 60%~80%, 60%~85%, 60%~90%, 60%~95%, 60%~100%, 65%~80%, 65%~85% , 65%~90%, 65%~95%, 65%~100%, 70%~80%, 70%~85%, 70%~90%, 70%~95%, 70%~100%, 75 %~80%, 75%~85%, 75%~90%, 75%~95%, 75%~100%, 80%~85%, 80%~90%, 80%~95%, 80%~ 100%, 85% to 90%, 85% to 95%, 85% to 100%, 90% to 95%, 90% to 100%, 50%, 60%, 65%, 70%, 75%, 80% , 85%, 90%, 95%, or 100%, etc.) are modified internucleotide linkages.

469. The oligonucleotide of any one of the preceding embodiments, wherein each modified internucleotide linkage in the second subdomain is independently a chiral internucleotide linkage.

470. The oligonucleotide of any one of the preceding embodiments, wherein each modified internucleotide linkage in the second subdomain is independently a phosphorothioate internucleotide linkage or a non-negatively charged internucleotide linkage.

471. The oligonucleotide of any one of the preceding embodiments, wherein each modified internucleotide linkage in the second subdomain is independently a phosphorothioate internucleotide linkage or a neutral internucleotide linkage.

472. The method of any one of the preceding embodiments, wherein at least about 1-50 (e.g., about 5, 6, 7, 8, 9, or 10 to about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 40 or 50, or about 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 40 or 50, etc., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, etc.) chiral internucleotide linkages are chirally controlled.

473. The method of any one of the preceding embodiments, wherein at least 5% to 100% (e.g., about 10% to 100%, 20 to 100%, 30% to 100%, 40% to 100%, 50% to 80%, 50% to 85%, 50% to 90%, 50% to 95%, 60% to 80%, 60% to 85%, 60% to 90% , 60%~95%, 60%~100%, 65%~80%, 65%~85%, 65%~90%, 65%~95%, 65%~100%, 70%~80%, 70% %~85%, 70%~90%, 70%~95%, 70%~100%, 75%~80%, 75%~85%, 75%~90%, 75%~95%, 75%~ 100%, 80% to 85%, 80% to 90%, 80% to 95%, 80% to 100%, 85% to 90%, 85% to 95%, 85% to 100%, 90% to 95% , 90%~100%, 10%, 20%, 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%, etc. ) is a chirally controlled oligonucleotide.

474. The oligonucleotide of any one of the above embodiments, wherein each chiral internucleotide linkage in the second subdomain is independently a chiral control internucleotide linkage.

475. The method of any one of the preceding embodiments, wherein at least about 1-50 (e.g., about 5, 6, 7, 8, 9, or 10 to about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 40 or 50, or about 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 40 or 50, etc., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, etc.) chiral internucleotide linkages are Sp .

476. The method of any one of the preceding embodiments, wherein at least about 1-50 (e.g., about 5, 6, 7, 8, 9, or 10 to about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 40 or 50, or about 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 40 or 50, etc., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, etc.) chiral internucleotide linkages are R p .

477. The method of any one of the preceding embodiments, wherein at least 5% to 100% (e.g., about 10% to 100%, 20 to 100%, 30% to 100%, 40% to 100%, 50% to 80%, 50% to 85%, 50% to 90%, 50% to 95%, 60% to 80%, 60% to 85%, 60% to 90% , 60%~95%, 60%~100%, 65%~80%, 65%~85%, 65%~90%, 65%~95%, 65%~100%, 70%~80%, 70% %~85%, 70%~90%, 70%~95%, 70%~100%, 75%~80%, 75%~85%, 75%~90%, 75%~95%, 75%~ 100%, 80% to 85%, 80% to 90%, 80% to 95%, 80% to 100%, 85% to 90%, 85% to 95%, 85% to 100%, 90% to 95% , 90%~100%, 10%, 20%, 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%, etc. ) is S p, an oligonucleotide.

478. The oligonucleotide according to any one of the above embodiments, wherein each chiral internucleotide linkage in the second subdomain is Sp .

479. The oligonucleotide of any one of the preceding embodiments, wherein each internucleotide linkage in the second subdomain is independently a modified internucleotide linkage.

480. The oligonucleotide according to any one of embodiments 1 to 478, wherein the second subdomain comprises one or more natural phosphate linkages.

481. The oligonucleotide according to any one of embodiments 1 to 478, wherein the opposite nucleoside is linked to the 5' adjacent nucleoside via a natural phosphate linkage.

482. The oligonucleotide according to any one of embodiments 1 to 480, wherein the opposite nucleoside is linked to the 5' adjacent nucleoside via a modified internucleotide linkage.

483. The oligonucleotide according to any one of embodiments 1 to 482, wherein the opposite nucleoside is linked to the 3' adjacent nucleoside via a modified internucleotide linkage.

484. The method according to any one of embodiments 1 to 483, wherein the 3' adjacent nucleoside (position -1) to the opposite nucleoside (position 0) is 3' adjacent to the opposite nucleoside (position -1) via a modified internucleotide linkage. An oligonucleotide linked to a nucleoside (position -2).

485. The oligonucleotide according to any one of embodiments 482 to 484, wherein the modified internucleotide linkage is a chiral internucleotide linkage.

486. The oligonucleotide according to any one of embodiments 482 to 485, wherein the modified internucleotide linkages are phosphorothioate internucleotide linkages.

487. The oligonucleotide according to any one of embodiments 482 to 485, wherein the modified internucleotidic linkage is a non-negatively charged internucleotidic linkage.

488. The oligonucleotide according to any one of embodiments 482 to 485, wherein the modified internucleotide linkage is a neutrally charged internucleotide linkage.

489. The oligonucleotide according to any one of embodiments 485 to 488, wherein the chiral internucleotide linkages are chirally controlled.

490. The oligonucleotide according to any one of embodiments 485 to 489, wherein the chiral internucleotidic linkage is R p.

491. The oligonucleotide according to any one of embodiments 485 to 489, wherein the chiral internucleotidic linkage is Sp .

492. The oligonucleotide according to any one of embodiments 481 to 491, wherein the 5' adjacent nucleoside comprises a modified sugar.

493. The oligonucleotide according to any one of embodiments 481 to 491, wherein the 5' adjacent nucleoside comprises a modified sugar comprising a 2'-F modification.

494. The oligonucleotide according to any one of embodiments 481 to 491, wherein the 5' adjacent nucleoside comprises a sugar comprising two 2'-Hs (eg, a natural DNA sugar).

495. The oligonucleotide according to any one of embodiments 1-478 and 480-494, wherein the opposite nucleoside is linked to the 3' adjacent nucleoside via a natural phosphate linkage.

496. The oligonucleotide according to any one of embodiments 1-478 and 480-494, wherein the opposite nucleoside is linked to the 3' adjacent nucleoside via a modified internucleotide linkage.

497. The oligonucleotide of embodiment 496, wherein the modified internucleotidic linkage is a chiral internucleotidic linkage.

498. The oligonucleotide of embodiment 496 or 497, wherein the modified internucleotide linkages are phosphorothioate internucleotide linkages.

499. The oligonucleotide of embodiment 496 or 497, wherein the modified internucleotidic linkage is a non-negatively charged internucleotidic linkage.

500. The oligonucleotide of embodiment 496 or 497, wherein the modified internucleotide linkage is a neutrally charged internucleotide linkage.

501. The oligonucleotide according to any one of embodiments 497 to 500, wherein the chiral internucleotide linkages are chirally controlled.

502. The oligonucleotide according to any one of embodiments 497 to 501, wherein the chiral internucleotidic linkage is R p.

503. The oligonucleotide according to any one of embodiments 497 to 501, wherein the chiral internucleotidic linkage is Sp .

504. The oligonucleotide according to any one of the preceding embodiments, wherein the 3' adjacent nucleosides comprise a modified sugar.

505. The oligonucleotide of embodiment 503, wherein the 3' flanking nucleoside comprises a modified sugar comprising a 2'-F modification.

506. The oligonucleotide of embodiment 503, wherein the 3' flanking nucleoside comprises a sugar comprising two 2'-Hs (eg, a natural DNA sugar).

507. The oligonucleotide of any one of the preceding embodiments, wherein the 3' adjacent nucleoside comprises a base other than G.

508. The oligonucleotide of any one of the preceding embodiments, wherein the 3' adjacent nucleoside comprises a base less steric than G.

509. The oligonucleotide of any one of the preceding embodiments, wherein the 3' adjacent nucleoside comprises a nucleobase that is or comprises ring BA having the structure of formula BA-VI.

510. The oligonucleotide of any one of embodiments 507-509, wherein the ring BA is the ring BA of any one of embodiments 232-298.

511. The method of any one of embodiments 507 to 510, wherein the nucleobase is Phosphorus, oligonucleotide.

512. The method of any one of embodiments 507 to 510, wherein the nucleobase is Phosphorus, oligonucleotide.

513. The oligonucleotide according to any one of embodiments 507 to 510, wherein the nucleobase is hypoxanthine.

514. The oligonucleotide according to any one of the preceding embodiments, wherein the target nucleic acid comprises 5'-CA-3' and A is a target adenosine.

515. The sugar of the 5 'direction adjacent nucleoside according to any one of the above embodiments An oligonucleotide that is or comprises the same.

516. The sugar of any one of embodiments 1 to 514, wherein the 5 'direction adjacent nucleoside is An oligonucleotide that is or comprises the same.

517. The sugar of any one of embodiments 1 to 514, wherein the 5 'direction adjacent nucleoside is An oligonucleotide that is or comprises the same.

518. The sugar of the nucleoside opposite to the target nucleoside according to any one of the above embodiments An oligonucleotide that is or comprises the same.

519. according to any one of embodiments 1 to 517, wherein the sugar of the nucleoside opposite the target nucleoside is An oligonucleotide that is or comprises the same.

520. according to any one of embodiments 1 to 517, wherein the sugar of the nucleoside opposite the target nucleoside is An oligonucleotide that is or comprises the same.

521. The sugar of the 3 'direction adjacent nucleoside according to any one of the above embodiments An oligonucleotide that is or comprises the same.

522. The sugar of any one of embodiments 1 to 520, wherein the 3 'direction adjacent nucleoside is An oligonucleotide that is or comprises the same.

523. The sugar of any one of embodiments 1 to 520, wherein the sugar of the 3 'direction adjacent nucleoside is An oligonucleotide that is or comprises the same.

524. The oligonucleotide of any one of the preceding embodiments, wherein the second subdomain enables, promotes, or contributes to recruitment of an ADAR protein to the target nucleic acid.

525. The oligonucleotide of any one of the preceding embodiments, wherein the second subdomain enables, facilitates, or contributes to interaction of the ADAR with the target nucleic acid.

526. The oligonucleotide according to any one of the preceding embodiments, wherein the second subdomain is in contact with a domain having enzymatic activity.

527. The oligonucleotide according to any one of the above embodiments, wherein the second subdomain is in contact with a domain having deaminase activity of ADAR1.

528. The oligonucleotide according to any one of the preceding embodiments, wherein the second subdomain is in contact with a domain having a deaminase activity of ADAR2.

529. The method of any one of the preceding embodiments, wherein the third subdomain is from about 1 to about 50 (e.g., from about 5, 6, 7, 8, 9, or 10 to about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 40 or 50, or about 5, 6, 7, 8, 9, 10, 11, 12 , 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 40 or 50, etc.) nucleobases in length.

530. The number of third subdomains according to any one of the preceding embodiments, from about 1 to about 10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10). An oligonucleotide having a length of nucleobases.

531. The method of any one of the preceding embodiments, wherein the third subdomain is one or more (e.g., 1-10, 1, 2, 3, 4, 5 , 6, 7, 8, 9, or 10, etc.) mismatches.

532. The oligonucleotide of any one of the preceding embodiments, wherein the third subdomain comprises two or more mismatches when the oligonucleotide is aligned with the target nucleic acid for complementarity.

533. The oligonucleotide of any one of embodiments 1-531, wherein the third subdomain comprises no more than one mismatch when the oligonucleotide is aligned with a target nucleic acid for complementarity.

534. The oligonucleotide of any one of embodiments 1-531, wherein the third subdomain comprises no more than two mismatches when the oligonucleotide is aligned with the target nucleic acid for complementarity.

535. The method of any one of the preceding embodiments, wherein the third subdomain is one or more (e.g., 1-10, 1, 2, 3, 4, 5 , 6, 7, 8, 9, or 10 bulges, etc.).

536. The oligonucleotide of embodiment 535, wherein each bulge independently comprises one or more base pairs that are not Watson-Crick or wobble pairs.

537. The method of any one of the preceding embodiments, wherein the third subdomain is one or more (eg, 1-10, 1, 2, 3, 4, 5 , 6, 7, 8, 9, or 10, etc.) wobble pairs.

538. The oligonucleotide of any one of the preceding embodiments, wherein the third subdomain comprises two or more wobble pairs when the oligonucleotide is aligned with a target nucleic acid for complementarity.

539. The oligonucleotide of any one of the preceding embodiments, wherein the third subdomain comprises no more than two wobble pairs when the oligonucleotide is aligned with a target nucleic acid for complementarity.

540. The oligonucleotide according to any one of embodiments 1 to 530, wherein the third subdomain is completely complementary to the target nucleic acid.

541. The method of any one of the above embodiments, wherein the third subdomain has about 1-50 (e.g., about 5, 6, 7, 8, 9, or 10 to about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 40 or 50, or about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 40 or 50, etc., about 1, 2 , 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 modified sugars, etc.).

542. The method of any one of the preceding embodiments, wherein about 5% to 100% (e.g., about 10% to 100%, 20 to 100%, 30% to 100%, 40%) of the sugars in the third subdomain %~100%, 50%~80%, 50%~85%, 50%~90%, 50%~95%, 60%~80%, 60%~85%, 60%~90%, 60%~ 95%, 60% to 100%, 65% to 80%, 65% to 85%, 65% to 90%, 65% to 95%, 65% to 100%, 70% to 80%, 70% to 85% , 70%~90%, 70%~95%, 70%~100%, 75%~80%, 75%~85%, 75%~90%, 75%~95%, 75%~100%, 80 %~85%, 80%~90%, 80%~95%, 80%~100%, 85%~90%, 85%~95%, 85%~100%, 90%~95%, 90%~ 100%, 10%, 20%, 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%) independently. Oligonucleotides, which are modified sugars with modifications other than 2'-F.

543. The method of any one of the above embodiments, wherein about 50%-100% (e.g., about 50%-80%, 50%-85%, 50%-90%, 50%~95%, 60%~80%, 60%~85%, 60%~90%, 60%~95%, 60%~100%, 65%~80%, 65%~85%, 65% ~90%, 65%~95%, 65%~100%, 70%~80%, 70%~85%, 70%~90%, 70%~95%, 70%~100%, 75%~80 %, 75%~85%, 75%~90%, 75%~95%, 75%~100%, 80%~85%, 80%~90%, 80%~95%, 80%~100%, 85% to 90%, 85% to 95%, 85% to 100%, 90% to 95%, 90% to 100%, 50%, 60%, 65%, 70%, 75%, 80%, 85% , 90%, 95%, or 100%, etc.) are independently modified sugars with modifications other than 2'-F.

544. The method of any one of the above embodiments, wherein the third subdomain is a bicyclic sugar (eg, LNA sugar), an acyclic sugar (eg, UNA sugar), a 2'-OR modified sugar, or a 2'- from about ₁ to about 50 (eg about 5, 6, 7, ₈ ; 9, or 10 to about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 40 or 50, or about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 40 or 50, etc., about 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 modified sugars, etc.) nucleotide.

545. The method of any one of the preceding embodiments, wherein about 5%-100% (e.g., about 10%-100%, 20-100%, 30%-100%, 40%) of the sugars in the third subdomain %~100%, 50%~80%, 50%~85%, 50%~90%, 50%~95%, 60%~80%, 60%~85%, 60%~90%, 60%~ 95%, 60% to 100%, 65% to 80%, 65% to 85%, 65% to 90%, 65% to 95%, 65% to 100%, 70% to 80%, 70% to 85% , 70%~90%, 70%~95%, 70%~100%, 75%~80%, 75%~85%, 75%~90%, 75%~95%, 75%~100%, 80 %~85%, 80%~90%, 80%~95%, 80%~100%, 85%~90%, 85%~95%, 85%~100%, 90%~95%, 90%~ 100%, 10%, 20%, 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%) independently. A bicyclic sugar (e.g. LNA sugar), an acyclic sugar (e.g. UNA sugar), a sugar with a 2'-OR strain, or a sugar with a 2'-N(R) ₂ strain (each R independently optionally substituted) an oligonucleotide that is a modified sugar selected from C _1-6 aliphatic).

546. The method of any one of the above embodiments, wherein about 50%-100% (e.g., about 50%-80%, 50%-85%, 50%-90%, 50%~95%, 60%~80%, 60%~85%, 60%~90%, 60%~95%, 60%~100%, 65%~80%, 65%~85%, 65% ~90%, 65%~95%, 65%~100%, 70%~80%, 70%~85%, 70%~90%, 70%~95%, 70%~100%, 75%~80 %, 75%~85%, 75%~90%, 75%~95%, 75%~100%, 80%~85%, 80%~90%, 80%~95%, 80%~100%, 85% to 90%, 85% to 95%, 85% to 100%, 90% to 95%, 90% to 100%, 50%, 60%, 65%, 70%, 75%, 80%, 85% , 90%, 95%, or 100%, etc.) are independently bicyclic sugars (e.g., LNA sugars), acyclic sugars (e.g., UNA sugars), sugars with 2'-OR modifications, or 2'-N (R ) ₂ modified sugars, wherein each R is independently an optionally substituted C _1-6 aliphatic oligonucleotide.

547. The method of any one _of the preceding embodiments, wherein the third subdomain comprises one _or more (eg, For example, about 1 to 20, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, etc.) An oligonucleotide containing a modified sugar.

548. The method of any one of _the preceding embodiments, wherein the third subdomain comprises one or more (eg, about 1-20, 1, 2, 3, 4, 5, 6 , 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, etc.) modified sugars.

549. The method of any one of the preceding embodiments, wherein the third subdomain is one or more (e.g., about 1-20, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, etc.) LNA sugars.

550. The method of any one of the above implementations, wherein the third subdomain is one or more (e.g., about 1-20, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, etc.) of acyclic sugars (eg, UNA sugars).

551. The method of any one of the preceding embodiments, wherein the third subdomain comprises one or more (e.g., about 1-20, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, etc.) modified sugars.

552. The method of any one of the preceding embodiments, wherein the third subdomain comprises one or more (e.g., about 1-20, 1, 2, 3, 4, 5, 6, 7 , 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, etc.) sugars.

553. The method of any one of the above embodiments, wherein the third subdomain comprises one or more (e.g., about 1-20, 1, 2, 3, 4, 5, 6 , 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, etc.) sugars.

554. The method of any one of the preceding embodiments, wherein the third subdomain comprises one or more (eg, about 1-20) comprising a 2'-OR variant (R is an optionally substituted C _1-6 aliphatic). , 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, etc.) oligonucleotide.

555. The method of any one of the preceding embodiments, wherein the third subdomain comprises one or more (e.g., about 1-20, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, etc.) modified sugars.

556. The method of any one of embodiments 1 to 546, wherein each sugar in the third subdomain is independently a 2'-OR variant (R is an optionally substituted C _1-6 aliphatic) or 2'-OL An oligonucleotide comprising ^{a B} -4' modification.

557. The method according to embodiment 556, wherein each sugar in the third subdomain is independently a 2'-OR variant (R is an optionally substituted C _1-6 aliphatic) or a 2'-OL ^B -4' variant (L ^B is an optionally substituted -CH ₂ -.

558. The oligonucleotide of embodiment 556, wherein each sugar in the third subdomain independently comprises 2'-OMe.

559. The oligonucleotide of any one of the preceding embodiments, wherein the third subdomain comprises a 5'-end portion having a length of about 1-8 nucleobases.

560. The oligonucleotide of embodiment 559, wherein the 5'-end has a length of about 1, 2, or 3 nucleobases.

561. The oligonucleotide of embodiment 559 or 560, wherein the 5'-end portion is linked to a second subdomain.

562. The oligonucleotide according to any one of embodiments 559 to 561, wherein one or more of the sugars at the 5'-terminus are independently modified sugars.

563. The method of embodiment 562, wherein the modified sugars are independently bicyclic sugars (eg LNA sugars), acyclic sugars (eg UNA sugars), sugars with 2'-OR modifications, or 2'-N(R) ₂ An oligonucleotide selected from modified sugars, wherein each R is independently an optionally substituted C _1-6 aliphatic.

564. The oligonucleotide of embodiment 562, wherein at least one of the modified sugars independently comprises 2'-F.

565. The oligonucleotide according to any one of embodiments 559 to 561, wherein at least one sugar at the 5'-end independently comprises two 2'-Hs (eg, per natural DNA).

566. The oligonucleotide according to any one of embodiments 559 to 565, wherein one or more sugars at the 5'-end independently comprise 2'-OH (eg, a native RNA sugar).

567. The sugar of any one of embodiments 559 to 561, wherein the sugar at the 5'-terminus independently comprises two 2'-Hs (eg per natural DNA) or comprises a 2'-OH (eg per natural DNA). per native RNA), oligonucleotides.

568. The oligonucleotide according to any one of embodiments 559 to 561, wherein the sugar at the 5'-end is independently a natural DNA or RNA sugar.

569. The oligonucleotide according to any one of embodiments 559 to 568, wherein the 5'-end comprises one or more mismatches.

570. The oligonucleotide according to any one of embodiments 559 to 569, wherein the 5'-end comprises one or more wobbles.

571. The method of any one of embodiments 559 to 570, wherein the 5'-end is about 60-100% (e.g., 66%, 70%, 75%, 80%, 85%, 90%) relative to the target nucleic acid %, 95% or more) complementary, oligonucleotides.

572. The oligonucleotide of any one of the preceding embodiments, wherein the third subdomain comprises a 3'-end portion having a length of about 1-8 nucleobases.

573. The oligonucleotide of embodiment 572, wherein the 3'-terminus has a length of about 1, 2, 3, or 4 nucleobases.

574. The oligonucleotide of embodiment 572 or 573, wherein the 3'-terminal portion comprises the 3'-terminal nucleobase of the third subdomain.

575. The oligonucleotide according to any one of embodiments 572 to 574, wherein at least one of the sugars at the 3'-terminus is independently a modified sugar.

576. The method of embodiment 575, wherein the modified sugars are independently bicyclic sugars (eg LNA sugars), acyclic sugars (eg UNA sugars), sugars with 2'-OR modifications, or 2'-N(R) ₂ An oligonucleotide selected from modified sugars, wherein each R is independently an optionally substituted C _1-6 aliphatic.

577. The oligonucleotide of embodiment 575 or 576, wherein the one or more modified sugars independently comprise 2'-F.

578. according to embodiment 575 or 576, at least 20%, 25%, 30%, 35%, 40%, 50%, 60%, 70%, 75%, 80%, 90% in the third subdomain; or 95% of the sugars independently comprise 2'-F.

579. according to any one of embodiments 575 to 578, wherein one or more sugars at the 3'-terminus are independently 2'-OR variants (R is an optionally substituted C _1-6 aliphatic) or 2'-OL ^B An oligonucleotide comprising a -4' modification.

580. is used in embodiment 579, wherein each sugar at the 3'-terminus independently comprises a 2'-OR modification (R is an optionally substituted C _1-6 aliphatic) or a 2'-OL ^B -4' modification. , oligonucleotides.

581. The oligonucleotide of embodiment 579 or 580, wherein L ^B is optionally substituted -CH ₂ -.

582. The oligonucleotide of embodiment 579 or 580, wherein L ^B is -CH ₂ -.

583. The oligonucleotide of embodiment 579, wherein each sugar at the 3'-terminus independently comprises 2'-OMe.

584. The oligonucleotide according to any one of embodiments 572 to 583, wherein the 3'-end comprises one or more mismatches.

585. The oligonucleotide according to any one of embodiments 572 to 584, wherein the 3'-end comprises one or more wobbles.

586. The method of any one of embodiments 572 to 585, wherein the 3'-end is about 60-100% (e.g., 66%, 70%, 75%, 80%, 85%, 90%) relative to the target nucleic acid %, 95% or more) complementary, oligonucleotides.

587. The method of any one of the preceding embodiments, wherein the third subdomain is from about 1 to about 50 (e.g., about 5, 6, 7, 8, 9, or 10 to about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 40 or 50, or about 5, 6, 7, 8, 9, 10, 11, 12 , 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 40 or 50, etc., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, etc.) modified internucleotide linkages.

588. The method of any one of the preceding embodiments, wherein about 5%-100% (e.g., about 10%-100%, 20-100%, 30%-100%) of the internucleotide linkages in the third subdomain %, 40%~100%, 50%~80%, 50%~85%, 50%~90%, 50%~95%, 60%~80%, 60%~85%, 60%~90%, 60%~95%, 60%~100%, 65%~80%, 65%~85%, 65%~90%, 65%~95%, 65%~100%, 70%~80%, 70% ~85%, 70%~90%, 70%~95%, 70%~100%, 75%~80%, 75%~85%, 75%~90%, 75%~95%, 75%~100 %, 80%~85%, 80%~90%, 80%~95%, 80%~100%, 85%~90%, 85%~95%, 85%~100%, 90%~95%, 90% to 100%, 10%, 20%, 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%, etc.) is an oligonucleotide that is a linkage between modified nucleotides.

589. The method of any one of the preceding embodiments, wherein about 50%-100% (e.g., about 50%-80%, 50%-85%, 50%- 90%, 50%~95%, 60%~80%, 60%~85%, 60%~90%, 60%~95%, 60%~100%, 65%~80%, 65%~85% , 65%~90%, 65%~95%, 65%~100%, 70%~80%, 70%~85%, 70%~90%, 70%~95%, 70%~100%, 75 %~80%, 75%~85%, 75%~90%, 75%~95%, 75%~100%, 80%~85%, 80%~90%, 80%~95%, 80%~ 100%, 85% to 90%, 85% to 95%, 85% to 100%, 90% to 95%, 90% to 100%, 50%, 60%, 65%, 70%, 75%, 80% , 85%, 90%, 95%, or 100%, etc.) are modified internucleotide linkages.

590. The oligonucleotide of any one of the preceding embodiments, wherein each modified internucleotide linkage is independently a chiral internucleotide linkage.

591. The oligonucleotide of any one of the above embodiments, wherein the internucleotide linkage between the last nucleoside and the penultimate nucleoside of the third subdomain is a non-negatively charged internucleotide linkage.

592. The oligonucleotide of any one of the preceding embodiments, wherein each modified internucleotide linkage is independently a phosphorothioate internucleotide linkage or a non-negatively charged internucleotide linkage.

593. The oligonucleotide of any one of the preceding embodiments, wherein each modified internucleotide linkage is independently a phosphorothioate internucleotide linkage or a neutral internucleotide linkage.

594. The method of any one of the preceding embodiments, wherein at least about 1-50 (e.g., about 5, 6, 7, 8, 9, or 10 to about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 40 or 50, or about 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 40 or 50, etc., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, etc.) chiral internucleotide linkages are chirally controlled.

595. The method of any one of the preceding embodiments, wherein at least 5%-100% (e.g., about 10%-100%, 20-100%, 30%- 100%, 40% to 100%, 50% to 80%, 50% to 85%, 50% to 90%, 50% to 95%, 60% to 80%, 60% to 85%, 60% to 90% , 60%~95%, 60%~100%, 65%~80%, 65%~85%, 65%~90%, 65%~95%, 65%~100%, 70%~80%, 70% %~85%, 70%~90%, 70%~95%, 70%~100%, 75%~80%, 75%~85%, 75%~90%, 75%~95%, 75%~ 100%, 80% to 85%, 80% to 90%, 80% to 95%, 80% to 100%, 85% to 90%, 85% to 95%, 85% to 100%, 90% to 95% , 90%~100%, 10%, 20%, 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%, etc. ) is a chirally controlled oligonucleotide.

596. The oligonucleotide of any one of the preceding embodiments, wherein the internucleotide linkage between the last nucleoside and the penultimate nucleoside of the third subdomain is chirally controlled.

597. The oligonucleotide of any one of the preceding embodiments, wherein each chiral internucleotidic linkage is independently a chiral control internucleotidic linkage.

598. The method of any one of the preceding embodiments, wherein at least about 1-50 (e.g., about 5, 6, 7, 8, 9, or 10 to about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 40 or 50, or about 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 40 or 50, etc., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, etc.) chiral internucleotide linkages are Sp .

599. The method of any one of the preceding embodiments, wherein at least 5%-100% (eg, about 10%-100%, 20-100%, 30%-100%) of the chiral internucleotide linkages in the third subdomain 100%, 40% to 100%, 50% to 80%, 50% to 85%, 50% to 90%, 50% to 95%, 60% to 80%, 60% to 85%, 60% to 90% , 60%~95%, 60%~100%, 65%~80%, 65%~85%, 65%~90%, 65%~95%, 65%~100%, 70%~80%, 70% %~85%, 70%~90%, 70%~95%, 70%~100%, 75%~80%, 75%~85%, 75%~90%, 75%~95%, 75%~ 100%, 80% to 85%, 80% to 90%, 80% to 95%, 80% to 100%, 85% to 90%, 85% to 95%, 85% to 100%, 90% to 95% , 90%~100%, 10%, 20%, 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%, etc. ) is S p, an oligonucleotide.

600. The oligonucleotide of any one of the preceding embodiments, wherein each chiral internucleotide linkage in the third subdomain is Sp .

601. The oligonucleotide according to any one of embodiments 1 to 599, wherein the internucleotide linkage between the last nucleoside and the penultimate nucleoside of the third subdomain is R p.

602. The method according to any one of the above embodiments, wherein the internucleotide linkage connecting the last nucleoside of the second subdomain and the first nucleoside of the third subdomain is a non-negatively charged internucleotide linkage, oligonucleotide.

603. The oligonucleotide according to any one of the above embodiments, wherein the internucleotidic linkage at position -2 is a non-negatively charged internucleotidic linkage.

604. The oligonucleotide of embodiment 602 or 603, wherein the non-negatively charged internucleotidic linkages are chirally controlled.

605. The oligonucleotide of embodiment 604, wherein the non-negatively charged internucleotidic linkage is R p.

606. The oligonucleotide of embodiment 604, wherein the non-negatively charged internucleotidic linkage is S p.

607. The oligonucleotide of any one of the preceding embodiments, wherein each internucleotide linkage in the third subdomain is independently a modified internucleotide linkage.

608. The oligonucleotide of any one of embodiments 1-606, wherein the third subdomain comprises one or more natural phosphate linkages.

609. The oligonucleotide according to any one of the preceding embodiments, wherein the third subdomain enables, promotes, or contributes to recruitment of the ADAR protein to the target nucleic acid.

610. The oligonucleotide of any one of the preceding embodiments, wherein the third subdomain enables, facilitates, or contributes to interaction of the ADAR with the target nucleic acid.

611. The oligonucleotide according to any one of the preceding embodiments, wherein the third subdomain is in contact with a domain having enzymatic activity.

612. The oligonucleotide according to any one of the above embodiments, wherein the third subdomain is in contact with a domain having deaminase activity of ADAR1.

613. The oligonucleotide according to any one of the above embodiments, wherein the third subdomain is in contact with a domain having a deaminase activity of ADAR2.

614. The oligonucleotide of any one of the preceding embodiments, wherein each wobble base pair is independently G-U, I-A, G-A, I-U, I-C, I-T, A-A, or inverted A-T.

615. The oligonucleotide of any one of the preceding embodiments, wherein each wobble base pair is independently G-U, I-A, G-A, I-U, or I-C.

616. The method of any one of the preceding embodiments, wherein each cyclic sugar or each sugar is independently optionally substituted Phosphorus, oligonucleotide.

617. The method of any one of the above embodiments, wherein each cyclic sugar or each sugar is independently An oligonucleotide having a structure of

618. The oligonucleotide of embodiment 617 comprising one or more sugars wherein R ^2s and R ^4s are H.

619. The oligonucleotide comprising one or more sugars according to embodiment 617 or 618, wherein R ^2s is -OR and R ^4s is H.

620. The oligonucleotide according to any one of embodiments 617 to 619, wherein the oligonucleotide comprises one or more sugars wherein R ^2s is -OR (R is an optionally substituted C _1-4 alkyl) and R ^4s is H.

621. The oligonucleotide according to any one of embodiments 617 to 620, comprising one or more sugars wherein R ^2s is -OMe and R ^4s is H.

622. The oligonucleotide according to any one of embodiments 617 to 621, comprising one or more sugars wherein R ^2s is -F and R ^4s is H.

623. according to any one of embodiments 617 to 622, wherein R ^4s and R ^2s comprise one or more sugars forming a crosslinker having the structure of optionally substituted 2'-O-CH ₂ -4' nucleotide.

624. The oligonucleotide according to any one of embodiments 617 to 622, wherein R ^4s and R ^2s comprise one or more sugars forming a crosslinker having the structure 2'-O-CH ₂ -4'.

625. The oligonucleotide according to any one of the preceding embodiments, comprising additional chemical moieties.

626. The oligonucleotide according to any one of the preceding embodiments, comprising a targeting moiety.

627. The oligonucleotide according to any one of the preceding embodiments, comprising a carbohydrate moiety.

628. The oligonucleotide according to any one of embodiments 623 to 627, wherein the moiety is or comprises a ligand for an asialoglycoprotein receptor.

629. The oligonucleotide according to any one of embodiments 623 to 628, wherein the moiety is or comprises GalNAc or a derivative thereof.

630. The method according to any one of embodiments 623 to 629, wherein the moiety is optionally substituted An oligonucleotide that is or comprises the same.

631. The method of any one of embodiments 623 to 629, wherein the moiety is optionally substituted An oligonucleotide that is or comprises the same.

632. The oligonucleotide according to any one of embodiments 623 to 631, wherein the moiety is connected to the oligonucleotide chain through a linker.

633. The oligonucleotide of embodiment 632, wherein the linker is or comprises L001.

634. The oligonucleotide of embodiment 633, wherein L001 is linked to the 5'-terminal 5'-carbon of the oligonucleotide chain via a phosphate group.

635. The oligonucleotide according to any one of the preceding embodiments, wherein the additional chemical moiety is or comprises a nucleic acid moiety.

636. The oligonucleotide of embodiment 635, wherein the nucleic acid is or comprises an aptamer.

637. The oligonucleotide according to any one of the preceding embodiments, in salt form.

638. The oligonucleotide according to any one of the preceding embodiments, in the form of a pharmaceutically acceptable salt.

639. The oligonucleotide according to any one of the preceding embodiments, in the form of a sodium salt.

640. The oligonucleotide according to any one of the preceding embodiments, in the form of an ammonium salt.

641. The oligonucleotide of any one of the preceding embodiments, wherein at least one or each neutral internucleotide linkage, if present, is independently n001.

642. The oligonucleotide of any one of the preceding embodiments, wherein each non-negatively charged internucleotide linkage, if present, is independently n001.

643. according to any one of the above embodiments, there are at most 5, 6, 7, 8, 9, 10, 11 or 12 nucleosides in the 3' direction to the nucleoside opposite the target adenosine, oligonucleotide.

644. according to any one of the above embodiments, there are at most 5, 6, 7, 8, 9, 10, 11 or 12 nucleosides in the 3' direction to the nucleoside opposite the target nucleoside and each nucleoside is independently optionally substituted A, T, C, G, U, or a tautomer thereof.

645. The method of any one of the above embodiments, wherein about 50% to 100% (e.g., about or at least about 50%, 55 %, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%) are each independently a linkage between the modified nucleotides.

646. The method of any one of the above embodiments, wherein about 50% to 100% (e.g., about or at least about 50%, 55 %, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%) are each independently a phosphorothioate internucleotide linkage or an oligonucleotide that is not negatively charged internucleotide linkage. .

647. The oligonucleotide of any one of the preceding embodiments, wherein at most 1, 2, or 3 internucleotide linkages in the 3' direction to the nucleoside opposite the target adenosine are natural phosphate linkages.

648. The oligonucleotide according to any one of the above embodiments, wherein at most 1, 2, or 3 internucleotide linkages in the 3' direction to the opposite nucleoside of the target adenosine are R p internucleotide linkages.

649. according to any one of the above embodiments, wherein at most 1, 2, or 3 internucleotide linkages in the 3 'direction to the opposite nucleoside of the target adenosine are R p phosphorothioate internucleotidic linkages, oligonucleotide.

650. The method according to any one of the above embodiments, wherein the internucleoside linkage between the nucleoside opposite the target nucleoside and its 3' adjacent nucleoside (considered position -1) is a sterically random phosphorothio An oligonucleotide, which is an eight internucleotide linkage.

651. The method according to any one of embodiments 1 to 649, wherein the internucleotide linkage between the nucleoside opposite the target nucleoside and its 3' adjacent nucleoside (considered position -1) is a chiral control R An oligonucleotide, which is a p phosphorothioate internucleotidic linkage.

652. The method according to any one of embodiments 1 to 649, wherein the internucleotide linkage between the nucleoside opposite the target nucleoside and its 3' adjacent nucleoside (considered position -1) is a chiral control S An oligonucleotide, which is a p phosphorothioate internucleotidic linkage.

653. according to any one of embodiments 1 to 649, wherein the internucleoside bonded to the nucleoside opposite the target nucleoside at the 3'-position (considered position -1) of the sugar is R p phosphorothio 8 internucleotidic linkage, optionally the only R p phosphorothioate internucleotidic linkage in the 3' direction to the nucleoside opposite to the target adenosine.

654. according to any one of embodiments 1 to 649, wherein the internucleoside linked to the nucleoside opposite the target nucleoside at the 3'-position of the sugar (considered position -1) is Sp phosphorothio An oligonucleotide, which is an eight internucleotide linkage.

655. according to any one of embodiments 1 to 649, wherein the internucleotide linkage linked to the nucleoside opposite the target nucleoside at the 3'-position of the sugar (considered position -1) is a sterically random phosphorothioate An oligonucleotide, which is an eight internucleotide linkage.

656. The method according to any one of embodiments 1 to 649, wherein an internucleotide linkage between the 3' adjacent nucleoside of the nucleoside opposite the target nucleoside and the next 3' adjacent nucleoside (e.g. For example, position -2 between N _-1 and N -2 of 5'-…N ₀ N _-1 N _-2 … _-3 ', where N ₀ represents the nucleoside opposite to the target nucleoside) is negative. An oligonucleotide, which is an uncharged internucleotide linkage.

657. The oligonucleotide of embodiment 656, wherein the non-negatively charged internucleotidic linkages are sterically random.

658. The oligonucleotide of embodiment 656, wherein the non-negatively charged internucleotidic linkages are chirally controlled.

659. The oligonucleotide of embodiment 656, wherein the non-negatively charged internucleotidic linkages are chirally controlled and are Sp .

660. The oligonucleotide of embodiment 656, wherein the non-negatively charged internucleotidic linkages are chirally controlled and R p.

661. The oligonucleotide according to any one of embodiments 656 to 660, wherein the non-negatively charged internucleotidic linkages are phosphoryl guanidine internucleotidic linkages.

662. The oligonucleotide according to any one of embodiments 656 to 660, wherein the non-negatively charged internucleotide linkage is n001.

663. The oligonucleotide according to any one of embodiments 656 to 660, wherein the non-negatively charged internucleotide linkage is n004, n008, n025 or n026.

664. The oligonucleotide of any one of the above embodiments, wherein the first internucleotidic linkage is a non-negatively charged internucleotidic linkage.

665. The oligonucleotide of embodiment 664, wherein the non-negatively charged internucleotidic linkages are sterically random.

666. The oligonucleotide of embodiment 664, wherein the non-negatively charged internucleotidic linkages are chirally controlled.

667. The oligonucleotide of embodiment 664, wherein the non-negatively charged internucleotidic linkages are chirally controlled and are Sp .

668. The oligonucleotide of embodiment 664, wherein the non-negatively charged internucleotidic linkages are chirally controlled and R p.

669. The oligonucleotide according to any one of embodiments 664 to 668, wherein the non-negatively charged internucleotidic linkages are phosphoryl guanidine internucleotidic linkages.

670. The oligonucleotide according to any one of embodiments 664 to 668, wherein the non-negatively charged internucleotidic linkage is n001.

671. The oligonucleotide according to any one of embodiments 664 to 668, wherein the non-negatively charged internucleotide linkages are n004, n008, n025, n026.

672. The oligonucleotide of any one of the above embodiments, wherein the last internucleotidic linkage is a non-negatively charged internucleotidic linkage.

673. The oligonucleotide of embodiment 672, wherein the non-negatively charged internucleotidic linkages are sterically random.

674. The oligonucleotide of embodiment 672, wherein the non-negatively charged internucleotidic linkages are chirally controlled.

675. The oligonucleotide of embodiment 672, wherein the non-negatively charged internucleotidic linkages are chirally controlled and are Sp .

676. The oligonucleotide of embodiment 672, wherein the non-negatively charged internucleotidic linkages are chirally controlled and R p.

677. The oligonucleotide according to any one of embodiments 672 to 676, wherein the non-negatively charged internucleotidic linkages are phosphoryl guanidine internucleotidic linkages.

678. The oligonucleotide according to any one of embodiments 672 to 676, wherein the non-negatively charged internucleotide linkages are n004, n008, n025, n026.

679. The oligonucleotide according to any one of embodiments 672 to 676, wherein the non-negatively charged internucleotide linkage is n001.

680. The oligonucleotide of any one of the preceding embodiments, wherein the internucleotide linkage at position -3 relative to the nucleoside opposite the target adenosine is not a R p phosphorothioate internucleotide linkage.

681. The oligonucleotide of any one of the preceding embodiments, wherein the internucleotide linkage at position -6 relative to the nucleoside opposite the target adenosine is not a R p phosphorothioate internucleotide linkage.

682. The method according to any one of the above embodiments, wherein the internucleotide linkage at positions -4 and / or -5 relative to the nucleoside opposite the target nucleoside is a modified internucleotide linkage, for example phosphorothioate An oligonucleotide, which is a linkage between nucleotides.

683. The method according to any one of the above embodiments, wherein the nucleoside opposite the target nucleoside is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 from the 5'-end , at position 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 or more.

684. The method according to any one of the above embodiments, wherein the nucleoside opposite the target nucleoside is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 from the 3'-end , at position 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 or more.

685. The oligonucleotide of embodiment 683 or 684, wherein the position is position 4.

686. The oligonucleotide of embodiment 683 or 684, wherein the position is position 5.

687. The oligonucleotide of embodiment 683 or 684, wherein the position is position 6.

688. The oligonucleotide of embodiment 683 or 684, wherein the position is position 7.

689. The oligonucleotide of embodiment 683 or 684, wherein position is position 8.

690. The oligonucleotide of embodiment 683 or 684, wherein the position is position 9.

691. The oligonucleotide of embodiment 683 or 684, wherein position is position 10.

692. The method of any one of the preceding embodiments, wherein about 50% to 100% (e.g., about or at least about 50%, 55 %, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%) are independently modified internucleotide linkages, optionally chirally controlled oligonucleotides.

693. The method of any one of the above embodiments, wherein about 50% to 100% of the phosphorothioate internucleoside linkages in the 5' direction to the nucleoside opposite the target nucleoside (eg, target adenosine) ( For example, about or at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%) are each chirally controlled oligonucleotide that is Sp . .

694. The oligonucleotide of any one of the preceding embodiments, wherein zero or at most 1, 2, or 3 internucleotide linkages in the 5' direction to the nucleoside opposite the target adenosine are natural phosphate linkages.

695. The method of any one of the above embodiments, wherein an internucleotide linkage at position +5 relative to the nucleoside opposite the target nucleoside (eg, N ₀ is the nucleoside opposite the target nucleoside) , …N ₊₅ N ₊₄ N ₊₃ N ₊₂ N ₊₁ N ₀ ... the internucleotide linkage connecting N ₊₄ and N ₊₅ ) is an oligo, not an R p phosphorothioate internucleotide linkage nucleotide.

696. The method according to any one of the above embodiments, wherein one or more or all internucleotide linkages at positions +6 to +8 relative to the nucleoside opposite to the target adenosine are each independently, optionally chirally controlled, modified internucleotide linkage Phosphorus, oligonucleotide.

697. The method of any one of the above embodiments, wherein one or more or all internucleotide linkages at positions +6 to +8 relative to the nucleoside opposite to the target adenosine are each independently, optionally chirally controlled phosphorothioate An oligonucleotide, which is a linkage between nucleotides.

698. The method of any one of the above embodiments, wherein one or more or all internucleotide linkages at positions +6, +7, +8, +9, and +11 relative to the nucleoside opposite the target adenosine are each independently R p phosphorothioate internucleotidic linkages, oligonucleotides.

699. The method of any one of the above embodiments, wherein one or more or all internucleotide linkages at positions +5, +6, +7, +8, and +9 relative to the nucleoside opposite to the target adenosine are each independently Oligonucleotides, which are S p phosphorothioate internucleotidic linkages.

700. The method of any one of the above embodiments, wherein about 50% to 100% (eg, about 50% to 80%, 50% to 85%, 50 %~90%, 50%~95%, 60%~80%, 60%~85%, 60%~90%, 60%~95%, 60%~100%, 65%~80%, 65%~ 85%, 65% to 90%, 65% to 95%, 65% to 100%, 70% to 80%, 70% to 85%, 70% to 90%, 70% to 95%, 70% to 100% , 75%~80%, 75%~85%, 75%~90%, 75%~95%, 75%~100%, 80%~85%, 80%~90%, 80%~95%, 80 % to 100%, 85% to 90%, 85% to 95%, 85% to 100%, 90% to 95%, 90% to 100%, or at least about 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% complementarity).

701. In any one of the above embodiments, the base sequence of the oligonucleotide is or comprises a sequence different from UCCCUUUCTCIUCGA at no more than 1, 2, 3, 4 or 5 positions, and each U is independently T Oligonucleotides that can be substituted and vice versa.

702. The method of any one of the above embodiments, wherein the base sequence of the oligonucleotide is or comprises a sequence different from UCCCUUUCTCGUCGA at no more than 1, 2, 3, 4 or 5 positions, and each U is independently represented by T Oligonucleotides that can be substituted and vice versa.

703. The oligonucleotide according to any one of the above embodiments, wherein the base sequence of the oligonucleotide is or comprises a sequence different from UCCCUUUCTCIUCGA at no more than 1, 2, 3, 4 or 5 positions.

704. The oligonucleotide according to any one of the above embodiments, wherein the base sequence of the oligonucleotide is or comprises a sequence different from UCCCUUUCTCGUCGA at no more than 1, 2, 3, 4 or 5 positions.

705. The oligonucleotide according to any one of the above embodiments, wherein the base sequence of the oligonucleotide is or comprises UCCCUUUCTCIUCGA, and each U can be independently replaced by T and vice versa.

706. The oligonucleotide according to any one of the above embodiments, wherein the base sequence of the oligonucleotide is or comprises UCCCUUUCTCGUCGA, and each U may be independently replaced by T and vice versa.

707. The oligonucleotide according to any one of the above embodiments, wherein the base sequence of the oligonucleotide is or comprises UCCCUUUCTCIUCGA.

708. The oligonucleotide according to any one of the above embodiments, wherein the base sequence of the oligonucleotide is or comprises UCCCUUUCTCGUCGA.

709. The method of any one of the above embodiments, wherein the base sequence of the oligonucleotide is or comprises a sequence different from UUCAGUCCCUUUCTCIUCGA at no more than 1, 2, 3, 4 or 5 positions, and each U is independently T Oligonucleotides that can be substituted and vice versa.

710. The method of any one of the above embodiments, wherein the base sequence of the oligonucleotide is or comprises a sequence different from UUCAGUCCCUUUCTCGUCGA at no more than 1, 2, 3, 4 or 5 positions, and each U is independently T Oligonucleotides that can be substituted and vice versa.

711. The oligonucleotide according to any one of the above embodiments, wherein the nucleotide sequence of the oligonucleotide is or comprises a sequence different from UUCAGUCCCUUUCTCIUCGA at no more than 1, 2, 3, 4 or 5 positions.

712. The oligonucleotide according to any one of the above embodiments, wherein the nucleotide sequence of the oligonucleotide is or comprises a sequence different from UUCAGUCCCUUUCTCGUCGA at no more than 1, 2, 3, 4 or 5 positions.

713. The oligonucleotide according to any one of the above embodiments, wherein the base sequence of the oligonucleotide is or comprises UUCAGUCCCUUUCTCIUCGA, and each U may be independently replaced with T and vice versa.

714. The oligonucleotide according to any one of the above embodiments, wherein the base sequence of the oligonucleotide is or comprises UUCAGUCCCUUUCTCGUCGA, and each U can be independently replaced by T and vice versa.

715. The oligonucleotide according to any one of the above embodiments, wherein the base sequence of the oligonucleotide is or comprises UUCAGUCCCUUUCTCIUCGA.

716. The oligonucleotide according to any one of the above embodiments, wherein the nucleotide sequence of the oligonucleotide is or comprises UUCAGUCCCUUUCTCGUCGA.

717. The method of any one of the above embodiments, wherein the base sequence of the oligonucleotide is or comprises a sequence different from CCCCAGCAGCUUCAGUCCCUUUCTCGUCGA at no more than 1, 2, 3, 4 or 5 positions, and each U is independently T Oligonucleotides that can be substituted and vice versa.

718. The method of any one of the above embodiments, wherein the base sequence of the oligonucleotide is or comprises a sequence different from CCCCAGCAGCUUCAGUCCCUUUCTCGUCGA at no more than 1, 2, 3, 4 or 5 positions, and each U is independently T Oligonucleotides that can be substituted and vice versa.

719. The oligonucleotide according to any one of the above embodiments, wherein the base sequence of the oligonucleotide is or comprises a sequence different from CCCCAGCAGCUUCAGUCCCUUUCTCGUCGA at no more than 1, 2, 3, 4 or 5 positions.

720. The oligonucleotide according to any one of the above embodiments, wherein the base sequence of the oligonucleotide is or comprises a sequence different from CCCCAGCAGCUUCAGUCCCUUUCTCGUCGA at no more than 1, 2, 3, 4 or 5 positions.

721. The oligonucleotide according to any one of the above embodiments, wherein the base sequence of the oligonucleotide is or comprises CCCCAGCAGCUUCAGUCCCUUUCTCGUCGA, and each U can be independently replaced by T and vice versa.

722. The oligonucleotide according to any one of the above embodiments, wherein the base sequence of the oligonucleotide is or comprises CCCCAGCAGCUUCAGUCCCUUUCTCGUCGA, and each U can be independently replaced by T and vice versa.

723. The oligonucleotide according to any one of the above embodiments, wherein the base sequence of the oligonucleotide is or comprises CCCCAGCAGCUUCAGUCCCUUUCTCGUCGA, and each U can be independently replaced by T and vice versa.

724. The oligonucleotide according to any one of the above embodiments, wherein the nucleotide sequence of the oligonucleotide is or comprises CCCCAGCAGCUUCAGUCCCUUUCTCGUCGA.

725. The oligonucleotide according to any one of the above embodiments, wherein the base sequence of the oligonucleotide is CCCCAGCAGCUUCAGUCCCUUUCTCGUCGA.

726. The method of any one of the above embodiments, wherein the nucleotide sequence of the oligonucleotide is or comprises a sequence that differs from CCCAGCAGCUUCAGUCCCUUUCTUIUCGAU at no more than 1, 2, 3, 4 or 5 positions, and each U is independently T Oligonucleotides that can be substituted and vice versa.

727. The method of any one of the above embodiments, wherein the base sequence of the oligonucleotide is or comprises a sequence different from CCCAGCAGCUUCAGUCCCUUUCTUIUCGAU at no more than 1, 2, 3, 4 or 5 positions, and each U is independently T Oligonucleotides that can be substituted and vice versa.

728. The oligonucleotide according to any one of the above embodiments, wherein the base sequence of the oligonucleotide is or comprises a sequence different from CCCAGCAGCUUCAGUCCCUUUCTUIUCGAU at no more than 1, 2, 3, 4 or 5 positions.

729. The oligonucleotide according to any one of the above embodiments, wherein the base sequence of the oligonucleotide is or comprises a sequence different from CCCAGCAGCUUCAGUCCCUUUCTUIUCGAU at no more than 1, 2, 3, 4 or 5 positions.

730. The oligonucleotide according to any one of the above embodiments, wherein the base sequence of the oligonucleotide is or comprises CCCAGCAGCUUCAGUCCCUUUCTUIUCGAU, and each U can be independently replaced by T and vice versa.

731. The oligonucleotide according to any one of the above embodiments, wherein the base sequence of the oligonucleotide is or comprises CCCAGCAGCUUCAGUCCCUUUCTUIUCGAU, and each U can be independently replaced by T and vice versa.

732. The oligonucleotide according to any one of the above embodiments, wherein the base sequence of the oligonucleotide is or comprises CCCAGCAGCUUCAGUCCCUUUCTUIUCGAU, and each U can be independently replaced by T and vice versa.

733. The oligonucleotide according to any one of the above embodiments, wherein the nucleotide sequence of the oligonucleotide is or comprises CCCAGCAGCUUCAGUCCCUUUCTUIUCGAU.

734. The oligonucleotide according to any one of the above embodiments, wherein the nucleotide sequence of the oligonucleotide is CCCAGCAGCUUCAGUCCCUUUCTUIUCGAU.

735. The oligonucleotide according to any one of embodiments 1 to 724, wherein the base sequence of the oligonucleotide is or comprises CCCAGCAGCUUCAGUCCCUUUCTUIUCGAU.

736. The oligonucleotide according to any one of embodiments 1 to 724, wherein the base sequence of the oligonucleotide is CCCAGCAGCUUCAGUCCCUUUCTUIUCGAU.

737. The oligonucleotide according to any one of embodiments 1 to 724, wherein the base sequence of the oligonucleotide is or comprises CCCAGCAGCUUCAGUCCCUUUCUAIUCGAU.

738. The oligonucleotide according to any one of embodiments 1 to 724, wherein the base sequence of the oligonucleotide is CCCAGCAGCUUCAGUCCCUUUCUAAIUCGAU.

739. Any one of the preceding embodiments, b001U, b002U, b003U, b004U, b005U, b006U, b007U, b008U, b009U, b011U, b012U, b013U, b001A, b002A, b003A, b001G, b002G, b001 C, b002C , b003C, b004C, b005C, b006C, b007C, b008C, b009C, b002I, b003I, b004I, b014I, and an optionally protected nucleobase of a nucleoside selected from zdnp.

740. Any one of the preceding embodiments, b001U, b002U, b003U, b004U, b005U, b006U, b008U, b002A, b001G, b004C, b007U, b001A, b001C, b002C, b003C, b002I, b003I, b009 U, b003A , and an optionally protected nucleobase of a nucleoside selected from b007C.

741. b001U, b002U, b003U, b004U, b005U, b006U, b007U, b008U, b009U, b011U, b012U, b013U, b001A, b002A, b003A, b001G, b002G, b001C, b002C, b00 3C, b004C, b005C, b006C, b007C, An oligonucleotide comprising an optionally protected nucleobase of a nucleoside selected from b008C, b009C, b002I, b003I, b004I, b014I, and zdnp.

742. b001U, b002U, b003U, b004U, b005U, b006U, b008U, b002A, b001G, b004C, b007U, b001A, b001C, b002C, b003C, b002I, b003I, b009U, b003A, and b0 Optionally protecting a nucleoside selected from 07C An oligonucleotide containing a nucleobase.

743. The oligonucleotide according to any one of the preceding embodiments, comprising an optionally protected nucleobase b001U.

744. The oligonucleotide according to any one of the preceding embodiments, comprising an optionally protected nucleobase b002U.

745. The oligonucleotide according to any one of the preceding embodiments, comprising an optionally protected nucleobase b003U.

746. The oligonucleotide according to any one of the preceding embodiments, comprising an optionally protected nucleobase b004U.

747. The oligonucleotide according to any one of the preceding embodiments, comprising an optionally protected nucleobase b005U.

748. The oligonucleotide according to any one of the preceding embodiments, comprising an optionally protected nucleobase b006U.

749. The oligonucleotide according to any one of the preceding embodiments, comprising an optionally protected nucleobase b007U.

750. The oligonucleotide according to any one of the preceding embodiments, comprising an optionally protected nucleobase b008U.

751. The oligonucleotide according to any one of the preceding embodiments, comprising an optionally protected nucleobase b009U.

752. The oligonucleotide according to any one of the preceding embodiments, comprising an optionally protected nucleobase b011U.

753. The oligonucleotide according to any one of the preceding embodiments, comprising an optionally protected nucleobase b012U.

754. The oligonucleotide according to any one of the preceding embodiments, comprising an optionally protected nucleobase b013U.

755. The oligonucleotide according to any one of the preceding embodiments, comprising an optionally protected nucleobase b001A.

756. The oligonucleotide according to any one of the preceding embodiments, comprising an optionally protected nucleobase b002A.

757. The oligonucleotide according to any one of the preceding embodiments, comprising an optionally protected nucleobase b003A.

758. The oligonucleotide according to any one of the preceding embodiments, comprising an optionally protected nucleobase b001G.

759. The oligonucleotide according to any one of the preceding embodiments, comprising an optionally protected nucleobase b002G.

760. The oligonucleotide according to any one of the preceding embodiments, comprising an optionally protected nucleobase b001C.

761. The oligonucleotide according to any one of the preceding embodiments, comprising an optionally protected nucleobase b002C.

762. The oligonucleotide according to any one of the preceding embodiments, comprising an optionally protected nucleobase b003C.

763. The oligonucleotide according to any one of the preceding embodiments, comprising an optionally protected nucleobase b004C.

764. The oligonucleotide according to any one of the preceding embodiments, comprising an optionally protected nucleobase b005C.

765. The oligonucleotide according to any one of the preceding embodiments, comprising an optionally protected nucleobase b006C.

766. The oligonucleotide according to any one of the preceding embodiments, comprising an optionally protected nucleobase b007C.

767. The oligonucleotide according to any one of the preceding embodiments, comprising an optionally protected nucleobase b008C.

768. The oligonucleotide according to any one of the preceding embodiments, comprising an optionally protected nucleobase b009C.

769. The oligonucleotide according to any one of the preceding embodiments, comprising an optionally protected nucleobase b002I.

770. The oligonucleotide according to any one of the preceding embodiments, comprising an optionally protected nucleobase b003I.

771. The oligonucleotide according to any one of the preceding embodiments, comprising an optionally protected nucleobase b004I.

772. The oligonucleotide according to any one of the preceding embodiments, comprising an optionally protected nucleobase b014I.

773. The oligonucleotide according to any one of the preceding embodiments, comprising an optionally protected nucleobase and zdnp.

774. The method of any one of the preceding embodiments, wherein aC, b001U, b002U, b003U, b004U, b005U, b006U, b007U, b008U, b009U, b010U, b011U, b012U, b013U, b001A, b001rA, b002A, b003A, b001G C sm11, Gsm11, Tsm11, b009Csm11, b009Csm12 , Gsm12, Tsm12, Csm12, rCsm13, rCsm14, Csm15, Csm16, Csm17, L034, zdnp, and an optionally protected nucleoside selected from Tsm18.

775. Any one of the preceding embodiments, b001U, b002U, b003U, b004U, b005U, b006U, b008U, b002A, b001G, b004C, b007U, b001A, b001C, b002C, b003C, b002I, b003I, b009 U, b003A , b007C, Asm01, Gsm01, 5MSfC, Usm04, 5MRdT, Csm15, Csm16, rCsm14, Csm17, and an optionally protected nucleoside selected from Tsm18.

776. aC, b001U, b002U, b003U, b004U, b005U, b006U, b007U, b008U, b009U, b010U, b011U, b012U, b013U, b001A, b001rA, b002A, b003A, b001G, b002G , b001C, b002C, b003C, b003mC, b004C, b005C, b006C, b007C, b008C, b002I, b003I, b004I, b014I, Asm01, Gsm01, 5MSfC, Usm04, 5MRdT, Csm04, Csm11, Gsm11, Tsm11, b009Csm11, b009Cs m12, Gsm12, Tsm12, Csm12, rCsm13, rCsm14, An oligonucleotide comprising an optionally protected nucleoside selected from Csm15, Csm16, Csm17, L034, zdnp, and Tsm18.

777. b001U, b002U, b003U, b004U, b005U, b006U, b008U, b002A, b001G, b004C, b007U, b001A, b001C, b002C, b003C, b002I, b003I, b009U, b003A, b00 7C, Asm01, Gsm01, 5MSfC, Usm04, An oligonucleotide comprising an optionally protected nucleoside selected from 5MRdT, Csm15, Csm16, rCsm14, Csm17, and Tsm18.

778. An oligonucleotide according to any one of the preceding embodiments comprising an optionally protected sugar of a nucleoside selected from Asm01, Gsm01, 5MSfC, Usm04, 5MRdT, Csm15, Csm16, rCsm14, Csm17 and Tsm18.

779. The oligonucleotide according to any one of the preceding embodiments, comprising an optionally protected nucleoside aC.

780. The oligonucleotide according to any one of the preceding embodiments, comprising the optionally protected nucleoside b001U.

781. The oligonucleotide according to any one of the preceding embodiments, comprising the optionally protected nucleoside b002U.

782. The oligonucleotide according to any one of the preceding embodiments, comprising the optionally protected nucleoside b003U.

783. The oligonucleotide according to any one of the preceding embodiments, comprising the optionally protected nucleoside b004U.

784. The oligonucleotide according to any one of the preceding embodiments, comprising the optionally protected nucleoside b005U.

785. The oligonucleotide according to any one of the preceding embodiments, comprising the optionally protected nucleoside b006U.

786. The oligonucleotide according to any one of the preceding embodiments, comprising the optionally protected nucleoside b007U.

787. The oligonucleotide according to any one of the preceding embodiments, comprising the optionally protected nucleoside b008U.

788. The oligonucleotide according to any one of the preceding embodiments, comprising the optionally protected nucleoside b009U.

789. The oligonucleotide according to any one of the preceding embodiments, comprising the optionally protected nucleoside b010U.

790. The oligonucleotide of any one of the preceding embodiments, comprising the optionally protected nucleoside b011U.

791. The oligonucleotide of any one of the preceding embodiments, comprising the optionally protected nucleoside b012U.

792. The oligonucleotide according to any one of the preceding embodiments, comprising the optionally protected nucleoside b013U.

793. The oligonucleotide according to any one of the preceding embodiments, comprising the optionally protected nucleoside b001A.

794. The oligonucleotide according to any one of the preceding embodiments, comprising the optionally protected nucleoside b001rA.

795. The oligonucleotide according to any one of the preceding embodiments, comprising the optionally protected nucleoside b002A.

796. The oligonucleotide according to any one of the preceding embodiments, comprising the optionally protected nucleoside b003A.

797. The oligonucleotide according to any one of the preceding embodiments, comprising the optionally protected nucleoside b001G.

798. The oligonucleotide according to any one of the preceding embodiments, comprising the optionally protected nucleoside b002G.

799. The oligonucleotide according to any one of the preceding embodiments, comprising the optionally protected nucleoside b001C.

800. The oligonucleotide of any one of the preceding embodiments, comprising the optionally protected nucleoside b002C.

801. The oligonucleotide according to any one of the preceding embodiments, comprising the optionally protected nucleoside b003C.

802. The oligonucleotide according to any one of the preceding embodiments, comprising the optionally protected nucleoside b003mC.

803. The oligonucleotide according to any one of the preceding embodiments, comprising the optionally protected nucleoside b004C.

804. The oligonucleotide according to any one of the preceding embodiments, comprising the optionally protected nucleoside b005C.

805. The oligonucleotide according to any one of the preceding embodiments, comprising the optionally protected nucleoside b006C.

806. The oligonucleotide according to any one of the preceding embodiments, comprising the optionally protected nucleoside b007C.

807. The oligonucleotide according to any one of the preceding embodiments, comprising the optionally protected nucleoside b008C.

808. The oligonucleotide according to any one of the preceding embodiments, comprising the optionally protected nucleoside b002I.

809. The oligonucleotide according to any one of the preceding embodiments, comprising the optionally protected nucleoside b003I.

810. The oligonucleotide according to any one of the preceding embodiments, comprising the optionally protected nucleoside b004I.

811. The oligonucleotide according to any one of the preceding embodiments, comprising the optionally protected nucleoside b014I.

812. An oligonucleotide according to any one of the preceding embodiments, comprising the optionally protected nucleoside Asm01.

813. The oligonucleotide according to any one of the preceding embodiments, comprising the optionally protected nucleoside Gsm01.

814. The oligonucleotide according to any one of the preceding embodiments, comprising the optionally protected nucleoside 5MSfC.

815. An oligonucleotide according to any one of the preceding embodiments, comprising the optionally protected nucleoside Usm04.

816. The oligonucleotide according to any one of the preceding embodiments, comprising the optionally protected nucleoside 5MRdT.

817. The oligonucleotide according to any one of the preceding embodiments, comprising the optionally protected nucleoside Csm04.

818. An oligonucleotide according to any one of the preceding embodiments, comprising the optionally protected nucleoside Csm11.

819. An oligonucleotide according to any one of the preceding embodiments, comprising the optionally protected nucleoside Gsm11.

820. An oligonucleotide according to any one of the preceding embodiments, comprising the optionally protected nucleoside Tsm11.

821. An oligonucleotide according to any one of the preceding embodiments, comprising the optionally protected nucleoside b009Csm11.

822. The oligonucleotide according to any one of the preceding embodiments, comprising the optionally protected nucleoside b009Csm12.

823. An oligonucleotide according to any one of the preceding embodiments, comprising the optionally protected nucleoside Gsm12.

824. The oligonucleotide according to any one of the preceding embodiments, comprising the optionally protected nucleoside Tsm12.

825. The oligonucleotide according to any one of the preceding embodiments, comprising the optionally protected nucleoside Csm12.

826. The oligonucleotide according to any one of the preceding embodiments, comprising the optionally protected nucleoside rCsm13.

827. The oligonucleotide according to any one of the preceding embodiments, comprising the optionally protected nucleoside rCsm14.

828. The oligonucleotide according to any one of the preceding embodiments, comprising the optionally protected nucleoside Csm15.

829. The oligonucleotide according to any one of the preceding embodiments, comprising the optionally protected nucleoside Csm16.

830. An oligonucleotide according to any one of the preceding embodiments, comprising the optionally protected nucleoside Csm17.

831. The oligonucleotide according to any one of the preceding embodiments, comprising an optionally protected abasic nucleoside.

832. The oligonucleotide according to any one of the preceding embodiments, comprising an optionally protected L010.

833. The oligonucleotide according to any one of the preceding embodiments, comprising the optionally protected nucleoside L034.

834. The oligonucleotide according to any one of the preceding embodiments, comprising the optionally protected nucleoside zdnp.

835. The oligonucleotide according to any one of the preceding embodiments, comprising the optionally protected nucleoside Tsm18.

836. The oligonucleotide according to any one of the preceding embodiments, wherein each optionally protected nucleobase or nucleoside is independently each optionally substituted nucleobase or nucleoside.

837. The oligonucleotide according to any one of the preceding embodiments, wherein each optionally protected or substituted nucleobase or nucleoside is each unprotected or unsubstituted.

838. The oligonucleotide according to any one of the preceding embodiments, comprising an internucleotidic linkage having the structure -YP(=W)(-XR ^L )-Z-.

839. An oligonucleotide comprising an internucleotide linkage having the structure -YP(=W)(-XR ^L )-Z-.

840. The oligonucleotide of embodiment 838 or 839, wherein W is O.

841. The oligonucleotide of embodiment 838 or 839, wherein W is S.

842. The oligonucleotide according to any one of embodiments 838 to 841, wherein Y is -O-.

843. The oligonucleotide according to any one of embodiments 838 to 842, wherein Z is a covalent bond.

844. The oligonucleotide according to any one of embodiments 838 to 842, wherein Z is -O-.

845. An oligonucleotide comprising an internucleotide linkage comprising -XR ^L.

846. The oligonucleotide according to any one of the preceding embodiments, comprising an internucleotide linkage comprising -XR ^L.

847. The method of any one of embodiments 838 to 846, wherein -XR ^L is -N(R')SO ₂ R" and R" is R', -OR', or -N(R') ₂ , oligonucleotides.

848. The oligonucleotide according to any one of embodiments 838 to 846, wherein -XR ^L is -NHSO ₂ R″ and R″ is an optionally substituted C _1-6 aliphatic.

849. The oligonucleotide according to any one of embodiments 838 to 846, wherein -XR ^L is -NHSO ₂ R" and R" is methyl.

850. The oligonucleotide according to any one of embodiments 838 to 846, wherein -XR ^L is -NHSO ₂ R" and R" is optionally substituted phenyl.

851. The oligonucleotide according to any one of the preceding embodiments, comprising n002.

852. The oligonucleotide according to any one of the preceding embodiments, comprising n006.

853. The oligonucleotide according to any one of the preceding embodiments, comprising n020.

854. The oligonucleotide according to any one of the preceding embodiments, comprising -OP(=0)(NHSO ₂ CH ₃ )O-.

855. The oligonucleotide according to any one of the preceding embodiments, wherein the first 1, 2 or 3 internucleotide linkages are each independently an internucleotide linkage of any one of embodiments 847-854.

856. The oligonucleotide according to any one of the preceding embodiments, wherein the last one, two or three internucleotide linkages are each independently an internucleotide linkage of any one of embodiments 847-854.

857. The oligonucleotide according to any one of the preceding embodiments, wherein the one or more internal internucleotide linkages are each independently an internucleotide linkage of any one of embodiments 847-854.

858. The method of any one of embodiments 838 to 846, wherein -XR ^L is -N(R')C(0)R" and R" is R', -OR', or -N(R') ₂ persons, oligonucleotide.

859. The oligonucleotide according to any one of embodiments 838 to 846, wherein -XR ^L is -NHC(O)R" and R" is an optionally substituted C _1-6 aliphatic.

860. The oligonucleotide according to any one of embodiments 838 to 846, wherein -XR ^L is -NHC(O)R" and R" is methyl.

861. The oligonucleotide according to any one of embodiments 838 to 846, wherein -XR ^L is -NHC(O)R" and R" is optionally substituted phenyl.

862. The oligonucleotide according to any one of embodiments 838 to 846, wherein -XR ^L is -NHC(O)R" and R" is -OR'.

863. The oligonucleotide according to any one of embodiments 838 to 846, wherein -XR ^L is -NHC(O)R" and R" is -N(R') ₂ .

864. The method of any one of embodiments 838 to 846, wherein -XR ^L is -N(R')P(O)(R") ₂ and each R" is independently R', -OR', or -N(R') ₂ , an oligonucleotide.

865. The method of any one of embodiments 838-846, wherein -XR ^L is -N(R')P(S)(R") ₂ and each R" is independently R', -OR', or -N(R') ₂ , an oligonucleotide.

866. The oligonucleotide according to any one of embodiments 838 to 846, wherein -XR ^L is selected from Table L-1, L-2, L-3, L-4, L-5 or L-6.

867. The method of any one of the preceding embodiments, wherein about 20% to 90% (e.g., about 20% to 80%, 20% to 70%, 30% to 90%, 30%) of all sugars in the oligonucleotide -80%, 30%-70%, 30%-60%, 30%-50%, about 30%, 40%, 50%, 60% or 70%) is a 2'-F modified sugar.

868. The method of any one of the preceding embodiments, wherein about 30% to 70% of all sugars in the oligonucleotide (e.g., about 30% to 60%, 30% to 50%, about 30%, 40%, 50%) %, 60% or 70%) is a 2'-F modified sugar.

869. The method of any one of the preceding embodiments, wherein about 30% to 60% (e.g., about 40% to 60%, 30% to 50%, about 30%, 40%, 50%) of all sugars in the oligonucleotide %, 60% or 70%) is a 2'-F modified sugar.

870. The oligonucleotide of any one of the preceding embodiments, wherein at least about 65% of all sugars in the oligonucleotide are 2'-F modified sugars.

871. The oligonucleotide of any one of the preceding embodiments, wherein at least about 70% of all sugars in the oligonucleotide are 2'-F modified sugars.

872. The oligonucleotide of any one of the preceding embodiments, wherein at least about 75% of all sugars in the oligonucleotide are 2'-F modified sugars.

873. The oligonucleotide of any one of the preceding embodiments, wherein at least about 80% of all sugars in the oligonucleotide are 2'-F modified sugars.

874. The oligonucleotide of any one of the preceding embodiments, wherein at least about 85% of all sugars in the oligonucleotide are 2'-F modified sugars.

875. The oligonucleotide of any one of the preceding embodiments, wherein at least about 90% of all sugars in the oligonucleotide are 2'-F modified sugars.

876. The method of any one of the preceding embodiments, wherein about 20% to 90% (e.g., about 20% to 80%, 20% to 70%, 30% to 90%, 30%) of all sugars in the oligonucleotide ~80%, 30%~70%, 30%~60%, 30%~50%, about 30%, 40%, 50%, 60% or 70%) each independently per 2'-OR variant (R is not -H).

877. The method of any one of the preceding embodiments, wherein about 30% to 70% of all sugars in the oligonucleotide (e.g., about 30% to 60%, 30% to 50%, about 30%, 40%, 50%) %, 60% or 70%) are each independently a 2'-OR modified sugar (R is not -H).

878. The method of any one of the preceding embodiments, wherein about 30% to 60% (e.g., about 40% to 60%, 30% to 50%, about 30%, 40%, 50%) of all sugars in the oligonucleotide %, 60% or 70%) are each independently a 2'-OR modified sugar (R is not -H).

879. The oligonucleotide according to any one of embodiments 876 to 878, wherein the 2'-OR modified sugar is a 2'-OR modified sugar wherein R is an optionally substituted C _1-6 aliphatic.

880. The oligonucleotide according to any one of the preceding embodiments, wherein the 2'-OR modified sugar is a 2'-OMe modified sugar.

881. The oligonucleotide according to any one of the preceding embodiments, wherein the 2'-OR modified sugar is a 2'-MOE modified sugar.

882. The oligonucleotide according to any one of the preceding embodiments, wherein the 2'-OR modified sugar is a bicyclic sugar.

883. The oligonucleotide according to any one of the preceding embodiments, wherein the 2'-OR modified sugar is an LNA sugar.

884. The oligonucleotide according to any one of the preceding embodiments, wherein the 2'-OR modified sugar is a cEt sugar.

885. The oligonucleotide according to any one of embodiments 876 to 878, wherein each 2'-OR modified sugar is independently a 2'-OR modified sugar wherein R is an optionally substituted C _1-6 aliphatic.

886. The oligonucleotide according to any one of embodiments 876 to 878, wherein each 2'-OR modified sugar is independently a 2'-OMe or 2'-MOE modified sugar.

887. The method according to any one of embodiments 876 to 878, wherein each 2'-OR modified sugar is independently a 2'-OMe or 2'-MOE modified sugar, and at least one is a 2'-OMe modified sugar and at least One is a 2'-MOE modified sugar, an oligonucleotide.

888. The oligonucleotide according to any one of embodiments 876 to 878, wherein each 2'-OR modified sugar is a 2'-OMe modified sugar.

889. The method of any one of the preceding embodiments, wherein the first domain is one or more (e.g., 1-20, 1-15, 1-14, 1-13, 1-12, 1-11, 1- 10, 2~20, 3~15, 4~15, 5~15, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 , 17, 18, 19, 20, etc.) of 2'-F blocks and one or more (e.g., 1-20, 1-15, 1-14, 1-13, 1-12, 1-11, 1 ~10, 2~20, 3~15, 4~15, 5~15, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, etc.), each sugar of each 2'-F block is independently a 2'-F modified sugar, and each sugar of each separation block is oligonucleotides, which are independently sugars other than 2'-F modified sugars.

890. The oligonucleotide of any one of the preceding embodiments, wherein the first domain has two or more 2'-F blocks.

891. The oligonucleotide of any one of the preceding embodiments, wherein the first domain has three or more 2'-F blocks.

892. The oligonucleotide of any one of the preceding embodiments, wherein the first domain has at least four 2'-F blocks.

893. The oligonucleotide of any one of the preceding embodiments, wherein the first domain has at least 5 2'-F blocks.

894. The oligonucleotide of any one of the preceding embodiments, wherein the first domain has two or more separating blocks.

895. The oligonucleotide of any one of the preceding embodiments, wherein the first domain has three or more separating blocks.

896. The oligonucleotide of any one of the preceding embodiments, wherein the first domain has 4 or more separating blocks.

897. The oligonucleotide of any one of the preceding embodiments, wherein the first domain has at least 5 separating blocks.

898. The oligonucleotide of any one of the preceding embodiments, wherein each sugar of each separating block is independently a 2'-modified sugar.

899. The oligonucleotide of any one of the preceding embodiments, wherein the sugars of the separating blocks are independently 2'-OR sugars (R is not -H).

900. The oligonucleotide of any one of the preceding embodiments, wherein each separating block independently comprises a 2'-OR modified sugar (R is not -H).

901. The oligonucleotide of any one of the preceding embodiments, wherein the sugars of the separating blocks are independently 2'-OR sugars, where R is an optionally substituted C _1-6 aliphatic.

902. The oligonucleotide of any one of the preceding embodiments, wherein each separating block independently comprises a 2'-OR modified sugar, wherein R is an optionally substituted C _1-6 aliphatic.

903. The oligonucleotide of any one of the preceding embodiments, wherein each sugar in each separating block is independently a 2'-OR modified sugar or a bicyclic sugar (R is an optionally substituted C _1-6 aliphatic). .

904. The oligonucleotide of any one of the preceding embodiments, wherein each sugar in the separating block is independently a 2'-OR modified sugar, wherein R is an optionally substituted C _1-6 aliphatic.

905. The oligonucleotide of any one of the preceding embodiments, wherein each sugar of each separating block is independently a 2'-OR modified sugar, wherein R is an optionally substituted C _1-6 aliphatic.

906. The oligonucleotide of any one of the preceding embodiments, wherein each sugar in the separating block is independently a 2'-OMe or 2'-MOE modified sugar.

907. The oligonucleotide of any one of the preceding embodiments, wherein each sugar in each separating block is independently a 2'-OMe or 2'-MOE modified sugar.

908. The oligonucleotide of any one of the preceding embodiments, wherein the sugar of the separating block is a 2'-MOE modified sugar.

909. The oligonucleotide of any one of the preceding embodiments, wherein each sugar in the separating block is independently a 2'-OMe modified sugar.

910. The oligonucleotide of any one of the preceding embodiments, wherein each sugar in the separating block is independently a 2'-MOE modified sugar.

911. The oligonucleotide of any one of embodiments 1-897, wherein each sugar of each separating block is independently a 2'-OMe modified sugar.

912. The oligonucleotide of any one of embodiments 1-897, wherein each sugar of each separating block is independently a 2'-MOE modified sugar.

913. The method of any one of embodiments 889 to 912, wherein each 2'-F block independently contains about 1-20 (e.g., 1-20, 1-15, 1-14, 1-13 , 1~12, 1~11, 1~10, 2~20, 3~15, 4~15, 5~15, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, etc.) 2'-F modified sugars.

914. The method of any one of embodiments 889 to 912, wherein each 2'-F block contains about 1-10, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 , or oligonucleotides with 10 2'-F modified sugars.

915. The oligonucleotide of any one of embodiments 889 to 912, wherein each 2'-F block has about 1, 2, 3, 4, or 5 2'-F modified sugars.

916. The oligonucleotide according to any one of embodiments 889 to 912, wherein each 2'-F block has about 1, 2, or 3 2'-F modified sugars.

917. The method of any one of embodiments 889-916, wherein each separation block independently contains about 1-20 (e.g., 1-20, 1-15, 1-14, 1-13, 1- 12, 1~11, 1~10, 2~20, 3~15, 4~15, 5~15, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 , 13, 14, 15, 16, 17, 18, 19, 20, etc.) sugars, oligonucleotides.

918. The method of any one of embodiments 889-916, wherein each separation block contains about 1-10, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 An oligonucleotide with two sugars.

919. The oligonucleotide of any one of embodiments 889 to 916, wherein each separating block has about 1, 2, 3, 4, or 5 sugars.

920. The oligonucleotide according to any one of embodiments 889 to 916, wherein each separation has about 1, 2, or 3 sugars.

921. The oligonucleotide according to any one of embodiments 889 to 920, wherein each block of the first domain linked to the 2'-F block of the first domain is a separate block.

922. The oligonucleotide according to any one of embodiments 889 to 921, wherein each block of the first domain linked to the separating block of the first domain is a 2'-F block.

923. The method of any one of the preceding embodiments, wherein the oligonucleotide comprises two or more blocks per 2'-F modification, each block per 2'-F modification independently of 2, 3, 4, 5, comprising or consisting of 6, 7, 8, 9 or 10 consecutive 2'-F variant sugars, each two consecutive 2'-F variant sugar blocks being independently separated by a separating block, such separation wherein the blocks contain one or more sugars that are not independently 2'-F modified sugars and no consecutive 2'-F modified sugars.

924. The method of any one of the preceding embodiments, wherein at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% of all phosphorothioate internucleotide linkages (e.g. 50%~100%, 60%~100%, 70~100%, 75%~100%, 80%~100%, 90%~100%, 95%~100%, 60%~95% %, 70%~95%, 75~95%, 80~95%, 85~95%, 90~95%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95 %, or 99%, etc.), or all phosphorothioate internucleotide linkages are Sp .

925. The oligonucleotide of any one of the preceding embodiments, wherein the first domain is 5' to the second domain.

926. The oligonucleotide of any one of the preceding embodiments, wherein the first domain is 3' to the second domain.

927. The oligo according to any one of the preceding embodiments, wherein in the second domain the first subdomain is on the 5' side of the second subdomain and the third subdomain is on the 3' side of the second subdomain. nucleotide.

928. The method of any one of the preceding embodiments, wherein the oligonucleotide comprises 5'-N ₁ N ₀ N _-1 -3', wherein each of N _-1 , N ₀ , and N ₁ is independently a nucleoside Phosphorus, oligonucleotide.

929. The method of any one of the preceding embodiments, wherein the oligonucleotide comprises 5'-N ₂ N ₁ N ₀ N _-1 N _-2 -3', N ₂ , N ₁ , N ₀ , N _-1 and N _-2 are each independently a nucleoside.

930. The method of any one of the preceding embodiments, wherein the oligonucleotide comprises 5'-N ₃ N ₂ N ₁ N ₀ N _-1 N _-2 N _-3 -3', N ₃ , N ₂ , N ₁ , N ₀ , N _-1 , N _-2 and N _-3 are each independently a nucleoside.

931. The method of any one of the preceding embodiments, wherein the oligonucleotide comprises 5'-N ₄ N ₃ N ₂ N ₁ N ₀ N _-1 N _-2 N _-3 N _-4 -3', and N ₄ , N ₃ , N ₂ , N ₁ , N ₀ , N _-1 , N _-2 , N _-3 and N _-4 are each independently a nucleoside.

932. The method of any one of the preceding embodiments, wherein the oligonucleotide is 5'-N ₅ N ₄ N ₃ N ₂ N ₁ N ₀ N _-1 N _-2 N _-3 N _-4 N _-5 -3' Including, N ₅ , N ₄ , N ₃ , N ₂ , N ₁ , N ₀ , N _-1 , N _-2 , N _-3 , N _-4 and N _-5 each independently a nucleoside, an oligo nucleotide.

933. The method of any one of the preceding embodiments, wherein the oligonucleotide is 5'-N ₆ N ₅ N ₄ N ₃ N ₂ N ₁ N ₀ N _-1 N _-2 N _-3 N _-4 N _-5 N _{- 6-3} ', N ₆ , N ₅ , N _{4 , N 3 , N 2 , N 1 , N 0} , N _-1 , N _-2 , N _-3 , N _-4 , N _-5 and N _-6 An oligonucleotide, each independently a nucleoside.

934. The method of any one of the preceding embodiments, wherein the second subdomain comprises 5'-N ₁ N ₀ N _-1 -3', and each of N _-1 , N ₀ , and N ₁ independently represents a new A cleoside, an oligonucleotide.

935. An oligonucleotide comprising 5'-N ₁ N ₀ N _-1 -3' as described herein.

936. The oligonucleotide according to any one of the above embodiments, wherein when the oligonucleotide is aligned with the target nucleic acid, N ₀ is opposite to the target adenosine.

937. The method of any one of the preceding embodiments, wherein N ₋₁ , N ₀ and N ₁ are each independently 2′-F modified sugars, native RNA sugars, or 2′ replacing 2′-OH of native RNA sugars. -Oligonucleotides having sugars without substituents.

938. The oligonucleotide of any one of the above embodiments, wherein N _-1 , N ₀ and N ₁ each independently have a 2'-F modified sugar, a native RNA sugar, or a 2'-substituent free sugar .

939. The oligonucleotide according to any one of the above embodiments, wherein N ₋₁ , N ₀ and N ₁ each independently have a 2′-F modified sugar, a natural RNA sugar, or a natural DNA sugar.

940. The oligonucleotide of any one of the preceding embodiments, wherein no more than one of N ₋₁ , N ₀ and N ₁ has a 2′-F modified sugar.

941. The oligonucleotide of any one of the preceding embodiments, wherein no more than one of N ₋₁ , N ₀ and N ₁ has a native RNA sugar.

942. The oligonucleotide according to any one of the preceding embodiments, wherein the sugar of N ₁ is a 2′-F modified sugar.

943. The oligonucleotide according to any one of embodiments 1 to 941, wherein the sugar of N ₀ is a sugar containing no substituents at the position corresponding to the 2'-OH of the native RNA sugar.

944. The oligonucleotide according to any one of embodiments 1 to 941, wherein the sugar of N ₀ is a sugar that does not contain a 2'-substituent.

945. The oligonucleotide according to any one of embodiments 1 to 941, wherein the sugar of N ₁ is a natural DNA sugar.

946. The oligonucleotide according to any one of embodiments 1 to 941, wherein the sugar of N1 is a native RNA sugar.

947. The oligonucleotide according to any one of the preceding embodiments, wherein the sugar of N ₀ is a modified sugar.

948. The oligonucleotide of any one of the preceding embodiments, wherein the sugar of N ₀ is a 2′-F modified sugar.

949. The oligonucleotide according to any one of embodiments 1 to 946, wherein the sugar of N ₀ is a sugar containing no substituents at the position corresponding to the 2'-OH of the native RNA sugar.

950. The oligonucleotide according to any one of embodiments 1 to 946, wherein the sugar of N0 is a sugar that does not contain a 2'-substituent.

951. The oligonucleotide of any one of embodiments 1-946, wherein the sugar of N ₀ is a 5'-modified sugar.

952. The oligonucleotide of any one of embodiments 1-946, wherein the sugar of N ₀ is a 5'-Me modified sugar.

953. The oligonucleotide of any one of embodiments 1-946, wherein the sugar of N ₀ is an acyclic sugar.

954. The oligonucleotide according to any one of embodiments 1 to 946, wherein the sugar of N ₀ is sm01.

955. The oligonucleotide according to any one of embodiments 1 to 946, wherein the sugar of N ₀ is sm15.

956. is any one of embodiments 1-946, wherein the sugar of N ₀ is a substituted natural DNA sugar wherein one of the 2'-H is substituted with -OH or -F and the other 2'-H is unsubstituted; oligonucleotide.

957. The oligonucleotide of any one of embodiments 1-946, wherein the sugar of N ₀ is a natural DNA sugar.

958. The oligonucleotide according to any one of embodiments 1-946, wherein the sugar of N ₀ is a native RNA sugar.

959. The oligonucleotide according to any one of embodiments 1-958, wherein the nucleobase of N ₀ is C.

960. The oligonucleotide according to any one of embodiments 1-958, wherein the nucleobase of N ₀ is hypoxanthine.

961. The oligonucleotide according to any one of embodiments 1-958, wherein the nucleobase of N ₀ is T.

962. The oligonucleotide according to any one of embodiments 1-958, wherein the nucleobase of N ₀ is A.

963. The oligonucleotide according to any one of embodiments 1-958, wherein the nucleobase of N ₀ is G.

964. The oligonucleotide according to any one of embodiments 1-958, wherein the nucleobase of N ₀ is U.

965. The oligonucleotide according to any one of embodiments 1-958, wherein the nucleobase of N ₀ is b001U.

966. The oligonucleotide according to any one of embodiments 1-958, wherein the nucleobase of N ₀ is b002U.

967. The oligonucleotide according to any one of embodiments 1-958, wherein the nucleobase of N ₀ is b003U.

968. The oligonucleotide according to any one of embodiments 1-958, wherein the nucleobase of N ₀ is b004U.

969. The oligonucleotide according to any one of embodiments 1-958, wherein the nucleobase of N ₀ is b005U.

970. The oligonucleotide according to any one of embodiments 1-958, wherein the nucleobase of N ₀ is b006U.

971. The oligonucleotide according to any one of embodiments 1-958, wherein the nucleobase of N ₀ is b007U.

972. The oligonucleotide according to any one of embodiments 1-958, wherein the nucleobase of N ₀ is b008U.

973. The oligonucleotide according to any one of embodiments 1-958, wherein the nucleobase of N ₀ is b009U.

974. The oligonucleotide according to any one of embodiments 1-958, wherein the nucleobase of N ₀ is b011U.

975. The oligonucleotide according to any one of embodiments 1-958, wherein the nucleobase of N ₀ is b012U.

976. The oligonucleotide according to any one of embodiments 1-958, wherein the nucleobase of N ₀ is b013U.

977. The oligonucleotide according to any one of embodiments 1-958, wherein the nucleobase of N ₀ is b001A.

978. The oligonucleotide according to any one of embodiments 1-958, wherein the nucleobase of N ₀ is b002A.

979. The oligonucleotide according to any one of embodiments 1-958, wherein the nucleobase of N ₀ is b003A.

980. The oligonucleotide according to any one of embodiments 1-958, wherein the nucleobase of N ₀ is b001G.

981. The oligonucleotide according to any one of embodiments 1-958, wherein the nucleobase of N ₀ is b002G.

982. The oligonucleotide according to any one of embodiments 1-958, wherein the nucleobase of N ₀ is b001C.

983. The oligonucleotide according to any one of embodiments 1-958, wherein the nucleobase of N ₀ is b002C.

984. The oligonucleotide according to any one of embodiments 1-958, wherein the nucleobase of N ₀ is b003C.

985. The oligonucleotide according to any one of embodiments 1-958, wherein the nucleobase of N ₀ is b004C.

986. The oligonucleotide according to any one of embodiments 1-958, wherein the nucleobase of N ₀ is b005C.

987. The oligonucleotide according to any one of embodiments 1-958, wherein the nucleobase of N ₀ is b006C.

988. The oligonucleotide according to any one of embodiments 1-958, wherein the nucleobase of N ₀ is b007C.

989. The oligonucleotide according to any one of embodiments 1-958, wherein the nucleobase of N ₀ is b008C.

990. The oligonucleotide according to any one of embodiments 1-958, wherein the nucleobase of N ₀ is b009C.

991. The oligonucleotide according to any one of embodiments 1 to 958, wherein the nucleobase of N ₀ is b002I.

992. The oligonucleotide according to any one of embodiments 1-958, wherein the nucleobase of N ₀ is b003I.

993. The oligonucleotide according to any one of embodiments 1-958, wherein the nucleobase of N ₀ is b004I.

994. The oligonucleotide according to any one of embodiments 1-958, wherein the nucleobase of N ₀ is b014I.

995. The oligonucleotide according to any one of embodiments 1-958, wherein the nucleobase of N ₀ is zndp.

996. The oligonucleotide according to any one of embodiments 1-946, wherein N ₀ is dC.

997. The oligonucleotide according to any one of embodiments 1-946, wherein N ₀ is fU.

998. The oligonucleotide according to any one of embodiments 1-946, wherein N ₀ is dU.

999. The oligonucleotide according to any one of embodiments 1-946, wherein N ₀ is fA.

1000. The oligonucleotide according to any one of embodiments 1-946, wherein N ₀ is dA.

1001. The oligonucleotide according to any one of embodiments 1-946, wherein N ₀ is fT.

1002. The oligonucleotide according to any one of embodiments 1-946, wherein N ₀ is dT.

1003. The oligonucleotide according to any one of embodiments 1-946, wherein N ₀ is fC.

1004. The oligonucleotide according to any one of embodiments 1-946, wherein N ₀ is fG.

1005. The oligonucleotide according to any one of embodiments 1-946, wherein N ₀ is dG.

1006. The oligonucleotide according to any one of embodiments 1-946, wherein N ₀ is dI.

1007. The oligonucleotide according to any one of embodiments 1-946, wherein N ₀ is fI.

1008. The oligonucleotide according to any one of embodiments 1-946, wherein N ₀ is aC.

1009. The oligonucleotide according to any one of embodiments 1-946, wherein N ₀ is m5dC.

1010. The oligonucleotide according to any one of embodiments 1-946, wherein N ₀ is 5MRm5dC.

1011. The oligonucleotide of any one of embodiments 1-946, wherein N ₀ is 5MSm5dC.

1012. The oligonucleotide according to any one of embodiments 1-946, wherein N ₀ is b001G.

1013. The oligonucleotide according to any one of embodiments 1-946, wherein N ₀ is b002G.

1014. The oligonucleotide according to any one of embodiments 1-946, wherein N ₀ is b001C.

1015. The oligonucleotide according to any one of embodiments 1-946, wherein N ₀ is b002C.

1016. The oligonucleotide of any one of embodiments 1-946, wherein N ₀ is b003C.

1017. The oligonucleotide according to any one of embodiments 1-946, wherein N ₀ is b003mC.

1018. The oligonucleotide according to any one of embodiments 1-946, wherein N ₀ is b004C.

1019. The oligonucleotide according to any one of embodiments 1-946, wherein N ₀ is b005C.

1020. The oligonucleotide according to any one of embodiments 1-946, wherein N ₀ is b006C.

1021. The oligonucleotide of any one of embodiments 1-946, wherein N ₀ is b007C.

1022. The oligonucleotide according to any one of embodiments 1-946, wherein N ₀ is b008C.

1023. The oligonucleotide according to any one of embodiments 1-946, wherein N ₀ is b009C.

1024. The oligonucleotide according to any one of embodiments 1-946, wherein N ₀ is b001A.

1025. The oligonucleotide according to any one of embodiments 1-946, wherein N ₀ is b002A.

1026. The oligonucleotide according to any one of embodiments 1-946, wherein N ₀ is b003A.

1027. The oligonucleotide according to any one of embodiments 1-946, wherein N ₀ is b001U.

1028. The oligonucleotide according to any one of embodiments 1-946, wherein N ₀ is b002U.

1029. The oligonucleotide according to any one of embodiments 1-946, wherein N ₀ is b003U.

1030. The oligonucleotide according to any one of embodiments 1-946, wherein N ₀ is b004U.

1031. The oligonucleotide according to any one of embodiments 1-946, wherein N ₀ is b005U.

1032. The oligonucleotide according to any one of embodiments 1-946, wherein N ₀ is b006U.

1033. The oligonucleotide according to any one of embodiments 1-946, wherein N ₀ is b007U.

1034. The oligonucleotide according to any one of embodiments 1-946, wherein N ₀ is b008U.

1035. The oligonucleotide according to any one of embodiments 1-946, wherein N ₀ is b009U.

1036. The oligonucleotide according to any one of embodiments 1-946, wherein N ₀ is b010U.

1037. The oligonucleotide according to any one of embodiments 1 to 946, wherein N ₀ is b011U.

1038. The oligonucleotide according to any one of embodiments 1-946, wherein N ₀ is b012U.

1039. The oligonucleotide according to any one of embodiments 1 to 946, wherein N ₀ is b013U.

1040. The oligonucleotide according to any one of embodiments 1 to 946, wherein N ₀ is b002I.

1041. The oligonucleotide according to any one of embodiments 1 to 946, wherein N ₀ is b003I.

1042. The oligonucleotide according to any one of embodiments 1 to 946, wherein N ₀ is b004I.

1043. The oligonucleotide according to any one of embodiments 1 to 946, wherein N ₀ is b014I.

1044. The oligonucleotide according to any one of embodiments 1-946, wherein N ₀ is Asm01.

1045. The oligonucleotide according to any one of embodiments 1-946, wherein N ₀ is Gsm01.

1046. The oligonucleotide according to any one of embodiments 1-946, wherein N ₀ is Tsm01.

1047. The oligonucleotide according to any one of embodiments 1-946, wherein N ₀ is 5MSfC.

1048. The oligonucleotide according to any one of embodiments 1-946, wherein N ₀ is Usm04.

1049. The oligonucleotide according to any one of embodiments 1-946, wherein N ₀ is 5MRdT.

1050. The oligonucleotide according to any one of embodiments 1-946, wherein N ₀ is Csm04.

1051. The oligonucleotide according to any one of embodiments 1-946, wherein N ₀ is Csm11.

1052. The oligonucleotide according to any one of embodiments 1-946, wherein N ₀ is Gsm11.

1053. The oligonucleotide according to any one of embodiments 1-946, wherein N ₀ is Tsm11.

1054. The oligonucleotide according to any one of embodiments 1-946, wherein N ₀ is b009Csm11.

1055. The oligonucleotide according to any one of embodiments 1-946, wherein N ₀ is b009Csm12.

1056. The oligonucleotide according to any one of embodiments 1-946, wherein N ₀ is Gsm12.

1057. The oligonucleotide according to any one of embodiments 1-946, wherein N ₀ is Tsm12.

1058. The oligonucleotide according to any one of embodiments 1-946, wherein N ₀ is Csm12.

1059. The oligonucleotide according to any one of embodiments 1-946, wherein N ₀ is rCsm13.

1060. The oligonucleotide according to any one of embodiments 1-946, wherein N ₀ is rCsm14.

1061. The oligonucleotide according to any one of embodiments 1-946, wherein N ₀ is Csm15.

1062. The oligonucleotide according to any one of embodiments 1-946, wherein N ₀ is Csm16.

1063. The oligonucleotide according to any one of embodiments 1-946, wherein N ₀ is Csm17.

1064. The oligonucleotide according to any one of embodiments 1-946, wherein N ₀ is abasic.

1065. The oligonucleotide according to any one of embodiments 1-946, wherein N ₀ is L010.

1066. The oligonucleotide according to any one of embodiments 1-946, wherein N ₀ is L034.

1067. The oligonucleotide according to any one of embodiments 1-946, wherein N ₀ is Csm15.

1068. The oligonucleotide according to any one of embodiments 1-946, wherein N ₀ is Tsm18.

1069. The oligonucleotide according to any one of embodiments 1-946, wherein N ₀ is b001rA.

1070. The method of any one of the above embodiments, wherein the nucleobases of N ₁ are A, T, C, G, U, hypoxanthine, b001U, b002U, b003U, b004U, b005U, b006U, b007U, b008U, b009U, b011U, b012U, b013U, b001A, b002A, b003A, b001G, b002G, b001C, b002C, b003C, b004C, b005C, b006C, b007C, b008C, b009C, b002I, b003I, b004I, b0 14I or zdnp, an oligonucleotide.

1071. The oligonucleotide of any one of the preceding embodiments, wherein the nucleobase of N ₁ is a modified nucleobase.

1072. The oligonucleotide of any one of the above embodiments, wherein the sugar of N ₁ is a 2′-F modified sugar.

1073. The oligonucleotide according to any one of embodiments 1 to 1070, wherein the sugar of N ₁ is a sugar containing no substituents at the position corresponding to the 2'-OH of the native RNA sugar.

1074. The oligonucleotide according to any one of embodiments 1 to 1070, wherein the sugar of N1 is a sugar that does not contain a 2'-substituent.

1075. The oligonucleotide according to any one of embodiments 1 to 1070, wherein the sugar of N ₁ is a natural DNA sugar.

1076. The oligonucleotide according to any one of embodiments 1 to 1070, wherein the sugar of N ₁ is a natural RNA sugar.

1077. The oligonucleotide according to any one of embodiments 1 to 1070, wherein N ₁ is dA, dT, dC, dG, dU, fA, fT, fC, fG or fU.

1078. The method of any one of embodiments 1 to 1076, wherein the nucleobases of N ₁ are A, T, C, G, U, hypoxanthine, b001U, b002U, b003U, b004U, b005U, b006U, b007U, b008U, b009U, b011U, b012U, b013U, b001A, b002A, b003A, b001G, b002G, b001C, b002C, b003C, b004C, b005C, b006C, b007C, b008C, b009C, b002I, b003I, b0 oligonucleotide, which is 04I, b014I or zdnp.

1079. The oligonucleotide according to any one of embodiments 1 to 1070, wherein N ₁ is b001A.

1080. The oligonucleotide according to any one of embodiments 1 to 1070, wherein N ₁ is b002A.

1081. The oligonucleotide according to any one of embodiments 1 to 1070, wherein N ₁ is b003A.

1082. The oligonucleotide according to any one of embodiments 1 to 1070, wherein N ₁ is b001C.

1083. The oligonucleotide according to any one of embodiments 1 to 1070, wherein N ₁ is b004C.

1084. The oligonucleotide according to any one of embodiments 1 to 1070, wherein N ₁ is b007C.

1085. The oligonucleotide according to any one of embodiments 1 to 1070, wherein N ₁ is b008C.

1086. The oligonucleotide according to any one of embodiments 1 to 1070, wherein N ₁ is b008U.

1087. The oligonucleotide according to any one of embodiments 1 to 1070, wherein N ₁ is b010U.

1088. The oligonucleotide according to any one of embodiments 1 to 1070, wherein N ₁ is b011U.

1089. The oligonucleotide according to any one of embodiments 1 to 1070, wherein N ₁ is b012U.

1090. The oligonucleotide according to any one of embodiments 1 to 1070, wherein N ₁ is Csm11.

1091. The oligonucleotide according to any one of embodiments 1 to 1070, wherein N ₁ is Csm12.

1092. The oligonucleotide according to any one of embodiments 1 to 1070, wherein N ₁ is Csm17.

1093. The oligonucleotide according to any one of embodiments 1 to 1070, wherein N ₁ is b009Csm11.

1094. The oligonucleotide according to any one of embodiments 1 to 1070, wherein N ₁ is b009Csm12.

1095. The oligonucleotide according to any one of embodiments 1 to 1070, wherein N ₁ is Gsm01.

1096. The oligonucleotide according to any one of embodiments 1 to 1070, wherein N ₁ is Gsm11.

1097. The oligonucleotide according to any one of embodiments 1 to 1070, wherein N ₁ is Gsm12.

1098. The oligonucleotide according to any one of embodiments 1 to 1070, wherein N ₁ is Tsm01.

1099. The oligonucleotide according to any one of embodiments 1 to 1070, wherein N ₁ is Tsm11.

1100. The oligonucleotide according to any one of embodiments 1 to 1070, wherein N ₁ is Tsm12.

1101. The oligonucleotide according to any one of embodiments 1 to 1070, wherein N ₁ is Tsm18.

1102. The oligonucleotide according to any one of embodiments 1 to 1070, wherein N ₁ is L010.

1103. The oligonucleotide according to any one of the preceding embodiments, wherein the sugar of N _-1 is a modified sugar.

1104. The oligonucleotide according to any one of the preceding embodiments, wherein the sugar of N _-1 is a 2'-F modified sugar.

1105. The oligonucleotide according to any one of embodiments 1 to 1102, wherein the sugar of N _-1 is a sugar containing no substituent at the position corresponding to the 2'-OH of the native RNA sugar.

1106. The oligonucleotide according to any one of embodiments 1 to 1102, wherein the sugar of N _-1 is a sugar that does not contain a 2'-substituent.

1107. The oligonucleotide according to any one of embodiments 1 to 1102, wherein the sugar of N _-1 is a natural DNA sugar.

1108. The oligonucleotide according to any one of embodiments 1 to 1102, wherein the sugar of N _-1 is a natural RNA sugar.

1109. The oligonucleotide according to any one of the above embodiments, wherein N ₁ and N ₋₁ are both complementary to their corresponding nucleosides when the oligonucleotide is aligned with a target nucleic acid.

1110. The oligonucleotide of any one of the preceding embodiments, wherein at least one of N ₁ and N ₋₁ independently produces a mismatch or wobble base pair when the oligonucleotide is aligned with a target nucleic acid.

1111. The method of embodiment 1110, wherein the oligonucleotide provides a similar or higher level of editing of the target adenosine compared to the reference oligonucleotide, wherein the reference oligonucleotide is otherwise identical, but when the reference oligonucleotide is aligned with the target nucleic acid, the corresponding new An oligonucleotide having N ₁ and N ₋₁ complementary to the cleoside, wherein the target adenosine is opposite to N ₀ when the oligonucleotide is aligned with the target nucleic acid.

1112. The method of any one of the above embodiments, wherein the nucleobases of N _-1 are A, T, C, G, U, hypoxanthine, b001U, b002U, b003U, b004U, b005U, b006U, b007U, b008U, b009U , b011U, b012U, b013U, b001A, b002A, b003A, b001G, b002G, b001C, b002C, b003C, b004C, b005C, b006C, b007C, b008C, b009C, b002I, b003I, b004I, oligonucleotide, which is b014I or zdnp.

1113. The oligonucleotide according to any one of the preceding embodiments, wherein the nucleobase of N _-1 is a modified nucleobase.

1114. The oligonucleotide according to any one of embodiments 1 to 1111, wherein the nucleobase of N _-1 is hypoxanthine.

1115. The oligonucleotide according to any one of embodiments 1 to 1111, wherein the nucleobase of N _-1 is C.

1116. The oligonucleotide according to any one of embodiments 1 to 1111, wherein the nucleobase of N _-1 is T.

1117. The oligonucleotide according to any one of embodiments 1 to 1111, wherein the nucleobase of N _-1 is A.

1118. The oligonucleotide according to any one of embodiments 1 to 1111, wherein the nucleobase of N _-1 is G.

1119. The oligonucleotide according to any one of embodiments 1 to 1111, wherein the nucleobase of N _-1 is U.

1120. The oligonucleotide according to any one of embodiments 1 to 1111, wherein the nucleobase of N _-1 is b001U.

1121. The oligonucleotide according to any one of embodiments 1 to 1111, wherein the nucleobase of N _-1 is b002U.

1122. The oligonucleotide according to any one of embodiments 1 to 1111, wherein the nucleobase of N _-1 is b003U.

1123. The oligonucleotide according to any one of embodiments 1 to 1111, wherein the nucleobase of N _-1 is b004U.

1124. The oligonucleotide according to any one of embodiments 1 to 1111, wherein the nucleobase of N _-1 is b005U.

1125. The oligonucleotide according to any one of embodiments 1 to 1111, wherein the nucleobase of N _-1 is b006U.

1126. The oligonucleotide according to any one of embodiments 1 to 1111, wherein the nucleobase of N _-1 is b007U.

1127. The oligonucleotide according to any one of embodiments 1 to 1111, wherein the nucleobase of N _-1 is b008U.

1128. The oligonucleotide according to any one of embodiments 1 to 1111, wherein the nucleobase of N _-1 is b009U.

1129. The oligonucleotide according to any one of embodiments 1 to 1111, wherein the nucleobase of N _-1 is b011U.

1130. The oligonucleotide according to any one of embodiments 1 to 1111, wherein the nucleobase of N _-1 is b012U.

1131. The oligonucleotide according to any one of embodiments 1 to 1111, wherein the nucleobase of N _-1 is b013U.

1132. The oligonucleotide according to any one of embodiments 1 to 1111, wherein the nucleobase of N _-1 is b001A.

1133. The oligonucleotide according to any one of embodiments 1 to 1111, wherein the nucleobase of N _-1 is b002A.

1134. The oligonucleotide according to any one of embodiments 1 to 1111, wherein the nucleobase of N _-1 is b003A.

1135. The oligonucleotide according to any one of embodiments 1 to 1111, wherein the nucleobase of N _-1 is b001G.

1136. The oligonucleotide according to any one of embodiments 1 to 1111, wherein the nucleobase of N _-1 is b002G.

1137. The oligonucleotide according to any one of embodiments 1 to 1111, wherein the nucleobase of N _-1 is b001C.

1138. The oligonucleotide according to any one of embodiments 1 to 1111, wherein the nucleobase of N _-1 is b002C.

1139. The oligonucleotide according to any one of embodiments 1 to 1111, wherein the nucleobase of N _-1 is b003C.

1140. The oligonucleotide according to any one of embodiments 1 to 1111, wherein the nucleobase of N _-1 is b004C.

1141. The oligonucleotide according to any one of embodiments 1 to 1111, wherein the nucleobase of N _-1 is b005C.

1142. The oligonucleotide according to any one of embodiments 1 to 1111, wherein the nucleobase of N _-1 is b006C.

1143. The oligonucleotide according to any one of embodiments 1 to 1111, wherein the nucleobase of N _-1 is b007C.

1144. The oligonucleotide according to any one of embodiments 1 to 1111, wherein the nucleobase of N _-1 is b008C.

1145. The oligonucleotide according to any one of embodiments 1 to 1111, wherein the nucleobase of N _-1 is b009C.

1146. The oligonucleotide according to any one of embodiments 1 to 1111, wherein the nucleobase of N _-1 is b002I.

1147. The oligonucleotide according to any one of embodiments 1 to 1111, wherein the nucleobase of N _-1 is b003I.

1148. The oligonucleotide according to any one of embodiments 1 to 1111, wherein the nucleobase of N _-1 is b004I.

1149. The oligonucleotide according to any one of embodiments 1 to 1111, wherein the nucleobase of N _-1 is b014I.

1150. The oligonucleotide according to any one of embodiments 1 to 1111, wherein the nucleobase of N _-1 is zndp.

1151. The oligonucleotide according to any one of embodiments 1 to 1111, wherein N ₋₁ is dC.

1152. The oligonucleotide according to any one of embodiments 1 to 1111, wherein N ₋₁ is fU.

1153. The oligonucleotide according to any one of embodiments 1 to 1111, wherein N ₋₁ is dU.

1154. The oligonucleotide according to any one of embodiments 1 to 1111, wherein N ₋₁ is fA.

1155. The oligonucleotide according to any one of embodiments 1 to 1111, wherein N ₋₁ is dA.

1156. The oligonucleotide according to any one of embodiments 1 to 1111, wherein N ₋₁ is fT.

1157. The oligonucleotide according to any one of embodiments 1 to 1111, wherein N ₋₁ is dT.

1158. The oligonucleotide according to any one of embodiments 1 to 1111, wherein N ₋₁ is fC.

1159. The oligonucleotide according to any one of embodiments 1 to 1111, wherein N ₋₁ is fG.

1160. The oligonucleotide according to any one of embodiments 1 to 1111, wherein N ₋₁ is dG.

1161. The oligonucleotide according to any one of embodiments 1 to 1111, wherein N ₋₁ is dI.

1162. The oligonucleotide according to any one of embodiments 1 to 1111, wherein N ₋₁ is fI.

1163. The oligonucleotide according to any one of embodiments 1 to 1111, wherein N ₋₁ is aC.

1164. The oligonucleotide according to any one of embodiments 1 to 1111, wherein N ₋₁ is m5dC.

1165. The oligonucleotide according to any one of embodiments 1 to 1111, wherein N ₋₁ is 5MRm5dC.

1166. The oligonucleotide according to any one of embodiments 1 to 1111, wherein N ₋₁ is 5MSm5dC.

1167. The oligonucleotide according to any one of embodiments 1 to 1111, wherein N _-1 is b001G.

1168. The oligonucleotide according to any one of embodiments 1 to 1111, wherein N _-1 is b002G.

1169. The oligonucleotide according to any one of embodiments 1 to 1111, wherein N _-1 is b001C.

1170. The oligonucleotide according to any one of embodiments 1 to 1111, wherein N _-1 is b002C.

1171. The oligonucleotide according to any one of embodiments 1 to 1111, wherein N _-1 is b003C.

1172. The oligonucleotide according to any one of embodiments 1 to 1111, wherein N _-1 is b003mC.

1173. The oligonucleotide according to any one of embodiments 1 to 1111, wherein N _-1 is b004C.

1174. The oligonucleotide according to any one of embodiments 1 to 1111, wherein N _-1 is b005C.

1175. The oligonucleotide according to any one of embodiments 1 to 1111, wherein N _-1 is b006C.

1176. The oligonucleotide according to any one of embodiments 1 to 1111, wherein N _-1 is b007C.

1177. The oligonucleotide according to any one of embodiments 1 to 1111, wherein N _-1 is b008C.

1178. The oligonucleotide according to any one of embodiments 1 to 1111, wherein N _-1 is b009C.

1179. The oligonucleotide according to any one of embodiments 1 to 1111, wherein N _-1 is b001A.

1180. The oligonucleotide according to any one of embodiments 1 to 1111, wherein N _-1 is b002A.

1181. The oligonucleotide according to any one of embodiments 1 to 1111, wherein N _-1 is b003A.

1182. The oligonucleotide according to any one of embodiments 1 to 1111, wherein N ₋₁ is b001U.

1183. The oligonucleotide according to any one of embodiments 1 to 1111, wherein N _-1 is b002U.

1184. The oligonucleotide according to any one of embodiments 1 to 1111, wherein N _-1 is b003U.

1185. The oligonucleotide according to any one of embodiments 1 to 1111, wherein N _-1 is b004U.

1186. The oligonucleotide according to any one of embodiments 1 to 1111, wherein N _-1 is b005U.

1187. The oligonucleotide according to any one of embodiments 1 to 1111, wherein N _-1 is b006U.

1188. The oligonucleotide according to any one of embodiments 1 to 1111, wherein N _-1 is b007U.

1189. The oligonucleotide according to any one of embodiments 1 to 1111, wherein N _-1 is b008U.

1190. The oligonucleotide according to any one of embodiments 1 to 1111, wherein N _-1 is b009U.

1191. The oligonucleotide according to any one of embodiments 1 to 1111, wherein N _-1 is b010U.

1192. The oligonucleotide according to any one of embodiments 1 to 1111, wherein N _-1 is b011U.

1193. The oligonucleotide according to any one of embodiments 1 to 1111, wherein N _-1 is b012U.

1194. The oligonucleotide according to any one of embodiments 1 to 1111, wherein N _-1 is b013U.

1195. The oligonucleotide according to any one of embodiments 1 to 1111, wherein N _-1 is b002I.

1196. The oligonucleotide according to any one of embodiments 1 to 1111, wherein N _-1 is b003I.

1197. The oligonucleotide according to any one of embodiments 1 to 1111, wherein N _-1 is b004I.

1198. The oligonucleotide according to any one of embodiments 1 to 1111, wherein N _-1 is b014I.

1199. The oligonucleotide according to any one of embodiments 1 to 1111, wherein N _-1 is Asm01.

1200. The oligonucleotide according to any one of embodiments 1 to 1111, wherein N _-1 is Gsm01.

1201. The oligonucleotide according to any one of embodiments 1 to 1111, wherein N ₋₁ is 5MSfC.

1202. The oligonucleotide according to any one of embodiments 1 to 1111, wherein N _-1 is Usm04.

1203. The oligonucleotide according to any one of embodiments 1 to 1111, wherein N ₋₁ is 5MRdT.

1204. The oligonucleotide according to any one of embodiments 1 to 1111, wherein N _-1 is Csm04.

1205. The oligonucleotide according to any one of embodiments 1 to 1111, wherein N _-1 is Csm11.

1206. The oligonucleotide according to any one of embodiments 1 to 1111, wherein N _-1 is Gsm11.

1207. The oligonucleotide according to any one of embodiments 1 to 1111, wherein N _-1 is Tsm11.

1208. The oligonucleotide according to any one of embodiments 1 to 1111, wherein N _-1 is b009Csm11.

1209. The oligonucleotide according to any one of embodiments 1 to 1111, wherein N _-1 is b009Csm12.

1210. The oligonucleotide according to any one of embodiments 1 to 1111, wherein N _-1 is Gsm12.

1211. The oligonucleotide according to any one of embodiments 1 to 1111, wherein N _-1 is Tsm12.

1212. The oligonucleotide according to any one of embodiments 1 to 1111, wherein N _-1 is Csm12.

1213. The oligonucleotide according to any one of embodiments 1 to 1111, wherein N _-1 is rCsm13.

1214. The oligonucleotide according to any one of embodiments 1 to 1111, wherein N _-1 is rCsm14.

1215. The oligonucleotide according to any one of embodiments 1 to 1111, wherein N _-1 is Csm15.

1216. The oligonucleotide according to any one of embodiments 1 to 1111, wherein N _-1 is Csm16.

1217. The oligonucleotide according to any one of embodiments 1 to 1111, wherein N _-1 is Csm17.

1218. The oligonucleotide according to any one of embodiments 1 to 1111, wherein N ₋₁ is abasic.

1219. The oligonucleotide according to any one of embodiments 1 to 1111, wherein N _-1 is L010.

1220. The oligonucleotide according to any one of embodiments 1 to 1111, wherein N _-1 is L034.

1221. The oligonucleotide according to any one of embodiments 1 to 1111, wherein N _-1 is Csm15.

1222. The oligonucleotide according to any one of embodiments 1 to 1111, wherein N _-1 is Tsm18.

1223. The oligonucleotide according to any one of embodiments 1 to 1111, wherein N _-1 is b001rA.

1224. The oligonucleotide of any one of the preceding embodiments, wherein the internucleotide linkage between N ₀ and N ₁ is a phosphorothioate internucleotide linkage.

1225. The oligonucleotide of any one of the preceding embodiments, wherein the internucleotide linkage between N ₀ and N ₁ is an S p phosphorothioate internucleotide linkage.

1226. The oligonucleotide of any one of the preceding embodiments, wherein the internucleotide linkage between N ₀ and N ₋₁ is a phosphorothioate internucleotide linkage.

1227. The oligonucleotide of any one of the preceding embodiments, wherein the internucleotide linkage between N ₀ and N ₋₁ is an S p phosphorothioate internucleotide linkage.

1228. The oligonucleotide of any one of the preceding embodiments, wherein the sugar of N ₂ is a modified sugar.

1229. The oligonucleotide of any one of the preceding embodiments, wherein the sugar of N ₂ is a 2′-F modified sugar.

1230. The oligonucleotide of any one of the above embodiments, wherein the internucleotide linkage between N ₁ and N ₂ is a phosphorothioate internucleotide linkage.

1231. The oligonucleotide of any one of the above embodiments, wherein the internucleotide linkage between N ₁ and N ₂ is an S p phosphorothioate internucleotide linkage.

1232. The oligonucleotide of any one of the preceding embodiments, wherein the sugar of N ₃ is a modified sugar.

1233. The oligonucleotide according to any one of the preceding embodiments, wherein the sugar of N ₃ is a 2'-OR modified sugar (R is an optionally substituted C _1-6 aliphatic) or a bicyclic sugar.

1234. The oligonucleotide according to any one of the above embodiments, wherein the sugar of N ₃ is a 2′-OR modified sugar (R is an optionally substituted C _1-6 aliphatic).

1235. The oligonucleotide according to any one of the above embodiments, wherein the sugar of N ₃ is a 2′-OMe modified sugar.

1236. The oligonucleotide of embodiment 1233, wherein the sugar of N ₃ is a 2′-MOE modified sugar.

1237. The oligonucleotide of any one of the preceding embodiments, wherein the internucleotide linkage between N ₂ and N ₃ is a natural phosphate linkage.

1238. The oligonucleotide of any one of the preceding embodiments, wherein the sugar of N ₄ is a modified sugar.

1239. The oligonucleotide of any one of the preceding embodiments, wherein the sugar of N ₄ is a 2′-OR modified sugar (R is an optionally substituted C _1-6 aliphatic) or a bicyclic sugar.

1240. The oligonucleotide of any one of the preceding embodiments, wherein the sugar of N ₄ is a 2′-OR modified sugar (R is an optionally substituted C _1-6 aliphatic).

1241. The oligonucleotide according to any one of the above embodiments, wherein the sugar of N ₄ is a 2′-OMe modified sugar.

1242. The oligonucleotide of embodiment 1239, wherein the sugar of N ₄ is a 2′-MOE modified sugar.

1243. The oligonucleotide of any one of the preceding embodiments, wherein the internucleotide linkage between N ₃ and N ₄ is a natural phosphate linkage.

1244. The oligonucleotide of any one of the preceding embodiments, wherein the sugar of N ₅ is a modified sugar.

1245. The oligonucleotide according to any one of the above embodiments, wherein the sugar of N ₅ is a 2′-F modified sugar.

1246. The oligonucleotide of any one of the above embodiments, wherein the internucleotide linkage between N ₄ and N ₅ is a non-negatively charged internucleotide linkage.

1247. The oligonucleotide of any one of the preceding embodiments, wherein the internucleotide linkage between N ₄ and N ₅ is a phosphoryl guanidine internucleotide linkage.

1248. The oligonucleotide of any one of the preceding embodiments, wherein the internucleotide linkage between N ₄ and N ₅ is n001.

1249. The oligonucleotide according to any one of the preceding embodiments, wherein the internucleotide linkage between N ₄ and N ₅ is R p n001.

1250. The oligonucleotide of any one of the above embodiments, wherein the sugar of N ₆ is a modified sugar.

1251. The oligonucleotide of any one of the above embodiments, wherein the sugar of N ₆ is a 2′-F modified sugar.

1252. The oligonucleotide of any one of the preceding embodiments, wherein the internucleotide linkage between N ₅ and N ₆ is a phosphorothioate internucleotide linkage internucleotide linkage.

1253. The oligonucleotide of any one of the above embodiments, wherein the internucleotide linkage between N ₅ and N ₆ is an S p phosphorothioate internucleotide linkage internucleotide linkage.

1254. The oligonucleotide according to any one of the preceding embodiments, wherein the sugar of N _-2 is a modified sugar.

1255. The oligonucleotide according to any one of the preceding embodiments, wherein the sugar of N ₋₂ is a 2′-OR modified sugar (R is an optionally substituted C _1-6 aliphatic) or a bicyclic sugar.

1256. The oligonucleotide according to any one of the preceding embodiments, wherein the sugar of N ₋₂ is a 2′-OR modified sugar (R is an optionally substituted C _1-6 aliphatic).

1257. The oligonucleotide according to any one of the above embodiments, wherein the sugar of N _-2 is a 2'-OMe modified sugar.

1258. The oligonucleotide of embodiment 1255, wherein the sugar of N _-2 is a 2'-MOE modified sugar.

1259. The oligonucleotide according to any one of the preceding embodiments, wherein the internucleotide linkage between N _-1 and N _-2 is a non-negatively charged internucleotide linkage.

1260. The oligonucleotide of any one of the preceding embodiments, wherein the internucleotide linkage between N _-1 and N _-2 is a phosphoryl guanidine internucleotide linkage.

1261. The oligonucleotide according to any one of the preceding embodiments, wherein the internucleotide linkage between N _-1 and N _-2 is n004, n008, n025, n026.

1262. The oligonucleotide according to any one of the preceding embodiments, wherein the internucleotide linkage between N _-1 and N _-2 is R p n004, n008, n025, n026.

1263. The oligonucleotide according to any one of the preceding embodiments, wherein the internucleotide linkage between N _-1 and N _-2 is Sp n004, n008, n025, n026.

1264. The oligonucleotide according to any one of the preceding embodiments, wherein the internucleotide linkage between N _-1 and N _-2 is n001.

1265. The oligonucleotide according to any one of the preceding embodiments, wherein the internucleotide linkage between N _-1 and N _-2 is R p n001.

1266. The oligonucleotide of embodiment 1264, wherein the internucleotide linkage between N _-1 and N _-2 is Sp n001.

1267. The oligonucleotide according to any one of the above embodiments, wherein the sugar of N _-3 is a modified sugar.

1268. The oligonucleotide according to any one of the above embodiments, wherein the sugar of N _-3 is a 2'-F modified sugar.

1269. The oligonucleotide according to any one of the preceding embodiments, wherein the internucleotide linkage between N _-2 and N _-3 is a natural phosphate linkage.

1270. The oligonucleotide of any one of the above embodiments, wherein the sugar of N _-4 is a modified sugar.

1271. The oligonucleotide according to any one of the above embodiments, wherein the sugar of N _-4 is a 2'-OR modified sugar (R is an optionally substituted C _1-6 aliphatic) or a bicyclic sugar.

1272. The oligonucleotide according to any one of the preceding embodiments, wherein the sugar of N _-4 is a 2'-OR modified sugar (R is an optionally substituted C _1-6 aliphatic).

1273. The oligonucleotide according to any one of the above embodiments, wherein the sugar of N _-4 is a 2'-OMe modified sugar.

1274. The oligonucleotide of embodiment 1271, wherein the sugar of N _-4 is a 2'-MOE modified sugar.

1275. The oligonucleotide of any one of the preceding embodiments, wherein the linkage between N _-3 and N _-4 is a phosphorothioate internucleotidic linkage.

1276. The oligonucleotide according to any one of the above embodiments, wherein the linkage between N _-3 and N _-4 is an S p phosphorothioate internucleotidic linkage.

1277. The oligonucleotide according to any one of the above embodiments, wherein the sugar of N _-5 is a modified sugar.

1278. The oligonucleotide according to any one of the above embodiments, wherein the sugar of N _-5 is a 2'-OR modified sugar (R is an optionally substituted C _1-6 aliphatic) or a bicyclic sugar.

1279. The oligonucleotide according to any one of the preceding embodiments, wherein the sugar of N _-5 is a 2'-OR modified sugar (R is an optionally substituted C _1-6 aliphatic).

1280. The oligonucleotide according to any one of the preceding embodiments, wherein the sugar of N _-5 is a 2'-OMe modified sugar.

1281. The oligonucleotide of embodiment 1278, wherein the sugar of N _-5 is a 2'-MOE modified sugar.

1282. The oligonucleotide according to any one of the preceding embodiments, wherein the linkage between N _-4 and N _-5 is a phosphorothioate internucleotidic linkage.

1283. The oligonucleotide according to any one of the preceding embodiments, wherein the linkage between N _-4 and N _-5 is an S p phosphorothioate internucleotidic linkage.

1284. The oligonucleotide according to any one of the above embodiments, wherein the sugar of N _-6 is a modified sugar.

1285. The oligonucleotide according to any one of the above embodiments, wherein the sugar of N _-6 is a 2'-OR modified sugar (R is an optionally substituted C _1-6 aliphatic) or a bicyclic sugar.

1286. The oligonucleotide according to any one of the above embodiments, wherein the sugar of N _-6 is a 2'-OR modified sugar (R is an optionally substituted C _1-6 aliphatic).

1287. The oligonucleotide according to any one of the above embodiments, wherein the sugar of N _-6 is a 2'-OMe modified sugar.

1288. The oligonucleotide of embodiment 1278, wherein the sugar of N _-6 is a 2'-MOE modified sugar.

1289. The oligonucleotide according to any one of the preceding embodiments, wherein the internucleotide linkage between N _-5 and N _-6 is a non-negatively charged internucleotide linkage.

1290. The oligonucleotide of any one of the preceding embodiments, wherein the internucleotide linkage between N _-5 and N _-6 is a phosphoryl guanidine internucleotide linkage.

1291. The oligonucleotide according to any one of the preceding embodiments, wherein the internucleotide linkage between N _-5 and N _-6 is n001.

1292. The oligonucleotide according to any one of the preceding embodiments, wherein the internucleotide linkage between N _-5 and N _-6 is R p n001.

1293. The method of any one of the preceding embodiments, wherein about 20%-80%, 30-70%, 30%-60%, 30%-50%, 40%-60%, 20% of the sugars of the oligonucleotide, 30%, 40%, 50%, 60%, 70% or 80% are each independently a 2'-F modified sugar.

1294. The oligonucleotide of any one of the preceding embodiments, wherein about 30% to 60% of the sugars of the oligonucleotide are each independently 2'-F modified sugars.

1295. The method of any one of the preceding embodiments, wherein about 20%-80%, 30-70%, 30%-60%, 30%-50%, 40%-60%, 20% of the sugars of the oligonucleotide 30%, 40%, 50%, 60%, 70% or 80% each independently 2'-OR, wherein R is an optionally substituted C _1-6 aliphatic oligonucleotide.

1296. The oligonucleotide of any one of the preceding embodiments, wherein about 30%-60% of the sugars of the oligonucleotide are each independently a 2'-OR modified sugar (R is an optionally substituted C _1-6 aliphatic). .

1297. The oligonucleotide of any one of the preceding embodiments, wherein about 30%-60% of the sugars of the oligonucleotide are each independently 2'-OMe or 2'-MOE modified sugars.

1298. The method of any one of the preceding embodiments, wherein about 20%-80%, 30-70%, 30%-60%, 30%-50%, 40%-60% of the sugars of the oligonucleotides in the first domain wherein %, 20%, 30%, 40%, 50%, 60%, 70%, or 80% are each independently 2'-OR, wherein R is an optionally substituted C _1-6 aliphatic oligonucleotide.

1299. The method of any one of the preceding embodiments, wherein about 30%-60% of the sugars of the oligonucleotide in the first domain are each independently a 2'-OR modified sugar (R is an optionally substituted C _1-6 aliphatic ), an oligonucleotide.

1300. The oligonucleotide of any one of the preceding embodiments, wherein about 30% to 60% of the sugars of the oligonucleotide in the first domain are each independently 2'-OMe or 2'-MOE modified sugars.

1301. The oligonucleotide of any one of the preceding embodiments, wherein the 3'-terminal nucleoside of the first domain is N ₂ .

1302. The oligonucleotide of any one of the above embodiments, wherein the 5'-terminal nucleoside of the first domain is the 5'-terminal nucleoside of the oligonucleotide.

1303. The method of any one of the preceding embodiments, wherein about or at least about 20%, 30%, 40%, 50%, 60%, 70% in an oligonucleotide or portion thereof, e.g., a first domain, a second domain, etc. %, 80% or 90% of the 2'-OR modified sugar, wherein R is an optionally substituted C _1-6 aliphatic, is independently linked to a natural phosphate linkage.

1304. The method of any one of the above embodiments, wherein each of one or more sugars of N _-2 , N _-3 , N _-4 , N _-5 , and N _-6 is independently a 2'-OR modified sugar (R is an optionally substituted C _1-6 aliphatic) or bicyclic sugar.

1305. The method of any one of the above embodiments, wherein each of one or more sugars of N _-2 , N _-3 , N _-4 , N _-5 , and N _-6 is independently a 2'-OR modified sugar (R is optionally substituted C _1-6 aliphatic).

1306. The oligonucleotide of any one of the preceding embodiments, wherein each sugar of one or more of N _-2 , N _-3 , N _-4 , N _-5 , and N _-6 is independently a 2'-OMe sugar.

1307. The oligonucleotide of any one of the preceding embodiments, wherein each sugar of one or more of N _-2 , N _-3 , N _-4 , N _-5 , and N _-6 is independently a 2'-MOE sugar.

1308. The method of any one of the preceding embodiments, wherein each of the sugars of one or more of N ₂ , N ₃ , N ₄ , N ₅ , N ₆ , N ₇ , and N ₈ is independently a 2′-OR modified sugar (R is an optionally substituted C _1-6 aliphatic) or bicyclic sugar.

1309. The method of any one of the preceding embodiments, wherein each of the sugars of one or more of N ₂ , N ₃ , N ₄ , N ₅ , N ₆ , N ₇ , and N ₈ is independently a 2′-OR modified sugar (R is an optionally substituted C _1-6 aliphatic).

1310. The oligonucleotide of any one of the preceding embodiments, wherein each sugar of one or more of N ₂ , N ₃ , N ₄ , N ₅ , N ₆ , N ₇ , and N ₈ is independently a 2′-OMe sugar. .

1311. The oligonucleotide of any one of the preceding embodiments, wherein each sugar of one or more of N ₂ , N ₃ , N ₄ , N ₅ , N ₆ , N ₇ , and N ₈ is independently a 2'-MOE sugar. .

1312. The method of any one of the above embodiments, wherein about or at least about 50% of the 2'-OR variant in the oligonucleotide or portion thereof, e.g., the first domain, the second domain, etc. (R is an optionally substituted C _1-6 aliphatic) are independently linked to natural phosphate linkages.

1313. The method of any one of the above embodiments, wherein at least 60%, 70%, 80% or 90% or all of the natural phosphate linkages are each independently a 2'-OR variant (R is optionally substituted C _{1- 6} aliphatic) or at least one modified sugar that is a bicyclic sugar.

1314. The method of any one of the preceding embodiments, wherein about 5% to 90%, about 10 to 80%, about 10 to 75%, about 10 to 70%, 10% to 60% of all internucleotide linkages of the oligonucleotide %, 10-50%, 10-40%, 10-30%, 15-40%, 20-30%, 25-30%, or about or at least about 5%, 10%, 15%, 20%, 25 %, 30%, 35%, 40%, 45% or 50% independently are natural phosphate linkages.

1315. The method of any one of the above embodiments, wherein about 5%-90%, about 10-80%, about 10-75%, about 10-70%, 10%- 60%, 10-50%, 10-40%, 10-30%, 15-40%, 20-30%, 25-30%, or about or at least about 5%, 10%, 15%, 20%, wherein 25%, 30%, 35%, 40%, 45% or 50% are independently natural phosphate linkages.

1316. The method of any one of the preceding embodiments, wherein +3 (between N ₊₄ N ₊₃ ), +4, +6, +8, +9, +12, +14, +15, +17 and + wherein the one or more internucleotide linkages at one or more of positions 18 are independently natural phosphate linkages.

1317. The method of any one of the preceding embodiments, wherein about 5% to 90%, about 10 to 80%, about 10 to 75%, about 10 to 70%, 10% to 60% of all internucleotide linkages of the oligonucleotide %, 10-50%, 10-40%, 10-30%, 15-40%, 20-30%, 25-30%, 30%-70%, 40-70%, 40%-65%, 40 % to 60%, or about or at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, or 65% oligonucleotides, which are independently phosphorothioate nucleotide linkages.

1318. The method of any one of the above embodiments, wherein about 5%-90%, about 10-80%, about 10-75%, about 10-70%, 10%- 60%, 10-50%, 10-40%, 10-30%, 15-40%, 20-30%, 25-30%, 30%-70%, 40-70%, 40%-65%, 40% to 60%, or about or at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, or 65% are independently phosphorothioate nucleotide linkages.

1319. The method of any one of the preceding embodiments, wherein +1 (between N ₊₁ N ₀ ), +2, +5, +6, +7, +8, +11, +14, +15, +16 , the one or more internucleotide linkages at one or more of positions +17, +19, +20, +21 and +22 are independently phosphorothioate internucleotide linkages.

1320. The method of any one of the preceding embodiments, wherein about 5%-90%, about 10-80%, about 10-75%, about 10-70%, 10%-60% of all internucleotide linkages of the oligonucleotide %, 10~50%, 10~40%, 10~30%, 10%~20%, 10~15%, 15~40%, 15%~35%, 15%~30%, 15~25%, 15-20%, or about or at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45% or 50% independently non-negatively charged internucleotide linkages Phosphorus, oligonucleotide.

1321. The method of any one of the above embodiments, wherein about 5% to 90%, about 10 to 80%, about 10 to 75%, about 10 to 70%, 10% to about 10% of all internucleotide linkages of the first domain 60%, 10-50%, 10-40%, 10-30%, 10%-20%, 10-15%, 15-40%, 15%-35%, 15%-30%, 15-25% , 15-20%, or about or at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45% or 50% independently non-negatively charged internucleotides A linker, an oligonucleotide.

1322. The method of any one of the preceding embodiments, wherein about 5% to 90%, about 10 to 80%, about 10 to 75%, about 10 to 70%, 10% to 60% of all internucleotide linkages of the oligonucleotide %, 10~50%, 10~40%, 10~30%, 10%~20%, 10~15%, 15~40%, 15%~35%, 15%~30%, 15~25%, 15-20%, or about or at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45% or 50% are independently phosphorylguanidine internucleotide linkages, oligonucleotide.

1323. The method of any one of the preceding embodiments, wherein about 5% to 90%, about 10 to 80%, about 10 to 75%, about 10 to 70%, 10% to about 10% to about 10% of all internucleotide linkages of the first domain 60%, 10-50%, 10-40%, 10-30%, 10%-20%, 10-15%, 15-40%, 15%-35%, 15%-30%, 15-25% , 15-20%, or about or at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45% or 50% are independently phosphorylguanidine internucleotide linkages , oligonucleotides.

1324. The method of any one of the preceding embodiments, wherein about 5% to 90%, about 10 to 80%, about 10 to 75%, about 10 to 70%, 10% to 60% of all internucleotide linkages of the oligonucleotide %, 10~50%, 10~40%, 10~30%, 10%~20%, 10~15%, 15~40%, 15%~35%, 15%~30%, 15~25%, 15-20%, or about or at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45% or 50% independently is n001.

1325. The method of any one of the above embodiments, wherein about 5% to 90%, about 10 to 80%, about 10 to 75%, about 10 to 70%, 10% to about 10% of all internucleotide linkages of the first domain 60%, 10-50%, 10-40%, 10-30%, 10%-20%, 10-15%, 15-40%, 15%-35%, 15%-30%, 15-25% , 15-20%, or about or at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45% or 50% independently is n001.

1326. The method of any one of the preceding embodiments, wherein the one or more internucleotide linkages at one or more or all of positions +5 (between N ₊₅ N ₊₄ ), +10, +13 or +23 are independently negative An oligonucleotide, which is an uncharged internucleotide linkage.

1327. The method of any one of the above embodiments, wherein the one or more internucleotide linkages at one or more or all of positions +5 (between N ₊₅ N ₊₄ ), +10, +13 or +23 are independently phosphorus An oligonucleotide, which is a polylguanidine internucleotidic linkage.

1328. The method of any one of the above embodiments, wherein the one or more internucleotide linkages at one or more or all of positions +5 (between N ₊₅ N ₊₄ ), +10, +13 or +23 are independently n001 Phosphorus, oligonucleotide.

1329. The oligonucleotide of any one of the preceding embodiments, wherein the sugar of N ₄ is a 2′-OR modified sugar (R is an optionally substituted C _1-6 aliphatic).

1330. The oligonucleotide of any one of the preceding embodiments, wherein the internucleotide linkage between N ₃ and N ₄ is a natural phosphate linkage.

1331. The oligonucleotide according to any one of the preceding embodiments, wherein there are 5 or more nucleosides on the 3' side of N ₀ .

1332. The oligonucleotide according to any one of the preceding embodiments, wherein there are 6 or more nucleosides on the 3' side of N ₀ .

1333. The oligonucleotide according to any one of the preceding embodiments, wherein there are 7 or more nucleosides on the 3' side of N ₀ .

1334. The oligonucleotide according to any one of the above embodiments, wherein there are 8 or more nucleosides on the 3' side of N ₀ .

1335. The oligonucleotide according to any one of embodiments 1 to 1330, wherein there are 3 nucleosides on the 3' side of N ₀ .

1336. The oligonucleotide according to any one of embodiments 1 to 1330, wherein there are 4 nucleosides on the 3' side of N ₀ .

1337. The oligonucleotide according to any one of embodiments 1 to 1330, wherein there are 5 nucleosides on the 3' side of N ₀ .

1338. The oligonucleotide according to any one of embodiments 1 to 1330, wherein there are 6 nucleosides on the 3' side of N ₀ .

1339. The oligonucleotide according to any one of embodiments 1 to 1330, wherein there are 7 nucleosides on the 3' side of N ₀ .

1340. The oligonucleotide according to any one of embodiments 1 to 1330, wherein there are 8 nucleosides on the 3' side of N ₀ .

1341. The oligonucleotide according to any one of embodiments 1 to 1330, wherein there are 9 nucleosides on the 3' side of N ₀ .

1342. The oligonucleotide according to any one of embodiments 1 to 1330, wherein there are 10 nucleosides on the 3' side of N ₀ .

1343. The method of any one of the above embodiments, wherein 5 _or more (eg, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 or more) nucleotides.

1344. The oligonucleotide according to any one of the preceding embodiments, wherein there are 8 or more nucleosides on the 5' side of N ₀ .

1345. The oligonucleotide according to any one of the preceding embodiments, wherein there are 10 or more nucleosides on the 5' side of N ₀ .

1346. The oligonucleotide according to any one of the preceding embodiments, wherein there are 15 or more nucleosides on the 5' side of N ₀ .

1347. The oligonucleotide according to any one of the above embodiments, wherein there are 16 or more nucleosides on the 5' side of N ₀ .

1348. The oligonucleotide according to any one of the preceding embodiments, wherein there are 17 or more nucleosides on the 5' side of N ₀ .

1349. The oligonucleotide according to any one of the above embodiments, wherein there are 18 or more nucleosides on the 5' side of N ₀ .

1350. The oligonucleotide according to any one of the preceding embodiments, wherein there are 19 or more nucleosides on the 5' side of N ₀ .

1351. The oligonucleotide according to any one of the preceding embodiments, wherein there are 20 or more nucleosides on the 5' side of N ₀ .

1352. The oligonucleotide according to any one of the preceding embodiments, wherein there are 21 or more nucleosides on the 5' side of N ₀ .

1353. The oligonucleotide according to any one of the preceding embodiments, wherein there are 22 or more nucleosides on the 5' side of N ₀ .

1354. The oligonucleotide according to any one of the preceding embodiments, wherein there are 23 or more nucleosides on the 5' side of N ₀ .

1355. The oligonucleotide according to any one of the preceding embodiments, wherein there are 24 or more nucleosides on the 5' side of N ₀ .

1356. The oligonucleotide according to any one of the preceding embodiments, wherein there are 25 or more nucleosides on the 5' side of N ₀ .

1357. The oligonucleotide according to any one of the preceding embodiments, wherein there are 26 or more nucleosides on the 5' side of N ₀ .

1358. The oligonucleotide according to any one of embodiments 1 to 1343, wherein there are 20 nucleosides on the 5' side of N ₀ .

1359. The oligonucleotide according to any one of embodiments 1 to 1343, wherein there are 21 nucleosides on the 5' side of N ₀ .

1360. The oligonucleotide according to any one of embodiments 1 to 1343, wherein there are 22 nucleosides on the 5' side of N ₀ .

1361. The oligonucleotide according to any one of embodiments 1 to 1343, wherein there are 23 nucleosides on the 5' side of N ₀ .

1362. The oligonucleotide according to any one of embodiments 1 to 1343, wherein there are 24 nucleosides on the 5' side of N ₀ .

1363. The oligonucleotide according to any one of embodiments 1 to 1343, wherein there are 25 nucleosides on the 5' side of N ₀ .

1364. The oligonucleotide according to any one of embodiments 1 to 1343, wherein there are 26 nucleosides on the 5' side of N ₀ .

1365. The oligonucleotide according to any one of embodiments 1 to 1343, wherein there are 27 nucleosides on the 5' side of N ₀ .

1366. The oligonucleotide according to any one of embodiments 1 to 1343, wherein there are 28 nucleosides on the 5' side of N ₀ .

1367. The oligonucleotide according to any one of embodiments 1 to 1343, wherein there are 29 nucleosides on the 5' side of N ₀ .

1368. The oligonucleotide according to any one of embodiments 1 to 1343, wherein there are 30 nucleosides on the 5' side of N ₀ .

1369. The oligonucleotide according to any one of the above embodiments, wherein the first 1, 2, 3, 4 or 5 sugars at the 5'-end of the oligonucleotide are each independently a sugar capable of increasing stability.

1370. The oligonucleotide according to any one of the above embodiments, wherein the first three sugars at the 5'-end of the oligonucleotide are each independently a sugar capable of increasing stability.

1371. The oligonucleotide according to any one of the above embodiments, wherein the first four sugars at the 5'-end of the oligonucleotide are each independently a sugar capable of increasing stability.

1372. The oligonucleotide according to any one of the above embodiments, wherein the first 5 sugars at the 5'-end of the oligonucleotide are each independently a sugar capable of increasing stability.

1373. The method according to any one of the above embodiments, wherein the first 1, 2, 3, 4 or 5 sugars at the 5'-end of the oligonucleotide are each independently a bicyclic sugar and a 2'-OR modified sugar (R is optionally substituted C _1-6 aliphatic).

1374. The method of any one of the above embodiments, wherein the first three sugars at the 5'-end of the oligonucleotide are each independently a bicyclic sugar and a 2'-OR modified sugar (R is an optionally substituted C _1-6 aliphatic an oligonucleotide selected from).

1375. The method of any one of the above embodiments, wherein the first four sugars at the 5'-end of the oligonucleotide are each independently a bicyclic sugar and a 2'-OR modified sugar (R is an optionally substituted C _1-6 aliphatic an oligonucleotide selected from).

1376. The method of any one of the above embodiments, wherein the first 5 sugars at the 5'-end of the oligonucleotide are each independently a bicyclic sugar and a 2'-OR modified sugar (R is an optionally substituted C _1-6 aliphatic an oligonucleotide selected from).

1377. The method of any one of the above embodiments, wherein the first three sugars at the 5'-end of the oligonucleotide are each independently a 2'-OR modified sugar (R is an optionally substituted C _1-6 aliphatic). , oligonucleotides.

1378. The method of any one of the above embodiments, wherein the first four sugars at the 5'-end of the oligonucleotide are each independently a 2'-OR modified sugar (R is an optionally substituted C _1-6 aliphatic). , oligonucleotides.

1379. The method of any one of the above embodiments, wherein the first 5 sugars at the 5'-end of the oligonucleotide are each independently a 2'-OR modified sugar (R is an optionally substituted C _1-6 aliphatic). , oligonucleotides.

1380. The method of any one of the above embodiments, wherein the first 1, 2, 3, 4 or 5 sugars at the 5'-end of the oligonucleotide are each independently a 2'-OMe or 2'-MOE modified sugar, oligonucleotide.

1381. The oligonucleotide according to any one of the above embodiments, wherein the first 1, 2, 3, 4 or 5 sugars at the 5'-end of the oligonucleotide are each independently a 2'-OMe modified sugar.

1382. The oligonucleotide according to any one of the above embodiments, wherein the first three sugars at the 5'-end of the oligonucleotide are each independently a 2'-OMe modified sugar.

1383. The oligonucleotide according to any one of the above embodiments, wherein the first four sugars at the 5'-end of the oligonucleotide are each independently a 2'-OMe modified sugar.

1384. The oligonucleotide according to any one of the above embodiments, wherein the first 5 sugars at the 5'-end of the oligonucleotide are each independently a 2'-OMe modified sugar.

1385. The oligonucleotide according to any one of embodiments 1 to 1380, wherein the first 1, 2, 3, 4 or 5 sugars at the 5'-end of the oligonucleotide are each independently a 2'-MOE modified sugar.

1386. The oligonucleotide according to any one of embodiments 1 to 1380, wherein the first three sugars at the 5'-end of the oligonucleotide are each independently a 2'-MOE modified sugar.

1387. The oligonucleotide according to any one of embodiments 1 to 1380, wherein the first four sugars at the 5'-end of the oligonucleotide are each independently a 2'-MOE modified sugar.

1388. The oligonucleotide according to any one of embodiments 1 to 1380, wherein the first 5 sugars at the 5'-end of the oligonucleotide are each independently a 2'-MOE modified sugar.

1389. The oligonucleotide according to any one of the above embodiments, wherein the last 1, 2, 3, 4 or 5 sugars at the 3'-end of the oligonucleotide are each independently a sugar capable of increasing stability.

1390. The oligonucleotide of any one of the above embodiments, wherein the last three sugars at the 3'-end of the oligonucleotide are each independently a sugar capable of increasing stability.

1391. The oligonucleotide according to any one of the above embodiments, wherein the last four sugars at the 3'-end of the oligonucleotide are each independently a sugar capable of increasing stability.

1392. The oligonucleotide according to any one of the above embodiments, wherein the last 5 sugars at the 3'-end of the oligonucleotide are each independently a sugar capable of increasing stability.

1393. The method according to any one of the above embodiments, wherein the last 1, 2, 3, 4 or 5 sugars at the 3'-end of the oligonucleotide are each independently a bicyclic sugar and a 2'-OR modified sugar (R is optionally substituted C _1-6 aliphatic).

1394. The method of any one of the above embodiments, wherein the last three sugars at the 3'-end of the oligonucleotide are each independently a bicyclic sugar and a 2'-OR modified sugar (R is an optionally substituted C _1-6 aliphatic an oligonucleotide selected from).

1395. The method of any one of the above embodiments, wherein the last four sugars at the 3'-end of the oligonucleotide are each independently a bicyclic sugar and a 2'-OR modified sugar (R is an optionally substituted C _1-6 aliphatic an oligonucleotide selected from).

1396. The method of any one of the above embodiments, wherein the last 5 sugars at the 3'-end of the oligonucleotide are each independently a bicyclic sugar and a 2'-OR modified sugar (R is an optionally substituted C _1-6 aliphatic an oligonucleotide selected from).

1397. The method of any one of the above embodiments, wherein the last three sugars at the 3'-end of the oligonucleotide are each independently a 2'-OR modified sugar (R is an optionally substituted C _1-6 aliphatic). , oligonucleotides.

1398. The method of any one of the above embodiments, wherein the last four sugars at the 3'-end of the oligonucleotide are each independently a 2'-OR modified sugar (R is an optionally substituted C _1-6 aliphatic). , oligonucleotides.

1399. The method of any one of the above embodiments, wherein the last 5 sugars at the 3'-end of the oligonucleotide are each independently a 2'-OR modified sugar (R is an optionally substituted C _1-6 aliphatic). , oligonucleotides.

1400. The method of any one of the above embodiments, wherein the last 1, 2, 3, 4 or 5 sugars at the 3'-end of the oligonucleotide are each independently a 2'-OMe or 2'-MOE modified sugar, oligonucleotide.

1401. The oligonucleotide according to any one of the above embodiments, wherein the last 1, 2, 3, 4 or 5 sugars at the 3'-end of the oligonucleotide are each independently a 2'-OMe modified sugar.

1402. The oligonucleotide according to any one of the above embodiments, wherein the last three sugars at the 3'-end of the oligonucleotide are each independently a 2'-OMe modified sugar.

1403. The oligonucleotide according to any one of the above embodiments, wherein the last four sugars at the 3'-end of the oligonucleotide are each independently a 2'-OMe modified sugar.

1404. The oligonucleotide according to any one of the above embodiments, wherein the last 5 sugars at the 3'-end of the oligonucleotide are each independently a 2'-OMe modified sugar.

1405. The oligonucleotide according to any one of embodiments 1 to 1400, wherein the last 1, 2, 3, 4 or 5 sugars at the 3'-end of the oligonucleotide are each independently a 2'-MOE modified sugar.

1406. The oligonucleotide according to any one of embodiments 1 to 1400, wherein the last three sugars at the 3'-end of the oligonucleotide are each independently a 2'-MOE modified sugar.

1407. The oligonucleotide according to any one of embodiments 1 to 1400, wherein the last four sugars at the 3'-end of the oligonucleotide are each independently a 2'-MOE modified sugar.

1408. The oligonucleotide according to any one of embodiments 1 to 1400, wherein the last 5 sugars at the 3'-end of the oligonucleotide are each independently a 2'-MOE modified sugar.

1409. The oligonucleotide of any one of the preceding embodiments, wherein the first internucleotide linkage from the 5'-end of the oligonucleotide is a non-negatively charged internucleotide linkage.

1410. The oligonucleotide of any one of the preceding embodiments, wherein the first internucleotide linkage from the 5'-end of the oligonucleotide is a neutral internucleotide linkage.

1411. The oligonucleotide of any one of the above embodiments, wherein the first internucleotide linkage from the 5'-end of the oligonucleotide is a phosphoryl guanidine internucleotide linkage.

1412. The oligonucleotide according to any one of the above embodiments, wherein the first internucleotide linkage from the 5'-end of the oligonucleotide is n004, n008, n025, n026.

1413. The oligonucleotide according to any one of the above embodiments, wherein the first internucleotide linkage from the 5'-end of the oligonucleotide is n001.

1414. The oligonucleotide according to any one of the above embodiments, wherein the first internucleotide linkage from the 5'-end is chirally controlled and R p .

1415. The oligonucleotide according to any one of embodiments 1 to 1413, wherein the first internucleotide linkage from the 5'-end is chirally controlled and is Sp .

1416. The oligonucleotide according to any one of the above embodiments, wherein both internucleoside linkages attached to the third nucleoside from the 5'-end are each independently a phosphorothioate internucleoside linkage.

1417. The oligonucleotide according to any one of the above embodiments, wherein both internucleoside linkages linked to the fourth nucleoside from the 5'-terminus are each independently a phosphorothioate internucleotide linkage.

1418. The oligonucleotide according to any one of the above embodiments, wherein both internucleoside linkages attached to the fifth nucleoside from the 5'-terminus are each independently a phosphorothioate internucleotide linkage.

1419. The oligonucleotide according to any one of embodiments 1416 to 1418, wherein each phosphorothioate internucleotide linkage is chirally controlled.

1420. The oligonucleotide of embodiment 1419, wherein each phosphorothioate internucleotide linkage is S p.

1421. The oligonucleotide of any one of the preceding embodiments, wherein the first internucleotide linkage from the 3'-end of the oligonucleotide is a non-negatively charged internucleotide linkage.

1422. The oligonucleotide of any one of the preceding embodiments, wherein the first internucleotide linkage from the 3'-end of the oligonucleotide is a neutral internucleotide linkage.

1423. The oligonucleotide of any one of the preceding embodiments, wherein the first internucleotide linkage from the 3'-end of the oligonucleotide is a phosphoryl guanidine internucleotide linkage.

1424. The oligonucleotide according to any one of the preceding embodiments, wherein the first internucleotide linkage from the 3'-end of the oligonucleotide is n004, n008, n025, n026.

1425. The oligonucleotide according to any one of the preceding embodiments, wherein the first internucleotide linkage from the 3'-end of the oligonucleotide is n001.

1426. The oligonucleotide according to any one of the preceding embodiments, wherein the first internucleotide linkage from the 3'-end is chirally controlled and R p .

1427. The oligonucleotide according to any one of embodiments 1 to 1426, wherein the first internucleotide linkage from the 3'-end is chirally controlled and is Sp .

1428. The oligonucleotide according to any one of the preceding embodiments, wherein the sugar of the nucleoside opposite the target adenosine (position 0, such nucleoside: N ₀ ) is a natural DNA sugar.

1429. The method of any one of the above embodiments, wherein the nucleoside at position +1 (the nucleoside to the adjacent 5' side of N ₀ ; ie, 5'-...N ₊₁ N ₀ ...-3' wherein the sugar of N ₊₁ ) is a natural DNA sugar.

1430. The method according to any one of embodiments 1 to 1428, wherein the nucleoside at position +1 (the nucleoside to the adjacent 5' side of N ₀ ; ie, 5'-...N ₊₁ N ₀ ...- An oligonucleotide wherein the sugar of N ₊₁ ) of 3' is a 2'-F modified sugar.

1431. The sugar of the nucleoside at position +2 (N +2 of 5'-...N ₊₂ N ₊₁ N ₀ ...-3') is _2' -F Modified sugars, oligonucleotides.

1432. The sugar of any one of the above embodiments, the nucleoside at position -1 (N -1 of 5'-...N ₊₂ N ₊₁ N ₀ N _-1 ...- ₃ ') is a natural DNA sugars, oligonucleotides.

1433. The method according to any one of the above embodiments, wherein the nucleoside at position -2 (5'-...N ₊₂ N ₊₁ N ₀ N _-1 N _-2 ... -3' of N _-2 ) An oligonucleotide, wherein the sugar is a sugar capable of increasing stability.

1434. The method according to any one of the above embodiments, wherein the nucleoside at position -2 (5'-...N ₊₂ N ₊₁ N ₀ N _-1 N _-2 ... -3' of N _-2 ) The oligonucleotide is a bicyclic sugar or a 2'-OR modified sugar (R is an optionally substituted C _1-6 aliphatic).

1435. The method of any one of the above embodiments, wherein the nucleoside at position -2 (5'-...N ₊₂ N ₊₁ N ₀ N _-1 N _-2 ...-3' of N _-2 ) An oligonucleotide in which the sugar is a bicyclic sugar.

1436. The method according to any one of embodiments 1 to 1434, wherein the nucleoside at position -2 (5'-...N ₊₂ N ₊₁ N ₀ N _-1 N _-2 ... -3 'N _-2 ) is a 2'-OR modified sugar (R is an optionally substituted C _1-6 aliphatic) oligonucleotide.

1437. The method according to any one of embodiments 1 to 1434, wherein the nucleoside at position -2 (5'-...N ₊₂ N ₊₁ N ₀ N _-1 N _-2 ... -3 'N _-2 ) is a 2'-OMe modified sugar.

1438. The method according to any one of embodiments 1 to 1434, wherein the nucleoside at position -2 (5'-...N ₊₂ N ₊₁ N ₀ N _-1 N _-2 ... -3 'N _-2 ) is a 2'-MOE modified sugar.

1439. The method according to any one of embodiments 1 to 1434, wherein the nucleoside at position -2 (5'-...N ₊₂ N ₊₁ N ₀ N _-1 N _-2 ... -3 'N _-2 ) is a 2'-MOE modified sugar.

1440. The method of any one of the above embodiments, wherein the nucleoside at position -3 (5'-...N ₊₂ N ₊₁ N ₀ N _-1 N _-2 N _-3 ... N -3 'N _- An oligonucleotide wherein the sugar in ₃ ) is a 2'-F modified sugar.

1441. The method of any one of the above embodiments, wherein each sugar of the nucleoside following N _-3 (eg, N _-4 , N _-5 , N _{-6 ,} etc.) independently increases stability. sugars, oligonucleotides.

1442. The method of any one of the above embodiments, wherein each sugar of the nucleoside following N _-3 (eg, N _-4 , N _-5 , N _{-6 ,} etc.) is independently a bicyclic sugar or 2 An oligonucleotide, wherein the '-OR modified sugar (R is an optionally substituted C _1-6 aliphatic).

1443. The oligonucleotide of any one of the preceding embodiments, wherein the sugar of the nucleoside following N _-3 (eg, N _-4 , N _-5 , N _{-6 ,} etc.) is a bicyclic sugar.

1444. The sugar of any one of the above embodiments, the nucleoside after N _-3 (eg, N _-4 , N _-5 , N _{-6 ,} etc.) is a 2'-OR modified sugar (R is an optionally substituted C _1-6 aliphatic).

1445. The oligonucleotide according to any one of the above embodiments, wherein the sugar of the nucleoside after N _-3 (eg, N _-4 , N _-5 , N _{-6 ,} etc.) is a 2'-OMe modified sugar. nucleotide.

1446. The oligonucleotide of any one of the above embodiments, wherein the sugar of the nucleoside following N _-3 (eg, N _-4 , N _-5 , N _{-6 ,} etc.) is a 2'-MOE modified sugar. nucleotide.

1447. The method of any one of embodiments 1 to 1442, wherein each sugar of the nucleoside following N _-3 (eg, N _-4 , N _-5 , N _-6 , etc.) is independently 2' -OR modified sugar (R is an optionally substituted C _1-6 aliphatic) oligonucleotide.

1448. The method of any one of embodiments 1 to 1442, wherein each sugar of the nucleoside following N _-3 (eg, N _-4 , N _-5 , N _-6 , etc.) is independently 2' -OMe modified sugars, oligonucleotides.

1449. The method of any one of embodiments 1 to 1442, wherein each sugar of the nucleoside following N _-3 (eg, N _-4 , N _-5 , N _-6 , etc.) is independently 2' -MOE modified sugars, oligonucleotides.

1450. The oligonucleotide of any one of the preceding embodiments, wherein each internucleotide linkage linked to N ₊₁ or N ₀ is independently a phosphorothioate internucleotide linkage.

1451. The oligonucleotide of any one of the preceding embodiments, wherein each internucleotide linkage linked to N ₊₁ or N ₀ is independently an S p phosphorothioate internucleotide linkage.

1452. The oligonucleotide according to any one of the preceding embodiments, wherein the internucleotide linkage between N _-1 and N _-2 is a non-negatively charged internucleotide linkage.

1453. The oligonucleotide according to any one of the preceding embodiments, wherein the internucleotide linkage between N _-1 and N _-2 is a neutral internucleotide linkage.

1454. The oligonucleotide according to any one of the preceding embodiments, wherein the internucleotide linkage between N _-1 and N _-2 is a phosphoryl guanidine internucleotide linkage.

1455. The oligonucleotide according to any one of the preceding embodiments, wherein the internucleotide linkage between N _-1 and N _-2 is n004, n008, n025, n026.

1456. The oligonucleotide according to any one of the preceding embodiments, wherein the internucleotide linkage between N _-1 and N _-2 is n001.

1457. The oligonucleotide according to any one of the preceding embodiments, wherein the internucleotide linkage between N _-1 and N _-2 is chirally controlled and R p .

1458. The oligonucleotide according to any one of embodiments 1 to 1456, wherein the internucleotide linkage between N _-1 and N _-2 is chirally controlled and is Sp .

1459. The oligonucleotide according to any one of the preceding embodiments, wherein the internucleotide linkage between N _-2 and N _-3 is a natural phosphate linkage.

1460. The method of any one of the above embodiments, wherein each internucleotide linkage linked to the nucleoside following N _-3 (eg, N _-4 , N _-5 , N _-6 , etc.) is 3 ' -oligonucleotides, which are independently phosphorothioate internucleotidic linkages except for the first internucleotidic linkage from the terminus.

1461. The oligonucleotide of embodiment 1460, wherein the phosphorothioate internucleotidic linkages are chirally controlled and are Sp .

1462. The oligonucleotide according to any one of the preceding embodiments, wherein the bicyclic sugar is an LNA sugar or a cEt sugar.

1463. The method of any one of the preceding embodiments, comprising from 1 to 10 (eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) natural phosphate linkages To, oligonucleotide.

1464. The oligonucleotide of any one of the preceding embodiments, comprising no more than 5 (eg, 1, 2, 3, 4, or 5) natural phosphate linkages.

1465. The oligonucleotide of any one of the preceding embodiments, comprising no more than 10 (eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10) n001 .

1466. The method of any one of the preceding embodiments, wherein no more than 10 (eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10) phosphoryl guanidine internucleotide linkages are Including, oligonucleotides.

1467. An oligonucleotide comprising a duplexing region and a targeting region, wherein the targeting region is or comprises a second region of any one of the above embodiments.

1468. An oligonucleotide comprising a duplexing region and a targeting region, wherein the targeting region is or comprises 5'-N ₁ N ₀ N _-1 -3' of any one of the above embodiments.

1469. The oligonucleotide of embodiment 1467 or 1468, wherein the duplexed region is capable of forming a duplex with a nucleic acid (duplexed nucleic acid).

1470. The oligonucleotide according to any one of embodiments 1467 to 1469, wherein the targeting region is capable of forming a duplex with a target nucleic acid comprising a target adenosine.

1471. The oligonucleotide according to any one of embodiments 1467 to 1470, wherein the duplexed nucleic acid is not a target nucleic acid.

1472. An oligonucleotide according to any one of embodiments 1467 to 1470, which is an oligonucleotide according to any one of embodiments 1 to 1466.

1473. The method of any one of embodiments 1467-1472, wherein the length of the targeting region is about or at least about 10% to 100%, or at least about 10, 11, 12, 13, 14, 15, 16, 17, 18 , 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleosides.

1474. The method of any one of embodiments 1467-1473, wherein the length of the duplexed region is about or at least about 10% to 100%, or at least about 10, 11, 12, 13, 14, 15, 16, 17, An oligonucleotide that is 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleosides.

1475. The method of any one of embodiments 1467-1474, wherein the length of the duplexed oligonucleotide is about or at least about 10% to 100%, or at least about 10, 11, 12, 13, 14, 15, 16, 17 , 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleosides.

1476. The oligonucleotide of any one of embodiments 1467-1475, wherein the duplexed oligonucleotide comprises a stem loop.

1477. The oligonucleotide according to any one of embodiments 1467 to 1476, comprising at least one modified sugar, at least one modified internucleotide linkage and at least one natural phosphate linkage.

1478. The oligonucleotide according to any one of embodiments 1467 to 1477, which is not chirally controlled.

1479. The oligonucleotide according to any one of embodiments 1467 to 1478, wherein the duplexed oligonucleotide comprises one or more modified sugars and one or more modified internucleotide linkages.

1480. The oligonucleotide according to any one of embodiments 1467 to 1479, wherein most or all sugars in the duplexed oligonucleotide are modified sugars.

1481. The oligonucleotide according to any one of embodiments 1467 to 1479, wherein most or all sugars in the duplexed oligonucleotide are 2'-F modified sugars.

1482. The oligonucleotide according to any one of embodiments 1467 to 1479, wherein most or all sugars in the duplexed oligonucleotide are 2'-OR modified sugars (R is an optionally substituted C _1-6 aliphatic).

1483. The oligonucleotide according to any one of embodiments 1467 to 1479, wherein most or all sugars in the duplexed oligonucleotide are 2'-OMe modified sugars.

1484. The oligonucleotide according to any one of embodiments 1467 to 1483, wherein most or all internucleotide linkages in the duplexed oligonucleotide are modified.

1485. The oligonucleotide according to any one of embodiments 1467 to 1484, wherein most or all internucleotide linkages in the duplexed oligonucleotide are phosphorothioate internucleotide linkages.

1486. The oligonucleotide according to any one of embodiments 1467 to 1485, wherein the duplexed oligonucleotide is chirally controlled.

1487. The oligonucleotide according to any one of embodiments 1467 to 1486, wherein most or all internucleotide linkages in the duplexed oligonucleotide are S p phosphorothioate internucleotide linkages.

1488. The oligonucleotide according to any one of embodiments 1467 to 1487, wherein the oligonucleotide and its duplexed oligonucleotide are administered as a duplex.

1489. The oligonucleotide according to any one of embodiments 1467 to 1487, wherein the oligonucleotide and its duplexed oligonucleotide are administered separately.

1490. The method according to any one of the preceding embodiments, wherein each sugar is a natural DNA sugar, a natural RNA sugar, a 2'-F modified sugar and a 2'-OR modified sugar (R is an optionally substituted C _1-6 aliphatic sugar). ), oligonucleotides independently selected from

1491. The oligonucleotide according to any one of the preceding embodiments, wherein each sugar is independently selected from natural DNA sugars, natural RNA sugars, 2'-F modified sugars, and 2'-OMe or 2'-MOE modified sugars. nucleotide.

1492. The oligonucleotide of any one of the above embodiments, wherein each internucleotidic linkage is independently selected from natural phosphate linkages, non-negatively charged internucleotidic linkages and phosphorothioate internucleotidic linkages.

1493. The oligonucleotide of any one of the above embodiments, wherein each internucleotidic linkage is independently selected from natural phosphate linkages, neutral internucleotide linkages and phosphorothioate internucleotide linkages.

1494. The oligonucleotide of any one of the preceding embodiments, wherein each internucleotidic linkage is independently selected from natural phosphate linkages, phosphorylguanidine internucleotide linkages and phosphorothioate internucleotide linkages.

1495. The oligonucleotide of any one of the preceding embodiments, wherein each internucleotidic linkage is independently selected from natural phosphate linkages, n001 and phosphorothioate internucleotidic linkages.

1496. Mod001L001mCn001RmC*SmC*SfA*SfG*SmCmA*SfG*SfCmU*SfUn001RmCfA*SfGn001RfUmC*SfC*SfC*SfU*SmUmUfC*ST*Sb008U*SIn001SmUfC*SmG*Sm An oligonucleotide having the structure of An001RmU.

1497. Mod001L001mCn001RmC*SmC*SfA*SfG*SmCmA*SfG*SfCmU*SfUn001RmCfA*SfGn001RfUmC*SfC*SfC*SfUn001RmUmUfC*ST*Sb008U*SIn001SmUfC*SmG* An oligonucleotide having the structure of SmAn001RmU.

1498. Mod001L001mCn001RmC*SmC*SfA*SfG*SmCmAfG*SfCmU*SfUn001RmCfA*SfGn001RfUmC*SfC*SmCfUn001RmUmUfC*ST*Sb008U*SIn001SmUfC*SmG*SmAn0 An oligonucleotide having a structure of 01RmU.

1499. Mod001L001mCn001RmC*SmC*SfA*SfG*SfCmA*SfG*SmCmU*SfUn001RmCfA*SfGn001RfUmC*SfC*SfC*SfUn001RfU*SmUfC*ST*Sb008U*SIn001SmUfC*Sm An oligonucleotide having a structure of G*SmAn001RmU.

1500. Mod001L001mCn001RmC*SmC*SfA*SfG*SfCmA*SfG*SfCmU*SfUn001RmCfA*SfGn001RfUmC*SmCfC*SfUn001RfU*SmUfC*ST*Sb008U*SIn001SmUfC*SmG* An oligonucleotide having the structure of SmAn001RmU.

1501. Mod001L001mCn001RmC*SmC*SfA*SfG*SmCmAfG*SfC*SfU*SfUn001RfC*SfAfGn001RfUmCmCfC*SfU*SmUmU*SfC*ST*Sb008U*SIn001SmUfC*SmG*SmAn0 An oligonucleotide having a structure of 01RmU.

1502. Mod001L001mCn001RmC*SmC*SfA*SfG*SfCmA*SfG*SfCmU*SfUn001RmCfA*SfGn001RfUmC*SfC*SfC*SfU*SfU*SmUfC*ST*Sb008U*SIn001SmUfC*SmG* An oligonucleotide having the structure of SmAn001RmU.

1503. Mod001L001mCn001RmC*SmC*SfA*SfG*Sm5CeoAeofG*SfC*STeofUn001RmCfA*SfGn001RfUmC*SfC*SfC*SfUn001RTeoTeofC*ST*Sb008U*SIn001SmUfC*SmG*S An oligonucleotide having the structure of mAn001RmU.

1504. Mod001L001mCn001RmC*SmC*SfA*SfG*SmCmAfG*SfC*SmUfUn001RmCfA*SmGn001RfUmC*SfC*SfC*SfUn001RmUmUfC*ST*Sb008U*SIn001SmUfC*SmG*Sm An oligonucleotide having the structure of An001RmU.

1505. Mod001L001mCn001RmC*SmC*SfA*SfG*Sm5CeoAeofG*SfC*STeofUn001RmCfA*SmGn001RfUmC*SfC*SfC*SfUn001RTeoTeofC*ST*Sb008U*SIn001SmUfC*SmG*S An oligonucleotide having the structure of mAn001RmU.

1506. Mod001L001mCn001RmC*SmC*SfA*SfG*Sm5CeoAeofG*SfC*STeofUn001RmCfA*SmGn001RfUm5Ceo*SfC*SfC*SfUn001RTeoTeofC*ST*Sb008U*SIn001SmUfC*SmG An oligonucleotide having the structure of *SmAn001RmU.

1507. Mod001L001mCn001RmC*SmC*SfA*SfG*Sm5CeoAeofG*SmCTeo*SmUn001RmCfA*SfGn001RmUmCmC*SfC*SfU*STeoTeofC*ST*Sb008U*SIn001SmUfC*SmG*SmAn00 An oligonucleotide having a structure of 1RmU.

1508. Mod001L001mCn001RmC*SmC*SfA*SfG*Sm5CeoAeofG*SmCTeo*SmUn001RmCfA*SfGn001RmUm5CeomC*SfC*SfU*STeoTeofC*ST*Sb008U*SIn001SmUfC*SmG*SmAn An oligonucleotide having a structure of 001RmU.

1509. Mod001L001mCn001RmC*SmC*SfA*SfG*Sm5CeoAeofG*Sm5CeoTeo*SmUn001Rm5CeofA*SfGn001RmUm5Ceom5Ceo*SfC*SfU*STeoTeofC*ST*Sb008U*SIn001SmUfC* An oligonucleotide having a structure of SmG*SmAn001RmU.

1510. Mod001L001mCn001RmC*SmC*SfA*SfG*Sm5CeoAeofG*SfC*SmUmUn001RmCfA*SfGn001RfUm5Ceo*SfC*SmCmUn001RmUTeofC*ST*Sb008U*SIn001SmUfC*SmG* An oligonucleotide having the structure of SmAn001RmU.

1511. Mod001L001mCn001RmC*SmC*SfA*SfG*Sm5CeoAeofG*SfC*STeofUn001RmCfA*SfGn001RfUm5Ceo*SfC*SfC*SfUn001RTeoTeofC*ST*Sb008U*SIn001SmUfC*SmG An oligonucleotide having the structure of *SmAn001RmU.

1512. The oligonucleotide according to any one of the preceding embodiments, in salt form.

1513. The oligonucleotide according to any one of the above embodiments, in the form of a pharmaceutically acceptable salt.

1514. The method of any one of the preceding embodiments, wherein one or more (e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more) chiral linkage phosphorus centers are independently about or at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% , 98% or 99%, an oligonucleotide.

1515. The method of any one of the preceding embodiments, wherein one or more (e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more) chiral linkage phosphorus centers are independently about or at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% Phosphorus, oligonucleotide.

1516. The method of any one of the preceding embodiments, wherein the diastereomeric excess of each phosphorothioate-linked phosphorus is independently about or at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% oligonucleotide.

1517. The method of any one of the preceding embodiments, wherein the diastereomeric excess of each phosphorothioate-linked phosphorus is independently about or at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% oligonucleotide.

1518. The method of any one of the preceding embodiments, wherein the diastereomeric excess of each chiral linkage phosphorus center is independently about or at least about 80%, 85%, 90%, 91%, 92%, 93%, 94 %, 95%, 96%, 97%, 98% or 99% oligonucleotide.

1519. The method of any one of the preceding embodiments, wherein the diastereomeric excess of each chiral linkage phosphorus center is independently about or at least about 90%, 91%, 92%, 93%, 94%, 95%, 96 %, 97%, 98% or 99% oligonucleotide.

1520. The method of any one of the above embodiments, wherein about 10% to 100% (e.g., about 10% to 95%, 50% to 80%, 50% to 85%, 50% to 90%, 50 %~95%, 60%~80%, 60%~85%, 60%~90%, 60%~95%, 60%~100%, 65%~80%, 65%~85%, 65%~ 90%, 65% to 95%, 65% to 100%, 70% to 80%, 70% to 85%, 70% to 90%, 70% to 95%, 70% to 100%, 75% to 80% , 75%~85%, 75%~90%, 75%~95%, 75%~100%, 80%~85%, 80%~90%, 80%~95%, 80%~100%, 85 % to 90%, 85% to 95%, 85% to 100%, 90% to 95%, 90% to 100%, or about or at least about 10%, 20%, 30%, 40%, 50%, 60 %, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% purity, etc.).

1521. The method of any one of the above embodiments, wherein about 50% to 100% (e.g., about 50% to 80%, 50% to 85%, 50% to 90%, 50% to 95%, 60 %~80%, 60%~85%, 60%~90%, 60%~95%, 60%~100%, 65%~80%, 65%~85%, 65%~90%, 65%~ 95%, 65% to 100%, 70% to 80%, 70% to 85%, 70% to 90%, 70% to 95%, 70% to 100%, 75% to 80%, 75% to 85% , 75%~90%, 75%~95%, 75%~100%, 80%~85%, 80%~90%, 80%~95%, 80%~100%, 85%~90%, 85 % to 95%, 85% to 100%, 90% to 95%, 90% to 100%, or at least about 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, oligonucleotides with a purity of 95%, or 100%, etc.).

1522. A pharmaceutical composition comprising or delivering an effective amount of an oligonucleotide of any one of the preceding embodiments or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier.

1523. An oligonucleotide composition comprising a plurality of oligonucleotides, wherein the plurality of oligonucleotides

1) common base sequence and

2) one or more (e.g., about 1-50, 1-40, 1-30, 1-25, 1-20, 1-15, 1-10, 5-50, 5-40, 5-30; 5-25, 5-20, 5-15, 5-10, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 or more) chiral internucleotide linkages ("chiral controlled internucleotide linkages") that share stereochemistry, which is the same linkage independently;

Wherein each of the plurality of oligonucleotides is independently an oligonucleotide of any one of the above embodiments or an acid, base or salt form thereof.

1524. An oligonucleotide composition comprising a plurality of oligonucleotides, wherein the plurality of oligonucleotides

1) common base sequence and

2) one or more (e.g., about 1-50, 1-40, 1-30, 1-25, 1-20, 1-15, 1-10, 5-50, 5-40, 5-30; 5-25, 5-20, 5-15, 5-10, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 or more) chiral internucleotide linkages ("chiral controlled internucleotide linkages") that share stereochemistry, which is the same linkage independently;

Wherein each of the plurality of oligonucleotides is independently an oligonucleotide of any one of embodiments 1637-1662 or an acid, base or salt form thereof.

1525. An oligonucleotide composition comprising a plurality of oligonucleotides, wherein the plurality of oligonucleotides

1) common base sequence and

2) one or more (e.g., about 1-50, 1-40, 1-30, 1-25, 1-20, 1-15, 1-10, 5-50, 5-40, 5-30; 5-25, 5-20, 5-15, 5-10, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 or more) chiral internucleotide linkages ("chiral controlled internucleotide linkages") that share stereochemistry, which is the same linkage independently;

The consensus nucleotide sequence is complementary to the nucleotide sequence of the nucleic acid portion comprising the target adenosine, composition.

1526. The method of embodiment 1525, wherein the consensus base sequence is 0-10 that are not Watson-Crick base pairs (e.g., 0-1, 0-2, 0-3, 0-4, 0-5, 0-6 , 0~7, 0~8, 0~9, 0~10, 1~2, 1~3, 1~4, 1~5, 1~6, 1~7, 1~8, 1~9, 1 ~10, 2~3, 2~4, 2~5, 2~6, 2~7, 2~8, 2~9, 2~10, 3~4, 3~5, 3~6, 3~7 , 3 to 8, 3 to 9, 3 to 10, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mismatches, etc.) , composition.

1527. The composition of embodiment 1525, wherein the consensus base sequence is complementary to a base sequence of a nucleic acid portion with 0-5 mismatches that are not Watson-Crick base pairs.

1528. The composition of embodiment 1525, wherein the consensus base sequence is 100% complementary to the base sequence of the nucleic acid portion over the length of the consensus base sequence, excluding the nucleoside opposite the target adenosine.

1529. The composition of embodiment 1525, wherein the consensus base sequence is 100% complementary to the base sequence of the nucleic acid portion over the length of the consensus base sequence.

1530. The composition according to any one of embodiments 1523 to 1529, capable of editing target A to I when contacted with a nucleic acid in a system expressing an ADAR.

1531. The composition of any one of embodiments 1523-1530, wherein the target adenosine is a G to A mutation associated with a condition, disorder or disease.

1532. The composition of any one of embodiments 1523-1531, wherein the plurality of oligonucleotides share the same base and sugar modifications.

1533. The composition of any one of embodiments 1523-1532, wherein the plurality of oligonucleotides share the same backbone chiral center pattern.

1534. A composition according to any one of embodiments 1523 or 1533, wherein a plurality of oligonucleotides are enriched in comparison to a sterically random preparation of oligonucleotides in which internucleotidic linkages are not chirally controlled.

1535. The composition of any one of embodiments 1523-1533, wherein all oligonucleotides at nonrandom levels in the composition that share a common base sequence and the same base and sugar modifications are a plurality of oligonucleotides.

1536. The composition of any one of embodiments 1523-1533, wherein all oligonucleotides at nonrandom levels in the composition that share a common base sequence are a plurality of oligonucleotides.

1537. The composition of any one of embodiments 1523-1536, wherein the plurality of oligonucleotides are of the same oligonucleotide or one or more pharmaceutically acceptable salts thereof.

1538. The composition of any one of embodiments 1523-1536, wherein the plurality of oligonucleotides are one or more pharmaceutically acceptable salts of the same acid form oligonucleotide.

1539. The composition of any one of embodiments 1523-1536, wherein the plurality of oligonucleotides have the same composition.

1540. The composition of embodiment 1539, wherein all oligonucleotides at non-random levels in the composition that share the same base sequence as the plurality of oligonucleotides are a plurality of oligonucleotides.

1541. The composition of embodiment 1539, wherein all oligonucleotides at non-random levels in the composition that share the same composition are a plurality of oligonucleotides.

1542. The composition of any one of embodiments 1523-1536, wherein the plurality of oligonucleotides have the same structure.

1543. The composition of any one of embodiments 1523-1542, wherein the plurality of oligonucleotides are sodium salts.

1544. The composition of any one of embodiments 1523-1543, wherein the plurality of oligonucleotides share the same linkage stereochemistry at 10 or more chiral internucleotide linkages.

1545. The composition of any one of embodiments 1523-1544, wherein the plurality of oligonucleotides share the same linkage stereochemistry at each phosphorothioate internucleotide linkage.

1546. The composition of any one of embodiments 1523-1545, wherein the plurality of oligonucleotides do not share the same linkage stereochemistry at one or more or any internucleotide linkages that are not negatively charged.

1547. An oligonucleotide composition comprising one or more of a plurality of oligonucleotides, wherein each of the plurality of oligonucleotides is independently

1) common base sequence and

2) one or more (e.g., about 1-50, 1-40, 1-30, 1-25, 1-20, 1-15, 1-10, 5-50, 5-40, 5-30; 5-25, 5-20, 5-15, 5-10, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 or more) chiral internucleotide linkages ("chiral controlled internucleotide linkages") that share stereochemistry, which is the same linkage independently;

Wherein each of the plurality of oligonucleotides is independently an oligonucleotide of any one of the above embodiments or an acid, base or salt form thereof.

1548. An oligonucleotide composition comprising one or more of a plurality of oligonucleotides, wherein each of the plurality of oligonucleotides is independently

1) common base sequence and

2) one or more (e.g., about 1-50, 1-40, 1-30, 1-25, 1-20, 1-15, 1-10, 5-50, 5-40, 5-30; 5-25, 5-20, 5-15, 5-10, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 or more) chiral internucleotide linkages ("chiral controlled internucleotide linkages") that share stereochemistry, which is the same linkage independently;

Wherein each of the plurality of oligonucleotides is independently an oligonucleotide of any one of the embodiments and embodiments 1637 to 1662 or an acid, base or salt form thereof.

1549. An oligonucleotide composition comprising one or more of a plurality of oligonucleotides, wherein each of the plurality of oligonucleotides is independently

1) common base sequence and

2) one or more (e.g., about 1-50, 1-40, 1-30, 1-25, 1-20, 1-15, 1-10, 5-50, 5-40, 5-30; 5-25, 5-20, 5-15, 5-10, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 or more) chiral internucleotide linkages ("chiral controlled internucleotide linkages") that share stereochemistry, which is the same linkage independently;

Wherein each of the plurality of oligonucleotides is independently an oligonucleotide of any one of embodiments 1637-1662 or an acid, base or salt form thereof.

1550. An oligonucleotide composition comprising one or more of a plurality of oligonucleotides, wherein each of the plurality of oligonucleotides is independently

1) common base sequence and

2) one or more (e.g., about 1-50, 1-40, 1-30, 1-25, 1-20, 1-15, 1-10, 5-50, 5-40, 5-30; 5-25, 5-20, 5-15, 5-10, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, At least 18, 19, 20, 21, 22, 23, 24, or 25) chiral internucleotide linkages ("chiral controlled internucleotide linkages") share stereochemistry that is the same linkage independently;

Wherein each of the plurality of consensus nucleotide sequences is independently complementary to a nucleotide sequence of a nucleic acid portion comprising a target adenosine.

1551. In an embodiment, the consensus base sequence is 0-10 non-Watson-Crick base pairs (e.g., 0-1, 0-2, 0-3, 0-4, 0-5, 0-6, 0~7, 0~8, 0~9, 0~10, 1~2, 1~3, 1~4, 1~5, 1~6, 1~7, 1~8, 1~9, 1~ 10, 2~3, 2~4, 2~5, 2~6, 2~7, 2~8, 2~9, 2~10, 3~4, 3~5, 3~6, 3~7, 3-8, 3-9, 3-10, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, etc.) complementary to the base sequence of the nucleic acid portion with mismatches, composition.

1552. The composition of embodiment 1551, wherein each of the plurality of consensus base sequences is independently complementary to a base sequence of a nucleic acid portion having 0-5 mismatches that are not Watson-Crick base pairs.

1553. The composition of embodiment 1551, wherein each of the plurality of consensus sequences is independently 100% complementary to a sequence of bases in the nucleic acid portion over the length of the consensus sequence, excluding the nucleoside opposite the target adenosine.

1554. The composition of embodiment 1551, wherein each of the plurality of consensus base sequences is independently 100% complementary to the base sequence of the nucleic acid portion over the length of the consensus base sequence.

1555. The composition of any one of embodiments 1547-1554, wherein each of the plurality of oligonucleotides is capable of independently editing target A to I when contacted with a nucleic acid in a system expressing an ADAR.

1556. The composition of any one of embodiments 1547-1555, wherein the target adenosine is a G to A mutation associated with a condition, disorder or disease.

1557. The method according to any one of embodiments 1547 to 1556, wherein the plurality of oligonucleotides comprises two or more (eg, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) composition.

1558. The composition of any one of embodiments 1547-1557, wherein at least two of the plurality of common base sequences are different from each other.

1559. The composition of any one of embodiments 1547-1558, wherein the two plurality of oligonucleotides do not share the same common base sequence.

1560. The composition of any one of embodiments 1547-1559, wherein at least two of the plurality of oligonucleotides target different adenosines.

1561. The composition of any one of embodiments 1547-1560, wherein the two plurality of oligonucleotides do not target the same adenosine.

1562. The composition of any one of embodiments 1547-1561, wherein at least two of the plurality of oligonucleotides target different transcripts.

1563. The composition of any one of embodiments 1547-1562, wherein the two plurality of oligonucleotides do not target the same transcript.

1564. The composition of any one of embodiments 1547-1563, wherein at least two of the plurality of oligonucleotides target adenosine residues in transcripts from different polynucleotides.

1565. The composition of any one of embodiments 1547-1566, wherein the two plurality of oligonucleotides do not target transcripts from the same polynucleotide.

1566. The composition of any one of embodiments 1547-1565, wherein at least two of the plurality of oligonucleotides target adenosine residues in transcripts from different genes.

1567. The composition of any one of embodiments 1547 to 1566, wherein the two plurality of oligonucleotides do not target transcripts from the same gene.

1568. The composition of any one of embodiments 1547-1567, wherein each plurality of oligonucleotides independently share the same base and sugar modifications within the plurality.

1569. The composition of any one of embodiments 1547-1568, wherein each plurality of oligonucleotides independently share the same backbone chiral center pattern within the plurality.

1570. A composition according to any one of embodiments 1547 or 1569, wherein, independently for each plurality, a plurality of oligonucleotides are enriched relative to a sterically random preparation of the plurality of oligonucleotides in which internucleotide linkages are not chirally controlled.

1571. The method of any one of embodiments 1547 to 1570, wherein, independently for each plurality, all oligonucleotides at nonrandom levels in the composition that share a common base sequence and the same base and sugar modifications are a plurality of oligonucleotides. composition.

1572. The composition of any one of embodiments 1547-1570, wherein all oligonucleotides at nonrandom levels in the composition that share a common base sequence, independently for each plurality, are a plurality of oligonucleotides.

1573. The composition of any one of embodiments 1547-1572, wherein, independently for each plurality, the plurality of oligonucleotides are of the same oligonucleotide or one or more pharmaceutically acceptable salts thereof.

1574. The composition of any one of embodiments 1547-1573, wherein, independently for each plurality, the plurality of oligonucleotides are one or more pharmaceutically acceptable salts of the same acid form oligonucleotide.

1575. The composition of any one of embodiments 1547-1572, wherein independently for each plurality, the plurality of oligonucleotides have the same composition.

1576. The composition of embodiment 1575, wherein all oligonucleotides at non-random levels in the composition that share the same base sequence as the plurality of oligonucleotides, independently for each plurality, are oligonucleotides in the plurality.

1577. The composition of embodiment 1575, wherein all oligonucleotides at non-random levels in the composition that share the same composition, independently for each plurality, are a plurality of oligonucleotides.

1578. The composition of any one of embodiments 1547 to 1577, wherein, independently of one or two or all of the plurality, independently of each plurality, the plurality of oligonucleotides have the same structure.

1579. The composition of any one of embodiments 1547 to 1578, wherein, independently for one or two or all of the plurality, the plurality of oligonucleotides are each independently in the form of a pharmaceutically acceptable salt.

1580. The composition of any one of embodiments 1547 to 1578, wherein, independently for one or two or all of the plurality of oligonucleotides, the plurality of oligonucleotides are sodium salts.

1581. is according to any one of embodiments 1547 to 1580, wherein, independently for one or two or all of the plurality, the plurality of oligonucleotides share stereochemistry that is the same linkage at 10 or more chiral internucleotide linkages. composition.

1582. The composition of any one of embodiments 1547-1581, wherein, independently for each plurality, the plurality of oligonucleotides share stereochemistry that is the same linkage at 10 or more chiral internucleotide linkages.

1583. The method of any one of embodiments 1547 to 1582, wherein, independently of one or two or all of the plurality, the plurality of oligonucleotides share the same linkage stereochemistry at each phosphorothioate internucleotide linkage. Do, composition.

1584. The composition of any one of embodiments 1547-1583, wherein, independently for each plurality, the plurality of oligonucleotides share the same linkage stereochemistry at each phosphorothioate internucleotide linkage.

1585. The method of any one of embodiments 1547 to 1584, wherein, independently for one or two or all of the plurality, the plurality of oligonucleotides are identical linkages at one or more or any internucleotidic linkages that are not negatively charged. Compositions that do not share stereochemistry.

1586. The method according to any one of embodiments 1547 to 1585, wherein, independently for each plurality, the plurality of oligonucleotides do not share the same linkage stereochemistry at one or more or any internucleotidic linkages that are not negatively charged. , composition.

1587. A composition comprising a plurality of oligonucleotides of a particular oligonucleotide type, wherein the particular oligonucleotide type is

a) consensus sequence;

b) a common backbone connection pattern;

c) a common backbone chiral center pattern;

d) characterized by a deformation pattern that is a common backbone;

The composition is enriched in oligonucleotides of a particular oligonucleotide type relative to a substantially racemic preparation of oligonucleotides having the same common base sequence, backbone linkage pattern, and backbone phosphorus modification pattern, or within a composition that shares a common base sequence. chirally controlled in that all oligonucleotides at nonrandom levels are a plurality of oligonucleotides;

Wherein each of the plurality of oligonucleotides is independently an oligonucleotide of any one of the above embodiments or an acid, base or salt form thereof.

1588. A composition comprising a plurality of oligonucleotides of a particular oligonucleotide type, wherein the particular oligonucleotide type is

a) consensus sequence;

b) a common backbone connection pattern;

c) a common backbone chiral center pattern;

d) characterized by a deformation pattern that is a common backbone;

The composition is enriched in oligonucleotides of a particular oligonucleotide type relative to a substantially racemic preparation of oligonucleotides having the same common base sequence, backbone linkage pattern, and backbone phosphorus modification pattern, or within a composition that shares a common base sequence. chirally controlled in that all oligonucleotides at nonrandom levels are a plurality of oligonucleotides;

Wherein each of the plurality of oligonucleotides is independently an oligonucleotide of any one of embodiments 1637-1662 or an acid, base or salt form thereof.

1589. A composition comprising a plurality of oligonucleotides of a particular oligonucleotide type, wherein the particular oligonucleotide type is

a) consensus sequence;

b) a common backbone connection pattern;

c) a common backbone chiral center pattern;

d) characterized by a deformation pattern that is a common backbone;

The composition is enriched in oligonucleotides of a particular oligonucleotide type relative to a substantially racemic preparation of oligonucleotides having the same common base sequence, backbone linkage pattern, and backbone phosphorus modification pattern, or within a composition that shares a common base sequence. chirally controlled in that all oligonucleotides at nonrandom levels are a plurality of oligonucleotides;

The consensus nucleotide sequence is complementary to the nucleotide sequence of the nucleic acid portion comprising the target adenosine, composition.

1590. The method of embodiment 1589, wherein the consensus base sequence is 0-10 that are not Watson-Crick base pairs (e.g., 0-1, 0-2, 0-3, 0-4, 0-5, 0-6 , 0~7, 0~8, 0~9, 0~10, 1~2, 1~3, 1~4, 1~5, 1~6, 1~7, 1~8, 1~9, 1 ~10, 2~3, 2~4, 2~5, 2~6, 2~7, 2~8, 2~9, 2~10, 3~4, 3~5, 3~6, 3~7 , 3 to 8, 3 to 9, 3 to 10, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mismatches, etc.) , composition.

1591. The composition of embodiment 1589, wherein the consensus base sequence is complementary to a base sequence of a nucleic acid portion with 0-5 mismatches that are not Watson-Crick base pairs.

1592. The composition of embodiment 1589, wherein the consensus sequence is 100% complementary to a sequence of bases in the nucleic acid portion over the length of the consensus sequence except for the nucleoside opposite the target adenosine.

1593. The composition of embodiment 1589, wherein the consensus base sequence is 100% complementary to the base sequence of the nucleic acid portion over the length of the consensus base sequence.

1594. The composition according to any one of embodiments 1587 to 1593, capable of editing target A to I when contacted with a nucleic acid in a system expressing an ADAR.

1595. The composition of any one of embodiments 1587-1594, wherein the target adenosine is a G to A mutation associated with a condition, disorder or disease.

1596. A composition according to any one of embodiments 1587 to 1595, wherein oligonucleotides of a particular oligonucleotide type are enriched relative to substantially racemic preparations of oligonucleotides having the same consensus base sequence, backbone linkage pattern, and backbone phosphorus modification pattern. .

1597. A composition comprising a plurality of oligonucleotides, wherein each oligonucleotide of the plurality is independently a particular oligonucleotide or a salt thereof, wherein the particular oligonucleotide is an oligonucleotide of any one of embodiments 1-1513.

1598. A composition comprising a plurality of oligonucleotides, wherein each oligonucleotide of the plurality is independently a particular oligonucleotide or a salt thereof, wherein the particular oligonucleotide is an oligonucleotide of any one of embodiments 1 to 1513, At least about 5%-100%, 10%-100%, 20-100%, 30%-100%, 40%-100%, 50%-100%, 5% of all oligonucleotides in the composition that share a base sequence ~90%, 10%~90%, 20~90%, 30%~90%, 40%~90%, 50%~90%, 5%~85%, 10%~85%, 20~85%, 30%~85%, 40%~85%, 50%~85%, 5%~80%, 10%~80%, 20~80%, 30%~80%, 40%~80%, 50%~ 80%, 5%~75%, 10%~75%, 20~75%, 30%~75%, 40%~75%, 50%~75%, 5%~70%, 10%~70%, 20-70%, 30%-70%, 40%-70%, 50%-70%, 5%-65%, 10%-65%, 20-65%, 30%-65%, 40%-65 %, 50%~65%, 5%~60%, 10%~60%, 20~60%, 30%~60%, 40%~60%, 50%~60%, 5%, 10%, 20 %, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% is a plurality of oligonucleotides.

1599. A composition comprising a plurality of oligonucleotides, wherein each oligonucleotide of the plurality is independently a particular oligonucleotide or a salt thereof, wherein the particular oligonucleotide is an oligonucleotide of any one of embodiments 1 to 1513, and wherein the particular oligonucleotide or At least about 5%-100%, 10%-100%, 20-100%, 30%-100%, 40%-100%, 50%-100%, 5% of all oligonucleotides in the composition that share a salt configuration thereof %~90%, 10%~90%, 20~90%, 30%~90%, 40%~90%, 50%~90%, 5%~85%, 10%~85%, 20~85% , 30%~85%, 40%~85%, 50%~85%, 5%~80%, 10%~80%, 20~80%, 30%~80%, 40%~80%, 50% ~80%, 5%~75%, 10%~75%, 20~75%, 30%~75%, 40%~75%, 50%~75%, 5%~70%, 10%~70% , 20~70%, 30%~70%, 40%~70%, 50%~70%, 5%~65%, 10%~65%, 20~65%, 30%~65%, 40%~ 65%, 50%~65%, 5%~60%, 10%~60%, 20~60%, 30%~60%, 40%~60%, 50%~60%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% , or 99% are a plurality of oligonucleotides.

1600. A composition comprising a plurality of oligonucleotides, wherein each oligonucleotide of the plurality is independently a specific oligonucleotide or a salt thereof, wherein the specific oligonucleotide is an oligonucleotide of Table 1.

1601. The composition of any one of embodiments 1587-1600, wherein all oligonucleotides at nonrandom levels in the composition that share a common base sequence are a plurality of oligonucleotides.

1602. The method of any one of embodiments 1523-1601, wherein the oligonucleotide level of the plurality of oligonucleotides in the composition that share a plurality of common base sequences is about or at least about (DS) ^nc (DS is from about 85% to about 85%). 100% (e.g., about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater than 99.5%) and nc is the number of chiral control internucleotide linkages), composition.

1603. The method of any one of embodiments 1523 to 1601, wherein for each plurality of oligonucleotides, the oligonucleotide level of the plurality of oligonucleotides in the composition that share a common base sequence is independently about or at least about (DS) ^nc Phosphorus (DS is about 85% to 100% (e.g., about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%) %, 97%, 98%, 99%, or 99.5%) and nc is the number of chiral control internucleotide linkages), composition.

1604. The method of any one of embodiments 1523-1601, wherein the oligonucleotide level of the plurality of oligonucleotides in the composition sharing a common composition is about or at least about (DS) ^nc (DS is about 85%-100% ( For example, about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% or greater) and nc is the number of chiral controlled internucleotide linkages), the composition.

1605. The method of any one of embodiments 1523 through 1601, wherein for each plurality of oligonucleotides, the oligonucleotide level of the plurality of oligonucleotides in the composition that share a common configuration is independently about or at least about (DS) ^nc (DS is about 85% to 100% (e.g., about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96% , 97%, 98%, 99%, or 99.5%) and nc is the number of chiral control internucleotide linkages), the composition.

1606. The method of any one of embodiments 1523-1605, wherein the DS is between about 90% and 100% (e.g., about 90%, 91%, 92%, 93%, 94%, 95%, 96%, at least 97%, 98%, 99%, or 99.5%).

1607. The method of any one of embodiments 1602-1606, wherein nc is from about 5 to about 40 (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 , 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37 , 38, 39, or 40) or higher.

1608. The method of any one of embodiments 1523-1601, wherein the level is at least about 10% to 100%, or at least about 10%, 20%, 30%, 40%, 50%, 60%, 65%, 70% %, 75%, 80%, 85%, 90%, or 95%.

1609. The method of any one of embodiments 1523-1601, wherein the level is at least about 50%-100%, or at least about 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90% %, or 95%, composition.

1610. A composition comprising a specific oligonucleotide, wherein about 10% to 100% (e.g., about 10% to 100%, 20 to 100%, 30%) of all oligonucleotides in the composition that share the base sequence of the oligonucleotide ~100%, 40%~100%, 50%~80%, 50%~85%, 50%~90%, 50%~95%, 60%~80%, 60%~85%, 60%~90 %, 60%~95%, 60%~100%, 65%~80%, 65%~85%, 65%~90%, 65%~95%, 65%~100%, 70%~80%, 70%~85%, 70%~90%, 70%~95%, 70%~100%, 75%~80%, 75%~85%, 75%~90%, 75%~95%, 75% ~100%, 80%~85%, 80%~90%, 80%~95%, 80%~100%, 85%~90%, 85%~95%, 85%~100%, 90%~95 %, 90% to 100%, 10%, 20%, 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% etc.) are independently specific oligonucleotides or salts thereof.

1611. A composition comprising a specific oligonucleotide, wherein about 30% to 90% of all oligonucleotides in the composition that share a base sequence of the oligonucleotide are independently specific oligonucleotides or salts thereof.

1612. A composition comprising a specific oligonucleotide, wherein about 40% to 90% of all oligonucleotides in the composition that share a base sequence of the oligonucleotide are independently specific oligonucleotides or salts thereof.

1613. The composition of any one of embodiments 1610-1612, wherein the particular oligonucleotide is an oligonucleotide of any one of embodiments 1-1521.

1614. The composition of any one of embodiments 1610 to 1613, wherein the particular oligonucleotide is an oligonucleotide selected from Table 1.

1615. The method of any one of embodiments 1610-1614, wherein the specific oligonucleotide is about or at least about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 , 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more chiral internucleotide linkages.

1616. The composition of any one of embodiments 1610-1615, wherein each salt is independently a pharmaceutically acceptable salt.

1617. The composition of any one of embodiments 1523-1616, wherein the target adenosine residue is modified when the composition is contacted with a sample comprising a target nucleic acid and an adenosine deaminase.

1618. The composition of embodiment 1617, wherein the modification is or comprises a modification performed by ADAR1.

1619. The composition of embodiment 1617 or 1618, wherein the modification is or comprises a modification performed by ADAR2.

1620. The composition of any one of embodiments 1617-1619, wherein the modification is performed in vitro.

1621. The composition of any one of embodiments 1617-1619, wherein the sample is a cell.

1622. The composition of any one of embodiments 1617-1621, wherein the target adenosine is converted to inosine.

1623. The composition of any one of embodiments 1617-1622, wherein the target adenosine is modified to a greater degree than that observed in a comparable reference oligonucleotide composition.

1624. The composition of embodiment 1623, wherein the reference oligonucleotide composition comprises no or lower levels of the plurality of oligonucleotides.

1625. The composition of embodiment 1623 or 1624, wherein the reference composition does not contain oligonucleotides having the same composition as the plurality of oligonucleotides.

1626. The composition of any one of embodiments 1623-1625, wherein the reference composition does not contain oligonucleotides having the same structure as the plurality of oligonucleotides.

1627. The composition of embodiment 1623, wherein the reference oligonucleotide composition is a composition wherein an oligonucleotide having the same base sequence as the plurality of oligonucleotides comprises a lower level of 2'-F modification than the plurality of oligonucleotides.

1628. The method according to any one of embodiments 1623 to 1627, wherein the reference oligonucleotide composition comprises an oligonucleotide having the same base sequence as the plurality of oligonucleotides comprising a lower level of 2'-OMe modification than the plurality of oligonucleotides. A composition that is a composition.

1629. The composition of any one of embodiments 1623-1628, wherein the reference oligonucleotide composition is a composition wherein an oligonucleotide having the same base sequence as the plurality of oligonucleotides has a different sugar modification pattern compared to the plurality of oligonucleotides. .

1630. The reference oligonucleotide composition of any one of embodiments 1623 to 1629, wherein an oligonucleotide having the same base sequence as the plurality of oligonucleotides comprises a lower level of modified internucleotide linkages than the plurality of oligonucleotides. A composition, a composition.

1631. The reference oligonucleotide composition of any one of embodiments 1623 to 1630, wherein the oligonucleotide having the same base sequence as the plurality of oligonucleotides has a lower level of phosphorothioate internucleotide linkages than the plurality of oligonucleotides A composition comprising a, composition.

1632. The composition of any one of embodiments 1623-1631, wherein the reference composition is a stereorandom oligonucleotide composition.

1633. The composition of embodiment 1623, wherein the reference composition is a stereorandom oligonucleotide composition of a plurality of oligonucleotides and oligonucleotides of identical composition.

1634. The method of any one of the preceding embodiments, which does not cause significant degradation of the nucleic acid (e.g., up to about 5%-100% (e.g., up to about 10-100%, 20-100%, 30%~100%, 40%~100%, 50%~80%, 50%~85%, 50%~90%, 50%~95%, 60%~80%, 60%~85%, 60% ~90%, 60%~95%, 60%~100%, 65%~80%, 65%~85%, 65%~90%, 65%~95%, 65%~100%, 70%~80 %, 70%~85%, 70%~90%, 70%~95%, 70%~100%, 75%~80%, 75%~85%, 75%~90%, 75%~95%, 75% to 100%, 80% to 85%, 80% to 90%, 80% to 95%, 80% to 100%, 85% to 90%, 85% to 95%, 85% to 100%, 90% ~95%, 90%~100%, 10%, 20%, 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% etc.)) composition.

1635. The method of any one of the above embodiments, which does not cause significant exon skipping or altered exon inclusion in the target nucleic acid (e.g., at most about 5%-100% (e.g., at most about 10%-100%) , 20-100%, 30%-100%, 40%-100%, 50%-80%, 50%-85%, 50%-90%, 50%-95%, 60%-80%, 60% ~85%, 60%~90%, 60%~95%, 60%~100%, 65%~80%, 65%~85%, 65%~90%, 65%~95%, 65%~100 %, 70%~80%, 70%~85%, 70%~90%, 70%~95%, 70%~100%, 75%~80%, 75%~85%, 75%~90%, 75% to 95%, 75% to 100%, 80% to 85%, 80% to 90%, 80% to 95%, 80% to 100%, 85% to 90%, 85% to 95%, 85% ~100%, 90%~95%, 90%~100%, 10%, 20%, 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90 %, 95%, or 100%, etc)) composition.

1636. The composition of any one of embodiments 1523-1635, wherein the composition is a pharmaceutical composition and further comprises a pharmaceutically acceptable carrier.

1637. An oligonucleotide identical to the oligonucleotide of any one of the above embodiments except that at the position of the modified internucleotide linkage there is a linkage having the structure -O ⁵ -P ^L (R ^CA )-O ³ -

(Wherein, P ^L is P or P(=W);

W is O, S, or W ^N ;

R ^CA is or contains an optionally substituted or capped chiral auxiliary moiety,

O ⁵ is an oxygen bonded to the 5'-carbon of the sugar;

O ³ is the oxygen attached to the 3'-carbon of the sugar).

1638. The oligonucleotide of embodiment 1637, wherein the chiral auxiliary is removed and the linkage is converted to a modified internucleotidic linkage.

1639. The oligonucleotide of embodiment 1637, wherein the modified internucleotide linkages are phosphorothioate internucleotide linkages.

1640. The oligonucleotide of embodiment 1639, wherein when W is replaced with -SH and R ^CA is replaced with O, ^PL has the same configuration as the linkage phosphorus of a phosphorothioate internucleotidic linkage.

1641. The oligonucleotide according to any one of embodiments 1637 to 1640, wherein the modified internucleotide linkages are neutral internucleotide linkages.

1642. The oligonucleotide according to any one of embodiments 1637 to 1640, wherein the modified internucleotide linkages are phosphoryl guanidine internucleotide linkages.

1643. The oligonucleotide according to any one of embodiments 1637 to 1640, wherein the modified internucleotide linkages are n004, n008, n025, n026.

1644. The oligonucleotide according to any one of embodiments 1637 to 1640, wherein the modified internucleotide linkage is n001.

1645. according to any one of embodiments 1637 to 1644, wherein each position of the phosphorothioate internucleotidic linkage is independently a linkage having the structure -O ⁵ -P ^L (W)(R ^CA )-O ³ - , oligonucleotides.

1646. is according to any one of embodiments 1637 to 1644, wherein at each position of the modified internucleotide linkage independently there is a linkage having the structure -O ⁵ -P ^L (W)(R ^CA )-O ³ -, oligonucleotide.

1647. The oligonucleotide according to any one of embodiments 1637 to 1646, wherein one or each W is S.

1648. The oligonucleotide according to any one of embodiments 1637 to 1647, wherein only one P ^L is P.

1649. The method of any one of embodiments 1637 to 1648, wherein each R ^CA is independently or Phosphorus, oligonucleotide.

1650. The method of any one of embodiments 1637 to 1648, wherein each R ^CA is independently or , R ^C1 is R, -Si(R) ₃ or -SO ₂ R, and R ^C2 and R ^C3 are optionally substituted having 0 to 2 heteroatoms in addition to the nitrogen atom together with the atoms intervening therebetween. An oligonucleotide that forms a 3-7 membered saturated or partially unsaturated ring, and R ^C4 is -H or -C(O)R'.

1651. The oligonucleotide of embodiment 1649 or 1650, wherein R ^C4 in the linkage is -C(O)R and ^PL is P.

1652. The oligonucleotide of embodiment 1650 or 1651, wherein in the linkage R ^C4 is -C(O)R and W is S.

1653. The oligonucleotide according to any one of embodiments 1650 to 1652, wherein at each linkage where W is S, R ^C4 is -C(O)R'.

1654. The oligonucleotide according to any one of embodiments 1650 to 1653, wherein R ^C4 is -C(O)CH ₃ .

1655. The oligonucleotide of embodiment 1650, wherein R ^C4 in the linkage is -H and ^PL is P.

1656. is according to any one of embodiments 1650 to 1655, wherein R ^C2 and R ^C3 together with the atoms intervening between them form an optionally substituted 5-membered ring having no heteroatoms other than nitrogen atoms; nucleotide.

1657. The method of any one of embodiments 1650 to 1656, wherein each R ^CA is independently or Phosphorus, oligonucleotide.

1658. The oligonucleotide according to any one of embodiments 1650 to 1657, wherein R ^C1 is -SiPh ₂ Me.

1659. The oligonucleotide according to any one of embodiments 1650 to 1657, wherein R ^C1 is -SO ₂ R.

1660. The oligonucleotide according to any one of embodiments 1650 to 1657, wherein R ^C1 is -SO ₂ R and R is an optionally substituted C _1-10 aliphatic.

1661. The oligonucleotide according to any one of embodiments 1650 to 1657, wherein R ^C1 is -SO ₂ R and R is optionally substituted phenyl.

1662. The oligonucleotide according to any one of embodiments 1650 to 1657, wherein R ^C1 is -SO ₂ R and R is phenyl.

1663. A phosphoramidite wherein the nucleobase is the nucleobase of any one of embodiments 1 to 1521 or a tautomer thereof, wherein the nucleobase or tautomer thereof is optionally substituted or protected.

1664. A phosphoramidite wherein the nucleobase is or comprises ring BA or a tautomer of ring BA, and ring BA is BA-I, BA-I-a, BA-I-b, BA- II, BA-II-a, BA-II-b, BA-III, BA-III-a, BA-III-b, BA-IV, BA-IV-a, BA-IV-b, BA-V, A phosphoramidite having the structure BA-V-a, BA-V-b, or BA-VI, wherein the nucleobases are optionally substituted or protected.

1665. The phosphoramidite of embodiment 1663 or 1664, wherein the sugar of the phosphoramidite is the sugar of any one of embodiments 1-1521, and the sugar is optionally protected.

1666. is according to any one of embodiments 1663 to 1665, and has the structure of R ^NS -P(OR)N(R) ₂ , R ^NS is an optionally protected nucleoside moiety, and each R is used herein Phosphoamidites, as described in.

1667. The phosphoramidite according to any one of embodiments 1663 to 1665, having the structure R ^NS -P(OCH ₂ CH ₂ CN)N(i-Pr) ₂ .

1668. The phosphoramidite according to any one of embodiments 1663 to 1665, comprising a chiral auxiliary moiety, wherein phosphorus is bonded to oxygen and nitrogen atoms of the chiral auxiliary moiety.

1669. according to any one of embodiments 1663 to 1665 or 1668, , , , or , or a phosphoramidite having a salt structure thereof.

1670. The method of any one of embodiments 1663 to 1665 or 1668, wherein the phosphoramidite is or has the structure of, R ^NS is an optionally protected nucleoside moiety, R ^C1 is R, -Si(R) ₃ or -SO ₂ R, and R ^C2 and R ^C3 are atoms intervening therebetween and Phosphoamidites, which together form an optionally substituted 3-7 membered saturated or partially unsaturated ring having 0-2 heteroatoms in addition to nitrogen atoms.

1671. The phosphoramidite according to embodiment 1669 or 1670, wherein R ^C2 and R ^C3 together with the atoms intervening therebetween form an optionally substituted 5-membered saturated ring having no heteroatoms other than nitrogen atoms. .

1672. The method of any one of embodiments 1669 to 1671, , , , , , or , or a phosphoramidite having a salt structure thereof.

1673. The method of any one of embodiments 1669 to 1671, , , , , , or , or a phosphoramidite having a salt structure thereof.

1674. according to any one of embodiments 1669 to 1671, , , , or , or a phosphoramidite having a salt structure thereof.

1675. The method of any one of embodiments 1669 to 1671, wherein or Phosphoamidite having the structure of.

1676. The phosphoramidite according to any one of embodiments 1669 to 1675, wherein R ^C1 is -SiPh ₂ Me.

1677. The phosphoramidite according to any one of embodiments 1669 to 1675, wherein R ^C1 is -SO ₂ R.

1678. The phosphoramidite according to any one of embodiments 1669 to 1675, wherein R ^C1 is —SO ₂ R and R is an optionally substituted C _1-10 aliphatic.

1679. The phosphoramidite according to any one of embodiments 1669 to 1675, wherein R ^C1 is -SO ₂ R and R is optionally substituted phenyl.

1680. The phosphoramidite according to any one of embodiments 1669 to 1675, wherein R ^C1 is -SO ₂ R and R is phenyl.

1681. or a salt structure thereof, wherein R ^NS is an optionally substituted/protected nucleoside, X ^C is O or S, and each of R ^C5 and R ^C6 is independently R.

1682. The compound of embodiment 1681, wherein X ^C is O.

1683. The compound of embodiment 1681, wherein X ^C is S.

1684. The compound according to any one of embodiments 1681 to 1683, wherein one R ^C5 is not hydrogen.

1685. The compound according to any one of embodiments 1681 to 1684, wherein one R ^C5 is hydrogen.

1686. The compound according to any one of embodiments 1681 to 1685, wherein one R ^C6 is not hydrogen.

1687. The compound according to any one of embodiments 1681 to 1686, wherein one R ^C6 is hydrogen.

1688. The method of any one of embodiments 1681 to 1687, wherein one R ^C5 and one R ^C6 are optionally substituted 3-20 (eg, 3-20) having 0-5 heteroatoms together with intervening atoms. 15, 3~10, 5~10, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20) single rings, A compound that forms a bicyclic or polycyclic ring.

1689. The compound according to any one of embodiments 1681 to 1687, wherein one R ^C5 and one R ^C6 together with intervening atoms form an optionally substituted cyclohexyl ring.

1690. The compound of embodiment 1681, wherein -X ^C -C(R ^C5 ) ₂ -C(R ^C6 ) ₂ -S- is -OCH(CH ₃ )CH(CH ₃ )S-.

1691. The compound of embodiment 1681, wherein -X ^C -C(R ^C5 ) ₂ -C(R ^C6 ) ₂ -S- is -SCH(CH ₃ )CH(CH ₃ )S-.

1692. The phosphoramidite or compound according to any one of embodiments 1666 to 1691, wherein the hydroxyl group of R ^NS is protected.

1693. The phosphoramidite or compound according to any one of embodiments 1666 to 1691, wherein the hydroxyl group of R ^NS is protected as -ODMTr.

1694. The phosphoramidite or compound according to any one of embodiments 1666 to 1691, wherein the 5'-OH of R ^NS is protected.

1695. The phosphoramidite or compound of embodiment 1694, wherein the 5'-OH of R ^NS is protected as -ODMTr.

1696. The method of any one of embodiments 1666 to 1695, wherein R ^NS is , , , , , , , , , , , , , , and , or a salt thereof, wherein BA ^s is a phosphoramidite or compound as described herein.

1697. The method of any one of embodiments 1666 to 1696, wherein R ^NS is , , , , , , , , , , , , , , and , or a salt thereof, wherein BA ^s is a phosphoramidite or compound as described herein.

1698. The method of any one of embodiments 1666 or 1697, wherein R ^NS is , , , , , , , , , , , , , , and , or salts thereof, wherein BA ^s is an optionally substituted or protected nucleobase, and each —OH is optionally and independently substituted or protected.

1699. The method of any one of embodiments 1666 to 1698, wherein R ^NS is , , , , , , , , , , , , , , and , or a salt thereof, wherein BA ^s is an optionally substituted or protected nucleobase, each -OH of a nucleoside is independently protected, and at least one -OH is protected as DMTrO-. Dite or Compound.

1700. The method of any one of embodiments 1666 to 1699, wherein R ^NS is , , , , , , , , , , , , , , and , or salts thereof, wherein BA ^s is an optionally protected nucleobase selected from A, T, C, G, U and tautomers thereof, each -OH of the nucleoside is independently protected, and at least one -OH of is protected as DMTrO-, phosphoramidite or compound.

1701. The method of any one of embodiments 1666 to 1700, wherein the phosphoramidite or compound comprises a nucleobase or a tautomer thereof of any one of embodiments 1 to 1521, wherein the nucleobase or tautomer thereof is An optionally substituted or protected phosphoramidite or compound.

1702. The method according to any one of embodiments 1666 to 1701, wherein the phosphoramidite or compound comprises a nucleobase, and the nucleobase is Ring BA or a tautomer of Ring BA or a tautomer of Ring BA or Ring BA. and the ring BA is BA-I, BA-I-a, BA-I-b, BA-II, BA-II-a, BA-II-b, BA-III, BA-III-a, BA-III-b, Phosphoamidai having the structure BA-IV, BA-IV-a, BA-IV-b, BA-V, BA-V-a, BA-V-b, or BA-VI, wherein the nucleobases are optionally substituted or protected t or compound.

1703. The method according to any one of embodiments 1666 to 1702, wherein the phosphoramidite or compound comprises a nucleobase, and the nucleobase is Ring BA or a tautomer of Ring BA or a tautomer of Ring BA or Ring BA. and the ring BA is BA-I, BA-I-a, BA-I-b, BA-II, BA-II-a, BA-II-b, BA-III, BA-III-a, BA-III-b, Phosphoamidai having the structure BA-IV, BA-IV-a, BA-IV-b, BA-V, BA-V-a, BA-V-b, or BA-VI, wherein the nucleobases are optionally substituted or protected t or compound.

1704. The method of any one of embodiments 1666 to 1703, wherein BA ^s is BA-I, BA-Ia, BA-Ib, BA-II, BA-II-a, BA-II-b, BA-III, BA-III-a, BA-III-b, BA-IV, BA-IV-a, BA-IV-b, BA-V, BA-Va, BA-Vb, or BA-VI, or tautomerism of ring BA A phosphoramidite or compound having an isomeric structure, wherein the nucleobases are optionally substituted or protected.

1705. A phosphoramidite or compound according to any one of embodiments 1666 to 1701 comprising hypoxanthine.

1706. A phosphoramidite or compound according to any one of embodiments 1666 to 1701 comprising an O ⁶ -protected hypoxanthine.

1707. according to any one of embodiments 1666 to 1701, comprising O ⁶ -protected hypoxanthine, wherein the O ⁶ protecting group is -CH ₂ CH ₂ Si(R) ₃ , wherein -CH ₂ CH ₂ - is optionally substituted, wherein each R is other than -H.

1708. The phosphoramidite or compound of any one of embodiments 1666-1701, comprising O ⁶ -protected hypoxanthine, wherein the O ⁶ protecting group is —CH ₂ CH ₂ Si(Me) ₃ .

1709. A phosphoramidite or compound according to any one of embodiments 1666-1708, comprising a sugar that is a sugar of any one of embodiments 1-1521.

1710. The phosphoramidite or compound according to any one of embodiments 1666 to 1695, wherein R ^NS is an optionally substituted or protected nucleoside selected from A, T, C, G and U.

1711. The method of any one of embodiments 1666 to 1695, wherein R ^NS is b001U, b002U, b003U, b004U, b005U, b006U, b008U, b002A, b001G, b004C, b007U, b001A, b001C, b002C, b003C, b 002I, A phosphoramidite or compound that is an optionally substituted or protected nucleoside selected from b003I, b009U, b003A, b007C, Asm01, Gsm01, 5MSfC, Usm04, 5MRdT, Csm15, Csm16, rCsm14, Csm17 and Tsm18.

1712. The phosphoramidite or compound according to any one of embodiments 1666 to 1711, wherein R ^NS is bonded to phosphorus through 3'-0-.

1713. The phosphoramidite of any one of embodiments 1669-1712, wherein the phosphoramidite is at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% pure. Dite or composition.

1714. A method of preparing an oligonucleotide or composition comprising coupling an -OH group of an oligonucleotide or nucleoside with a phosphoramidite or compound of any one of embodiments 1663-1713.

1715. A method of preparing an oligonucleotide or composition comprising coupling the 5'-OH of an oligonucleotide or nucleoside with a phosphoramidite or compound of any one of embodiments 1663-1713.

1716. A method of preparing an oligonucleotide or composition comprising removing a chiral auxiliary moiety from an oligonucleotide of any one of embodiments 1523-1662.

1717. The method of any one of embodiments 1714-1716, wherein the oligonucleotide, or oligonucleotide in the composition, comprises a sugar comprising 2'-OH.

1718. The method of any one of embodiments 1714 to 1717, wherein the oligonucleotide, or oligonucleotide in the composition, comprises a sugar comprising 2'-OH, and the sugar is linked to a chiral control internucleotidic linkage.

1719. The oligonucleotide, composition or method of any one of the preceding embodiments, wherein each heteroatom is independently selected from nitrogen, oxygen, silicon, phosphorus, and sulfur.

1720. The oligonucleotide, composition or method of any one of the preceding embodiments, wherein each nucleobase independently comprises an optionally substituted ring having at least one nitrogen.

1721. As a method,

Evaluating an agent or composition thereof in a cell, tissue or animal, wherein the cell, tissue or animal is or comprises a cell, tissue or organ associated with or of a condition, disorder or disease, and/or the condition, disorder, or disease; comprising a nucleotide sequence associated with a disorder or disease; and

A method comprising administering to a subject susceptible to or suffering from a condition, disorder or disease an effective amount of an agent or composition for preventing or treating the condition, disorder or disease.

1722. As a method,

administering to a subject susceptible to or suffering from a condition, disorder or disease an effective amount of an agent or composition for preventing or treating the condition, disorder or disease, wherein the agent or composition is evaluated in a cell, tissue or animal , wherein the cell, tissue or animal is or comprises a cell, tissue or organ associated with or associated with the condition, disorder or disease and/or comprises a nucleotide sequence associated with the condition, disorder or disease.

1723. The method of embodiment 1721 or 1722, wherein the subject is a human.

1724. The method of any one of embodiments 1721 to 1723, wherein the condition, disorder or disease is associated with a G to A mutation.

1725. The method of any one of embodiments 1721 to 1724, wherein the condition, disorder or disease is associated with a 1024 G>A(E342K) mutation in the human SERPINA1 gene.

1726. The method of any one of embodiments 1721-1725, wherein the condition, disorder or disease is alpha-1 antitrypsin deficiency.

1727. The method of any one of embodiments 1721-1723, wherein the condition, disorder or disease is cancer.

1728. A method for characterizing an oligonucleotide or composition comprising:

A method comprising administering an oligonucleotide or composition to a cell or population comprising or expressing an ADAR1 polypeptide or feature thereof, or a polynucleotide encoding an ADAR1 polypeptide or feature thereof.

1729. The method of any one of embodiments 1721 to 1728, wherein the cells are rodent cells.

1730. The method of any one of embodiments 1721-1728, wherein the cells are rat cells.

1731. The method of any one of embodiments 1721 to 1728, wherein the cells are mouse cells.

1732. The method of any one of embodiments 1721 to 1731, wherein the genome of the cell comprises a polynucleotide encoding an ADAR1 polypeptide or feature thereof.

1733. A method for characterizing an oligonucleotide or composition comprising:

A method comprising administering an oligonucleotide or composition to a non-human animal or population comprising or expressing an ADAR1 polypeptide or feature thereof, or a polynucleotide encoding an ADAR1 polypeptide or feature thereof.

1734. The method of embodiment 1733, wherein the animal is a mouse.

1735. The method of embodiment 1733 or 1734, wherein the animal's genome comprises a polynucleotide encoding an ADAR1 polypeptide or feature thereof.

1736. The method of embodiment 1733 or 1734, wherein the germline genome of the animal comprises a polynucleotide encoding an ADAR1 polypeptide or feature thereof.

1737. The method of any one of embodiments 1721 to 1736, wherein the ADAR1 polypeptide or feature thereof is one or both of the human ADAR1 Z-DNA binding domains or comprises one or both of the human ADAR1 Z-DNA binding domains. .

1738. The method of any one of embodiments 1721 to 1737, wherein the ADAR1 polypeptide or feature thereof is one or more or all of the human ADAR1 dsRNA binding domains or comprises one or more or all of the human ADAR1 dsRNA binding domains.

1739. The method of any one of embodiments 1721-1738, wherein the ADAR1 polypeptide or feature thereof is or comprises a human deaminase domain.

1740. The method of any one of embodiments 1721-1739, wherein the ADAR1 polypeptide or feature thereof is or comprises a human ADAR1.

1741. The method of any one of embodiments 1721-1740, wherein the ADAR1 polypeptide or feature thereof is or comprises human ADAR1 p110.

1742. The method of any one of embodiments 1721-1740, wherein the ADAR1 polypeptide or feature thereof is or comprises human ADAR1 p150.

1743. The method of any one of embodiments 1721 to 1742, wherein the level of activity of the oligonucleotide or composition observed in the cells or cells of the animal, or population thereof, is observed in the cells prior to manipulation or in the cells of the animal, or population thereof. A method more similar to that observed in comparable human cells or populations thereof compared to

1744. The method of embodiment 1743, wherein the comparable human cell is of the same type as the cell or animal cell.

1745. The method of any one of embodiments 1721-1744, wherein the cell, tissue or animal is or comprises a cell, tissue or organ associated with or of a condition, disorder or disease.

1746. The method of embodiment 1745, wherein a cell, tissue or organ associated with or of the condition, disorder or disease is or comprises a tumor.

1747. The method of any one of embodiments 1721-1746, wherein the cell, tissue or animal comprises a nucleotide sequence associated with a condition, disorder or disease.

1748. The method of embodiment 1747, wherein the nucleotide sequence associated with the condition, disorder or disease is homozygous.

1749. The method of embodiment 1747, wherein the nucleotide sequence associated with the condition, disorder or disease is heterozygous.

1750. The method of embodiment 1747, wherein the nucleotide sequence associated with the condition, disorder or disease is hemizygous.

1751. The method of any one of embodiments 1747-1750, wherein the nucleotide sequence associated with the condition, disorder or disease is in a genome.

1752. The method of any one of embodiments 1747 to 1751, wherein the nucleotide sequence associated with the condition, disorder or disease is in the genome of some cells but not all cells.

1753. The method of any one of embodiments 1747-1752, wherein the nucleotide sequence associated with the condition, disorder or disease is in the germline genome.

1754. The method of any one of embodiments 1747-1753, wherein the nucleotide sequence associated with the condition, disorder or disease is a mutation.

1755. The method of any one of embodiments 1747-1754, wherein the nucleotide sequence associated with the condition, disorder or disease is a G to A mutation.

1756. The method of any one of embodiments 1747-1755, wherein the nucleotide sequence associated with the condition, disorder or disease is a G to A mutation of SERPINA1.

1757. The method of any one of embodiments 1747-1756, wherein the nucleotide sequence associated with the condition, disorder or disease is the 1024 G>A(E342K) mutation of human SERPINA1.

1758. The method of any one of embodiments 1721 to 1756, wherein the cell, tissue or animal comprises the 1024 G>A(E342K) mutation of the human SERPINA1 gene.

1759. The method of embodiment 1758, wherein the cell, tissue or animal comprises NOD.Cg-Prkdcscid Il2rgtm1Wjl Tg(SERPINA1*E342K)#Slcw/SzJ.

1760. The method of any one of embodiments 1721 to 1759, wherein the subject comprises the 1024 G>A(E342K) mutation of human SERPINA1.

1761. The method of embodiment 1760, wherein the subject is homozygous for the 1024 G>A(E342K) mutation of human SERPINA1.

1762. The method of embodiment 1760, wherein the subject is heterozygous for the 1024 G>A(E342K) mutation of human SERPINA1.

1763. The method of embodiment 1760, wherein the subject is heterozygous for the 1024 G>A(E342K) mutation of SERPINA1 and one allele is wild type.

1764. A method of modifying a target adenosine in a target nucleic acid comprising contacting the target nucleic acid with an oligonucleotide or composition of any one of the preceding embodiments.

1765. A method of deamination of a target adenosine in a target nucleic acid comprising contacting the target nucleic acid with an oligonucleotide or composition of any one of the above embodiments.

1766. A method of producing a product of a specific nucleic acid, or restoring or increasing the level of a product of a specific nucleic acid, comprising contacting a target nucleic acid with an oligonucleotide or composition of any one of the above embodiments, wherein the target nucleic acid is a target A method comprising adenosine and different from a target nucleic acid in that certain nucleic acids have an I or G in place of the target adenosine.

1767. A method of reducing the product level of a target nucleic acid comprising contacting the target nucleic acid with an oligonucleotide or composition of any one of the above embodiments, wherein the target nucleic acid comprises target adenosine.

1768. The method of embodiment 1766 or 1767, wherein the product is a protein.

1769. The method of embodiment 1766 or 1767, wherein the product is mRNA.

1770. The method of any one of embodiments 1764 to 1769, wherein the oligonucleotide or oligonucleotide composition in the base sequence of the oligonucleotide is substantially complementary to the base sequence of the target nucleic acid.

1771. The method of any one of embodiments 1764-1770, wherein the target nucleic acid is in a sample.

1772. As a method,

Contacting the oligonucleotide or composition of any one of the above embodiments with a sample comprising a target nucleic acid and an adenosine deaminase,

The base sequence of the oligonucleotide in the oligonucleotide or oligonucleotide composition is substantially complementary to the base sequence of the target nucleic acid,

the target nucleic acid comprises a target adenosine;

wherein the target adenosine is modified.

1773. As a method,

1) obtaining a modification of a first level of target adenosine in a target nucleic acid (the level observed when the first oligonucleotide composition is contacted with a sample comprising the target nucleic acid and adenosine deaminase), wherein the first oligonucleotide composition comprises: Comprising a first plurality of oligonucleotides sharing the same base sequence that is substantially complementary to the base sequence of the target nucleic acid, and

2) obtaining a modification of the reference level of target adenosine in the target nucleic acid (the level observed when the reference oligonucleotide composition is contacted with a sample comprising the target nucleic acid and adenosine deaminase), wherein the reference oligonucleotide composition is Comprising a plurality of reference oligonucleotides sharing the same base sequence that is substantially complementary to the base sequence,

At this time,

the first plurality of oligonucleotides comprises more sugars with 2'-F modifications, sugars with 2'-OR modifications (R is not -H), and/or chiral internucleotidic linkages than the reference plurality of oligonucleotides; do,

The method of claim 1 , wherein the first oligonucleotide composition provides a higher level of modification relative to the oligonucleotides of the reference oligonucleotide composition.

1774. As a method,

obtaining a modification of a first level of target adenosine in the target nucleic acid (a level observed when the first oligonucleotide composition is contacted with a sample comprising the target nucleic acid and an adenosine deaminase), wherein the first oligonucleotide composition comprises: A first plurality of oligonucleotides sharing the same base sequence that is substantially complementary to the base sequence of the target nucleic acid,

The first level of modification of the target adenosine is higher than the reference level of modification of the target adenosine, the reference level observed when the reference oligonucleotide composition is contacted with a sample comprising a target nucleic acid and an adenosine deaminase, wherein the reference oligonucleotide composition is It includes a plurality of reference oligonucleotides that share the same base sequence that is substantially complementary to the base sequence of the target nucleic acid,

At this time,

the first plurality of oligonucleotides comprises more sugars with 2'-F modifications, sugars with 2'-OR modifications (R is not -H), and/or chiral internucleotidic linkages than the reference plurality of oligonucleotides; How to.

1775. As a method,

1) obtaining a modification of a first level of target adenosine in a target nucleic acid (the level observed when the first oligonucleotide composition is contacted with a sample comprising the target nucleic acid and adenosine deaminase), wherein the first oligonucleotide composition comprises: Comprising a first plurality of oligonucleotides sharing the same base sequence that is substantially complementary to the base sequence of the target nucleic acid, and

2) obtaining a modification of the reference level of target adenosine in the target nucleic acid (the level observed when the reference oligonucleotide composition is contacted with a sample comprising the target nucleic acid and adenosine deaminase), wherein the reference oligonucleotide composition is Comprising a plurality of reference oligonucleotides sharing the same base sequence that is substantially complementary to the base sequence,

At this time,

The first plurality of oligonucleotides may have more sugars with 2'-F modifications, sugars with 2'-OR modifications (R is not -H), and/or chiral controlled chiral internucleotidic linkages than the reference plurality of oligonucleotides. contains a lot,

The method of claim 1 , wherein the first oligonucleotide composition provides a higher level of modification relative to the oligonucleotides of the reference oligonucleotide composition.

1776. As a method,

obtaining a modification of a first level of target adenosine in the target nucleic acid (a level observed when the first oligonucleotide composition is contacted with a sample comprising the target nucleic acid and an adenosine deaminase), wherein the first oligonucleotide composition comprises: A first plurality of oligonucleotides sharing the same base sequence that is substantially complementary to the base sequence of the target nucleic acid,

The first level of modification of the target adenosine is higher than the reference level of modification of the target adenosine, the reference level observed when the reference oligonucleotide composition is contacted with a sample comprising a target nucleic acid and an adenosine deaminase, wherein the reference oligonucleotide composition is It includes a plurality of reference oligonucleotides that share the same base sequence that is substantially complementary to the base sequence of the target nucleic acid,

At this time,

The first plurality of oligonucleotides may have more sugars with 2'-F modifications, sugars with 2'-OR modifications (R is not -H), and/or chiral controlled chiral internucleotidic linkages than the reference plurality of oligonucleotides. A lot inclusive, way.

1777. As a method,

1) obtaining a modification of a first level of target adenosine in a target nucleic acid (the level observed when the first oligonucleotide composition is contacted with a sample comprising the target nucleic acid and adenosine deaminase), wherein the first oligonucleotide composition comprises: Comprising a first plurality of oligonucleotides sharing the same base sequence that is substantially complementary to the base sequence of the target nucleic acid, and

2) obtaining a modification of the reference level of target adenosine in the target nucleic acid (the level observed when the reference oligonucleotide composition is contacted with a sample comprising the target nucleic acid and adenosine deaminase), wherein the reference oligonucleotide composition is Comprising a plurality of reference oligonucleotides sharing the same base sequence that is substantially complementary to the base sequence,

At this time,

the first plurality of oligonucleotides comprises one or more chiral controlled chiral internucleotidic linkages;

the reference plurality of oligonucleotides does not contain chiral controlled chiral internucleotide linkages (the reference oligonucleotide composition is a stereorandom composition);

The method of claim 1 , wherein the first oligonucleotide composition provides a higher level of modification relative to the oligonucleotides of the reference oligonucleotide composition.

1778. As a method,

obtaining a modification of a first level of target adenosine in the target nucleic acid (a level observed when the first oligonucleotide composition is contacted with a sample comprising the target nucleic acid and an adenosine deaminase), wherein the first oligonucleotide composition comprises: A first plurality of oligonucleotides sharing the same base sequence that is substantially complementary to the base sequence of the target nucleic acid,

The first level of modification of the target adenosine is higher than the reference level of modification of the target adenosine, the reference level observed when the reference oligonucleotide composition is contacted with a sample comprising a target nucleic acid and an adenosine deaminase, wherein the reference oligonucleotide composition is It includes a plurality of reference oligonucleotides that share the same base sequence that is substantially complementary to the base sequence of the target nucleic acid,

At this time,

the first plurality of oligonucleotides comprises one or more chiral controlled chiral internucleotidic linkages;

wherein the reference plurality of oligonucleotides does not contain chiral controlled chiral internucleotide linkages (the reference oligonucleotide composition is a stereorandom composition).

1779. The method of any one of embodiments 1773-1778, wherein the first oligonucleotide composition is the oligonucleotide composition of any one of the preceding embodiments.

1780. The method of any one of embodiments 1773-1779, wherein the reference oligonucleotide composition is the reference oligonucleotide composition of any one of embodiments 1624-1633.

1781. The method of any one of embodiments 1764-1780, wherein the deaminase is an ADAR enzyme.

1782. The method of any one of embodiments 1764-1780, wherein the deaminase is ADAR1.

1783. The method of any one of embodiments 1764-1780, wherein the deaminase is ADAR2.

1784. The method of any one of embodiments 1764 to 1783, wherein the target nucleic acid is or comprises RNA.

1785. The method of any one of embodiments 1764-1784, wherein the sample is a cell.

1786. The method of any one of embodiments 1764 to 1785, wherein the target nucleic acid has an I or G at the position of the target adenosine instead of the target adenosine, compared to a nucleic acid that differs from the target nucleic acid, and is more distinct from the condition, disorder or disease. A method that is related to, is more related to a decrease in a desired property or function, or is more related to an increase in an undesirable property or function.

1787. The method of any one of embodiments 1764 to 1785, wherein the target adenosine is a G to A mutation.

1788. A method of preventing or treating a condition, disorder or disease comprising administering or delivering to a subject susceptible to or suffering from the condition, disorder or disease an effective amount of an oligonucleotide or composition of any one of the foregoing embodiments.

1789. A method of preventing or treating a condition, disorder or disease prone to G to A mutation, comprising administering or delivering to a subject predisposed to or suffering from it an effective amount of an oligonucleotide or composition of any one of the foregoing embodiments. How to include steps to do.

1790. A method of preventing or treating a condition, disorder or disease prone to G to A mutation comprising administering to a subject predisposed to or suffering from it an effective amount of an oligonucleotide or composition of any one of the foregoing embodiments. How to include.

1791. A method of increasing the level and/or activity of an alpha-1 antitrypsin (A1AT) polypeptide in the serum or blood of a subject comprising administering to the subject an effective amount of an oligonucleotide or composition of any one of the above embodiments. How to include.

1792. The method of embodiment 1791, wherein the A1AT polypeptide provides one or more higher activities compared to a reference A1AT polypeptide.

1793. The method of embodiment 1791 or 1792, wherein the A1AT polypeptide is a wild-type A1AT polypeptide.

1794. A method according to any one of embodiments 1791 to 1793, wherein the amount of A1AT polypeptide in serum is increased.

1795. The method according to any one of embodiments 1791 to 1793, wherein the amount of standard A 1AT polypeptide in serum is reduced.

1796. The method according to any one of embodiments 1791 to 1795, wherein the ratio of the A1AT polypeptide to the reference A1AT polypeptide in serum or blood is increased.

1797. The method of any one of embodiments 1791 to 1796, wherein the reference A1AT polypeptide is mutated.

1798. The method of any one of embodiments 1791 to 1797, wherein the reference A1AT polypeptide is an E342K A1AT polypeptide.

1799. A method of reducing the level and/or activity of a mutant alpha-1 antitrypsin (A1AT) polypeptide in the serum or blood of a subject, comprising administering to the subject an effective amount of an oligonucleotide or composition of any one of the above embodiments. How to include steps.

1800. The method of embodiment 1799, wherein the mutant A1AT polypeptide is an E342KA1AT polypeptide.

1801. The method of any one of embodiments 1791-1800, wherein the subject is predisposed to or suffering from a condition, disorder or disease.

1802. A method of preventing or treating a condition, disorder or disease associated with a G to A mutation, comprising administering or delivering to a subject predisposed to or suffering from it an effective amount of an oligonucleotide or composition of any one of the foregoing embodiments. How to include steps.

1803. A method of preventing or treating a condition, disorder or disease associated with a G to A mutation comprising administering to a subject predisposed to or suffering from it an effective amount of an oligonucleotide or composition of any one of the above embodiments. How to include.

1804. The method of any one of embodiments 1788-1803, wherein the base sequence of the oligonucleotide in the oligonucleotide or oligonucleotide composition is substantially complementary to the base sequence of a target nucleic acid comprising the target adenosine that is a mutant.

1805. The method of embodiment 1803 or 1804, wherein the condition, disorder or disease is prone to A to G transformation or A to I transformation.

1806. The method of any one of embodiments 1788-1805, wherein the cell associated with the condition, disorder or disease comprises or expresses an ADAR protein.

1807. The method of any one of embodiments 1788-1805, wherein the cell associated with the condition, disorder or disease comprises or expresses ADAR1.

1808. The method of any one of embodiments 1788-1805, wherein the cell associated with the condition, disorder or disease comprises or expresses ADAR2.

1809. The method of any one of embodiments 1788 to 1808, wherein the subject is a human subject.

1810. The method of any one of embodiments 1788-1809, wherein the condition, disorder or disease is or is associated with alpha-1 antitrypsin deficiency.

1811. The method according to any one of embodiments 1764 to 1810, comprising converting a target adenosine to I.

1812. The method of any one of embodiments 1764 to 1811, wherein two or more different adenosines are targeted and edited.

1813. The method of any one of embodiments 1764 to 1811, wherein two or more different transcripts are targeted and edited.

1814. The method of any one of embodiments 1764 to 1811, wherein transcripts from two or more different polynucleotides are targeted and edited.

1815. The method of any one of embodiments 1764 to 1811, wherein transcripts from two or more genes are targeted and edited.

1816. The method of any one of embodiments 1812 to 1815, comprising administering two or more oligonucleotides, each oligonucleotide independently targeting a different target, and each oligonucleotide independently targeting a different target according to embodiment 1 to 1521, or a salt thereof, of any one of embodiments.

1817. The method of any one of embodiments 1812 to 1815, comprising administering two or more oligonucleotide compositions, wherein each oligonucleotide composition independently targets at least one different target, and wherein each oligonucleotide composition is independently a composition of any one of embodiments 1522-1636.

1818. The method of any one of embodiments 1812-1817 comprising administering the composition of any one of embodiments 1547-1636.

1819. The method of any one of embodiments 1812-1818, wherein two or more oligonucleotides or compositions are administered simultaneously.

1820. The method of any one of embodiments 1812-1819, wherein two or more oligonucleotides or compositions are administered simultaneously as a single composition.

1821. The method of any one of embodiments 1812 to 1819, wherein the two or more oligonucleotides or compositions are administered as separate compositions.

1822. The method of any one of embodiments 1812 to 1818, wherein the one or more oligonucleotides or compositions are administered before or after one or more other oligonucleotides or compositions.

1823. The method of any one of embodiments 1788 to 1822, wherein the subject comprises the 1024 G>A(E342K) mutation of human SERPINA1.

1824. The method of embodiment 1823, wherein the subject is homozygous for the 1024 G>A(E342K) mutation of human SERPINA1.

1825. The method of embodiment 1823, wherein the subject is heterozygous for the 1024 G>A(E342K) mutation of human SERPINA1.

1826. The method of embodiment 1823, wherein the subject is heterozygous for the 1024 G>A(E342K) mutation of human SERPINA1 and one allele is wild type.

1827. The method of any one of embodiments 1788-1826, wherein the condition, disorder or disease is associated with a G to A mutation of SERPINA1.

1828. The method of any one of embodiments 1788 to 1827, wherein the condition, disorder or disease is associated with the 1024 G>A(E342K) mutation of human SERPINA1.

1829. The method according to any one of embodiments 1788 to 1828, wherein the condition, disorder or disease is alpha-1 antitrypsin deficiency.

1830. The method of any one of embodiments 1788-1829, wherein the subject has a heterozygous ZZ genotype.

1831. The method of any one of embodiments 1788-1829, wherein the subject has a homozygous ZZ genotype.

1832. The method according to any one of embodiments 1788 to 1831, which increases or restores the level or activity of wild type in the liver.

1833. The method of any one of embodiments 1788-1832, which reduces Z-AAT aggregation.

1834. A method according to any one of embodiments 1788 to 1833, which reduces or prevents liver damage.

1835. The method of any one of embodiments 1788 to 1834, which reduces or prevents cirrhosis of the liver.

1836. The method of any one of embodiments 1788-1835, which increases the level of wild-type AAT in the blood.

1837. The method of any one of embodiments 1788 to 1836, which increases the level of circulating lung-bound wild-type AAT in the blood.

1838. The method of any one of embodiments 1788 to 1837, which reduces or prevents lung damage.

1839. The method according to any one of embodiments 1788 to 1838, which reduces or prevents lung damage from proteases.

1840. A method according to any one of embodiments 1788 to 1839, which reduces or prevents pulmonary inflammation.

1841. The method of any one of embodiments 1788-1826, wherein the condition, disorder or disease is a recessive or dominant genetically defined condition, disorder or disease.

1842. The method of any one of embodiments 1788-1826, wherein the condition, disorder or disease is a liver condition, disorder or disease.

1843. The method of any one of embodiments 1788-1826, wherein the condition, disorder or disease is a metabolic liver condition, disorder or disease.

1844. The method of any one of embodiments 1788-1826, wherein the condition, disorder or disease is a neurological condition, disorder or disease.

1845. The method of any one of embodiments 1788-1826, wherein the condition, disorder or disease is a neurodevelopmental condition, disorder or disease.

1846. The method of any one of embodiments 1788-1826, wherein the condition, disorder or disease is a condition, disorder or disease associated with ion channel permeability.

1847. The method of any one of embodiments 1788-1826, wherein the condition, disorder or disease is familial epilepsy.

1848. A method according to any one of embodiments 1788 to 1826, wherein the condition, disorder or disease is neuropathic pain.

1849. The method according to any one of embodiments 1788 to 1826, wherein the condition, disorder or disease is a haplodeletion mutation condition, disorder or disease.

1850. The method of any one of embodiments 1788-1826, wherein the condition, disorder or disease is a neuromuscular condition, disorder or disease.

1851. A method according to any one of embodiments 1788 to 1826, wherein the condition, disorder or disease is dementia.

1852. The method of any one of embodiments 1788-1851, wherein the oligonucleotide administered to the subject comprises a targeting moiety.

1853. The method of any one of embodiments 1788-1851, wherein the oligonucleotide administered to the subject comprises a targeting moiety that targets the liver.

1854. The method of any one of embodiments 1788-1851, wherein the oligonucleotide administered to the subject comprises one or more ligands that target one or more receptors expressed in the liver.

1855. The method of any one of embodiments 1788-1851, wherein the oligonucleotide administered to the subject comprises one or more ligands targeting an asialoglycoprotein receptor.

1856. The method of any one of embodiments 1788-1851, wherein the oligonucleotide administered to the subject is a GalNAc conjugated oligonucleotide.

1857. The method of any one of embodiments 1788-1851, wherein the oligonucleotide administered to the subject comprises one or more ligands that target one or more receptors expressed in the liver.

1858. Use of the oligonucleotide or composition of any one of the above embodiments for altering mRNA splicing, wherein the target adenosine of the mRNA is edited.

1859. The use of embodiment 1860, wherein an exon is skipped, an exon is included, or a frame is restored.

1860. Use of the oligonucleotide or composition of any one of the above embodiments for altering mRNA splicing, wherein the target adenosine of the mRNA is edited.

1861. The use of embodiment 1860, wherein the level of RNA and/or polypeptide encoded thereby is reduced.

1862. Use of the oligonucleotide or composition of any one of the above embodiments for silencing protein expression, wherein the target adenosine of mRNA encoding the protein is edited.

1863. The use of embodiment 1862, wherein the expression, level and/or activity of the protein is increased or restored.

1864. Use of the oligonucleotide or composition of any one of the above embodiments for correcting a nonsense mutation, wherein the target adenosine of the RNA is edited to correct the nonsense mutation.

1865. The use of embodiment 1864, wherein the expression, level and/or activity of the protein is increased or restored.

1866. Use of the oligonucleotide or composition of any one of the above embodiments for correcting a missense mutation, wherein the target adenosine of the RNA is edited to correct the missense mutation.

1867. The use of embodiment 1866, wherein the expression, level and/or activity of the protein is increased or restored.

1868. Use of an oligonucleotide or composition of any one of the above embodiments for editing a target adenosine at a codon.

1869. The use of embodiment 1868, wherein the sequence, expression, level and/or activity of the protein is altered.

1870. Use of an oligonucleotide or composition of any one of the above embodiments for editing a target adenosine in an upstream ORF.

1871. The use of embodiment 1870, wherein the expression, level and/or activity of the protein is increased.

1872. A method for modulating protein-protein interactions in a system wherein a protein is translated from its coding RNA, comprising contacting the coding RNA with an oligonucleotide or composition of any one of the foregoing embodiments, comprising: wherein adenosine in is edited, a protein is translated from the encoded mRNA ("edited protein"), and the edited protein differs from the unedited protein in amino acid residues involved in protein-protein interactions.

1873. A method of modulating an interaction between a protein and its partner protein in a system, comprising administering to the system of any one of the above embodiments, wherein the oligonucleotide or composition is a nucleic acid encoding the protein or its partner protein. wherein the adenosine can be edited, and the edited nucleic acid encodes a protein that is different from the protein encoded by the unedited nucleic acid at at least one amino acid residue involved in the interaction between the protein and its partner protein.

1874. The method according to any one of embodiments 1872 to 1873, wherein the edited adenosine is in a codon encoding an amino acid residue involved in an interaction between a protein and its partner protein.

1875. is according to any one embodiment of embodiment 1874, wherein the edited adenosine is in a codon encoding an amino acid residue involved in an interaction between the protein and its partner protein, and the editing is to change the amino acid to a different amino acid. method.

1876. The method of any one of embodiments 1872-1875, wherein protein-protein interactions are reduced or disrupted.

1877. The method of any one of embodiments 1872-1876, wherein the protein is a transcription factor.

1878. The method of any one of embodiments 1872-1877, wherein the level of protein is increased.

1879. The method according to any one of embodiments 1872 to 1878, wherein the expression of one or more nucleic acids regulated by a protein is regulated.

1880. The method of any one of embodiments 1872 to 1879, wherein expression of one or more nucleic acids regulated by a protein is increased.

1881. The method of any one of embodiments 1872-1880, wherein the protein is NRF2.

1882. The method of any one of embodiments 1872-1881, wherein the editing of NRF2 is Glu82 (eg to Gly), Glu79 (eg to Gly), Glu78 (eg to Gly), Asp76 (eg to Gly) , Editing codons encoding Ile28 (as Val), Asp27 (eg as Gly) or Gln26 (eg as Arg), or comprising the same.

1883. The method of any one of embodiments 1872-1882, wherein the partner protein is Keap1.

1884. The method of any one of embodiments 1872-1883, wherein editing of Keap1 is Ser603 (eg to Gly), Tyr572 (eg to Cys), Tyr525 (eg to Cys), Ser508 (eg to Gly) , His436 (eg to Arg), Asn382 (eg to Asp), Arg380 (eg to Gly), or editing codons encoding Tyr334.

1885. The method of any one of embodiments 1872-1883, wherein the system is or comprises a cell.

1886. The method of any one of embodiments 1872-1883, wherein the system is or comprises a tissue.

1887. The method of any one of embodiments 1872-1883, wherein the system is or comprises an organ.

1888. The method of any one of embodiments 1872-1883, wherein the system is or comprises an organism.

1889. A method of transcript editing in an immune cell comprising administering to the immune cell an effective amount of an oligonucleotide or composition of any one of the above embodiments.

1890. The method of embodiment 1889, wherein the immune cells are PBMCs.

1891. The method of embodiment 1889, wherein the immune cells are CD4+ cells.

1892. The method of embodiment 1889, wherein the immune cells are CD8+ cells.

1893. The method of embodiment 1889, wherein the immune cells are CD14+ cells.

1894. The method of embodiment 1889, wherein the immune cells are CD19+ cells.

1895. The method of embodiment 1889, wherein the immune cells are NK cells.

1896. The method of embodiment 1889, wherein the immune cells are Treg cells.

1897. The method of any one of embodiments 1889-1896, wherein a cell is activated.

1898. The method of any one of embodiments 1889 to 1896, wherein the cells are inactivated.

1899. The method of any one of embodiments 1889-1898, wherein the oligonucleotide or composition targets and edits FAS, BID, CTLA4, PDCD1, CBLB, PTPN6, TRAC or TRBC.

1900. A method of improving the level of editing of an oligonucleotide comprising incorporating a structural element recited in any one of the above embodiments.

1901. A compound, oligonucleotide, composition, nucleobase, sugar, nucleoside, internucleoside linkage or method described herein.

1902. An oligonucleotide comprising a nucleobase as described herein.

1903. An oligonucleotide comprising a sugar as described herein.

1904. An oligonucleotide comprising an internucleotide linkage as described herein.

1905. An oligonucleotide comprising an internucleotide linkage as described herein and a sugar (eg, sm01n001) linked to the internucleotide linkage as described herein.

Example

Specific examples of the technology provided (compounds (oligonucleotides, reagents, etc.), compositions, methods (methods of manufacture, methods of use, methods of evaluation, etc.), etc.) are presented herein. A person skilled in the art knows many techniques, for example US 9982257, US 20170037399, US 20180216108, US 20180216107, US 9598458, WO 2017/062862, WO 2018/067973, WO 2017/160741, WO 2017/1 92679, WO 2017/210647, WO 2018/098264, WO 2018/223056, WO 2018/237194, WO 2019/032607, WO 2019/055951, WO 2019/075357, WO 2019/200185, WO 2019/217784, WO 2019/032 612, WO 2020/191252; It is understood that those described in WO 2021/071858 and the like can be used to prepare and/or evaluate the properties and/or activity of provided techniques in accordance with the present invention.

Example 1. A Useful Technique for Evaluating Adenosine Editing.

Oligonucleotide design can be evaluated using a variety of systems. In some embodiments, cLuc oligonucleotides were prepared and evaluated in HEK293T cells. In some embodiments, oligonucleotides targeting cLuc (Cypridina) are evaluated in 293T cells transfected with plasmids for human ADAR1 or human ADAR2 and a cLuc luciferase reporter plasmid. The cLuc reporter plasmid consisted of (Gaussia)gLuc-p2A-cLuc(W85X) for luciferase. The cLuc reporter was activated by ADAR-mediated A>I editing. The editing activity of oligonucleotides was calculated using the following equation:

Fold change = treated oligonucleotide (cLuc/gLuc) / mock (cLuc/gLuc)

In some embodiments, the reporter plasmid and the ADAR1 or ADAR2 plasmid were co-transfected into HEK293T cells using the Lipofectamine 2000 transfection protocol (Thermo 11668030). After an appropriate period of time, eg 24 hours, HEK293T cells expressing the reporter and ADAR plasmids were reverse transfected with the appropriate amount of oligonucleotides for each experiment. cLuc and gLuc activities were measured after 48, 72, and/or 96 hours using the Pierce™ Gaussia Luciferase Glow Assay Kit (Pierce™ 16161) or Pierce™ Cypridina Luciferase Glow Assay Kit (Pierce™ 16170), respectively.

In some embodiments, oligonucleotides and compositions are evaluated and found to confer editing in a variety of cells, such as mouse or human primary hepatocytes, primary human retinal pigment epithelial cells, cell lines, and the like. In some embodiments, oligonucleotides and compositions are evaluated and identified as contributing to editing in a subject. In some embodiments, oligonucleotides and compositions have been evaluated and identified as conferring editing in animals, such as mice, non-human primates (eg, Cynomolgus macaques), and the like. In some embodiments, the animal is a transgenic animal, eg, a mouse expressing human ADAR1. In some embodiments, the animal is a model animal comprising a target adenosine associated with a condition, disorder or disease, eg, a G to A mutation in many cases. In some embodiments, provided technologies can provide efficient editing with or without the use of exogenous ADAR polypeptides. In some embodiments, provided techniques can provide efficient editing without the use of exogenous ADAR1 or ADAR2. In some embodiments, oligonucleotides and compositions are delivered by transfection (eg, using a transfection composition such as Lipofectamine RNAimax). In some embodiments, oligonucleotides and compositions are delivered by gymnotic free update. In particular, the invention provides techniques for evaluating agents, e.g., oligonucleotides and compositions thereof, for editing, e.g., A to I(G) editing. In some embodiments, the invention is useful for evaluating agents (e.g., oligonucleotides) and compositions thereof that interact with and/or modulate or utilize one or more functions of an ADAR polypeptide as described herein, e.g., an ADAR1 polypeptide. provide technology. In some embodiments, the invention provides non-human animal cells and/or non-human animals engineered to contain and/or express an ADAR1 polypeptide or feature thereof, or a polynucleotide encoding an ADAR1 polypeptide or feature thereof. In some embodiments, the ADAR1 polypeptide or feature thereof is or comprises a primate ADAR1 or feature thereof. In some embodiments, an ADAR1 polypeptide or feature thereof is or comprises a primate ADAR1. In some embodiments, an ADAR1 polypeptide or feature thereof is a primate ADAR1. In some embodiments, the primate is a non-human primate. In some embodiments, the primate is a human. In some embodiments, the ADAR1 polypeptide or feature thereof is or comprises a human p110 ADAR1 or feature thereof. In some embodiments, an ADAR1 polypeptide or feature thereof is or comprises a human p110 ADAR1. In some embodiments, an ADAR1 polypeptide or feature thereof is a human p110 ADAR1. In some embodiments, the ADAR1 polypeptide or feature thereof is or comprises a human p150 ADAR1 or feature thereof. In some embodiments, an ADAR1 polypeptide or feature thereof is or comprises a human p150 ADAR1. In some embodiments, an ADAR1 polypeptide or feature thereof is a human p150 ADAR1. In some embodiments, the non-human animal is a rodent. In some embodiments, it is a rat. In some embodiments, it is a mouse. In some embodiments, the present invention provides mice engineered to express human ADAR1. In some embodiments, the present invention provides mouse cells engineered to express human ADAR1.

In particular, this Example demonstrates that the provided technology is particularly useful for evaluating agents, eg, oligonucleotides, and compositions thereof useful for editing, eg, adenosine editing, as described in the Examples. In particular, various agents (e.g., oligonucleotides) and compositions thereof capable of conferring editing in a variety of human cells are identified in certain cells (e.g., mouse cells) that do not contain or express human ADARs (e.g., human ADAR1) and rodents (e.g., It is provided herein and confirmed in this example that certain animals, such as mice), may exhibit no or much lower levels of editing. In particular, mice, a commonly used animal model, are used to evaluate various agents (e.g., oligonucleotides) for editing in humans, as agents active in humans may show no activity or very low levels of activity. this may be limited. In some embodiments, the invention provides cells engineered to express human ADAR1 (eg, human ADAR1 p110, p150, etc.) and non-human animals (eg, rodents such as mice), and oligonucleotides and compositions thereof, such as In particular, such engineered cells and/or animals will exhibit more correlated and/or predictive activity in human cells than cells and/or animals that have not been so engineered. can

Generation of Non-Human Mice Expressing Human ADAR1: A variety of techniques can be used in accordance with the present invention to provide mice engineered to express human ADAR1 polypeptides or features thereof. Certain useful techniques are described in the present invention and in the priority applications, the entirety of each of which is independently incorporated by reference.

In some embodiments, in mouse cells and animals engineered to express human ADAR1, the various oligonucleotides exhibit activity profiles very similar to those in human cells compared to reference mouse cells and animals not engineered to express human ADAR1. showed up For example, many oligonucleotides do not exhibit activity in reference mouse cells and animals not engineered to express human ADAR1 as compared to human cells expressing human ADAR1 and/or mouse cells and animals engineered to express human ADAR1. or showed much lower levels of activity.

A variety of useful techniques for generating transgenic systems comprising animals are available to those skilled in the art and can be used in accordance with the present invention, including those described in priority applications and WO 2021/071858, the entire contents of each of which are herein incorporated by reference. is incorporated by reference in

As described herein, an animal engineered to comprise an ADAR1 polypeptide or feature thereof, or whose sequence comprises and/or expresses a polynucleotide encoding an ADAR1 polypeptide or feature thereof, can be used in a variety of animals (e.g., in a variety of conditions, an animal model of a disorder or disease), inter alia, a characteristic element associated with various conditions, disorders or diseases, and an ADAR1 polypeptide, or a characteristic thereof, or a sequence thereof, comprising both an ADAR1 polypeptide or a polynucleotide encoding a characteristic thereof. Animal models are provided. In some embodiments, the animal is a model animal comprising SERPINA1-Pi*Z. In some embodiments, the animal comprises a 1024 G>A(E342K) mutation of human SERPINA1 and a polynucleotide whose sequence encodes an ADAR1 polypeptide or feature thereof. Among other things, these animals are useful for evaluating various agents, such as oligonucleotides, for editing the 1024 G>A (E342K) mutation of human SERPINA1. Among other things, the techniques provided, eg, non-human animals engineered to contain or express an ADAR1 polypeptide or feature thereof, are particularly useful for evaluating agents for adenosine editing.

In some embodiments, a huADAR mouse as described herein is crossed with another mouse comprising a nucleotide sequence of interest (eg, a mutation associated with a condition, disorder or disease). In certain embodiments, such crosses are performed using in vitro fertilization as is known in the art in accordance with the present invention. In certain embodiments, such mice comprise a human serpin family A member 1 (SERPINA1) polynucleotide sequence or feature thereof. In certain embodiments, such mice are SERPINA1-Pi*Z mice comprising a human SERPINA1 gene comprising a G to A mutation corresponding to a 1024 G>A (E342K) mutation. In some embodiments, the resulting progeny are a human SERPINA1-Pi*Z polynucleotide sequence or feature thereof (eg, a portion comprising a mutation associated with a condition, disorder or disease, eg, 1024 G>A) and Both huADAR1 polynucleotide sequences or fragments thereof. In some embodiments, double transgenic animals (e.g., comprising a human ADAR1 sequence or features thereof and sequences associated with a condition, disorder, or disease) are also heterozygous to allow them to be humanized (ie, to an immunodeficient phenotype). . ^_ _ ^_ _ Suitable humanized mouse strains are known in the art. In some embodiments, a mouse whose sequence comprises a polynucleotide encoding an ADAR1 polypeptide or feature thereof has a SERPINA1 mutation (eg, a condition, disorder or disease (eg, alpha 1-antitrypsin (A1AT) deficiency)) 1024 G>A) related to. In some embodiments, the second mouse to be crossed is The Jackson Laboratory Stock No: 028842; NSG-PiZ (see also Borel F; Tang Q; Gernoux G; Greer C; Wang Z; Barzel A; Kay MA; Shultz LD; Greiner DL; Flotte TR; Brehm MA; Mueller C. 2017. Survival Advantage of Both Human Hepatocyte Xenografts and Genome-Edited Hepatocytes for Treatment of alpha-1 Antitrypsin Deficiency.Mol Ther 25(11):2477-2489PubMed:29032169MGI: J:243726] and Li S;Ling C;Zhong L;Li M;Su Q He R; Tang Q; Greiner DL; Shultz LD; Brehm MA; Flotte TR; Mueller C; Srivastava A; Gao G. 2015. Efficient and Targeted Transduction of Nonhuman Primate Liver With Systemically Delivered Optimized AAV3B Vectors. Mol Ther 23(12 ):1867-76 PubMed: 26403887MGI: J:230567). As described herein, in some embodiments, a huADAR mouse is engineered such that its sequence comprises and/or expresses a polynucleotide encoding a human ADAR1 p110 polypeptide or features thereof. In some embodiments, the huADAR mouse is engineered such that its sequence comprises and/or expresses a polynucleotide encoding a human ADAR1 p150 polypeptide or feature thereof.

In some embodiments, a huADAR mouse as described herein has been crossed with another mouse comprising the nucleotide sequence of interest. In some embodiments, a mouse whose sequence comprises a polynucleotide encoding an ADAR1 polypeptide is a SERPINA1 mutation (eg, 1024 G associated with a condition, disorder or disease (eg, alpha 1-antitrypsin (A1AT) deficiency)). >A) were crossed with mice containing. In some embodiments, such crosses were performed using in vitro fertilization as known in the art in accordance with the present invention. In some embodiments, such mice comprise a human serpin family A member 1 (SERPINA1) polynucleotide sequence or features thereof. In some embodiments, such mice are SERPINA1-Pi*Z cells comprising a human SERPINA1 gene comprising a G to A mutation corresponding to, for example, a 1024 G>A(E342K) mutation or a genetic feature corresponding thereto. it was a mouse In some embodiments, the resulting progeny comprised both a human SERPINA1-Pi*Z polynucleotide sequence and a huADAR1 polynucleotide sequence. In some embodiments, the double transgenic animal also has an additional heterozygous, hemizygous, and/or homozygous (wild-type or mutant) form that renders them humanized (e.g., having an immunodeficiency phenotype) in the mutant form. Background mutations or alleles were included. In some embodiments, these genotypes included NOD.Cg -Prkdc ^scid Il2rgtm1 ^Wjl / SzJ.

As will be appreciated by those skilled in the art, a variety of techniques may be used for crossbreeding in accordance with the present invention. In some embodiments, the technique is or involves IVF (eg, using sperm from a heterozygous or homozygous huADAR mouse and oocytes from another mouse, or vice versa). In some embodiments, the technique is or involves natural mating (eg, using sperm from a heterozygous or homozygous huADAR mouse and oocytes from another mouse, or vice versa).

For example, in some embodiments, heterozygous sperm from male huADAR mice and oocytes from NOD.Cg-Prkdcscid Il2rgtm1Wjl Tg(SERPINA1*E342K)#Slcw/SzJ (NSG-PiZ, Stock #028842) female mice are For example, using via IVF, Prkdcscid heterozygous/Il2rgtm1Wjl heterozygous/Tg(SERPINA1*E342K)#Slcw heterozygous/hADAR heterozygous female mice and Prkdcscid heterozygous/Il2rgtm1Wjl hemizygous/Tg(SERPINA1*E342K)# Slcw heterozygous/hADAR heterozygous male mice are generated. In some embodiments, homozygous sperm from a huADAR male mouse and oocytes from a NOD.Cg-Prkdcscid Il2rgtm1Wjl Tg(SERPINA1*E342K)#Slcw/SzJ (NSG-PiZ, Stock #028842) female mouse are, for example, Utilized via IVF, Prkdcscid heterozygous/Il2rgtm1Wjl heterozygous/Tg(SERPINA1*E342K)#Slcw heterozygous/hADAR heterozygous female mice and Prkdcscid heterozygous/Il2rgtm1Wjl hemizygous/Tg(SERPINA1*E342K)#Slcw heterozygous/ hADAR heterozygous male mice are generated. In some embodiments, homozygous sperm from male mice of breed "hADAR" and oocytes from NOD.Cg-Prkdcscid Il2rgtm1Wjl Tg(SERPINA1*E342K)#Slcw/SzJ(NSG-PiZ, Stock #028842) female mice are used. and the resulting mice were mated with, for example, NOD/ShiLtJ (The Jackson Laboratory Stock #001976) mice to establish a series of colonies. In some embodiments, the resulting mouse has HET HET HET HET, HET WILD HET HET, WILD HET HET HET, WILD WILD HET HET, HET HEMI HET HET, HET HEMI HET WILD, HET HET HET WILD, and/or WILD HEMI HET HET. One skilled in the art will understand that male or female gametes may be provided from either breed, for example, in some embodiments oocytes may be provided from the huADAR line, while sperm may be of a different genotype, eg, NOD.Cg. -Prkdc ^scid Il2rg ^tm1Wjl Tg(SERPINA1*E342K)#Slcw/SzJ(NSG-PiZ, Stock #028842). In some embodiments, a huADAR (or hADAR) mouse is engineered such that its sequence comprises and/or expresses a polynucleotide encoding an ADAR1 polypeptide or feature thereof. In some embodiments, an animal comprises a polynucleotide whose sequence encodes in its genome an ADAR1 polypeptide or feature thereof. In some embodiments, an animal comprises a polynucleotide whose sequence encodes in the germline genome an ADAR1 polypeptide or feature thereof. In some embodiments, the ADAR1 polypeptide is human ADAR1. In some embodiments, human ADAR1 is human ADAR1 p110. In some embodiments, human ADAR1 is human ADAR1 p150. As an example, a number of animals containing the human ADAR1 p110 and 1024 G>A (E342K) mutations in human SERPINA1 are generated using one or more of the protocols described herein (e.g., using heterozygous hADAR1 sperm and IVF). It became. As will be appreciated by those skilled in the art, in some embodiments, the resulting animal may be further bred to produce an animal of a desired genotype, eg, a heterozygous, hemizygous or homozygous mouse. In some embodiments, using IVF, heterozygous sperm from male huADAR mice and oocytes from NOD.Cg-Prkdcscid Il2rgtm1Wjl Tg(SERPINA1*E342K)#Slcw/SzJ (NSG-PiZ, Stock #028842) female mice were mated to Prkdcscid heterozygous/Il2rgtm1Wjl heterozygous/Tg(SERPINA1*E342K)#Slcw heterozygous/hADAR heterozygous female mice and Prkdcscid heterozygous/Il2rgtm1Wjl hemizygous/Tg(SERPINA1*E342K)#Slcw heterozygous/hADAR heterozygous mice. Conjugated male mice were generated. Additionally, pups were genotyped (assuming gene order Prkdcscid/Il2rgtm1Wjl/Tg(SERPINA1*E342K)#Slcw/hADAR) HET HET HET HET, HET WILD HET HET, WILD HET HET HET, WILD WILD HET HET, HET HEMI HET HET , HET HEMI HET WILD, HET HET HET WILD, and/or WILD HEMI HET HET. A number of animals containing the human ADAR1 p110 and 1024 G>A(E342K) mutations in human SERPINA1 were generated using one or more of the protocols described herein (eg, using heterozygous hADAR1 sperm and IVF).

In some embodiments, provided technologies, e.g., oligonucleotides and compositions thereof, are evaluated in such animal models. In some embodiments, the desired product (e.g., as an appropriate amount in serum) in an observed amount (e.g., ng/mL in serum) and/or relatively (e.g., as a percentage of total protein or total A1AT protein). The level, nature and/or activity of wild-type A1AT protein) is increased and/or the level, nature and/or activity of an undesirable product (eg, mutant (eg E342K) A1AT protein in serum) this is reduced

Provided technology can provide activity such as adenosine editing in various types of cells, tissues, organs, organisms, etc. (eg, liver, kidney, CNS, nerve cells, astrocytes, hepatocytes, etc.). In some embodiments, editing is confirmed in immune cells, eg, CD8+ T cells (in some instances, pre-stimulated with cytokines, eg, for 24 or 96 hours). In some embodiments, editing has been identified in a fibroblast cell line. In some embodiments, the editing was identified ex vivo in the NHP eye (retina). Editing of the target adenosine of various target transcripts was observed confirming the general applicability of the provided technology. Specific target transcripts are described herein and for example in priority applications and WO 2021/071858.

Oligonucleotides and compositions can be delivered using a number of techniques in accordance with the present invention. For example, in some embodiments, they are delivered by transfection. In some embodiments, they are delivered by zymotic absorption. In some embodiments, oligonucleotides include moieties capable of facilitating delivery. For example, in some embodiments, the moiety is a ligand for a polypeptide, eg, in many cases a receptor on the surface of a cell. In some embodiments, the polypeptide is expressed at a higher level by a type or population of cells, tissues, etc., so that it can be used for delivery. In some embodiments, the ligand is an ASGPR ligand. In some embodiments, a ligand is or comprises GalNAc or a derivative thereof. In some embodiments, an oligonucleotide can include two or more ligand moieties that are each independently a ligand of a polypeptide. In some embodiments, an oligonucleotide comprises two or more copies of a ligand moiety. In some embodiments, a moiety targets one or more properties of a location or environment (eg, pH, redox, etc.).

In some implementations, techniques of a given technique may provide increased stability, higher levels of editing, and the like. In some embodiments, provided techniques are administered over an extended period of time, e.g., about or at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, It can provide the desired editing activity for 40, 45 days or more. In some embodiments, the desired editing activity/level of editing is maintained for a prolonged period of time, e.g., about or at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, It can be maintained for 30, 35, 40, 45 days or more.

In some embodiments, provided technologies can provide high levels of selectivity. In some embodiments, about or at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 95%, 96%, 97%, 98%, 99%, 99.5% , or 99.9% of the observed adenosine editing is on the target adenosine. In some embodiments, about or at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the observed adenosine editing in the coding region is on a target adenosine. In some embodiments, about or at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% of the observed adenosine in a target nucleic acid (eg, a transcript of a target gene) Editing is on target adenosine. In some embodiments, about or at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the coding region of a target nucleic acid (e.g., a transcript of a target gene) The observed adenosine editing of is at the target adenosine. A variety of techniques are available to those skilled in the art to assess selectivity, such as, for example, RNA-Seq; These specific techniques are described herein or in priority application or WO 2021/071858, the entire contents of each of which are independently incorporated herein by reference. In some embodiments, the percentage for selectivity described herein is at least about 80%. In some embodiments, the percentage is at least about 85%. In some embodiments, the percentage is at least about 90%. In some embodiments, the percentage is at least about 95%. In some embodiments, the percentage is at least about 96%. In some embodiments, the percentage is at least about 97%. In some embodiments, the percentage is at least about 98%. In some embodiments, the percentage is at least about 99%. In some embodiments, the percentage is at least about 99.5%. In some embodiments, the percentage is at least about 99.9%. In some embodiments, the percentage is about 100%. In some embodiments, no off-target editing is observed. In some embodiments, provided technologies provide high in vivo selectivity.

In some embodiments, the present invention provides multiple editing. In some embodiments, multiple target adenosines are edited together, and one or more or each are independently edited at a similar level compared to when edited individually.

As examples illustrating the various advantages and advantages that provided technology can provide, various results are presented, for example, in the figures and tables herein.

As demonstrated herein, among other things, the present invention provides oligonucleotides comprising a variety of modifications (e.g., nucleobase modifications, sugar modifications, linkage modifications, etc., and combinations and patterns thereof) capable of providing efficient editing. to provide.

In some embodiments, the use of specific sugars at and/or near the editing site, eg, natural DNA sugars, 2'-F modified sugars, etc., provides editing activity. In some embodiments, the oligonucleotide comprises 5'-N ₁ N ₀ N _-1 -3', each of N ₁ , N ₀ , and N _-1 is independently a nucleoside, and N ₁ and N ₀ are binds to an internucleotide linkage as described herein, N _-1 and N ₀ bind to an internucleotide linkage as described herein, and N ₀ is opposite the target adenosine. In some embodiments, each sugar of N ₁ , N ₀ , and N ₋₁ is independently a natural DNA sugar. In some embodiments, the sugar of N ₁ is a 2′-modified sugar (eg, a 2′-F modified sugar), and each sugar of N ₀ and N ₋₁ is independently a natural DNA sugar. In some embodiments, such oligonucleotides provide high levels of editing. In some embodiments, the 2'-OR variant (where R is not -H) is used outside the second subdomain or editing region, e.g., in the first domain, the first subdomain, and/or the third subdomain. do. These modified sugars can be used at various positions in these domains/subdomains and are well tolerated and in various cases can improve the properties and/or activity of the oligonucleotide.

As demonstrated herein, the provided technology can provide efficient editing using significantly shorter oligonucleotides compared to various previously reported technologies. In some embodiments, oligonucleotides of varying length, eg, 27, 28, 29, 20, 31, 32 or more nucleosides, may provide for editing.

In some embodiments, the base sequence of the oligonucleotide has sufficient complementarity to the base sequence of the target nucleic acid such that the oligonucleotide can form a duplex under suitable conditions, eg, in vivo or in vitro editing conditions. In some embodiments, oligonucleotides selectively duplex with target nucleic acids over non-target nucleic acids. Although a certain level of complementarity to the target nucleic acid is desirable or necessary for a variety of applications, including targeted adenosine editing, complete complementarity is generally not required. In some embodiments, there are one or more inconsistencies, bulges, etc., as described herein. In some embodiments, the nucleobase of the nucleoside N ₀ opposite the target adenosine is not complementary to the target adenosine. In some embodiments, hypoxanthine is used in place of G, especially when it is close to or next to N ₀ . In some implementations, the first domain, first subdomain and/or third subdomain comprises one or more mismatches, for example 1, 2, 3, 4 or more mismatches.

In some embodiments, oligonucleotides are provided as chirally controlled oligonucleotide compositions. In some embodiments, as exemplified herein, chirally controlled oligonucleotide compositions provide a variety of desired properties and/or activities. In some embodiments, a chirally controlled oligonucleotide composition has improved properties and/or activity compared to a corresponding stereorandom oligonucleotide composition (e.g., of an oligonucleotide having the same configuration but not being chirally controlled at the chiral linkage phosphorus). to provide.

Among other things, Applicants believe that a composition of oligonucleotides comprising various modifications can provide targeted editing and that the nucleoside opposite the target adenosine is located at various positions of the oligonucleotide (e.g., in some cases 5, 6, 7, 8, 9 or more positions). It has also been confirmed that different versions of GalNAc can be used to provide delivery and/or activity (eg, in Mod001 or L025). As recognized by those skilled in the art and described and identified herein, edited nucleobases can perform various functions of G after editing (in some cases editing may be referred to as A to G). In various embodiments, a native RNA sugar may be used in a provided oligonucleotide, and in some cases the nucleoside opposite the target adenosine. In some embodiments, RNA or DNA nucleosides are utilized in the 3' adjacent position (N _-1 ) and have hypoxanthine as their nucleobase. In some embodiments, the 3' adjacent I or dI nucleoside is linked to the 3' adjacent nucleoside via a negatively charged S p internucleoside, such as a phosphorylguanidine internucleotide linkage, such as n001. Among other things, it has been found that various numbers of non-negatively charged internucleotidic linkages can be used in various parts according to the present invention. In some embodiments, non-complementary base pairs (eg, wobbles and/or mismatches) are used in addition to the editing region or second subdomain. In some embodiments, it has been found that removing non-complementary base pairs (eg, wobbles and/or mismatches) can improve editing efficiency. In some embodiments, it has been observed that certain nucleobases provide improved properties and/or activity. Among other things, it has been found that in some embodiments oligonucleotides comprising various modified nucleobases (or free base nucleosides) at N ₀ can provide editing. In some embodiments, oligonucleotides comprising specific base modifications, such as b001A, b002A, b008U, etc., have been observed to increase editing activity when compared to a reference composition. In some embodiments, oligonucleotides comprising specific base modifications such as b001A, b002A, b008U, etc. in N ₀ have been observed to increase editing activity when compared to a reference composition. In some embodiments, provided oligonucleotides include free base moieties between nucleosides comprising nucleobases. A variety of oligonucleotides containing one or more free base units in place of nucleosides containing nucleobases were evaluated and found to be capable of conferring editing activity. In some embodiments, it has been observed that free base units at certain positions provide higher activity than other positions. In some embodiments, it has been observed that oligonucleotides may provide different absolute and/or relative editing levels for ADAR1-p110, ADAR1-p150 and ADAR2 in certain circumstances.

In some embodiments, an oligonucleotide is completely complementary to a sequence of the same length in a target nucleic acid.

The provided technology can provide robust editing in the presence of ADAR1 and/or ADAR2. The provided technology can provide robust editing in the presence of ADAR1-p110 and/or ADAR1-p150.

Data identifying the various features, activities, advantages, etc. of the present technology are provided by way of example in various examples and figures, including those of the Priority Application, the entire contents of each of which are independently incorporated herein by reference. Certain techniques useful in accordance with the present invention, such as structural elements, assays, targets, etc., are described in WO 2021/071858, the entire contents of which are incorporated herein by reference.

Example 2. Oligonucleotide and Composition Preparation Techniques.

Various technologies (eg, phosphoramidites, nucleobases, nucleosides, etc.) ) is described in, for example, US 9982257, US 20170037399, US 20180216108, US 20180216107, US 9598458, WO 2017/062862, WO 2018/067973, WO 2017/160741, WO 2017/1926 79, WO 2017/210647, WO 2018/098264 , WO 2018/223056, WO 2018/237194, WO 2019/032607, WO 2019/055951, WO 2019/075357, WO 2019/200185, WO 2019/217784, WO 2019/032612, WO 2020/1 91252, and/or WO 2021/071858 that can be used in accordance with the present invention, including methods and reagents, each of which is incorporated herein by reference. In some embodiments, the present invention provides useful techniques for making oligonucleotides and compositions thereof.

In some embodiments, useful compounds or salts thereof are provided, including those described below. In some embodiments, compounds were prepared using the techniques described in the priority application and WO 2021/071858, the entire contents of each of which are incorporated herein by reference.

Certain useful techniques for preparing a variety of additional useful compounds are described by way of example below.

3-((2R,4S,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-hydroxytetrahydrofuran-2-yl)pyrimidine-2,4 (1H,3H)-dione (WV-NU-096) and 3-((2S,4S,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-hydride Synthesis of oxytetrahydrofuran-2-yl) pyrimidine-2,4 (1H, 3H) -dione (WV-NU-096A)

In some embodiments, the present invention provides compounds and methods for preparing nucleobases, sugars, nucleosides, and the like. In some embodiments, the compound has the structure NH(R′) ₂ or a salt thereof, where each R′ is as described herein. In some embodiments, two R' together with the nitrogen to which they are attached form an optionally substituted ring. In some embodiments, the ring formed is an optionally substituted monocyclic saturated, partially unsaturated or aromatic ring having 0-2 heteroatoms other than nitrogen. In some embodiments, NH(R') ₂ is a nucleobase. In some embodiments, the compound am. In some embodiments, the NH(R′) or nucleobase is suitably protected so that the reaction occurs selectively at the desired amino group. In some embodiments, the compound am. In some embodiments, the compound has the structure of, wherein LG is a leaving group, each R ^RA is independently a substituted C _6-10 aryl or a C _5-10 heteroaryl having 1 to 6 heteroatoms, wherein at least one substituent is independently is an electron withdrawing group. In some embodiments, each substituent is independently an electron withdrawing group. In some embodiments, R ^RA is a substituted aryl wherein the substituent is an electron withdrawing group. In some embodiments, each R ^RA is independently a substituted aryl, wherein the substituent is an electron withdrawing group. In some embodiments, the electron withdrawing group is -Cl. In some embodiments, R ^RA is p-chlorophenyl. In some embodiments, each R ^RA is p-chlorophenyl. In some embodiments, the leaving group is -Cl. One skilled in the art understands that a variety of electron withdrawing and leaving groups may be utilized in accordance with the present invention. In some embodiments, the compound , and each variable is independently as described herein. In some embodiments, the compound am. In some embodiments, the compound am. In some embodiments, the compound am. In some embodiments, the compound am. In some embodiments, the compound am. In some embodiments, the compound am. In some embodiments, the compound am. In some embodiments, the compound am. In some embodiments, the compound am. In some embodiments, the compound am. In some embodiments, the compound am. In some embodiments, the compound am. In some embodiments, the compound am. In some embodiments, the compound am. In some embodiments, the compound am. In some embodiments, the compound am.

In some embodiments, the present invention provides a compound selected from compounds having the structure of NH(R′) ₂ , a nucleobase and an amine, or a salt thereof (eg, )second A compound having the structure of (e.g., , etc.) or by reacting with a salt thereof a compound having the structure of etc.) or a salt thereof. In some embodiments, the reaction is performed under basic conditions, for example in the presence of NaH. In some embodiments, a suitable solvent is MeCN. In some embodiments, a suitable temperature is 0 to 65 °C. In some embodiments, provided methods a compound having the structure of etc.) or a salt thereof a compound having the structure of etc.) or a step of converting to a salt thereof. In some embodiments, the conversion is performed under ester hydrolysis conditions. In some embodiments, conversion is performed with a base (eg, NaOMe) in a suitable solvent (eg, an alcohol such as MeOH). It includes contacting a compound having a structure of or a salt thereof. In some embodiments, the method A compound having the structure of (e.g., etc.) or by protecting the 5'-OH of its salt a compound having the structure of etc.) or a salt thereof, wherein PGO is a protected —OH group. In some embodiments, PGO is DMTrO.

Step 1. To a solution of pyrimidine-2,4(1H,3H)-dione (100 g, 892.17 mmol, 1 equiv) in pyridine (1000 mL) was added Ac ₂ O (546.48 g, 5.35 mol, 501.36 mL, 6 equiv). ) was added. The mixture was stirred at 120 °C for 3 hours. The reaction mixture was concentrated under reduced pressure to give crude, the residue was washed with EtOAc (100 mL), filtered and the cake was dried under reduced pressure to give the product. 1-Acetylpyrimidine-2,4(1H,3H)-dione (100 g, 648.83 mmol, 72.73% yield) was obtained as a white solid. ¹ HNMR (400 MHz, DMSO-d ₆ ) δ = 11.55 (br s, 1H), 8.12 (d, J = 8.4 Hz, 1H), 5.80 (dd, J = 2.2, 8.5 Hz, 1H), 2.70 - 2.55 (m, 3H); TLC (petroleum ether: ethyl acetate = 0:1), Rf = 0.72.

Step 2. Charge 1-acetylpyrimidine-2,4(1H,3H)-dione (17 g, 110.30 mmol, 1 eq.) into a clean and dry three-necked 3-liter round bottom flask and dry MeCN (1700 mL) was dissolved. The reaction mixture was cooled to 0 °C using an ice bath. NaH (6.62 g, 165.45 mmol, 60% purity, 1.5 equiv) was added portionwise to the reaction mixture and stirred at 0° C. for 30 min. (2R,3S)-5-chloro-2-(((4-chlorobenzoyl)oxy)methyl)tetrahydrofuran-3-yl 4-chlorobenzoate (65.88 g, 153.32 mmol, 1.39 equiv) was added in portions and The reaction mixture was stirred at 0 °C for 30 minutes and at 65 °C for 3 hours. TLC (petroleum ether:ethyl acetate=1:1, Rf=0.24) showed that reactant 1 was consumed and a new spot was formed. The reaction mixture was then cooled to room temperature and filtered through a sintered funnel using Whatman filter paper. The filtrate was concentrated under reduced pressure to give the crude product. The crude product was purified by silica gel column chromatography (100-200 mesh). After the product was eluted with 50% to 80% EtOAc:petroleum ether, the solid was triturated with DCM (30 mL) to yield a mixture of compound WV-NU-096b and compound WV-NU-096c (50 g) as a yellow solid. got it with

Step 3. To a solution of a mixture of WV-NU-096b and WV-NU-096c (45 g, 89.06 mmol, 1 equiv) in MeOH (500 mL) was added NaOMe (12.03 g, 222.65 mmol, 2.5 equiv). The mixture was stirred at 15 °C for 2 h. 12.03 g of NH4Cl was added to the mixture, stirred for 30 minutes, filtered, and the filtrate was concentrated under reduced pressure to obtain a residue. The residue was purified by column chromatography (SiO ₂ , petroleum ether/ethyl acetate=1/1 to 0/1, then ethyl acetate/methanol=5/1) to obtain WV-NU-096d (20 g, 87.64 mmol, 98.41 % yield) was obtained as a yellow solid. LCMS: (M + H ⁺ ) = 227.0

Step 4. To a solution of WV-NU-096d (20.00 g, 87.64 mmol, 1 equiv) in pyridine (200 mL) was added DMTC1 (35.26 g, 104.07 mmol, 1.19 equiv). The mixture was stirred at 15 °C for 12 hours. The reaction mixture was quenched with water (200 mL) and extracted with 400 mL of ethyl acetate (200 mL * 2). The combined organic layers were washed with 50 mL of saturated brine, dried over Na ₂ SO ₄ , filtered, and concentrated under reduced pressure to give a residue. The residue was purified by preparative HPLC (column: Phenomenex Titank C18 bulk 250*70 mm 10u; mobile phase: [water (10 mM NH ₄ HCO ₃ )-ACN]; B%: 45%-75%, 20 min). WV-NU-096 (30 g, 55.27 mmol, 63.06% yield, 97.75% purity) and WV-NU-096A (5 g, 9.20 mmol, 10.50% yield, 97.61% purity) were obtained as white solids. WV-NU-096: ¹ HNMR (400 MHz, DMSO-d ₆ ) δ = 11.14 - 10.94 (m, 1H), 7.47 - 7.31 (m, 3H), 7.27 - 7.21 (m, 6H), 7.20 - 7.13 ( m, 1H), 6.86 - 6.77 (m, 4H), 6.61 - 6.52 (m, 1H), 5.57 - 5.49 (m, 1H), 5.08 - 5.02 (m, 1H), 4.29 - 4.19 (m, 1H), 3.87 - 3.76 (m, 1H), 3.74 - 3.69 (m, 6H), 3.24 - 3.16 (m, 1H), 3.08 - 3.01 (m, 1H), 2.62 - 2.52 (m, 1H), 2.04 - 1.92 (m , 1H); LCMS (M-H+): 529.2, LCMS purity: 97.75%. WV-NU-096A: ¹ H NMR (400 MHz, DMSO-d ₆ ) δ = 11.25 - 11.01 (m, 1H), 7.49 - 7.43 (m, 1H), 7.41 - 7.35 (m, 2H), 7.33 - 7.28 (m, 2H), 7.27 - 7.17 (m, 5H), 6.95 - 6.84 (m, 4H), 6.57 - 6.44 (m, 1H), 5.63 - 5.56 (m, 1H), 5.28 - 5.19 (m, 1H) ( m, 1H), 2.38 - 2.30 (m, 1H); LCMS (M-H+): 529.2, LCMS purity: 97.61%.

3-((2R,4S,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-hydroxytetrahydrofuran-2-yl)pyrimidine-2,4 (1H,3H)-dione (WV-NU-096) and 3-((2S,4S,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-hydride Synthesis of oxytetrahydrofuran-2-yl) pyrimidine-2,4 (1H, 3H) -dione (WV-NU-096A)

Step 1. To a solution of pyrimidine-2,4(1H,3H)-dione (100 g, 892.17 mmol, 1 equiv) in pyridine (1000 mL) was added Ac ₂ O (546.48 g, 5.35 mol, 501.36 mL, 6 equiv). ) was added. The mixture was stirred at 120 °C for 3 hours. The reaction mixture was concentrated under reduced pressure to give crude, the residue was washed with EtOAc (100 mL), filtered and the cake was dried under reduced pressure to give the product. 1-Acetylpyrimidine-2,4(1H,3H)-dione (100 g, 648.83 mmol, 72.73% yield) was obtained as a white solid. ¹ HNMR (400 MHz, DMSO-d ₆ ) δ = 11.55 (br s, 1H), 8.12 (d, J = 8.4 Hz, 1H), 5.80 (dd, J = 2.2, 8.5 Hz, 1H), 2.70 - 2.55 (m, 3H); TLC (petroleum ether: ethyl dacetate = 0:1), Rf = 0.72.

Step 2. Charge 1-acetylpyrimidine-2,4(1H,3H)-dione (17 g, 110.30 mmol, 1 eq.) into a clean and dry three-necked 3-liter round bottom flask and dry MeCN (1700 mL) was dissolved. The reaction mixture was cooled to 0 °C using an ice bath. NaH (6.62 g, 165.45 mmol, 60% purity, 1.5 equiv) was added portionwise to the reaction mixture and stirred at 0° C. for 30 min. (2R,3S)-5-chloro-2-(((4-chlorobenzoyl)oxy)methyl)tetrahydrofuran-3-yl 4-chlorobenzoate (65.88 g, 153.32 mmol, 1.39 equiv) was added in portions and The reaction mixture was stirred at 0 °C for 30 minutes and at 65 °C for 3 hours. TLC (petroleum ether:ethyl acetate=1:1, Rf=0.24) showed that reactant 1 was consumed and a new spot was formed. The reaction mixture was then cooled to room temperature and filtered through a sintered funnel using Whatman filter paper. The filtrate was concentrated under reduced pressure to give the crude product. The crude product was purified by silica gel column chromatography (100-200 mesh). After the product was eluted with 50% to 80% EtOAc:petroleum ether, the solid was triturated with DCM (30 mL) to yield a mixture of compound WV-NU-096b and compound WV-NU-096c (50 g) as a yellow solid. got it with

Step 3. To a solution of a mixture of WV-NU-096b and WV-NU-096c (45 g, 89.06 mmol, 1 equiv) in MeOH (500 mL) was added NaOMe (12.03 g, 222.65 mmol, 2.5 equiv). The mixture was stirred at 15 °C for 2 hours. 12.03 g of NH4Cl was added to the mixture, stirred for 30 minutes, filtered, and the filtrate was concentrated under reduced pressure to obtain a residue. The residue was purified by column chromatography (SiO ₂ , petroleum ether/ethyl acetate=1/1 to 0/1, then ethyl acetate/methanol=5/1) to give WV-NU-096d (20 g, 87.64 mmol, 98.41 % yield) was obtained as a yellow solid. LCMS: (M + H ⁺ ) = 227.0

Step 4. To a solution of WV-NU-096d (20.00 g, 87.64 mmol, 1 equiv) in pyridine (200 mL) was added DMTC1 (35.26 g, 104.07 mmol, 1.19 equiv). The mixture was stirred at 15 °C for 12 hours. The reaction mixture was quenched with water (200 mL) and extracted with 400 mL of ethyl acetate (200 mL * 2). The combined organic layers were washed with 50 mL of saturated brine, dried over Na ₂ SO ₄ , filtered, and concentrated under reduced pressure to give a residue. The residue was purified by preparative HPLC (column: Phenomenex Titank C18 bulk 250*70 mm 10u; mobile phase: [water (10 mM NH ₄ HCO ₃ )-ACN]; B%: 45%-75%, 20 min). WV-NU-096 (30 g, 55.27 mmol, 63.06% yield, 97.75% purity) and WV-NU-096A (5 g, 9.20 mmol, 10.50% yield, 97.61% purity) were obtained as white solids. WV-NU-096: ¹ HNMR (400 MHz, DMSO-d ₆ ) δ = 11.14 - 10.94 (m, 1H), 7.47 - 7.31 (m, 3H), 7.27 - 7.21 (m, 6H), 7.20 - 7.13 ( m, 1H), 6.86 - 6.77 (m, 4H), 6.61 - 6.52 (m, 1H), 5.57 - 5.49 (m, 1H), 5.08 - 5.02 (m, 1H), 4.29 - 4.19 (m, 1H), 3.87 - 3.76 (m, 1H), 3.74 - 3.69 (m, 6H), 3.24 - 3.16 (m, 1H), 3.08 - 3.01 (m, 1H), 2.62 - 2.52 (m, 1H), 2.04 - 1.92 (m , 1H); LCMS (M-H+): 529.2, LCMS purity: 97.75%. WV-NU-096A: ¹ H NMR (400 MHz, DMSO-d ₆ ) δ = 11.25 - 11.01 (m, 1H), 7.49 - 7.43 (m, 1H), 7.41 - 7.35 (m, 2H), 7.33 - 7.28 (m, 2H), 7.27 - 7.17 (m, 5H), 6.95 - 6.84 (m, 4H), 6.57 - 6.44 (m, 1H), 5.63 - 5.56 (m, 1H), 5.28 - 5.19 (m, 1H) ( m, 1H), 2.38 - 2.30 (m, 1H); LCMS (M-H+): 529.2, LCMS purity: 97.61%.

1-(1-((2R,4S,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-hydroxytetrahydrofuran-2-yl)-2- Synthesis of oxo-1,2-dihydropyrimidin-4-yl)-3-phenylurea (WV-NU-187)

Step 1. 4-Amino-1-((2R,4S,5R)-4-((tert-butyldimethylsilyl)oxy)-5-(((tert-butyldimethylsilyl)oxy) in ACN (1000 mL) To a solution of methyl)tetrahydrofuran-2-yl)pyrimidin-2(1H)-one (98 g, 215.04 mmol, 1 equiv) isocyanatobenzene (29.93 g, 251.26 mmol, 27.21 mL, 1.17 equiv) was added. added. The mixture was stirred at 20 °C for 6 hours. The reaction mixture was filtered to obtain a solid. The filtrate was quenched by adding 100 mL of water. The solid was washed with ACN (300 mL*3). 1-(1-((2R,4S,5R)-4-((tert-butyldimethylsilyl)oxy)-5-(((tert-butyldimethylsilyl)oxy)methyl)tetrahydrofuran-2-yl) Obtained -2-oxo-1,2-dihydropyrimidin-4-yl)-3-phenylurea (90 g, crude) as a white solid. LCMS (MH ⁺ ): 573.2

Step 2. 1-(1-((2R,4S,5R)-4-((tert-butyldimethylsilyl)oxy)-5-(((tert-butyldimethylsilyl)oxy)methyl in THF (900 mL) To a solution of )tetrahydrofuran-2-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)-3-phenylurea (90 g, 156.56 mmol, 1 equiv.) was added TBAF (1 M, 391.40 mL, 2.5 eq) was added. The mixture was stirred at 20 °C for 3 hours. TLC (petroleum ether: ethyl acetate = 0:1, Rf = 0.1) showed that the starting material was completely consumed and one new spot was formed. The reaction mixture was concentrated under reduced pressure to remove the solvent. The residue was purified by column chromatography (SiO ₂ , ethyl acetate/methanol=1/0 to 3/1) to obtain 1-(1-((2R,4S,5R)-4-hydroxy-5-(hydroxy Methyl)tetrahydrofuran-2-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)-3-phenylurea (54 g, crude) was obtained as a white solid.

Step 3. 1-(1-((2R,4S,5R)-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-2-oxo-1 in pyridine (500 mL) To a solution of 2-dihydropyrimidin-4-yl)-3-phenylurea (53 g, 153.03 mmol, 1 equiv) was added DMTC1 (77.78 g, 229.55 mmol, 1.5 equiv). The mixture was stirred at 20 °C for 5 hours. The reaction mixture was quenched by adding 200 mL of methanol, and concentrated under reduced pressure to obtain a residue. The residue was purified by column chromatography to give WV-NU-187 (26 g, 39.79 mmol, 76.56% yield, 99.28% purity) as a yellow solid. ¹ HNMR (400 MHz, chloroform-d) δ = 11.57 - 10.79 (m, 2H), 8.18 (d, J = 7.7 Hz, 1H), 7.68 (br d, J = 7.8 Hz, 2H), 7.41 (br d, J = 7.6 Hz, 2H), 7.36 - 7.22 (m, 9H), 7.17 (d, J = 8.8 Hz, 1H), 7.04 (br t, J = 7.3 Hz, 1H), 6.92 - 6.79 (m, 4H), 6.30 (br t, J = 5.4 Hz, 1H), 4.45 (br d, J = 5.0 Hz, 1H), 4.10 - 4.05 (m, 1H), 3.80 (s, 6H), 3.59 - 3.35 (m, 2H), 2.68 - 2.55 (m, 1H) ), 2.34 - 2.19 (m, 2H); LCMS (MH ⁺ ): 647.3; Purity: 99.28%.

1-(1-((2R,4S,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-hydroxytetrahydrofuran-2-yl)-2- Synthesis of oxo-1,2-dihydropyrimidin-4-yl)-3-(naphthalen-2-yl)urea (WV-NU-188)

Step 1. Two batches: 4-amino-1-((2R,4S,5R)-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)pyrimidine in DCM (250 mL) To a solution of -2(1H)-one (25 g, 110.03 mmol, 1 equiv) was added imidazole (59.92 g, 880.22 mmol, 8 equiv) and TBSCl (66.33 g, 440.11 mmol, 53.93 mL, 4 equiv). . The mixture was stirred at 20 °C for 12 hours. The reaction mixture was diluted with 500 mL of water and extracted with dichloromethane (500 mL * 2). The combined organic layers were dried over Na ₂ SO ₄ , filtered and concentrated under reduced pressure to give a residue of 4-amino-1-((2R,4S,5R)-4-((tert-butyldimethylsilyl)oxy)-5 Obtained -(((tert-butyldimethylsilyl)oxy)methyl)tetrahydrofuran-2-yl)pyrimidin-2(1H)-one (100 g, crude) as a colorless oil. LCMS (MH ⁺ ):454.5, purity: 99.93%

Step 2 . 2 batches: 4-amino-1-((2R,4S,5R)-4-((tert-butyldimethylsilyl)oxy)-5-(((tert-butyldimethylsilyl)oxy in MeCN (500 mL) To a solution of )methyl)tetrahydrofuran-2-yl)pyrimidin-2(1H)-one (46.5 g, 102.03 mmol, 1 eq.) 1-isocyanatonaphthalene (17.26 g, 102.03 mmol, 14.63 mL, 1 equivalent) was added. The mixture was stirred at 20 °C for 12 hours. The reaction mixture was diluted with 500 mL of water and extracted with DCM (200 mL *2). The combined organic layers were dried over Na ₂ SO ₄ , filtered, and concentrated under reduced pressure to yield 1-(1-((2R,4S,5R)-4-((tert-butyldimethylsilyl)oxy)-5-(((tert- Butyldimethylsilyl) oxy) methyl) tetrahydrofuran-2-yl) -2-oxo-1,2-dihydropyrimidin-4-yl) -3- (naphthalen-2-yl) urea (127 g) Obtained as a white solid. ¹ HNMR (400 MHz, DMSO-d6) δ = 12.51 (br s, 1H), 10.48 (s, 1H), 8.46 - 7.90 (m, 4H), 7.71 - 7.45 (m, 4H), 6.36 - 6.13 (m, 2H), 4.39 (br d, J = 4.5 Hz, 1H), 3.92 - 3.69 (m, 3H), 2.39 - 2.17 (m, 2H), 0.88 (br d, J = 7.5 Hz, 18H), 0.08 (br d, J = 1.1 Hz, 12H); LCMS (MH ⁺ ):622.9, purity: 85.7%

Step 3 . Two batches: 1-(1-((2R,4S,5R)-4-((tert-butyldimethylsilyl)oxy)-5-(((tert-butyldimethylsilyl)oxy) in THF (600 mL) Methyl) tetrahydrofuran-2-yl) -2-oxo-1,2-dihydropyrimidin-4-yl) -3- (naphthalen-2-yl) urea (63.5 g, 101.61 mmol, 1 eq.) TBAF (1 M, 254.03 mL, 2.5 eq) was added to the solution. The mixture was stirred at 20 °C for 2 h. The reaction mixture was concentrated under reduced pressure to give a residue. 500 ml of ethyl acetate was added to the reaction mixture and stirred at 25° C. for 30 minutes to precipitate a solid, which was then filtered to obtain 1-(1-((2R,4S,5R)-4-hydroxy-5-(hydroxymethyl) Tetrahydrofuran-2-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)-3-(naphthalen-2-yl)urea (80 g) was obtained as a white solid. ^1HNMR (400MHz, DMSO-d6) δ = 8.65 - 8.56 (m, 1H), 8.38 (d, J = 7.6 Hz, 1H), 8.08 (br d, J = 7.3 Hz, 1H), 7.89 (br dd, J = 2.9, 6.5 Hz, 1H) , 7.58 - 7.39 (m, 4H), 6.36 - 6.18 (m, 2H), 4.33 - 4.25 (m, 1H), 3.81 (br d, J = 3.5 Hz, 1H), 3.69 - 3.57 (m, 2H), 2.27 - 2.18 (m, 1H), 2.05 (td, J = 6.3, 13.0 Hz, 1H); LCMS (MH ⁺ ):395.1, purity: 97.74%.

Step 4 . Two batches: 1-(1-((2R,4S,5R)-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-2-oxo-1 in pyridine (400 mL) To a solution of ,2-dihydropyrimidin-4-yl)-3-(naphthalen-2-yl)urea (40 g, 100.91 mmol, 1 equiv) was added DMTC1 (51.29 g, 151.36 mmol, 1.5 equiv). . The mixture was stirred at 25 °C for 12 hours. The reaction mixture was diluted with 800 mL of water and extracted with ethyl acetate (400 mL *4). The combined organic layers were washed with 400 mL of brine, dried over Na ₂ SO ₄ , filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, petroleum ether/ethyl acetate = 10:1 to 0:1, 5%TEA) to give WV-NU-188 (82 g, 117.35 mmol, 58.57% yield) as a white solid. Got it. ^1HNMR (400MHz, DMSO-d6) δ = 10.53 (s, 1H), 8.44 (br d, J = 8.0 Hz, 1H), 8.29 (d, J = 7.5 Hz, 1H), 8.12 (d, J = 7.4 Hz, 1H), 7.98 - 7.94 ( m, 1H), 7.69 (d, J = 8.3 Hz, 1H), 7.64 - 7.55 (m, 2H), 7.50 (t, J = 7.9 Hz, 1H), 7.43 - 7.37 (m, 2H), 7.37 - 7.22 (m, 7H), 6.91 (dd, J = 1.0, 8.9 Hz, 4H), 6.25 - 6.13 (m, 2H), 5.40 (d, J = 4.6 Hz, 1H), 4.34 (quin, J = 5.3 Hz, 1H), 3.74 (s, 6H), 3.30 (br d, J = 3.6 Hz, 2H), 2.44 - 2.35 (m, 1H), 2.28 - 2.19 (m, 1H); LCMS (MH ⁺ ): 697.3; Purity: 99.66%.

N-(5-((2R,4S,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-hydroxytetrahydrofuran-2-yl)-6- Synthesis of oxo-1,6-dihydropyrimidin-2-yl)acetamide (WV-NU-189)

Step 1 . To a solution of BSA (73.19 g, 359.80 mmol, 88.94 mL, 3.1 equiv) in DMF (500 mL) under argon atmosphere, N-(5-iodo-6-oxo-1,6-dihydropyrimidin-2-yl ) was added dropwise to a suspension of acetamide (80.97 g, 290.16 mmol, 2.5 equiv). After stirring for 1 hour, the reaction becomes a clear solution. Then DIPEA (46.50 g, 359.80 mmol, 62.67 mL, 3.1 equiv) and tert-butyl (((2R,3S)-3-((tert-butyldimethylsilyl)oxy)-2,3-dihydrofuran-2 -yl)methoxy)dimethylsilane (40 g, 116.06 mmol, 1 eq) was added. In a separate flask, Pd(OAc) ₂ (1.82 g, 8.12 mmol, 0.07 equiv) was added to a solution of triphenylaric acid (14.22 g, 46.43 mmol, 0.4 equiv) in stirred DMF (500 mL). After 30 minutes, this solution was slowly added to the first flask and the mixture was stirred at 80° C. for 12 hours. The reaction was quenched by the addition of H ₂ O (30 mL) and the solvent was evaporated under reduced pressure. The residue was redissolved in EtOAc (500 mL) and washed with H ₂ O (2*100 mL) and brine (200 mL). The organic layer was dried over MgSO ₄ , filtered and concentrated under reduced pressure. The residue was purified by column chromatography (SiO ₂ , petroleum ether/ethyl acetate = 100/1 to 0/1) to obtain N-(5-((2R,5R)-4-((tert-butyldimethylsilyl)oxy )-5-(((tert-butyldimethylsilyl)oxy)methyl)-2,5-dihydrofuran-2-yl)-6-oxo-1,6-dihydropyrimidin-2-yl)acetamide (25 g, 50.43 mmol, 43.45% yield) as a white solid. ¹ H NMR (chloroform-d, 400 MHz) : δ = 8.27 (d, J = 2.0 Hz, 1H), 8.22 (br d, J = 8.0 Hz, 2H), 7.98 (dd, J = 8.6, 2.3 Hz, 1H), 7.30–7.39 (m, 4H), 5.69 (dd, J = 3.8, 1.4 Hz, 1H), 4.75 (s, 1H), 4.58 (tt, J = 3.7, 1.9 Hz, 1H), 3.85–3.92 (m, 1H), 3.75–3.81 (m, 1H) ), 2.22–2.24 (m, 4H), 0.86–0.98 (m, 19H), 0.22 (d, J = 6.6 Hz, 6H), 0.05 ppm (d, J = 2.5 Hz, 6H).

Step 2 . N-(5-((2R,5R)-4-((tert-butyldimethylsilyl)oxy)-5-(((tert-butyldimethylsilyl)oxy)methyl)-2,5-dihydrofuran-2 To a solution of -yl)-6-oxo-1,6-dihydropyrimidin-2-yl)acetamide (23 g, 46.39 mmol, 1 equiv.) in THF (200 mL) pyridine:hydrogen fluoride (23.65 g, 167.02 mmol, 21.50 mL, 70% purity, 3.6 equiv). The reaction was stirred at 25 °C for 12 hours. The suspension was diluted with acetic acid (30 mL) and the volatiles removed under reduced pressure. N-(5-((2R,5R)-5-(hydroxymethyl)-4-oxotetrahydrofuran-2-yl)-6-oxo-1,6-dihydropyrimidin-2-yl)acetate The amide (12.40 g, 46.40 mmol, 100.00 % yield) was obtained as a white solid, which was used in the next step without further purification; LCMS (M+H+): 268.3.

Step 3 . N-(5-((2R,5R)-5-(hydroxymethyl)-4-oxotetrahydrofuran-2-yl)-6-oxo-1,6-dihydropyrimidin-2-yl)acetate Amide (12.4 g, 46.40 mmol, 1 equiv) was dissolved in a mixture of MeCN (66 mL)/AcOH (66 mL) (1:1 v/v, ) and the mixture was cooled to -15 °C, followed by NaBH (OAc ) ₃ (23.11 g, 109.04 mmol, 2.35 equiv) was added in portions. The mixture was stirred at -15 °C for 2 hours. The mixture was evaporated to dryness under reduced pressure. The residue was purified by column chromatography (SiO ₂ , petroleum ether: ethyl acetate = 100/1 to 5/1) to obtain N-(5-((2R,4S,5R)-4-hydroxy-5-(hydroxy Oxymethyl)tetrahydrofuran-2-yl)-6-oxo-1,6-dihydropyrimidin-2-yl)acetamide (11 g, 40.85 mmol, 88.05% yield) was obtained as a white solid.

Step 4 . N-(5-((2R,4S,5R)-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-6-oxo-1,6-di in pyridine (100 mL) To a solution of hydropyrimidin-2-yl)acetamide (9 g, 33.43 mmol, 1 equiv) was added DMTC1 (11.33 g, 33.43 mmol, 1 equiv). The mixture was stirred at 15 °C for 12 hours. The residue was diluted with 200 mL of H ₂ O and extracted with 1500 mL of EtOAc (500 mL * 3). The combined organic layers were washed with 30 mL of brine (10 mL * 3), dried over Na ₂ SO ₄ , filtered, and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO ₂ , DCM: MeOH = 100/1 to 5/1) to give WV-NU-189 (15 g, 26.24 mmol, 78.51% yield) as a white solid. ¹ HNMR (chloroform-d, 400 MHz) : δ = 7.94 (br s, 1H), 7.43 (br d, J = 7.3 Hz, 2H), 7.27 (s, 7H), 7.22 (br d, J = 6.8 Hz, 1H), 6.83 (br d, J = 8.8 Hz, 4H), 5.17 (br s, 1H), 4.40 (br s, 1H), 4.03 (br s, 1H), 3.78 (s, 6H), 3.21-3.36 (m, 2H), 2.48 (br s, 1H), 2.18 (br s, 3H), 1.95 ppm (br s, 1H); LCMS (M-H+): 570.3, LCMS purity: 91.61%.

3-((2R,4S,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-hydroxytetrahydrofuran-2-yl)pyridin-2(1H) Synthesis of -one (WV-NU-197)

Step 1 . A suspension of 3-iodopyridin-2(1H)-one (16.03 g, 72.54 mmol, 2.5 equiv) in DMF (100 mL) under an argon atmosphere in a solution of BSA (18.30 g, 89.95 mmol, 22.23 mL, 3.1 equiv). was added to After stirring for 1 hour, the reaction becomes a clear solution. Then DIEA (11.63 g, 89.95 mmol, 15.67 mL, 3.1 eq) and tert-butyl (((2R,3S)-3-((tert-butyldimethylsilyl)oxy)-2,3-dihydrofuran-2 -yl)methoxy)dimethylsilane (10 g, 29.02 mmol, 1 eq) was added. In a separate flask, Pd(OAc) ₂ (456.01 mg, 2.03 mmol, 0.07 equiv) was added to a solution of triphenylaric acid (3.55 g, 11.61 mmol, 0.4 equiv) in stirred DMF (100 mL). After 30 minutes, this solution was slowly added to the first flask and the mixture was stirred at 80° C. for 12 hours. The reaction was quenched by the addition of H ₂ O (300 mL) and the solvent was evaporated under reduced pressure. The residue was redissolved in EtOAc (300 mL) and washed with H ₂ O (2*100 mL) and brine (30 mL). The organic layer was dried over MgSO ₄ , filtered and concentrated under reduced pressure. The residue was purified by column chromatography (SiO2, petroleum ether/ethyl acetate = 100/1 to 0/1) to give 3-((2R,5R)-4-((tert-butyldimethylsilyl)oxy)-5- (((tert-butyldimethylsilyl)oxy)methyl)-2,5-dihydrofuran-2-yl)pyridin-2(1H)-one (12 g, 27.41 mmol, 94.48% yield) was obtained as a white solid . ¹ HNMR (chloroform-d, 400 MHz): δ ¹ = 12.66 (brs, 1H), 7.81-7.85 (m, 1H), 7.22-7.29 (m, 2H), 6.22 (t, J = 6.7 Hz, 1H), 5.86 (d, J = 3.3 Hz, 1H), 4.95 (t, J = 1.6 Hz, 1H), 4.49–4.59 (m, 1H), 3.85 (dd, J = 11.3, 2.1 Hz, 1H), 3.69 (dd , J = 11.2, 3.7 Hz, 1H), 0.78–0.90 (m, 17H), 0.14 (d, J = 16.4 Hz, 6H), −0.01 ppm (d, J = 8.6 Hz, 6H); LCMS: M+H ⁺ = 438.7.

Step 2 . 3-((2R,5R)-4-((tert-butyldimethylsilyl)oxy)-5-(((tert-butyldimethylsilyl)oxy)methyl)-2,5-dihydro in THF (120 mL) To a solution of furan-2-yl)pyridin-2(1H)-one (12 g, 27.41 mmol, 1 equiv) was added pyridine; hydrogen fluoride (11.89 g, 95.95 mmol, 10.81 mL, 80% pure, 3.5 equiv) ) was degassed and purged with N ₂ three times, and then the mixture was stirred at 15° C. for 12 hours under N ₂ atmosphere. The filtrate was concentrated in vacuo to give crude 3-((2R,5R)-5-(hydroxymethyl)-4-oxotetrahydrofuran-2-yl)pyridin-2(1H)-one (5.74 g, 27.44 mmol, 100.00% yield) was obtained. LCMS : M+H ⁺ = 210.1 and M+Na ⁺ = 232.1.

Step 3 . MeCN in a solution of 3-((2R,5R)-5-(hydroxymethyl)-4-oxotetrahydrofuran-2-yl)pyridin-2(1H)-one (5.74 g, 27.44 mmol, 1 eq) (70 mL)/AcOH (70 mL), then NaBH(OAc) ₃ (13.67 g, 64.48 mmol, 2.35 equiv) was added portionwise. The mixture was stirred at 15 °C for 2 h. The mixture was evaporated to dryness under reduced pressure. The residue was purified by column chromatography (SiO ₂ , petroleum ether: ethyl acetate = 100/1 to 5/1) to give 3-((2R,4S,5R)-4-hydroxy-5-(hydroxymethyl) Tetrahydrofuran-2-yl)pyridin-2(1H)-one (3.1 g, 14.68 mmol, 53.49% yield) was obtained as a white solid. ¹ H NMR (chloroform-d, 400 MHz): δ ¹ = 7.72-7.78 (m, 1H), 7.35 (dd, J = 6.5, 2.0 Hz, 1H), 6.40 (t, J = 6.7 Hz, 1H), 5.16 (dd, J = 10.0, 5.9 Hz, 1H), 4.26–4.33 (m, 1H), 3.94 (td, J = 4.4, 2.7 Hz, 1H), 3.61–3.72 (m, 2H), 2.33 (ddd, J = 13.0, 5.9, 2.0 Hz, 1H), 1.87-2.00 ppm (m, 1H); LCMS : (M+H ⁺ ): 212.

Step 4. 3-((2R,4S,5R)-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)pyrimidin-2(1H)-one in pyridine (30 mL) To a solution of 3.10 mg, 14.68 mmol, 1 equiv.) was added DMTrCl (4.48 g, 13.21 mmol, 0.9 equiv.). The mixture was stirred at 15 °C for 2 hours. The reaction mixture was diluted with 50 mL of H ₂ O and extracted with 180 mL of EAOAC (60 mL * 3). The combined organic layers were washed with 15 mL of brine (5 mL * 3), dried over Na ₂ SO ₄ , filtered, and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO ₂ , DCM: MeOH = 100:1 to 5:1) to give WV-NU-197 (5.2 g) as a white solid. ^1HNMR (DMSO-d6, 400MHz): δ ¹ = 11.59 (br s, 1H), 7.39-7.50 (m, 3H), 7.18-7.35 (m, 8H), 6.89 (d, J = 8.5 Hz, 4H) , 6.15 (t, J = 6.7 Hz, 1H), 5.06 (d, J = 4.1 Hz, 1H), 5.00 (dd, J = 9.2, 6.0 Hz, 1H), 4.00–4.16 (m, 1H), 3.82– 3.95 (m, 1H), 3.73 (s, 6H), 2.99-3.17 (m, 3H), 2.26 (ddd, J = 12.7, 6.0, 2.5 Hz, 1H), 1.58 ppm (ddd, J = 12.7, 9.3, 6.1 Hz, 1H); LCMS: M+H ⁺ : 513.6, LCMS purity 100.0%

N-((3aR,5R,6R,6aS)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-6-hydroxy-3a,5,6,6a-tetrahydrofuro Synthesis of [2,3-d]oxazol-2-yl)acetamide (WV-NU-194)

Step 1 . (2S,3R,4R)-2,3,4,5-tetrahydroxypentanal (80 g, 532.87 mmol, 1 equiv) and added KHCO ₃ (2.80 g, 27.97 mmol, 5.25 mmol) in DMF (500 mL) e-2 equiv) and NH ₂ CN (26.80 g, 637.49 mmol, 26.80 mL, 1.20 equiv) was stirred at 90 °C for 1 h. After cooling to room temperature, the mixture was evaporated to half volume under reduced pressure and the resulting solution was stored at 5° C. for 20 hours. The precipitate obtained was filtered and 96% aq. Recrystallized from EtOH (600 ml) to (3aR,5R,6R,6aS)-2-amino-5-(hydroxymethyl)-3a,5,6,6a-tetrahydrofuro[2,3-d]oxa Zol-6-ol (50 g) was obtained as a white solid. ¹ HNMR (400 MHz, DMSO-d6) δ = 6.36 (br s, 2H), 5.66 (d, J = 5.6 Hz, 1H), 5.46 (br s, 1H), 4.75 (br s, 1H), 4.53 (br d, J = 5.5 Hz, 1H), 4.00 (br s, 1H), 3.67 - 3.59 (m, 1H), 3.40 (s, 1H), 3.33 - 3.19 (m, 2H).

Step 2. (3aR,5R,6R,6aS)-2-amino-5-(hydroxymethyl)-3a,5,6,6a-tetrahydrofuro[2,3-d]oxa in DCM (200 mL) To a solution of zol-6-ol (20 g, 114.84 mmol, 1 equiv) was added imidazole (46.91 g, 689.04 mmol, 6 equiv) followed by TBSCl (60.58 g, 401.94 mmol, 49.25 mL, 3.5 equiv). added. The mixture was stirred at 30 °C for 10 h. Both batches of reaction mixture were filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO ₂ , ethyl acetate:methanol = 0:1 to 5:1) to obtain (3aR,5R,6R,6aS)-6-((tert-butyldimethylsilyl)oxy)-5 -(((tert-butyldimethylsilyl)oxy)methyl)-3a,5,6,6a-tetrahydrofuro[2,3-d]oxazol-2-amine (91 g, 135.59 mmol, 55.15% yield, 60% purity) was obtained as a white solid. ¹ HNMR (400 MHz, chloroform-d) δ = 5.87 (d, J = 5.6 Hz, 1H), 4.64 (d, J = 5.6 Hz, 1H), 4.32 (d, J = 2.5 Hz, 1H), 3.91 - 3.81 (m, 1H), 3.63 (dd, J = 5.1, 10.7 Hz, 1H), 3.46 (dd, J = 7.6, 10.6 Hz, 1H), 0.91 - 0.86 (m, 19H), 0.11 (d, J = 8.0 Hz, 6H), 0.03 (s, 6H); LCMS (M+H ⁺ ) : 403.3, purity: 79.78%.

Step 3 . (3aR,5R,6R,6aS)-6-((tert-butyldimethylsilyl)oxy)-5-(((tert-butyldimethylsilyl)oxy)methyl)-3a,5, in pyridaben (400 mL) Ac ₂ O (7.10 g, 69.54 mmol, 6.51 mL, 0.7 equiv) to a solution of a mixture of 6,6a-tetrahydrofuro[2,3-d]oxazol-2-amine (40 g, 99.34 mmol, 1 equiv) was added dropwise. The mixture was stirred at 25 °C for 12 hours. The reaction mixture was filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, petroleum ether:ethyl acetate = 50:1 to 15:1) to obtain N-((3aR,5R,6R,6aS)-6-((tert-butyldimethylsilyl)oxy )-5-(((tert-butyldimethylsilyl)oxy)methyl)-3a,5,6,6a-tetrahydrofuro[2,3-d]oxazol-2-yl)acetamide (33 g, 74.21 mmol, 74.70% yield) as a yellow oil. ^1H NMR (400 MHz, chloroform-d) δ = 5.91 (d, J = 5.8 Hz, 1H), 4.81 (dd, J = 1.0, 5.8 Hz, 1H), 4.49 (dd, J = 0.9, 2.8 Hz, 1H), 3.98 (ddd, J = 2.9, 4.8, 7.4 Hz, 1H), 3.61 (dd, J = 5.0, 10.9 Hz, 1H), 3.44 (dd, J = 7.4, 10.9 Hz, 1H), 2.16 (s , 3H), 0.90 - 0.88 (m, 9H), 0.87 - 0.85 (m, 9H), 0.12 (d, J = 9.6 Hz, 6H), 0.02 (d, J = 3.8 Hz, 6H); LCMS (M+H) ⁺ : 445.4, purity: 92.67%.

Step 4 . N-((3aR,5R,6R,6aS)-6-((tert-butyldimethylsilyl)oxy)-5-(((tert-butyldimethylsilyl)oxy)methyl)-3a in THF (300 mL); TBAF (1 M, 111.31 mL, 1.5 equiv) was added to a solution of 5,6,6a-tetrahydrofuro[2,3-d]oxazol-2-yl)acetamide (33 g, 74.21 mmol, 1 equiv). added. The mixture was stirred at 25 °C for 1 hour. The reaction mixture was filtered and concentrated under reduced pressure to give a residue. The crude product was purified by reverse phase HPLC (column: C18 20-35 um 100A 100 g; mobile phase: [water-ACN]; B%: 0%-0% @ 30 mL/min), after purification LCMS (ET35599 -347-P2A1) N-((3aR,5R,6R,6aS)-6-hydroxy-5-(hydroxymethyl)-3a,5,6,6a-tetrahydrofuro[2,3- d]oxazol-2-yl)acetamide (11 g, 50.88 mmol, 68.75% yield) was obtained as a white solid. ¹ H NMR (400 MHz, deuterium oxide) δ = 4.43 - 4.36 (m, 1H), 4.14 - 3.98 (m, 3H), 3.84 - 3.61 (m, 3H), 3.56 (dd, J = 4.8, 12.4 Hz, 1H), 3.49 - 3.41 (m, 1H), 2.09 (s, 3H); LCMS (M+H ⁺ ): 217.2, purity: 99.41%.

Step 5 . N-((3aR,5R,6R,6aS)-6-hydroxy-5-(hydroxymethyl)-3a,5,6,6a-tetrahydrofuro[2,3-d] in DCM (50 mL) To a solution of oxazol-2-yl)acetamide (10 g, 46.26 mmol, 1 equiv) was added pyridine (7.32 g, 92.51 mmol, 7.47 mL, 2 equiv) and DMTrCl (9.40 g, 27.75 mmol, 0.6 equiv) to 0 was added at °C. The mixture was stirred at 20 °C for 2 h. The reaction mixture was quenched by adding 200 mL of water and then extracted with DCM (200 mL * 3). The combined organic layers were dried over Na ₂ SO ₄ , filtered and concentrated under reduced pressure to give a residue. The crude product was purified by reverse phase HPLC (column: C18 20-35 um 100A 100 g; mobile phase: [water (10 mM NH ₄ HCO ₃ )-ACN]; B%: 40%-65%, 20 min) to obtain WV -NU-194 (5.1 g, 9.83 mmol, 21.26% yield) was obtained as a white solid. ¹ H NMR (400 MHz, chloroform-d) δ = 9.60 (br s, 1H), 7.37 (d, J = 7.5 Hz, 2H), 7.29 - 7.19 (m, 7H), 7.16 - 7.10 (m, 1H) , 6.75 (dd, J = 4.4, 8.8 Hz, 4H), 5.89 (d, J = 6.0 Hz, 1H), 4.96 (dd, J = 1.6, 5.9 Hz, 1H), 4.42 (br d, J = 4.9 Hz) , 1H), 4.11 - 4.06 (m, 1H), 3.74 (d, J = 2.1 Hz, 6H), 3.29 - 3.19 (m, 2H), 2.01 (s, 3H); LCMS (MH ⁺ ):517, purity: 100%.

1-((2R,4S,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-hydroxytetrahydrofuran-2-yl)-3-methylpyrimidine Synthesis of -2,4(1H,3H)-dione (WV-NU-203)

Step 1 . 1-((2R,4S,5R)-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)pyrimidine 2,4(1H,3H)-dione ( To a solution of 20 g, 87.64 mmol, 1 equiv.) was added Mel (31.10 g, 219.10 mmol, 13.64 mL, 2.5 equiv.) and K ₂ CO ₃ (36.34 g, 262.93 mmol, 3 equiv.). The mixture was stirred at 55 °C for 2 hours. The reaction mixture was filtered and the filtrate was concentrated under reduced pressure to give a residue which was then extracted with DCM 200 mL * 2. The combined organic layers were dried, filtered, and concentrated under reduced pressure to give 1-((2R,4S,5R)-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-3-methylpyrimidine -2,4(1H,3H)-dione (15 g) was obtained as a white solid. LCMS: (M+H ⁺ ) 243.2.

Step 2 . 1-((2R,4S,5R)-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-3-methylpyrimidine-2,4(1H, To a solution of 3H)-dione (15 g, 61.93 mmol, 1 equiv) was added DMTC1 (23.08 g, 68.12 mmol, 1.1 equiv). The mixture was stirred at 15 °C for 1 hour. The reaction mixture was extracted with 150 mL * 2 of ethyl acetate. The combined organic layers were concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO ₂ , petroleum ether/ethyl acetate = 1/0 to 0/1) to give WV-NU-203 (13 g, 23.87 mmol, 38.55% yield) as a yellow solid. ¹ HNMR (400 MHz, DMSO-d ₆ ) δ = 7.42 - 7.34 (m, 2H), 7.31 (t, J = 7.6 Hz, 2H), 7.26 - 7.18 (m, 5H), 6.92 - 6.84 (m, 4H) ( m, 3H), 2.25 - 2.16 (m, 2H); LCMS: purity: 92.72%, (M - H ⁺ ): 543.59.

N-(9-((2R,4S,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-hydroxytetrahydrofuran-2-yl)-8- Synthesis of oxo-8,9-dihydro-7H-purin-6-yl)benzamide (WV-NU-137).

Step 1. After 3 hours, to a solution of Na (9.99 g, 434.67 mmol) in BnOH (391.84 g, 3.62 mol) (2R,3S,5R)-5-(6-amino-8-bromo-9H-purine -9-yl)-2-(hydroxymethyl)tetrahydrofuran-3-ol (25 g, 75.73 mmol) was added. The mixture was stirred at 15 °C for 12 hours. The reaction mixture was quenched at 0 °C by adding 800 mL of HCl (1 M), then saturated NaHCO ₃ aq. was added to pH ~9, extracted with EtOAc (1000 mL * 3) and dried over Na ₂ SO ₄ Worked, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO ₂ , petroleum ether/ethyl acetate = 5/1 to ethyl acetate: methanol = 10/1) to give (2R,3S,5R)-5-(6-amino-8- (Benzyloxy)-9H-purin-9-yl)-2-(hydroxymethyl)tetrahydrofuran-3-ol (35 g, 64.67% yield) was obtained as a yellow oil.LCMS: (M+H+) : 358.2

Step 2. (2R,3S,5R)-5-(6-amino-8-(benzyloxy)-9H-purin-9-yl)-2-(hydroxymethyl)tetrahydrofuran-3-ol (50 g, 139.91 mmol) (dried by azeotropic distillation on a rotary evaporator with pyridine (200 mL*3)) was added HMDS (338.72 g, 2.10 mol). The mixture was stirred at 150 °C for 12 hours. The reaction mixture was concentrated under reduced pressure to remove the solvent. 8-(benzyloxy)-9-((2R,4S,5R)-4-((trimethylsilyl)oxy)-5-(((trimethylsilyl)oxy)methyl)tetrahydrofuran-2-yl)-9H -Purin-6-amine (70.2 g, crude) was obtained as a yellow oil without purification.

Step 3. 8-(benzyloxy)-9-((2R,4S,5R)-4-((trimethylsilyl)oxy)-5-(((trimethylsilyl)oxy)methyl)tetra in pyridine (500 mL) To a solution of hydrofuran-2-yl)-9H-purin-6-amine (70.2 g) was added BzCl (29.50 g). The mixture was stirred at 20 °C for 2 h. MeOH (500 mL) and water (500 mL) were added, after 10 min NH ₃ .H ₂ O (250 mL) was added, after 30 min H ₂ O (500 mL) was added and EtOAc (500 mL * 4), dried over Na ₂ SO ₄ , filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO ₂ , petroleum ether/ethyl acetate = 1/0 to 0/1, then ethyl acetate/methanol = 10/1) to give N-(8-(benzyloxy)-9-( (2R,4S,5R)-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-9H-purin-6-yl)benzamide (36 g, 55.76% yield) was obtained as a yellow solid was obtained as LCMS: (M+H+): 462.2

Step 4. N-(8-(benzyloxy)-9-((2R,4S,5R)-4-hydroxy-5-(hydroxymethyl)tetrahydro in THF (500 mL) and MeOH (500 mL) To a solution of furan-2-yl)-9H-purin-6-yl)benzamide (36 g, 78 mmol) was added Pd/C (9 g, 39.01 mmol, 10% purity). The mixture was stirred at H ₂ (15 psi), 15° C. for 3 hours. The mixture was filtered and the filtrate was concentrated under reduced pressure to obtain N-(9-((2R,4S,5R)-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-8-oxo- 8,9-dihydro-7H-purin-6-yl)benzamide (28.9 g, crude) was obtained as a yellow solid. LCMS: (M+H+): 372.2.

Step 5. N-(9-((2R,4S,5R)-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-8-oxo-8 in pyridine (300 mL) To a solution of 9-dihydro-7H-purin-6-yl)benzamide (28.9 g, 77.82 mmol) was added DMTC1 (26.37 g, 77.82 mmol) and the mixture was stirred at 15 °C for 12 h. The reaction mixture was quenched by the addition of 0 °C water (200 mL) and extracted with EtOAc (300 mL* 3). Dried over Na ₂ SO ₄ , filtered and concentrated under reduced pressure to give a residue. The residue was purified by silica gel chromatography (petroleum ether/ethyl acetate = 10/1, 1/4, 5%TEA) to obtain N-(9-((2R,4S,5R)-5-((bis(4- Methoxyphenyl) (phenyl) methoxy) methyl) -4-hydroxytetrahydrofuran-2-yl) -8-oxo-8,9-dihydro-7H-purin-6-yl) benzamide (WV- NU-137) (32 g, 57.75% yield) as a white solid. ¹ HNMR (400 MHz, 400 MHz, DMSO-d6) δ = 8.38 - 8.24 (m, 1H), 8.12 - 8.00 (m, 2H), 7.67 - 7.60 (m, 1H), 7.58 - 7.51 (m, 2H), 7.38 - 7.33 (m, 2H), 7.26 - 7.13 (m, 7H), 6.81 (dd, J = 9.0, 13.3 Hz, 4H), 6.25 (t, J = 6.8 Hz, 1H), 5.29 (d, J = 4.6 Hz, 1H), 4.56 - 4.49 (m, 1H), 3.95 (q, J = 4.9 Hz, 1H), 3.71 (d, J = 4.4 Hz, 6H), 3.20 - 3.15 (m, 2H), 3.08 (td, J = 6.5, 13.0 Hz, 1H), 2.21 - 2.10 (m, 1H); LCMS (MH ⁺ ): 672.2.

N-(9-((2R,4S,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-(((1S,3S,3aS)-3-( (methyldiphenylsilyl)methyl)tetrahydro-1H,3H-pyrrolo[1,2-c][1,3,2]oxazaphosphol-1-yl)oxy)tetrahydrofuran-2-yl)- Synthesis of 8-oxo-8,9-dihydro-7H-purin-6-yl)benzamide

dry in rbf N-[9-[(2R,4R,5R)-5-[[bis(4-methoxyphenyl)-phenyl-methoxy]methyl]-4-hydroxy-tetrahydrofuran-2-yl ]-8-oxo-7H-purin-6-yl]benzamide (4.0 g, 5.94 mmol) was dissolved in THF (50 mL). To the clear solution was added triethylamine (5.59 mL, 40.08 mmol). [(3S,3aS)-1-chloro-3a,4,5,6-tetrahydro-3H-pyrrolo[1,2-c][1,3,2]oxazaphosphol-3-yl]methyl- Methyl-diphenyl-silane (0.96 M solution in THF, 11.16 mL, 10.69 mmol) was added dropwise. The reaction solution was stirred at room temperature for 2 hours. TLC showed the reaction to be complete. Anhydrous MgSO4 (708 mg) was added. Stir for 1 minute. The mixture was filtered and the filtrate was concentrated. The resulting crude product was purified by normal phase column chromatography applying 0-100% EtOAc in hexanes (each mobile phase contained 1.5% triethylamine) as a gradient to obtain N-(9-((2R,4S, 5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-(((1S,3S,3aS)-3-((methyldiphenylsilyl)methyl)tetrahydro- 1H,3H-pyrrolo[1,2-c][1,3,2]oxazaphosphol-1-yl)oxy)tetrahydrofuran-2-yl)-8-oxo-8,9-dihydro- 7H-purin-6-yl)benzamide was obtained as a white foam (4.45 g, 74.0% yield). ^1H NMR (600 MHz, CDCl ₃ ) δ 9.42 (s, 1H), 8.59 (s, 1H), 8.17 (s, 1H), 7.98 - 7.93 (m, 2H), 7.68 - 7.62 (m, 1H), 7.58 - 7.53 (m, 2H), 7.53 - 7.46 (m, 4H), 7.45 - 7.40 (m, 2H), 7.33 - 7.26 (m, 7H), 7.24 - 7.17 (m, 5H), 7.16 - 7.11 (m , 1H), 6.76 - 6.69 (m, 4H), 6.30 (dd, J = 7.3, 6.1 Hz, 1H), 5.05 (ddt, J = 8.9, 6.9, 4.5 Hz, 1H), 4.85 (dt, J = 8.9 , 5.7 Hz, 1H), 4.03 (q, J = 5.0 Hz, 1H), 3.73 (d, J = 4.5 Hz, 6H), 3.49 (ddt, J = 14.6, 10.6, 7.6 Hz, 1H), 3.40 (ddt , J = 12.6, 7.0, 5.5 Hz, 1H), 3.34 (dd, J = 10.1, 4.9 Hz, 1H), 3.25 (dd, J = 10.1, 5.9 Hz, 1H), 2.97 (tdd, J = 10.8, 8.8 , 4.3 Hz, 1H), 2.83 (dt, J = 13.3, 6.6 Hz, 1H), 2.08 (ddd, J = 13.5, 7.4, 4.6 Hz, 1H), 1.84 (ddt, J = 12.2, 8.5, 4.3 Hz, 1H), 1.70 - 1.63 (m, 1H), 1.55 (dd, J = 14.7, 8.9 Hz, 1H), 1.45 - 1.38 (m, 2H), 1.30 - 1.20 (m, 1H), 0.65 (s, 3H) ; ³¹ P NMR (243 MHz, CDCl ₃ ) δ 148.40; MS (ESI), 1013.18 [M + H] ⁺ .

N-(9-((2R,4S,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-(((1S,3S,3aS)-3-( (phenylsulfonyl)methyl)tetrahydro-1H,3H-pyrrolo[1,2-c][1,3,2]oxazaphosphol-1-yl)oxy)tetrahydrofuran-2-yl)-8 Synthesis of -oxo-8,9-dihydro-7H-purin-6-yl)benzamide

dry N-[9-[(2R,4R,5R)-5-[[bis(4-methoxyphenyl)-phenyl-methoxy]methyl]-4-hydroxy-tetrahydrofuran in THF (30 mL) To a solution of -2-yl]-8-oxo-7H-purin-6-yl]benzamide (3.0 g, 4.45 mmol) was added triethylamine (1.55 mL, 11.13 mmol). (3S,3aS)-3-(benzenesulfonylmethyl)-1-chloro-3a,4,5,6-tetrahydro-3H-pyrrolo[1,2-c][1,3,2]oxazapo Spall (0.9 M in THF, 8.91 mL, 8.02 mmol) was added dropwise. The resulting off-white slurry was stirred at room temperature for 2 hours. TLC and LCMS showed the reaction to be complete. The reaction was quenched with water (80 uL). Anhydrous MgSO4 (1.07 g) was added. The mixture was filtered through celite and the filtrate was concentrated to give the crude product as an off-white foam. The crude product was purified by normal phase column chromatography applying a gradient of 20-100% EtOAc in hexanes (each mobile phase contained 2.5% triethylamine) to afford the title compound as a white foam (2.979 g, 69.9% yield). was obtained as ¹ H NMR (600 MHz, CDCl ₃ ) δ 9.45 (s, 1H), 8.60 (s, 1H), 8.24 (s, 1H), 7.97 - 7.92 (m, 2H), 7.92 - 7.88 (m, 2H), 7.67 - 7.62 (m, 1H), 7.62 - 7.57 (m, 1H), 7.57 - 7.48 (m, 4H), 7.45 - 7.40 (m, 2H), 7.34 - 7.28 (m, 4H), 7.21 (dd, J = 8.3, 6.7 Hz, 2H), 7.19 - 7.13 (m, 1H), 6.79 - 6.72 (m, 4H), 6.39 (t, J = 6.8 Hz, 1H), 5.09 (ddt, J = 14.7, 6.9, 4.9 Hz, 2H), 4.08 - 4.03 (m, 1H), 3.76 (s, 3H), 3.75 (s, 3H), 3.69 (dq, J = 9.8, 5.9 Hz, 1H), 3.52 - 3.42 (m, 2H) , 3.37 (ddd, J = 12.2, 5.4, 2.4 Hz, 2H), 3.34 - 3.24 (m, 2H), 3.03 (tdd, J = 10.3, 8.8, 4.1 Hz, 1H), 2.30 (ddd, J = 13.5, 7.3, 4.5 Hz, 1H), 1.87 (dt, J = 11.4, 5.9 Hz, 1H), 1.80 - 1.72 (m, 1H), 1.70 - 1.63 (m, 1H), 1.12 (dtd, J = 11.7, 10.1, 8.5 Hz, 1H); ³¹ P NMR (243 MHz, CDCl ₃ ) δ 149.85; MS (ESI), 955.37 [M - H] ^- .

N-(9-((2R,4S,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-hydroxytetrahydrofuran-2-yl)-8- Synthesis of oxo-8,9-dihydro-7H-purin-6-yl)-2-phenoxyacetamide (WV-NU-195)

Step 1 . Three batches: (2R,3S,5R)-5-(6-amino-9H-purin-9-yl in dioxane (400 mL) and AcONa (0.5 M, 480 mL, 2.01 equiv) buffer, pH 4.7 To a solution of )-2-(hydroxymethyl)tetrahydrofuran-3-ol (30 g, 119.41 mmol, 1 equiv.), Br ₂ (22.90 g, 143.29 mmol, 7.39 mL, 1.2 equivalent) was added dropwise while stirring. The mixture was stirred at 15 °C for 12 hours. Three batches were combined for work-up. Concentrated Na ₂ S ₂ O ₅ was added to the mixture until the red color disappeared. The mixture was neutralized to pH 7.0 with 0.5 M NaOH. The residue was evaporated when a white solid precipitated. The solid was filtered off, washed with cold 1,4-dioxane (50 mL), and dried under high vacuum to (2R,3S,5R)-5-(6-amino-8-bromo-9H-purine-9 -yl)-2-(hydroxymethyl)tetrahydrofuran-3-ol (100 g, 302.90 mmol, 84.56% yield) was obtained as a yellow solid. ^1HNMR (400MHz, DMSO-d6) δ = 8.22 - 7.98 (m, 1H), 7.53 (br s, 2H), 6.29 (dd, J=6.5, 7.9 Hz, 1H), 5.35 (br d, J=12.3 Hz, 2H), 4.58 - 4.38 ( m, 1H), 3.95 - 3.82 (m, 1H), 3.65 (dd, J=4.5, 11.9 Hz, 1H), 3.48 (br dd, J=4.5, 11.7 Hz, 1H), 3.36 (br s, 1H) , 3.24 (ddd, J=6.1, 7.8, 13.4 Hz, 1H), 2.19 (ddd, J=2.6, 6.4, 13.1 Hz, 1H); LCMS (M+H+): 330.1.

Step 2 . (2R,3S,5R)-5-(6-amino-8-bromo-9H-purin-9-yl)-2-(hydroxymethyl)tetrahydrofuran-3-ol in pyridine (1500 mL) To a solution of 55 g, 166.60 mmol, 1 equiv.) was added NaOAc (24.87 g, 303.21 mmol, 1.82 equiv.) and (2-phenoxyacetyl) 2-phenoxyacetate (267.08 g, 932.94 mmol, 5.6 equiv.). The mixture was stirred at 80 °C for 2 hours. The reaction mixture was quenched by adding 100 mL of H ₂ O and the mixture was left at room temperature for 10 minutes. The mixture was evaporated then diluted with 1000 mL of DCM and 1000 mL of saturated NaHCO ₃ and extracted with DCM (1000 mL * 2). The combined organic layers were washed with brine (1000 mL * 2), dried over Na ₂ SO ₄ , filtered, and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO ₂ , petroleum ether/(DCM/EtOAc = 1:1) = 1/0 to 0/1) then some solid precipitated from the column and the column was washed with 2 L of DCM Concentration gave crude product. The crude product was triturated with 1000 mL of methanol. (2R,3S,5R)-5-(8-oxo-6-(2-phenoxyacetamido)-7,8-dihydro-9H-purin-9-yl)-2-((2-phenoxy Obtained cyacetoxy)methyl)tetrahydrofuran-3-yl 2-phenoxyacetate (60 g, 67.20 mmol, 40.34% yield, 75% purity) as a brown solid. LCMS (MH ⁺ ): 668.2.

Step 3 . Two batches: (2R,3S,5R)-5-(8-oxo-6-(2-phenoxyacet) in a mixed solvent of TEA (270 mL), pyridine (270 mL) and H ₂ O (810 mL) Amido)-7,8-dihydro-9H-purin-9-yl)-2-((2-phenoxyacetoxy)methyl)tetrahydrofuran-3-yl 2-phenoxyacetate (27 g, 40.32 mmol, 1 eq.) in a solution. The mixture was stirred at 15 °C for 1.5 h. The reaction mixture was concentrated under reduced pressure to remove the solvent. The two batches of crude were combined and purified by recrystallization from 500 mL of methanol to obtain N-(9-((2R,4S,5R)-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2 Obtained -yl)-8-oxo-8,9-dihydro-7H-purin-6-yl)-2-phenoxyacetamide (18 g, 44.85 mmol, 55.61% yield) as a brown solid. LCMS (MH ⁺ ): 400.1.

Step 4 . N-(9-((2R,4S,5R)-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-8-oxo-8,9-di in pyridine (300 mL) To a solution of hydro-7H-purin-6-yl)-2-phenoxyacetamide (16 g, 39.86 mmol, 1 equiv) was added DMTC1 (18.91 g, 55.81 mmol, 1.4 equiv). The mixture was stirred at 15 °C for 10 h. The reaction mixture was quenched by adding 50 mL of water, then sat. It was diluted with 500 mL of NaHCO ₃ and extracted with 1500 mL of ethyl acetate (500 mL *3). The combined organic layers were washed with 500 mL of saturated brine, dried over Na ₂ SO ₄ , filtered, and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO ₂ , petroleum ether/ethyl acetate = 1/0 to 0/1, 5% TEA) to give WV-NU-195 (20 g, 27.63 mmol, 69.30% yield, 97.21% purity) as a white solid. ¹ H NMR (400 MHz, DMSO-d ₆ ) δ = 10.94 (br s, 1H), 10.50 (br s, 1H), 8.26 (s, 1H), 7.38 - 7.28 (m, 4H), 7.26 - 7.11 ( m, 7H), 7.06 - 6.94 (m, 3H), 6.79 (dd, J = 8.9, 14.1 Hz, 4H), 6.23 (t, J = 6.8 Hz, 1H), 5.27 (d, J = 4.6 Hz, 1H) ), 4.84 (s, 2H), 4.58 - 4.44 (m, 1H), 3.97 - 3.91 (m, 1H), 3.70 (d, J = 5.0 Hz, 6H), 3.22 - 3.11 (m, 2H), 3.05 ( td, J = 6.4, 13.0 Hz, 1H), 2.14 (ddd, J = 4.9, 7.6, 12.9 Hz, 1H); LCMS (MH) ^- : 702.3; Purity: 97.21%.

N-(9-((2R,3R,4R,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-3-((tert-butyldimethylsilyl)oxy)- Synthesis of 4-hydroxytetrahydrofuran-2-yl)-8-oxo-8,9-dihydro-7H-purin-6-yl)benzamide

Step 1. After 3 h, to a solution of Na (21 g, 913.45 mmol, 21.65 mL, 8.43 equiv) in BnOH (1000 mL) (2R,3R,4S,5R)-2-(6-amino-8-bromide) Mo-9H-purin-9-yl)-5-(hydroxymethyl)tetrahydrofuran-3,4-diol (37.5 g, 108.34 mmol, 1.0 equiv) was added. The mixture was stirred at 15 °C for 12 hours. The mixture was poured into cold 1N HCl (2500 mL) and extracted with EtOAc (1500 mL). To the aqueous phase was added saturated NaHCO ₃ (aq) until pH>8 and the white cake was separated, filtered and concentrated to give crude. (2R,3R,4S,5R)-2-(6-amino-8-(benzyloxy)-9H-purin-9-yl)-5-(hydroxymethyl)tetrahydrofuran-3,4-diol ( 80 g, crude) as a white solid. LCMS: (M+H ⁺ ):374.4.

Step 2. (2R,3R,4S,5R)-2-(6-amino-8-(benzyloxy)-9H-purin-9-yl)-5-(hydroxymethyl)tetra in HMDS (400 mL) To a solution of hydrofuran-3,4-diol (39.0 g, 104.46 mmol, 1.0 equiv) the mixture was stirred at 130 °C for 12 h. The reaction mixture was concentrated under reduced pressure to give a residue. N-(8-(benzyloxy)-9-((2R,3R,4R,5R)-4-hydroxy-3-((trimethylsilyl)oxy)-5-(((trimethylsilyl)oxy)methyl) Tetrahydrofuran-2-yl)-9H-purin-6-yl)benzamide (61.62 g, crude) was obtained as a brown solid.

Step 3. N-(8-(benzyloxy)-9-((2R,3R,4R,5R)-4-hydroxy-3-((trimethylsilyl)oxy)-5-( in pyridine (460 mL) To a solution of ((trimethylsilyl)oxy)methyl)tetrahydrofuran-2-yl)-9H-purin-6-yl)benzamide (46.0 g, 77.98 mmol, 1 equiv.) was added benzoyl chloride (21.92 g, 155.96 mmol, 18.12 mL, 2.0 eq) was added. The mixture was stirred at 20 °C for 1 hour. 500 mL of MeOH : H ₂ O (1:1) was added to the reaction mixture and stirred at 15 °C for 10 minutes. NH ₃ .H ₂ O (150 mL) was then added to the mixture and stirred at 15° C. for 10 minutes. Then, the mixture was diluted with 200 mL of H ₂ O and extracted with 800 mL of EtOAc (200 mL*4). 200 mL of brine was added to the mixture and dried over Na ₂ SO ₄ . The mixture was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography. N-(8-(benzyloxy)-9-((2R,3R,4S,5R)-3,4-dihydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-9H-purine Obtained -6-yl)benzamide (33.99 g, 71.19 mmol, 91.29% yield) as a yellow solid. LCMS: (M+H ⁺ ): 478.4.

Step 4. N-(8-(benzyloxy)-9-((2R,3R,4S,5R)-3,4-dihydroxy-5-(hydroxy) in MeOH (1500 mL) and THF (500 mL) _Pd /C (7.0 g, 10 g, 10 % purity) was added. The mixture was stirred at 20 °C for 1 hour. The reaction was filtered and concentrated under reduced pressure to give a residue. The residue was purified without N-(9-((2R,3R,4S,5R)-3,4-dihydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-8-oxo Obtained -8,9-dihydro-7H-purin-6-yl)benzamide (19.6 g, 50.60 mmol, 68.83% yield) as a brown solid. LCMS: (M+H ⁺ ):388.2.

Step 5. N-(9-((2R,3R,4S,5R)-3,4-dihydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-8 in pyridine (150 mL) To a solution of -oxo-8,9-dihydro-7H-purin-6-yl)benzamide (14.8 g, 38.21 mmol, 1 equiv) was added DMTC1 (15.54 g, 45.85 mmol, 1.2 equiv). The mixture was stirred at 20 °C for 2 h. The reaction mixture was diluted with 10 mL of H ₂ O and extracted with ethyl acetate. The combined organic layers were washed with brine (100 mL), dried over Na ₂ SO ₄ , filtered, and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (petroleum ether/ethyl acetate = 100/1 to 0/1). N-(9-((2R,3R,4S,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-3,4-dihydroxytetrahydrofuran-2- Obtained yl)-8-oxo-8,9-dihydro-7H-purin-6-yl)benzamide (13.2 g, 19.14 mmol, 50.09% yield) as a brown solid. LCMS: (M+H ⁺ ): 690.5.

Step 6. N-(9-((2R,3R,4S,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-3,4- in DMF (100 mL) To a solution of dihydroxytetrahydrofuran-2-yl)-8-oxo-8,9-dihydro-7H-purin-6-yl)benzamide (10.20 g, 14.79 mmol, 1 eq.) imidazole (3.02 g, 44.37 mmol, 3.00 equiv) and TBSCl (2.01 g, 13.31 mmol, 1.63 mL, 0.9 equiv) were added. . The mixture was stirred at 15 °C for 10 h. The mixture was diluted with ethyl acetate and washed with NaHCO ₃ solution. The combined organic layers were dried over Na ₂ SO ₄ , filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (petroleum ether/ethyl acetate = 100/1 to 1/1). N-(9-((2R,3R,4R,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-3-((tert-butyldimethylsilyl)oxy)- Obtained 4-hydroxytetrahydrofuran-2-yl)-8-oxo-8,9-dihydro-7H-purin-6-yl)benzamide (3.82 g, 4.75 mmol, 32.13% yield) as a yellow solid. did ¹ HNMR (400 MHz, chloroform-d) δ = 9.51 (s, 1H), 8.57 (s, 1H), 8.26 (s, 1H), 8.03 (s, 1H), 7.96 (d, J = 7.5 Hz, 2H) , 7.70 - 7.63 (m, 1H), 7.61 - 7.54 (m, 2H), 7.48 (d, J = 7.3 Hz, 2H), 7.36 (dd, J = 2.0, 8.9 Hz, 4H), 7.26 - 7.16 (m , 3H), 6.78 (d, J = 8.7 Hz, 4H), 5.99 (d, J = 4.6 Hz, 1H), 5.32 - 5.27 (m, 1H), 4.48 (q, J = 5.5 Hz, 1H), 4.13 - 4.08 (m, 1H), 3.78 (s, 6H), 3.46 (dd, J = 3.9, 10.3 Hz, 1H), 3.32 (dd, J = 5.3, 10.3 Hz, 1H), 2.70 (d, J = 5.9 Hz, 1H), 2.06 (s, 1H), 1.58 (s, 2H), 1.27 (t, J = 7.2 Hz, 1H), 0.89 (s, 9H), 0.05 (s, 3H), -0.01 (s, 3H); LCMS: (MH ^- ):802.3.

N-(9-((2R,3R,4R,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-3-((tert-butyldimethylsilyl)oxy)- 4-(((1S,3S,3aS)-3-((phenylsulfonyl)methyl)tetrahydro-1H,3H-pyrrolo[1,2-c][1,3,2]oxazaphosphole-1 Synthesis of -yl)oxy)tetrahydrofuran-2-yl)-8-oxo-8,9-dihydro-7H-purin-6-yl)benzamide

Dry N-[9-[(2R,3S,5R)-5-[[bis(4-methoxyphenyl)-phenyl-methoxy]methyl]-3-[tert-butyl(dimethyl) in THF (35 mL) To a solution of )silyl]oxy-4-hydroxy-tetrahydrofuran-2-yl]-8-oxo-7H-purin-6-yl]benzamide (3.5 g, 4.35 mmol) triethylamine (1.52 mL, 10.88 mmol) was added. (3S,3aS)-3-(benzenesulfonylmethyl)-1-chloro-3a,4,5,6-tetrahydro-3H-pyrrolo[1,2-c][1,3,2]oxazapo Spall (0.9 M in THF, 8.71 mL, 7.84 mmol) was added dropwise. The resulting cloudy solution was stirred at room temperature for 3.5 hours. TLC and LCMS showed the reaction to be complete. The reaction was quenched with water (78 uL). Anhydrous MgSO ₄ (1.05 g) was added. The mixture was filtered through celite and the filtrate was concentrated to give the crude product as an off-white foam. The crude product was purified by normal phase column chromatography applying a gradient of 20-100% EtOAc in hexanes (each mobile phase contained 2.5% triethylamine) to afford the title compound as a white foam (3.512 g, 74.2% yield). was obtained as ¹ H NMR (600 MHz, CDCl ₃ ) δ 9.48 (s, 1H), 8.62 (s, 1H), 8.24 (s, 1H), 7.96 - 7.92 (m, 2H), 7.90 - 7.85 (m, 2H), 7.67 - 7.61 (m, 1H), 7.57 (td, J = 7.2, 1.2 Hz, 1H), 7.54 (t, J = 7.8 Hz, 2H), 7.50 - 7.43 (m, 4H), 7.38 - 7.32 (m, 4H), 7.22 (dd, J = 8.4, 6.9 Hz, 2H), 7.19 - 7.13 (m, 1H), 6.79 - 6.72 (m, 4H), 6.01 (d, J = 5.4 Hz, 1H), 5.33 (t , J = 5.3 Hz, 1H), 5.00 (q, J = 6.2 Hz, 1H), 4.78 (dt, J = 10.8, 4.7 Hz, 1H), 4.06 (q, J = 4.4 Hz, 1H), 3.76 (s , 6H), 3.67 (dq, J = 11.4, 5.8 Hz, 1H), 3.49 - 3.34 (m, 4H), 3.19 (dd, J = 10.4, 4.9 Hz, 1H), 3.01 (qd, J = 9.5, 4.0 Hz, 1H), 1.85 (t, J = 5.8 Hz, 1H), 1.77 - 1.70 (m, 1H), 1.68 - 1.62 (m, 1H), 1.16 - 1.06 (m, 1H), 0.83 (s, 9H) , 0.02 (s, 3H), -0.09 (s, 3H); ³¹ P NMR (243 MHz, CDCl ₃ ) δ 152.12; MS (ESI), 1086.13 [M - H] ^- .

(1S,3S,3aS)-1-(((2R,3S)-3-((bis(4-methoxyphenyl)(phenyl)methoxy)tetrahydrofuran-2-yl)methoxy)-3- Synthesis of ((phenylsulfonyl)methyl)tetrahydro-1H,3H-pyrrolo[1,2-c][1,3,2]oxazaphosphole

To a white slurry of dry (2R,3R)-3-[bis(4-methoxyphenyl)-phenyl-methoxy]tetrahydrofuran-2-yl]methanol (10.0 g, 23.78 mmol) in THF (150 mL) Triethylamine (17.9 mL, 128.42 mmol) was added. (3S,3aS)-3-(benzenesulfonylmethyl)-1-chloro-3a,4,5,6-tetrahydro-3H-pyrrolo[1,2-c][1,3,2]oxazapo Spall (0.9 M in THF, 47.56 mL, 42.81 mmol) was added dropwise. DCM (50 mL) was added. The white slurry was stirred at room temperature for 3.5 hours. TLC and LCMS showed the reaction to be complete. The reaction was quenched with water (428 uL). Anhydrous MgSO ₄ (5.7 g) was added. The mixture was filtered through celite and the filtrate was concentrated to give the crude product as an off-white foam. The crude product was purified by normal phase column chromatography applying a gradient of 20-100% EtOAc in hexanes (each mobile phase contained 5% triethylamine) to afford the title compound as a white foam (13.08 g, 78.2% yield). was obtained as ¹ H NMR (600 MHz, CDCl ₃ ) δ 7.92 (dd, J = 8.2, 1.4 Hz, 2H), 7.64 (tt, J = 7.4, 1.3 Hz, 1H), 7.54 (t, J = 7.8 Hz, 2H) , 7.46 (dd, J = 8.6, 1.3 Hz, 2H), 7.35 (d, J = 8.6 Hz, 4H), 7.29 (t, J = 7.6 Hz, 2H), 7.22 (tt, J = 7.3, 1.3 Hz, 1H), 6.84 (d, J = 8.9 Hz, 4H), 4.99 (q, J = 6.1 Hz, 1H), 4.07 (dt, J = 6.2, 1.9 Hz, 1H), 3.89 (ddd, J = 10.1, 8.1 , 5.9 Hz, 1H), 3.82 (td, J = 8.0, 2.8 Hz, 1H), 3.79 (s, 6H), 3.79 - 3.75 (m, 1H), 3.61 (dq, J = 9.7, 5.9 Hz, 1H) , 3.47 (dd, J = 14.5, 6.8 Hz, 1H), 3.45 - 3.38 (m, 2H), 3.34 (dd, J = 14.5, 5.6 Hz, 1H), 3.29 (ddd, J = 11.1, 8.7, 4.6 Hz) , 1H), 3.00 (qd, J = 10.5, 4.1 Hz, 1H), 1.83 (dtt, J = 11.9, 7.7, 3.3 Hz, 1H), 1.74 (dq, J = 11.9, 7.5 Hz, 1H), 1.61 ( qd, J = 7.7, 6.6, 3.0 Hz, 1H), 1.56 (dddd, J = 13.7, 10.0, 5.8, 3.9 Hz, 1H), 1.38 (ddt, J = 13.0, 5.4, 2.1 Hz, 1H), 1.07 ( dq, J = 11.5, 9.8 Hz, 1H); ³¹ P NMR (243 MHz, CDCl ₃ ) δ 152.11; MS (ESI), 704.87 [M + H] ⁺ .

(S)—N-(1-(3-(bis(4-methoxyphenyl)(phenyl)methoxy)-2-hydroxypropyl)-2-oxo-1,2-dihydropyrimidine-4- 1) Synthesis of benzamide (WV-NU-175)

To a solution of (S)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)oxirane (34.30 g, 159.39 mmol, 1 equiv) in DMF (300 mL) NaH (1.27 g, 31.88 mmol, 60% purity, 0.2 eq) was added and the mixture was stirred at 20° C. for 2 h, then (2S)-2-[[bis(4-methoxyphenyl)-phenyl-methoxy]methyl] Oxirane (60 g, 159.39 mmol, 1 eq) was added. The mixture was stirred at 115 °C for 4 hours. TLC (petroleum ether: ethyl acetate = 3:1, Rf = 0.05) showed that compound 2 was consumed and one new spot was formed. The solution was then cooled to 20° C. and partitioned between 1000 mL of saturated brine and EtOAc (200 mL*3). The organic phase was separated, washed twice with saturated brine, dried over Na ₂ SO ₄ and concentrated in vacuo. The residue was purified by column chromatography (SiO ₂ , petroleum ether/ethyl acetate = 1/0 to 0/1, 5% TEA). WV-NU-175 (14.9 g, 24.45 mmol, 15.34% yield, 97.094% purity) was obtained as a yellow solid. ¹ H NMR (400 MHz, DMSO- d ₆ ) δ ppm 2.90 - 3.03 (m, 2 H) 3.54 - 3.63 (m, 1 H) 3.73 (d, J = 1.50 Hz, 6 H) 4.02 (s, 1 H) ) 4.20 (br dd, J = 12.82, 3.06 Hz, 1 H) 5.31 (d, J = 5.88 Hz, 1 H) 6.90 (dd, J = 8.88, 1.75 Hz, 4 H) 7.19 - 7.36 (m, 8 H) ) 7.43 (d, J = 7.38 Hz, 2 H) 7.48 - 7.55 (m, 2 H) 7.61 (d, J = 7.38 Hz, 1 H) 7.96 - 8.05 (m, 2 H) 11.15 (br s, 1 H) ); LCMS (MH ⁺ ): 590.3; Purity: 98.72%.

(R)-N-(1-(3-(bis(4-methoxyphenyl)(phenyl)methoxy)-2-hydroxypropyl)-2-oxo-1,2-dihydropyrimidine-4- 1) Synthesis of benzamide (WV-NU-176)

Step 1 . To a solution of [(2S)-oxiran-2-yl]methanol [(2S)-oxiran-2-yl]methanol (35.7 g, 481.92 mmol, 31.88 mL, 1 eq.) 179.62 g, 530.11 mmol, 1.1 equiv) was added. The mixture was stirred at 15 °C for 10 h. TLC (petroleum ether: ethyl acetate = 3:1, Rf = 0.70) showed that reactant 1 was completely consumed and three new spots were formed. A few drops of 30 ml methanol were added to hydrolyze any unreacted DMTrC1 and the mixture was stirred for 10 minutes. The product was washed with H ₂ O (8000 ml) and extracted with EAOAC (500 mL * 3). The combined organic layers were washed with NaCl (50 mL * 3), dried over Na ₂ SO ₄ , filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO ₂ , petroleum ether/ethyl acetate = 50/1 to 3/1, 5%TEA) to obtain (R)-2-((bis(4-methoxyphenyl)phenyl)methyl Toxy)methyl)oxirane (240 g, 637.55 mmol, 66.15% yield) was obtained as a yellow oil. ¹ HNMR (400 MHz, DMSO-d6) δ = 7.43 - 7.38 (m, 2H), 7.34 - 7.19 (m, 7H), 6.89 (d, J = 8.8 Hz, 4H), 5.31 (d, J = 5.5 Hz, 1H), 3.84 (qd, J = 5.4, 10.4 Hz, 1H), 3.75 - 3.72 (m, 6H), 3.65 - 3.59 (m, 1H), 3.39 - 3.38 (m, 1H), 3.06 - 2.94 (m, 2H).

Step 2 . K 2 CO 3 (K ₂ CO ₃ ( 18.36 g, 132.82 mmol, 2 eq) was added. The mixture was stirred at 85° C. for 2 h and (2R)-2-[[bis(4-methoxyphenyl)-phenyl-methoxy]methyl]oxirane (25 g, 66.41 mmol, 1 eq.) was added. . The mixture was stirred at 85 °C for 12 hours. The mixture was concentrated in vacuo. The residue was quenched by saturated aqueous NaHCO ₃ (500 mL) then extracted with EtOAc (600 mL * 3). The combined organic phases were washed with brine (300 mL), dried over anhydrous Na ₂ SO ₄ , filtered and concentrated in vacuo. The residue was purified by silica gel column chromatography eluting with (petroleum ether/ethyl acetate = 50/1, 3/1) to give WV-NU-176 (24 g, 40.56 mmol, 11.75% yield) as a white solid. . ¹ HNMR (chloroform-d, 400 MHz): δ = 7.94 (s, 2H), 7.83 (br d, J = 7.4 Hz, 2H), 7.50-7.58 (m, 2H), 7.45 (t, J = 7.6 Hz, 2H), 7.34 (br d, J = 7.6 Hz, 3H), 7.20-7.29 (m, 7H), 7.12-7.20 (m, 2H), 6.76 (d, J = 8.8 Hz, 4H), 4.28 (dd, J = 13.6, 2.5 Hz, 1H), 4.14 (br s, 1H), 3.74–3.81 (m, 1H), 3.71 (s, 6H), 3.11–3.26 (m, 1H), 3.05 (dd, J = 9.6 , 6.0 Hz, 1H), 1.19 ppm (t, J = 7.1 Hz, 2H); LCMS: (MH ⁺ ): 590.2, LCMS purity 99.56%.

(S)—N-(1-(3-(bis(4-methoxyphenyl)(phenyl)methoxy)-2-hydroxypropyl)-4-oxo-1,4-dihydropyrimidine-2- 1) Synthesis of benzamide (WV-NU-199)

K ₂ CO ₃ (9.18 g, 66.41 mmol, 0.5 equiv.) was added dropwise at 85° C., and the mixture was stirred at this temperature for 30 minutes, then (S)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)oxy Egg (50 g, 132.82 mmol, 1 eq.) was added dropwise at 85°C. The resulting mixture was stirred at 85 °C for 48 hours. The reaction mixture was quenched at 15° C. by the addition of 150 mL of water, extracted with 1000 mL of ethyl acetate (500 mL * 2), dried over anhydrous Na ₂ SO ₄ , filtered, and concentrated under vacuum. The residue was purified by column chromatography (SiO ₂ , petroleum ether: ethyl acetate = 1:0 to 0:1) to give WV-NU-199 (14.23 g, 24.05 mmol, 18.11% yield) as a white solid. ¹ HNMR (400 MHz, DMSO-d6) δ = 13.40 - 13.08 (m, 1H), 8.17 (d, J = 7.9 Hz, 2H), 7.81 (d, J = 8.0 Hz, 1H), 7.53 - 7.42 (m, 3H) , 7.33 - 7.20 (m, 9H), 6.85 (d, J = 8.8 Hz, 4H), 5.94 (dd, J = 2.2, 7.9 Hz, 1H), 5.37 (d, J = 5.6 Hz, 1H), 4.67 ( dd, J = 3.1, 13.3 Hz, 1H), 4.34 - 4.18 (m, 1H), 3.75 - 3.68 (m, 7H), 3.15 (br dd, J = 4.9, 9.0 Hz, 1H), 2.96 (br t, J = 8.1 Hz, 1H); LCMS (M-H+): 592.24, Purity: 94.76%.

(R)—N-(1-(3-(bis(4-methoxyphenyl)(phenyl)methoxy)-2-hydroxypropyl)-4-oxo-1,4-dihydropyrimidine-2- 1) Synthesis of benzamide (WV-NU-200)

K ₂ CO ₃ (5.51 g, 39.85 mmol, 0.5 eq.) was added at 85° C. and the mixture was stirred at this temperature for 30 min, then (R)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)oxirane (30.00 g, 79.69 mmol, 1 eq.) was added dropwise at 85°C. The resulting mixture was stirred at 85 °C for 48 hours. The reaction mixture was quenched at 15° C. by adding 50 mL of water, extracted with 200 mL of ethyl acetate (100 mL * 2), dried over anhydrous Na ₂ SO ₄ , filtered, and concentrated under vacuum. The residue was purified by column chromatography (SiO ₂ , petroleum ether: Purification with ethyl acetate = 1:0 to 0:1) gave WV-NU-200 (7.95 g, 13.44 mmol, 16.86% yield) as a white solid. ¹ HNMR (400 MHz, DMSO-d6) δ = 13.23 (d, J = 2.0 Hz, 1H), 8.17 (d, J = 7.3 Hz, 2H), 7.81 (d, J = 8.0 Hz, 1H), 7.53 - 7.41 (m , 3H), 7.32 - 7.19 (m, 9H), 6.84 (d, J = 8.9 Hz, 4H), 5.93 (dd, J = 2.4, 8.0 Hz, 1H), 5.36 (d, J = 5.6 Hz, 1H) , 4.67 (dd, J = 3.1, 13.3 Hz, 1H), 4.33 - 4.19 (m, 1H), 3.75 - 3.67 (m, 7H), 3.14 (dd, J = 4.9, 9.1 Hz, 1H), 2.96 (br t, J = 8.1 Hz, 1H); LCMS (M-H+): 592.24, purity: 93.75%.

(S)-1-(3-(bis(4-methoxyphenyl)(phenyl)methoxy)-2-hydroxypropyl)-5-methylpyrimidine-2,4(1H,3H)-dione (WV -NU-180) Synthesis

To a solution of 5-methyl-1H-pyrimidine-2,4-dione (16.75 g, 132.82 mmol, 1 equiv) in DMF (100 mL) was added K ₂ CO ₃ (7.34 g, 53.13 mmol, 0.4 equiv) at 85 °C. , (S)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)oxirane (50 g, 132.82 mmol, 1 equiv) was added. The mixture was stirred at 85 °C for 24 hours. The mixture was diluted with 500 mL of H ₂ O and extracted with 500 mL of EtOAc*3. The organic phase was dried over Na ₂ SO ₄ , filtered and concentrated under reduced pressure to give a residue. The silica gel column was washed with 600 mL of petroleum ether (5% Et ₃ N) and 600 mL of petroleum ether. The residue was purified by column chromatography (SiO ₂ , petroleum ether/ethyl acetate = 100/1 to 1/1) to give WV-NU-180 (13.8 g, 26.33 mmol, 19.83% yield, 95.9% purity) as a yellow solid. got it with ^1HNMR (400MHz, DMSO-d6) δ = 11.20 (s, 1H), 7.50 - 7.13 (m, 10H), 6.88 (dd, J = 1.5, 8.8 Hz, 4H), 5.25 (d, J = 5.6 Hz, 1H), 3.95 - 3.86 (m, 2H), 3.77 - 3.70 (m, 6H), 3.53 - 3.40 (m, 1H), 3.01 - 2.93 (m, 1H), 2.91 - 2.82 (m, 1H), 2.75 - 2.71 (m, 1H), 2.73 ( s, 1H), 1.70 (s, 3H). LCMS: (M ^- H ⁺ ):501.1, LCMS purity : 95.9%.

(R)-1-(3-(bis(4-methoxyphenyl)(phenyl)methoxy)-2-hydroxypropyl)-5-methylpyrimidine-2,4(1H,3H)-dione (WV -NU-205) Synthesis

Two batches: K to a solution of (R)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)oxirane (60 g, 159.39 mmol, 1 equiv) in DMF (600 mL). ₂ CO ₃ (11.01 g, 79.69 mmol, 0.5 equiv) was added at 85 °C over 30 min followed by 5-methyl-1H-pyrimidine-2,4-dione (20.10 g, 159.39 mmol, 1 equiv). . The mixture was stirred at 85 °C for 12 hours. The reaction mixture was quenched at 15° C. by adding 500 mL of water, extracted with 2000 mL of ethyl acetate (1000 mL * 2), dried over anhydrous Na ₂ SO ₄ , filtered, and concentrated under vacuum. The residue was purified by column chromatography (SiO2, petroleum ether/ethyl acetate = 1:0 to 0:1) to give WV-NU-205 (10 g, 19.90 mmol) as a white solid. ¹ H NMR (400 MHz, DMSO-d6) δ = 11.18 (s, 1H), 7.42 (br d, J = 7.5 Hz, 2H), 7.36 - 7.21 (m, 9H), 6.88 (dd, J = 1.3, 8.7 Hz, 4H), 5.24 (d, J = 5.5 Hz, 1H), 3.95 - 3.86 (m, 2H), 3.74 (s, 6H), 3.46 (br dd, J = 9.4, 14.5 Hz, 1H), 3.01 - 2.85 (m, 2H), 1.70 (s, 3H); LCMS (M-H+): 502.56, purity: 96.97%.

(S)-N-(9-(3-(bis(4-methoxyphenyl)(phenyl)methoxy)-2-hydroxypropyl)-6-oxo-6,9-dihydro-1H-purine- Synthesis of 2-yl)isobutyramide (WV-NU-177)

3 batches: K ₂ to a solution of 2-methyl-N-(6-oxo-1,9-dihydropurin-2-yl)propanamide (11.75 g, 53.13 mmol, 1 equiv) in DMF (200 mL). CO ₃ (3.67 g, 26.56 mmol, 0.5 equiv) was added at 85° C. for 30 min, (S)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)oxirane(20 g, 53.13 mmol, 1 eq) was added. The mixture was stirred at 85 °C for 12 hours. Three reactants were combined for work-up. The reaction mixture was diluted with 500 mL of water and extracted with EtOAc (500 mL * 4). The combined organic layers were dried over Na ₂ SO ₄ , filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO ₂ , dichloromethane: methanol = 0:1 to 30:1). A mixture of 22 g of the crude material was prepared on a preparative-HPLC column (Phenomenex Titank C18 bulk 250*100 mm 10u; mobile phase: [water (10 mM NH ₄ HCO ₃ )-ACN]; B%: 45%-65%, 20 minutes) to give compound WV-NU-177 (8.1 g, 13.55 mmol, 36.82% yield) as a pale yellow solid. ¹ HNMR (400 MHz, DMSO-d6) δ = 12.03 (s, 1H), 11.56 (s, 1H), 7.89 - 7.86 (m, 1H), 7.43 - 7.37 (m, 2H), 7.33 - 7.18 (m, 7H) ), 6.90 - 6.84 (m, 4H), 5.42 (d, J = 5.0 Hz, 1H), 4.15 - 4.08 (m, 2H), 3.73 (d, J = 0.8 Hz, 6H), 3.34 (s, 1H) , 3.01 - 2.96 (m, 1H), 2.89 (dd, J = 4.1, 9.4 Hz, 1H), 2.78 (quin, J = 6.8 Hz, 1H), 1.11 (dd, J = 2.6, 6.9 Hz, 6H); LCMS (M-H+): 597.26, LCMS Purity: 97.82%.

(R)-N-(9-(3-(bis(4-methoxyphenyl)(phenyl)methoxy)-2-hydroxypropyl)-6-oxo-6,9-dihydro-1H-purine- Synthesis of 2-yl)isobutyramide (WV-NU-178)

5 batches: K to a solution of (R)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)oxirane (14 g, 37.19 mmol, 1 equiv) in DMF (100 mL). ₂ CO ₃ (2.06 g, 14.88 mmol, 0.4 equiv) was added and 2-methyl-N-(6-oxo-1,9-dihydropurin-2-yl) propanamide (8.23 g, 37.19 mmol, 1 equivalent) was added. The mixture was stirred at 85 °C for 12 hours. The reaction mixture was quenched at 15° C. by adding 500 mL of water, and extracted with 1000 mL of ethyl acetate (500 mL *2). The combined organic phases were washed with brine (150 mL), dried over anhydrous Na ₂ SO ₄ , filtered and concentrated in vacuo. The residue was purified by preparative HPLC (column: Phenomenex Titank C18 bulk 250*100 mm 10u; mobile phase: [water (10 mM NH ₄ HCO ₃ )-ACN]; B%: 50%-70%, 20 min). Compound WV-NU-178 (8 g, 13.39 mmol, 32.01% yield) was obtained as a white solid. ¹ HNMR (chloroform-d, 400 MHz): δ = 7.59 (s, 1H), 7.46 (d, J = 7.8 Hz, 2H), 7.30-7.36 (m, 6H), 7.22-7.27 (m, 1H), 6.85 (d, J = 8.8 Hz, 4H), 4.26 (br d, J = 11.3 Hz, 2H), 4.10 (br dd, J = 14.8, 8.3 Hz, 2H), 3.82 (s, 6H), 3.16–3.29 ( m, 2H), 2.57–2.65 (m, 1H), 1.30 ppm (dd, J = 6.8, 4.8 Hz, 6H). LCMS: MH ⁺ : 596.6, LCMS purity 99.48%.

(2R,3S,4R,5R)-2-(4-acetamido-2-oxopyrimidin-1(2H)-yl)-5-((bis(4-methoxyphenyl)(phenyl)methyl Synthesis of Toxy)methyl)-4-hydroxytetrahydrofuran-3-yl acetate (WV-NU-207)

Step 1. 4-Amino-1-((2R,3S,4S,5R)-3,4-dihydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)pyridine in pyridine (460 mL) Chloro-[ _chloro (diisopropyl)silyl]oxy-diisopropyl-silane (136.95 g, 136.95 g, 434.18 mmol, 138.90 mL, 1.1 eq) is added dropwise. And 2 hours, the mixture was stirred at 0 ~ 20 ℃ for 10 hours. The reaction mixture was concentrated in vacuo to give the crude product. The residue was purified by column chromatography (SiO ₂ , petroleum ether: ethyl acetate = 10:1 to 0:1) to obtain 4-amino-1-((6aR,8R,9S,9aS)-9-hydroxy-2 ,2,4,4-tetraisopropyltetrahydro-6H-furo[3,2-f][1,3,5,2,4]trioxadisilosin-8-yl)pyrimidine-2(1H )-one (170 g, 350.00 mmol, 88.67% yield) was obtained as a white solid. LCMS (M+H ⁺ ): 486.3.

Step 2. 4-Amino-1-((6aR,8R,9S,9aS)-9-hydroxy-2,2,4,4-tetraisopropyltetrahydro-6H-furo[3 in pyridine (1700 mL) DMAP ( 85.52 g, 699.99 mmol, 2 equiv) and Ac ₂ O (142.92 g, 1.40 mol, 131.12 mL, 4 equiv) were added. The mixture was stirred at 25 °C for 10 h. The reaction mixture was diluted with 1000 mL of water and then the organic phases were separated and collected. The combined organic layers were dried over Na ₂ SO ₄ , filtered, and concentrated under reduced pressure to (6aR,8R,9S,9aR)-8-(4-acetamido-2-oxopyrimidin-1(2H)-yl )-2,2,4,4-tetraisopropyltetrahydro-6H-furo[3,2-f][1,3,5,2,4]trioxadisilosin-9-yl acetate (199 g , crude) was obtained as a yellow oil. LCMS (M+H ⁺ ): 570.4.

Step 3. 3 batches: (6aR,8R,9S,9aR)-8-(4-acetamido-2-oxopyrimidin-1(2H)-yl)-2,2 in THF (600 mL) ,4,4-tetraisopropyltetrahydro-6H-furo[3,2-f][1,3,5,2,4]trioxadisilosin-9-yl acetate (66.3 g, 116.36 mmol, 1 equiv) was added TBAF (1 M, 174.54 mL, 1.5 equiv) and AcOH (6.99 g, 116.36 mmol, 6.66 mL, 1 equiv). The mixture was stirred at 20 °C for 2 h. The reaction mixture was filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO ₂ , ethyl acetate: MeOH = 20:1 to 1:1). After concentration under reduced pressure, 1 L ethyl acetate was stirred for 10 minutes and filtered to give (2R,3S,4R,5R)-2-(4-acetamido-2-oxopyrimidin-1(2H)-yl)- 4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-3-yl acetate (64 g, 195.55 mmol, 64.00% yield) was obtained as a white solid. ¹ HNMR (400 MHz, DMSO-d6) δ = 10.89 (br s, 1H), 8.21 (d, J = 7.5 Hz, 1H), 7.22 (d, J = 7.5 Hz, 1H), 6.17 (d, J = 4.8 Hz, 1H), 5.85 (br d , J = 4.1 Hz, 1H), 5.26 (t, J = 4.3 Hz, 1H), 5.12 (br s, 1H), 4.09 (br s, 2H), 3.87 (q, J = 4.8 Hz, 1H), 3.63 - 3.57 (m, 1H), 3.16 (d, J = 4.4 Hz, 2H), 2.10 (s, 3H), 1.85 (s, 3H); LCMS (M+H ⁺ ): 328.2; Purity: 73.59%.

Step 4 . Two batches: (2R,3S,4R,5R)-2-(4-acetamido-2-oxopyrimidin-1(2H)-yl)-4-hydroxy-5 in pyridine (500 mL) To a solution of -(hydroxymethyl)tetrahydrofuran-3-yl acetate (26 g, 79.44 mmol, 1 equiv) was added DMTC1 (26.92 g, 79.44 mmol, 1 equiv). The mixture was stirred at 25 °C for 20 hours. The reaction mixture was filtered and concentrated under reduced pressure to give a residue which was purified by column chromatography (SiO ₂ , ethyl acetate: MeOH = 20:1 to 1:1, 5%TEA) to give WV-NU-207 (46.5 g, 73.85 mmol, 46.50% yield) as a yellow solid. ¹ HNMR (400 MHz, DMSO-d6) δ = 10.90 (s, 1H), 7.87 (d, J = 7.5 Hz, 1H), 7.43 - 7.39 (m, 2H), 7.36 - 7.22 (m, 8H), 7.11 (d, J = 7.5 Hz, 1H), 6.91 (dd, J = 1.3, 8.8 Hz, 4H), 6.20 (d, J = 4.8 Hz, 1H), 5.94 (d, J = 4.6 Hz, 1H), 5.26 - 5.22 (m, 1H), 4.17 - 4.09 (m, 2H), 3.74 (s, 6H), 3.32 - 3.28 (m, 2H), 2.10 (s, 3H), 1.74 (s, 3H); LCMS (MH ⁺ ):628.2; Purity: 96.49%.

N-(1-((2R,3R,4S)-4-(bis(4-methoxyphenyl)(phenyl)methoxy)-3-hydroxytetrahydrofuran-2-yl)-2-oxo-1 Synthesis of ,2-dihydropyrimidin-4-yl) acetamide (WV-NU-088)

Step 1. (3R,4S)-2-acetoxy-4-((tert-butyldiphenylsilyl)oxy)tetrahydrofuran-3-yl benzoate (27.5 g, 54.49 mmol, 1 equiv) and N-(2-oxo-1H-pyrimidin-4-yl)acetamide (8.76 g, 57.22 mmol, 1.05 equiv) was added BSA (23.28 g, 114.44 mmol, 28.29 mL, 2.1 equiv). and the mixture was stirred at 60 °C for 30 min. TMSOTf (19.38 g, 87.19 mmol, 15.76 mL, 1.6 equiv.) was added dropwise, and stirring was continued at 60° C. for another 2 h. The mixture was cooled to room temperature, diluted with 100 mL of EtOAc, poured into 200 mL of cold saturated aqueous NaHCO ₃ solution, and the mixture was extracted with DCM (500 mL*2). The organic layer was separated, washed with H ₂ O (100 mL) and brine (100 mL), dried over MgSO ₄ and concentrated under reduced pressure to (2R,3R,4S)-2-(4-acetamido-2-oxophyte Rimidin-1(2H)-yl)-4-((tert-butyldiphenylsilyl)oxy)tetrahydrofuran-3-yl benzoate (30 g, crude) was obtained as a yellow solid. The mixture was used directly without further purification. LCMS : (M+H ⁺ ): 598.3.

Step 2. (2R,3R,4S)-2-(4-acetamido-2-oxopyrimidin-1(2H)-yl)-4-((tert-butyldiphenyl) in THF (240 mL) To a solution of silyl)oxy)tetrahydrofuran-3-yl benzoate (30 g, 50.19 mmol, 1 equiv) was added TBAF (1 M, 75.28 mL, 1.5 equiv). The mixture was stirred at 0 °C for 1 hour. TLC (ethyl acetate/petroleum ether = 2:1, R _f = 0.25) showed one major spot. The solvent was evaporated under reduced pressure and the residue was dissolved in 600 mL of EtOAc. The organic layer was separated, washed with H ₂ O (100 mL*2) and brine (100 mL), dried over MgSO ₄ and concentrated under reduced pressure to give the crude product. The residue was purified by column chromatography (SiO ₂ , petroleum ether / ethyl acetate = 5/1 to 1/2) to give (2R,3R,4S)-2-(4-acetamido-2-oxopyrimidine -1(2H)-yl)-4-hydroxytetrahydrofuran-3-yl benzoate (12 g, 33.40 mmol, 66.54% yield) was obtained as a yellow solid.

Step 3: (2R,3R,4S)-2-(4-acetamido-2-oxopyrimidin-1(2H)-yl)-4-hydroxytetrahydrofuran-3-yl benzoate (11 g, 30.61 mmol, 1 equiv), DMTC1 (15.56 g, 45.92 mmol, 1.5 equiv), and DMAP (373.98 mg, 3.06 mmol, 0.1 equiv) were coevaporated twice with 20 mL of anhydrous pyridine. The mixture was dissolved in anhydrous pyridine (80 mL) and stirred under argon at 80° C. for 16 hours. The mixture was concentrated to give crude product. The residue was purified by silica gel chromatography (petroleum ether/ethyl acetate = 10/1, 3/1, 5%TEA) to give (2R,3R,4S)-2-(4-acetamido-2-oxophy Rimidin-1(2H)-yl)-4-(bis(4-methoxyphenyl)(phenyl)methoxy)tetrahydrofuran-3-yl benzoate (18.5 g, crude) was obtained as a white solid . LCMS: (MH ⁺ ): 660.2.

Step 4 . (2R,3R,4S)-2-(4-acetamido-2-oxopyrimidin-1(2H)-yl)-4-(bis(4-methoxyphenyl)( To a solution of phenyl)methoxy)tetrahydrofuran-3-yl benzoate (18.5 g, 27.96 mmol, 1 equiv) was added LiOH.H ₂ O (1.41 g, 33.55 mmol, 1.2 equiv) at 0 °C. After adding water (500 mL), the organic solvent was removed by concentration under reduced pressure. The aqueous phase was extracted with EtOAc (250 mL*3), dried over Na ₂ SO ₄ , filtered and concentrated under reduced pressure to give 4-amino-1-((2R,3R,4S)-4-(bis(4-methyl) Obtained 14.4 g, crude) of hydroxyphenyl)(phenyl)methoxy)-3-hydroxytetrahydrofuran-2-yl)pyrimidin-2(1H)-one as a yellow solid.

Step 5. 4-Amino-1-((2R,3R,4S)-4-(bis(4-methoxyphenyl)(phenyl)methoxy)-3-hydroxytetrahydrofuran- in DMF (100 mL) To a solution of 2-yl)pyrimidin-2(1H)-one (14.4 g, 27.93 mmol, 1 equiv) was added Ac ₂ O (3.14 g, 30.72 mmol, 2.88 mL, 1.1 equiv) and the mixture was heated to 20 °C. was stirred for 12 hours. Water (500 mL) was added, extracted with EtOAc (500 mL*2), the organics were dried over Na ₂ SO ₄ , filtered and concentrated to give the crude product. The mixture was purified by silica gel chromatography (DCM/ethyl acetate = 20/1, 1/1, ethyl acetate:methanol = 20:1, 5% TEA) to give compound WV-NU-088 (8.3 g, 14.45 mmol, 51.73 % yield, 97.07% purity) as a white solid. ¹ HNMR (400 MHz, chloroform-d) δ = 9.23 (br s, 1H), 8.04 (d, J=7.5 Hz, 1H), 7.54 (d, J=7.4 Hz, 1H), 7.38 - 7.33 (m, 2H), 7.31 - 7.18 (m, 8H) ), 6.86 - 6.78 (m, 4H), 4.34 - 4.24 (m, 3H), 3.80 (d, J=2.4 Hz, 6H), 3.69 (dd, J=4.4, 9.9 Hz, 1H), 3.39 (dd, J=2.3, 9.8 Hz, 1H), 2.34 (s, 3H), 2.00 (s, 1H) ; LCMS purity: 97.07%, 556.2 (MH) ^- .

1-((2R,4S,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-(((1S,3S,3aS)-3-((phenylsulfone) phonyl)methyl)tetrahydro-1H,3H-pyrrolo[1,2-c][1,3,2]oxazaphosphol-1-yl)oxy)tetrahydrofuran-2-yl)-1,3- Synthesis of dihydro-2H-imidazol-2-one

Dry 3-[(2R,4R,5R)-5-[[bis(4-methoxyphenyl)-phenyl-methoxy]methyl]-4-hydroxy-tetrahydrofuran-2- in THF (40 mL) To a solution of yl]-1H-imidazol-2-one (4.0 g, 7.96 mmol) was added triethylamine (4.99 mL, 35.82 mmol). Cooled down to 0 °C. (3S,3aS)-3-(benzenesulfonylmethyl)-1-chloro-3a,4,5,6-tetrahydro-3H-pyrrolo[1,2-c][1,3,2]oxazapo Spall (0.90 M in THF, 13.27 mL, 11.94 mmol) was added dropwise. The resulting slurry was stirred at 0° C. for 2.5 hours and then at room temperature for 1.5 hours. The reaction was quenched with water (72 μL). Anhydrous MgSO ₄ (960 mg) was added. The mixture was filtered through celite and the filtrate was concentrated to give the crude product as an off-white foam. The crude product was purified by normal phase column chromatography applying a gradient of 0-100% MeCN in EtOAc (each mobile phase contained 2.5% triethylamine) to afford the title compound as a white foam (2.389 g, 38.2% yield). was obtained as ¹ H NMR (600 MHz, CDCl ₃ ) δ 9.68 (s, 1H), 7.90 - 7.86 (m, 2H), 7.62 - 7.56 (m, 1H), 7.50 (t, J = 7.8 Hz, 2H), 7.45 - 7.41 (m, 2H), 7.31 (dd, J = 8.7, 5.5 Hz, 4H), 7.28 (t, J = 7.5 Hz, 2H), 7.21 (t, J = 7.4 Hz, 1H), 6.83 (d, J = 8.5 Hz, 4H), 6.34 (t, J = 2.6 Hz, 1H), 6.21 (t, J = 2.7 Hz, 1H), 6.07 (t, J = 7.0 Hz, 1H), 4.92 (q, J = 6.1 Hz, 1H), 4.75 (dq, J = 8.8, 3.8, 3.4 Hz, 1H), 3.96 (q, J = 3.4 Hz, 1H), 3.78 (s, 6H), 3.58 (dq, J = 11.8, 6.0 Hz) , 1H), 3.51 - 3.41 (m, 2H), 3.35 (dd, J = 14.6, 5.3 Hz, 1H), 3.31 (dd, J = 10.3, 3.9 Hz, 1H), 3.18 (dd, J = 10.3, 3.7 Hz, 1H), 3.09 (qd, J = 10.1, 3.9 Hz, 1H), 2.31 (dd, J = 7.0, 4.4 Hz, 2H), 1.87 (dh, J = 12.6, 4.7, 3.7 Hz, 1H), 1.81 - 1.71 (m, 1H), 1.65 - 1.62 (m, 1H), 1.15 - 1.05 (m, 1H); ³¹ P NMR (243 MHz, CDCl ₃ ) δ 152.36; MS (ESI) , 784.77 [M - H] ^- .

N-(1-((2R,3R,4R,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-((tert-butyldimethylsilyl)oxy)- 3-(((1S,3S,3aS)-3-((phenylsulfonyl)methyl)tetrahydro-1H,3H-pyrrolo[1,2-c][1,3,2]oxazaphosphole-1 Synthesis of -yl)oxy)tetrahydrofuran-2-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)benzamide

Dry N-[1-[(2R,3S,5R)-5-[[bis(4-methoxyphenyl)-phenyl-methoxy]methyl]-4-[tert-butyl(dimethyl) in THF (100 mL) To a solution of )silyl]oxy-3-hydroxy-tetrahydrofuran-2-yl]-2-oxo-pyrimidin-4-yl]benzamide (10.0 g, 13.09 mmol) was added triethylamine (9.85 mL, 70.69 mmol) was added. (3S,3aS)-3-(benzenesulfonylmethyl)-1-chloro-3a,4,5,6-tetrahydro-3H-pyrrolo[1,2-c][1,3,2]oxazapo Spall (0.90 M in THF, 26.18 mL, 23.56 mmol) was added dropwise. The resulting slurry was stirred at room temperature for 2.5 hours. TLC and LCMS showed the reaction to be complete. The reaction was quenched with water (234 μL). Anhydrous MgSO ₄ (3.12 g) was added. The mixture was filtered through celite and the filtrate was concentrated to give the crude product as an off-white foam. The crude product was purified by normal phase column chromatography applying a gradient of 20-70% EtOAc in hexanes (each mobile phase contained 5% triethylamine) to afford the title compound as an off-white foam (9.95 g, 72.6% yield). was obtained as ¹ H NMR (600 MHz, CDCl ₃ ) δ 8.57 (bs, 2H), 7.99 - 7.95 (m, 2H), 7.91 - 7.82 (m, 2H), 7.64 - 7.58 (m, 2H), 7.52 (dt, J = 15.0, 7.6 Hz, 4H), 7.41 (d, J = 7.4 Hz, 2H), 7.34 (t, J = 7.5 Hz, 2H), 7.30 (dd, J = 8.8, 3.2 Hz, 5H), 6.88 (d , J = 8.5 Hz, 4H), 5.93 (d, J = 1.6 Hz, 1H), 5.14 (q, J = 6.3 Hz, 1H), 4.51 - 4.45 (m, 1H), 4.20 (dd, J = 8.0, 4.3 Hz, 1H), 4.15 (dd, J = 6.7, 4.2 Hz, 1H), 3.83 (s, 6H), 3.79 - 3.69 (m, 3H), 3.59 - 3.54 (m, 1H), 3.51 (dd, J = 14.7, 6.9 Hz, 1H), 3.43 - 3.39 (m, 1H), 3.35 (dd, J = 11.0, 2.6 Hz, 1H), 3.20 (qd, J = 9.4, 4.1 Hz, 1H), 1.82 (tt, J = 8.3, 4.3 Hz, 1H), 1.79 - 1.74 (m, 1H), 1.65 (ddt, J = 12.2, 6.1, 3.0 Hz, 1H), 1.18 - 1.11 (m, 1H), 0.75 (s, 9H) , -0.03 (s, 3H), -0.12 (s, 3H); ³¹ P NMR (243 MHz, CDCl ₃ ) δ 155.49; MS (ESI) , 1045.67 [M - H] ^- .

1-(1-((2R,4S,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-(((1S,3S,3aS)-3-( (methyldiphenylsilyl)methyl)tetrahydro-1H,3H-pyrrolo[1,2-c][1,3,2]oxazaphosphol-1-yl)oxy)tetrahydrofuran-2-yl)- Synthesis of 2-oxo-1,2-dihydropyrimidin-4-yl)-3-phenylurea

dry 1-[1-[(2R,4R,5R)-5-[[bis(4-methoxyphenyl)-phenyl-methoxy]methyl]-4-hydroxy-tetrahydrofuran in THF (100 mL) To a solution of -2-yl]-2-oxo-pyrimidin-4-yl]-3-phenyl-urea (10.0 g, 15.42 mmol) was added triethylamine (11.6 mL, 83.24 mmol). [(3S,3aS)-1-chloro-3a,4,5,6-tetrahydro-3H-pyrrolo[1,2-c][1,3,2]oxazaphosphol-3-yl]methyl- Methyl-diphenyl-silane (0.9574 M in THF, 28.98 mL, 27.75 mmol) was added dropwise. The resulting off-white slurry was stirred at room temperature for 3.5 hours. TLC and LCMS showed the reaction to be complete. The reaction was quenched with water (277 μL). Anhydrous MgSO ₄ (3.7 g) was added. The mixture was filtered through celite and the filtrate was concentrated to give the crude product as an off-white foam. The crude product was purified by normal phase column chromatography applying a gradient of 20-100% EtOAc in hexanes (each mobile phase contained 2.5% triethylamine) to afford the title compound as an off-white foam (9.18 g, 60.3% yield). was obtained as ^1H NMR (600 MHz, CDCl ₃ ) δ 11.38 (s, 1H), 11.05 (s, 1H), 8.10 (d, J = 7.7 Hz, 1H), 7.68 (d, J = 8.0 Hz, 2H), 7.45 (t, J = 7.4 Hz, 4H), 7.36 (t, J = 8.6 Hz, 3H), 7.30 (m, 9H), 7.26 - 7.21 (m, 6H), 7.04 (t, J = 7.4 Hz, 1H) , 6.84 (d, J = 8.4 Hz, 4H), 6.28 (t, J = 6.3 Hz, 1H), 4.78 - 4.68 (m, 2H), 3.93 (q, J = 3.3 Hz, 1H), 3.77 (s, 6H), 3.52 (ddt, J = 15.1, 10.5, 7.6 Hz, 1H), 3.32 (qd, J = 10.6, 2.9 Hz, 3H), 3.08 (dt, J = 10.8, 6.8 Hz, 1H), 2.60 (ddd , J = 14.1, 6.3, 4.0 Hz, 1H), 2.05 (m, 1H), 1.84 (dh, J = 12.7, 4.7, 3.9 Hz, 1H), 1.70 - 1.62 (m, 1H), 1.56 - 1.52 (m , 1H), 1.40 (dq, J = 15.8, 7.2, 6.7 Hz, 2H), 1.23 - 1.18 (m, 1H), 0.58 (s, 3H); ³¹ P NMR (243 MHz, CDCl ₃ ) δ 153.22; MS (ESI) , 986.91 [M - H] ^- .

1-(1-((2R,4S,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-(((1S,3S,3aS)-3-( (methyldiphenylsilyl)methyl)tetrahydro-1H,3H-pyrrolo[1,2-c][1,3,2]oxazaphosphol-1-yl)oxy)tetrahydrofuran-2-yl)- Synthesis of 2-oxo-1,2-dihydropyrimidin-4-yl)-3-(naphthalen-2-yl)urea

dry 1-[1-[(2R,4R,5R)-5-[[bis(4-methoxyphenyl)-phenyl-methoxy]methyl]-4-hydroxy-tetrahydrofuran in THF (150 mL) To a solution of -2-yl]-2-oxo-pyrimidin-4-yl]-3-(2-naphthyl)urea (15.0 g, 21.47 mmol) was added triethylamine (16.16 mL, 115.92 mmol). . [(3S,3aS)-1-chloro-3a,4,5,6-tetrahydro-3H-pyrrolo[1,2-c][1,3,2]oxazaphosphol-3-yl]methyl- Methyl-diphenyl-silane (0.9574 M in THF, 40.36 mL, 38.64 mmol) was added dropwise. The resulting off-white slurry was stirred at room temperature for 3.5 hours. TLC and LCMS showed the reaction to be complete. The reaction was quenched with water (386 μL). Anhydrous MgSO ₄ (5.15 g) was added. The mixture was filtered through celite and the filtrate was concentrated to give the crude product as an off-white foam. The crude product was purified by normal phase column chromatography applying a gradient of 10-60% EtOAc in hexanes (each mobile phase contained 5% triethylamine) to afford the title compound as an off-white foam (15.91 g, 71.4% yield). was obtained as ^1H NMR (600 MHz, DMSO) δ 12.56 (s, 1H), 10.56 (s, 1H), 8.48 - 8.43 (m, 1H), 8.28 (d, J = 7.6 Hz, 1H), 8.02 (d, J = 7.4 Hz, 1H), 7.99 - 7.94 (m, 1H), 7.70 (d, J = 8.2 Hz, 1H), 7.62 - 7.56 (m, 2H), 7.54 - 7.45 (m, 5H), 7.39 - 7.27 ( m, 8H), 7.27 - 7.20 (m, 8H), 6.88 (dd, J = 8.5, 5.7 Hz, 4H), 6.17 (d, J = 6.7 Hz, 1H), 6.09 (t, J = 6.4 Hz, 1H) ), 4.68 - 4.63 (m, 1H), 4.63 - 4.57 (m, 1H), 3.84 (q, J = 4.0 Hz, 1H), 3.73 (s, 3H), 3.72 (s, 3H), 3.38 (ddt, J = 14.8, 10.2, 7.5 Hz, 1H), 3.30 (m, 1H), 3.23 (dd, J = 10.8, 3.4 Hz, 1H), 3.19 (dd, J = 10.7, 4.4 Hz, 1H), 2.81 (qd , J = 10.6, 4.3 Hz, 1H), 2.24 - 2.17 (m, 1H), 1.88 (dt, J = 13.5, 6.6 Hz, 1H), 1.77 (dq, J = 12.9, 4.5 Hz, 1H), 1.64 - 1.56 (m, 1H), 1.51 (dd, J = 14.8, 5.3 Hz, 1H), 1.45 (dt, J = 11.3, 8.0 Hz, 2H), 0.59 (s, 3H); ³¹ P NMR (243 MHz, DMSO) δ 146.05; MS (ESI), 1036.85 [M - H] ^- .

N-(1-((2R,3R,4S)-4-((bis(4-methoxyphenyl)(phenyl)methoxy)-3-(((1S,3S,3aS)-3-((methyl Diphenylsilyl)methyl)tetrahydro-1H,3H-pyrrolo[1,2-c][1,3,2]oxazaphosphol-1-yl)oxy)tetrahydrofuran-2-yl)-2- Synthesis of oxo-1,2-dihydropyrimidin-4-yl)acetamide

dry N-[1-[(2R,4R)-4-[[bis(4-methoxyphenyl)-phenyl-methoxy]-3-hydroxy-tetrahydrofuran-2-yl in THF (40 mL) To a solution of ]-2-oxo-pyrimidin-4-yl]acetamide (4.0 g, 7.17 mmol) was added triethylamine (5.4 mL, 38.74 mmol). [(3S,3aS)-1-chloro-3a,4,5,6-tetrahydro-3H-pyrrolo[1,2-c][1,3,2]oxazaphosphol-3-yl]methyl- Methyl-diphenyl-silane (0.9574 M in THF, 13.49 mL, 12.91 mmol) was added dropwise. The reaction slurry was stirred at room temperature for 3 hours. TLC and LCMS showed the reaction to be complete. The reaction was quenched with water (129 μL). Anhydrous MgSO ₄ (1.72 g) was added. The mixture was filtered through celite and the filtrate was concentrated to give the crude product as an off-white foam. The crude product was purified by normal phase column chromatography applying a gradient of 20-70% EtOAc in hexanes (each mobile phase contained 2.5% triethylamine) to afford the title compound as an off-white foam (4.4 g, 68.4% yield). was obtained as ^1H NMR (600 MHz, CDCl ₃ ) δ 9.18 (s, 1H), 7.95 (d, J = 7.5 Hz, 1H), 7.51 - 7.45 (m, 4H), 7.43 - 7.37 (m, 3H), 7.35 ( q, J = 6.8 Hz, 3H), 7.32 - 7.28 (m, 3H), 7.26 (d, J = 4.3 Hz, 3H), 7.21 (p, J = 4.2 Hz, 1H), 7.18 (dd, J = 8.7 , 4.3 Hz, 4H), 6.80 (t, J = 8.7 Hz, 4H), 5.65 (s, 1H), 4.76 (q, J = 6.8 Hz, 1H), 4.39 (d, J = 8.2 Hz, 1H), 4.09 (d, J = 3.6 Hz, 1H), 3.79 - 3.75 (m, 1H), 3.76 (s, 3H), 3.75 (s, 3H), 3.61 (ddt, J = 14.9, 10.4, 7.6 Hz, 1H) , 3.42 (d, J = 9.9 Hz, 1H), 3.36 (ddd, J = 13.3, 10.2, 5.8 Hz, 1H), 3.21 (dt, J = 11.0, 6.9 Hz, 1H), 2.29 (s, 3H), 1.83 (dp, J = 12.7, 4.7 Hz, 1H), 1.69 - 1.61 (m, 1H), 1.53 (dd, J = 14.6, 7.9 Hz, 1H), 1.41 (dd, J = 14.6, 7.0 Hz, 1H) , 1.38 - 1.32 (m, 1H), 1.21 (p, J = 10.2 Hz, 1H), 0.53 (s, 3H); ³¹ P NMR (243 MHz, CDCl ₃ ) δ 158.33; MS (ESI), 895.65 [MH] ^- .

N-(9-((2R,4S,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-(((1S,3S,3aS)-3-( (phenylsulfonyl)methyl)tetrahydro-1H,3H-pyrrolo[1,2-c][1,3,2]oxazaphosphol-1-yl)oxy)tetrahydrofuran-2-yl)-8 Synthesis of -oxo-8,9-dihydro-7H-purin-6-yl)-2-phenoxyacetamide

Dry N-[9-[(2R,4R,5R)-5-[[bis(4-methoxyphenyl)-phenyl-methoxy]methyl]-4-hydroxy-tetrahydrofuran in THF (37.5 mL) To a solution of -2-yl]-8-oxo-7H-purin-6-yl]-2-phenoxy-acetamide (7.5 g, 10.66 mmol) was added triethylamine (3.71 mL, 26.64 mmol). The reaction flask was set in a water bath. (3S,3aS)-3-(benzenesulfonylmethyl)-1-chloro-3a,4,5,6-tetrahydro-3H-pyrrolo[1,2-c][1,3,2]oxazapo Spall (0.90 M in THF, 21.31 mL, 19.18 mmol) was added dropwise. The bath was removed. The off-white slurry was stirred at room temperature for 3 hours. TLC and LCMS showed the reaction to be complete. The reaction was quenched with water (153 μL). Anhydrous MgSO ₄ (2.04 g) was added. The mixture was filtered through celite and the filtrate was concentrated to give the crude product as an off-white foam. The crude product was purified by normal phase column chromatography applying a gradient of 30-100% EtOAc in hexanes (each mobile phase contained 1% triethylamine) to afford the title compound as an off-white foam (8.35 g, 79.4% yield). was obtained as ^1H NMR (600 MHz, CDCl ₃ ) δ 9.35 (s, 1H), 8.95 (s, 1H), 8.24 (d, J = 1.5 Hz, 1H), 7.90 (d, J = 7.7 Hz, 2H), 7.59 (t, J = 7.5 Hz, 1H), 7.51 (t, J = 7.9 Hz, 2H), 7.42 (d, J = 7.8 Hz, 2H), 7.39 - 7.34 (m, 2H), 7.33 - 7.28 (m, 4H), 7.21 (t, J = 7.5 Hz, 2H), 7.16 (t, J = 7.3 Hz, 1H), 7.09 (t, J = 7.4 Hz, 1H), 7.00 (d, J = 8.1 Hz, 2H) , 6.75 (dd, J = 8.8, 7.0 Hz, 4H), 6.38 (t, J = 6.8 Hz, 1H), 5.09 (h, J = 6.2, 5.4 Hz, 2H), 4.67 (s, 2H), 4.06 ( q, J = 5.1 Hz, 1H), 3.763 (s, 3H), 3.756 (s, 3H), 3.69 (dq, J = 11.7, 6.1 Hz, 1H), 3.47 (dt, J = 14.7, 8.1 Hz, 2H) ), 3.37 (dd, J = 10.1, 5.1 Hz, 2H), 3.28 (ddd, J = 23.8, 11.7, 6.1 Hz, 2H), 3.03 (tt, J = 9.8, 5.1 Hz, 1H), 2.30 (dq, J = 12.6, 6.3, 5.3 Hz, 1H), 1.88 (ddt, J = 13.0, 9.3, 5.1 Hz, 1H), 1.77 (q, J = 10.8, 10.0 Hz, 1H), 1.66 (dt, J = 12.7, 6.4 Hz, 1H), 1.12 (p, J = 10.0 Hz, 1H); ³¹ P NMR (243 MHz, CDCl ₃ ) δ 149.94; MS (ESI), 985.68 [M - H] ^- .

3-((2S,4S,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-(((1S,3S,3aS)-3-((phenylsulfone) phonyl)methyl)tetrahydro-1H,3H-pyrrolo[1,2-c][1,3,2]oxazaphosphol-1-yl)oxy)tetrahydrofuran-2-yl)pyrimidine-2, Synthesis of 4(1H,3H)-dione

Dry 3-[(2S,4R,5R)-5-[[bis(4-methoxyphenyl)-phenyl-methoxy]methyl]-4-hydroxy-tetrahydrofuran-2- in THF (22.5 mL) To a solution of yl]-1H-pyrimidine-2,4-dione (3.0 g, 5.65 mmol) was added triethylamine (1.97 mL, 14.14 mmol). (3S,3aS)-3-(benzenesulfonylmethyl)-1-chloro-3a,4,5,6-tetrahydro-3H-pyrrolo[1,2-c][1,3,2]oxazapo Spall) (0.90 M in THF, 11.31 mL, 10.18 mmol) was added dropwise. The cloudy reaction solution was stirred at room temperature for 1 hour. TLC and LCMS showed the reaction to be complete. The reaction was quenched with water (81 μL). Anhydrous MgSO ₄ (1.08 g) was added. The mixture was filtered through celite and the filtrate was concentrated to give the crude product as an off-white foam. The crude product was purified by normal phase column chromatography applying a gradient of 50-100% EtOAc in hexanes (each mobile phase contained 1% triethylamine) to afford the title compound as an off-white foam (3.48 g, 75.6% yield). was obtained as ¹ H NMR (600 MHz, CDCl ₃ ) δ 9.51 (s, 1H), 7.90 - 7.85 (m, 2H), 7.62 - 7.57 (m, 1H), 7.55 - 7.47 (m, 2H), 7.47 - 7.40 (m , 2H), 7.37 - 7.32 (m, 4H), 7.29 - 7.24 (m, 2H), 7.21 - 7.16 (m, 1H), 7.13 (d, J = 7.7 Hz, 1H), 6.86 - 6.78 (m, 4H) ), 6.75 (t, J = 7.7 Hz, 1H), 5.68 (d, J = 7.7 Hz, 1H), 4.94 (q, J = 6.1 Hz, 1H), 4.75 (p, J = 8.4 Hz, 1H), 4.44 (ddd, J = 7.5, 3.9, 2.3 Hz, 1H), 3.767 (s, 3H), 3.765 (s, 3H), 3.59 (dq, J = 10.0, 5.9 Hz, 1H), 3.44 - 3.26 (m, 4H), 3.04 - 3.00 (m, 1H), 3.00 - 2.93 (m, 1H), 2.83 (dt, J = 12.4, 8.2 Hz, 1H), 2.53 (dt, J = 12.5, 7.9 Hz, 1H), 1.82 (s, 1H), 1.72 (d, J = 10.6 Hz, 1H), 1.65 - 1.55 (m, 1H), 1.06 (dq, J = 11.6, 9.9 Hz, 1H); ³¹ P NMR (243 MHz, CDCl ₃ ) δ 150.88; MS (ESI), 812.53 [M - H] ^- .

N-(5-((2R,4S,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-(((1S,3S,3aS)-3-( (phenylsulfonyl)methyl)tetrahydro-1H,3H-pyrrolo[1,2-c][1,3,2]oxazaphosphol-1-yl)oxy)tetrahydrofuran-2-yl)-6 Synthesis of -oxo-1,6-dihydropyrimidin-2-yl)acetamide

dry N-[5-[(2R,4R,5R)-5-[[bis(4-methoxyphenyl)-phenyl-methoxy]methyl]-4-hydroxy-tetrahydrofuran in THF (35 mL) To a solution of -2-yl]-6-oxo-1H-pyrimidin-2-yl]acetamide (7.0 g, 12.25 mmol) was added triethylamine (4.27 mL, 30.61 mmol). The reaction flask was set in a water bath. (3S,3aS)-3-(benzenesulfonylmethyl)-1-chloro-3a,4,5,6-tetrahydro-3H-pyrrolo[1,2-c][1,3,2]oxazapo Spall (0.90 M in THF, 24.49 mL, 22.04 mmol) was added dropwise. The bath was removed. The white slurry was stirred at room temperature for 2 hours. TLC and LCMS showed the reaction to be complete. The reaction was quenched with water (176 μL). Anhydrous MgSO ₄ (2.35 g) was added. The mixture was filtered through celite and the filtrate was concentrated to give the crude product as an off-white foam. The crude product was purified by normal phase column chromatography applying a gradient of 0-60% EtOAc in MeCN (each mobile phase contained 1% triethylamine) to afford the title compound as a white foam (8.22 g, 78.5% yield). was obtained as ¹ H NMR (600 MHz, DMSO) δ 11.56 (s, 2H), 7.90 - 7.86 (m, 2H), 7.86 - 7.81 (m, 1H), 7.67 - 7.61 (m, 1H), 7.55 (t, J = 7.8 Hz, 2H), 7.42 - 7.37 (m, 2H), 7.31 (t, J = 7.7 Hz, 2H), 7.28 - 7.24 (m, 4H), 7.24 - 7.21 (m, 1H), 6.91 - 6.85 (m , 4H), 4.87 (m, 1H), 4.82 (ddd, J = 9.1, 5.9, 3.1 Hz, 1H), 4.59 (m, 1H), 3.85 (td, J = 4.5, 2.4 Hz, 1H), 3.77 ( dd, J = 15.0, 3.1 Hz, 1H), 3.74 (s, 6H), 3.72 - 3.68 (m, 1H), 3.44 (dq, J = 11.8, 6.1 Hz, 1H), 3.38 - 3.29 (m, 1H) , 3.13 (dd, J = 10.1, 4.3 Hz, 1H), 3.04 (dd, J = 10.2, 4.7 Hz, 1H), 2.85 - 2.76 (m, 1H), 2.28 - 2.22 (m, 1H), 2.15 (s , 3H), 1.76 (dt, J = 12.1, 4.6 Hz, 2H), 1.61 (td, J = 13.5, 11.2, 6.2 Hz, 1H), 1.56 - 1.50 (m, 1H), 1.10 (dq, J = 11.7 , 9.5 Hz, 1H); ³¹ P NMR (243 MHz, DMSO) δ 146.56; MS (ESI), 853.57 [M - H] ^- .

3-((2R,4S,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-(((1S,3S,3aS)-3-((phenylsulfone) phonyl)methyl)tetrahydro-1H,3H-pyrrolo[1,2-c][1,3,2]oxazaphosphol-1-yl)oxy)tetrahydrofuran-2-yl)pyrimidine-2, Synthesis of 4(1H,3H)-dione

Dry 3-[(2R,4R,5R)-5-[[bis(4-methoxyphenyl)-phenyl-methoxy]methyl]-4-hydroxy-tetrahydrofuran-2- in THF (200 mL) To a solution of yl]-1H-pyrimidine-2,4-dione (29.8 g, 56.17 mmol) was added triethylamine (19.57 mL, 140.42 mmol). The reaction flask was set in a water bath. (3S,3aS)-3-(benzenesulfonylmethyl)-1-chloro-3a,4,5,6-tetrahydro-3H-pyrrolo[1,2-c][1,3,2]oxazapo Spall (0.89 M in THF, 113.59 mL, 101.1 mmol) was added dropwise. The bath was removed. The cloudy reaction solution was stirred at room temperature for 2.5 hours. TLC and LCMS showed the reaction to be complete. The reaction was quenched with water (794 μL). Anhydrous MgSO ₄ (10.6 g) was added. The mixture was filtered through celite and the filtrate was concentrated to give the crude product as an off-white foam. The crude product was purified by normal phase column chromatography applying a gradient of 50-100% EtOAc in hexanes (each mobile phase contained 1% triethylamine) to afford the title compound as an off-white foam (40.4 g, 88.4% yield). was obtained as ¹ H NMR (600 MHz, CDCl ₃ ) δ 9.28 (bs, 1H), 7.87 (dd, J = 8.3, 1.4 Hz, 2H), 7.63 - 7.57 (m, 1H), 7.53 - 7.44 (m, 4H), 7.37 - 7.33 (m, 4H), 7.23 (t, J = 7.8 Hz, 2H), 7.19 - 7.13 (m, 1H), 6.81 - 6.75 (m, 4H), 6.70 (dd, J = 8.4, 5.1 Hz, 1H), 6.64 (d, J = 7.7 Hz, 1H), 5.53 (d, J = 7.7 Hz, 1H), 4.96 (q, J = 6.1 Hz, 1H), 4.87 (dq, J = 12.8, 5.6 Hz, 1H), 3.93 (td, J = 5.9, 3.9 Hz, 1H), 3.752 (s, 3H), 3.749 (s, 3H), 3.62 (dq, J = 11.7, 6.0 Hz, 1H), 3.44 - 3.25 (m , 5H), 2.94 (qd, J = 10.0, 4.0 Hz, 1H), 2.85 (ddd, J = 13.2, 8.0, 5.2 Hz, 1H), 2.23 (ddd, J = 13.6, 8.6, 5.5 Hz, 1H), 1.85 - 1.79 (m, 1H), 1.76 - 1.69 (m, 1H), 1.65 - 1.59 (m, 1H), 1.12 - 1.02 (m, 1H); ³¹ P NMR (243 MHz, CDCl ₃ ) δ 149.14; MS (ESI), 812.53 [M - H] ^- .

N-((3aR,5R,6R,6aS)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-6-(((1S,3S,3aS)-3-(( phenylsulfonyl)methyl)tetrahydro-1H,3H-pyrrolo[1,2-c][1,3,2]oxazaphospholl-1-yl)oxy)-3a,5,6,6a-tetrahydro Synthesis of furo[2,3-d]oxazol-2-yl)acetamide

dry N-[(3aR,5R,6aR)-5-[[bis(4-methoxyphenyl)-phenyl-methoxy]methyl]-6-hydroxy-3a,5,6 in THF (36 mL); To a solution of 6a-tetrahydrofuro[2,3-d]oxazol-2-yl]acetamide (4.87 g, 9.38 mmol) was added triethylamine (3.27 mL, 23.45 mmol). The reaction flask was set in a water bath. (3S,3aS)-3-(benzenesulfonylmethyl)-1-chloro-3a,4,5,6-tetrahydro-3H-pyrrolo[1,2-c][1,3,2]oxazapo Spall (0.89 M in THF, 18.97 mL, 16.89 mmol) was added dropwise. The bath was removed. The resulting cloudy solution was stirred at room temperature for 1.5 hours. TLC and LCMS showed the reaction to be complete. The reaction was quenched with water (135 μL). Anhydrous MgSO ₄ (1.8 g) was added. The mixture was filtered through celite and the filtrate was concentrated to give the crude product as an off-white foam. The crude product was purified by normal phase column chromatography applying a gradient of 25-100% EtOAc in hexanes, each mobile phase containing 1% triethylamine, to afford the title compound as a white foam (5.75 g, 76.4% yield). was obtained as ^1H NMR (600 MHz, CDCl ₃ ) δ 9.55 (s, 1H), 7.93 (dt, J = 7.3, 1.3 Hz, 2H), 7.61 (t, J = 7.6 Hz, 1H), 7.52 (t, J = 7.8 Hz, 2H), 7.41 - 7.37 (m, 2H), 7.30 - 7.26 (m, 5H), 7.25 (s, 1H), 7.22 - 7.16 (m, 1H), 6.84 - 6.78 (m, 4H), 5.92 (s, 1H), 5.09 (s, 1H), 4.91 (d, J = 5.6 Hz, 1H), 4.82 - 4.77 (m, 1H), 4.20 (s, 1H), 3.78 (s, 6H), 3.69 ( dd, J = 10.0, 5.7 Hz, 1H), 3.53 (dd, J = 15.5, 5.8 Hz, 1H), 3.47 (dd, J = 14.5, 7.3 Hz, 1H), 3.37 (dd, J = 14.6, 5.0 Hz) , 1H), 3.18 (dd, J = 10.0, 6.2 Hz, 1H), 3.13 (s, 1H), 2.93 (dd, J = 10.0, 6.8 Hz, 1H), 2.13 (s, 3H), 1.89 (td, J = 8.2, 3.9 Hz, 1H), 1.80 (d, J = 10.2 Hz, 1H), 1.65 (m, 1H), 1.14 (p, J = 9.9 Hz, 1H); ³¹ P NMR (243 MHz, CDCl ₃ ) δ 152.57; MS (ESI), 802.49 [M + H] ⁺ .

1-((2R,4S,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-(((1S,3S,3aS)-3-((phenylsulfone) phonyl)methyl)tetrahydro-1H,3H-pyrrolo[1,2-c][1,3,2]oxazaphosphol-1-yl)oxy)tetrahydrofuran-2-yl)-3-methylpyr Synthesis of midine-2,4(1H,3H)-dione

Dry 1-[(2R,4R,5R)-5-[[bis(4-methoxyphenyl)-phenyl-methoxy]methyl]-4-hydroxy-tetrahydrofuran-2- in THF (45 mL) To a solution of yl]-3-methyl-pyrimidine-2,4-dione (6.2 g, 11.38 mmol) was added triethylamine (3.97 mL, 28.46 mmol). The reaction flask was set in a water bath. (3S,3aS)-3-(benzenesulfonylmethyl)-1-chloro-3a,4,5,6-tetrahydro-3H-pyrrolo[1,2-c][1,3,2]oxazapo Spall (0.89 M in THF, 19.19 mL, 17.08 mmol) was added dropwise. The bath was removed. The resulting cloudy solution was stirred at room temperature for 3 hours. TLC and LCMS showed the reaction to be complete. The reaction was quenched with water (102 μL). Anhydrous MgSO ₄ (1.366 g) was added. The mixture was filtered through celite and the filtrate was concentrated to give the crude product as an off-white foam. The crude product was purified by normal phase column chromatography applying a gradient of 20-70% EtOAc in hexanes (each mobile phase contained 1% triethylamine) to afford the title compound as a white foam (7.06 g, 74.9% yield). was obtained as ¹ H NMR (600 MHz, CDCl ₃ ) δ 7.91 - 7.86 (m, 2H), 7.75 (d, J = 8.1 Hz, 1H), 7.63 - 7.54 (m, 1H), 7.53 - 7.47 (m, 2H), 7.40 - 7.35 (m, 2H), 7.32 - 7.28 (m, 2H), 7.28 - 7.26 (m, 4H), 7.25 - 7.22 (m, 1H), 6.87 - 6.81 (m, 4H), 6.33 (t, J = 6.5 Hz, 1H), 5.42 (d, J = 8.1 Hz, 1H), 4.98 (dt, J = 6.9, 5.5 Hz, 1H), 4.80 (ddt, J = 9.6, 6.7, 3.4 Hz, 1H), 4.02 (q, J = 3.1 Hz, 1H), 3.788 (s, 3H), 3.786 (s, 3H), 3.66 - 3.58 (m, 1H), 3.54 - 3.47 (m, 1H), 3.47 - 3.41 (m, 2H) ), 3.39 - 3.34 (m, 2H), 3.32 (s, 3H), 3.14 (tdd, J = 10.3, 8.8, 4.0 Hz, 1H), 2.57 - 2.51 (m, 1H), 2.23 (dt, J = 13.6 , 6.6 Hz, 1H), 1.88 (td, J = 8.4, 4.1 Hz, 1H), 1.79 (q, J = 11.4, 10.3 Hz, 1H), 1.68 - 1.62 (m, 1H), 1.15 - 1.06 (m, 1H); ³¹ P NMR (243 MHz, CDCl ₃ ) δ 153.80; MS (ESI), 850.35 [M + Na] ⁺ .

N-(1-((R)-3-(bis(4-methoxyphenyl)(phenyl)methoxy)-2-(((1S,3S,3aS)-3-((methyldiphenylsilyl)methyl )tetrahydro-1H,3H-pyrrolo[1,2-c][1,3,2]oxazaphosphol-1-yl)oxy)propyl)-4-oxo-1,4-dihydropyrimidine- Synthesis of 2-yl)benzamide

dry N-[1-[(2R)-3-[bis(4-methoxyphenyl)-phenyl-methoxy]-2-hydroxy-propyl]-4-oxo-pyrimidine- in THF (37.5 mL) To a solution of 2-yl]benzamide (5.0 g, 8.45 mmol) was added triethylamine (5.3 mL, 38.03 mmol). The flask was set in a water bath. [(3S,3aS)-1-chloro-3a,4,5,6-tetrahydro-3H-pyrrolo[1,2-c][1,3,2]oxazaphosphol-3-yl]methyl- Methyl-diphenyl-silane (0.9574 M in THF, 15.89 mL, 15.21 mmol) was added dropwise. The bath was removed. The resulting cloudy reaction solution was stirred at room temperature for 1.5 hours. TLC and LCMS showed the reaction to be complete. The reaction was quenched with water (122 μL). Anhydrous MgSO ₄ (1.62 g) was added. The mixture was filtered through celite and the filtrate was concentrated to give the crude product as an off-white foam. The crude product was purified by normal phase column chromatography applying a gradient of 10-70% EtOAc in hexanes (each mobile phase contained 1% triethylamine) to afford the title compound as a white foam (5.3 g, 67.4% yield). was obtained as ^1H NMR (600 MHz, CDCl ₃ ) δ 13.31 (s, 1H), 8.19 (dd, J = 8.2, 1.4 Hz, 2H), 7.45 (tdt, J = 7.7, 2.7, 1.4 Hz, 7H), 7.37 - 7.26 (m, 12H), 7.24 (ddt, J = 10.2, 8.5, 1.6 Hz, 3H), 7.08 (d, J = 8.0 Hz, 1H), 6.80 - 6.74 (m, 4H), 5.57 (d, J = 8.0 Hz, 1H), 4.71 (dq, J = 11.2, 5.8, 4.4 Hz, 2H), 4.40 (dd, J = 13.8, 4.0 Hz, 1H), 3.75 (s, 6H), 3.58 (dd, J = 13.7 , 8.1 Hz, 1H), 3.50 - 3.40 (m, 1H), 3.33 - 3.27 (m, 1H), 3.22 - 3.15 (m, 2H), 2.96 (tdd, J = 10.4, 8.5, 5.0 Hz, 1H), 1.74 (qt, J = 8.4, 4.1 Hz, 1H), 1.67 - 1.59 (m, 1H), 1.54 (dd, J = 14.5, 8.1 Hz, 1H), 1.36 (dd, J = 14.6, 7.0 Hz, 1H) , 1.32 - 1.26 (m, 1H), 1.24 - 1.17 (m, 1H), 0.52 (s, 3H); ³¹ P NMR (243 MHz, CDCl ₃ ) δ 153.11; MS (ESI), 929.76 [M - H] ^- .

N-(1-((S)-3-(bis(4-methoxyphenyl)(phenyl)methoxy)-2-(((1S,3S,3aS)-3-((methyldiphenylsilyl)methyl )tetrahydro-1H,3H-pyrrolo[1,2-c][1,3,2]oxazaphosphol-1-yl)oxy)propyl)-4-oxo-1,4-dihydropyrimidine- Synthesis of 2-yl)benzamide

dry N-[1-[(2S)-3-[bis(4-methoxyphenyl)-phenyl-methoxy]-2-hydroxy-propyl]-4-oxo-pyrimidine- in THF (52.5 mL) To a solution of 2-yl]benzamide (7.0 g, 11.83 mmol) was added triethylamine (5.94 mL, 42.59 mmol). The flask was set in a water bath. [(3S,3aS)-1-chloro-3a,4,5,6-tetrahydro-3H-pyrrolo[1,2-c][1,3,2]oxazaphosphol-3-yl]methyl- Methyl-diphenyl-silane (0.9574 M in THF, 22.24 mL, 21.3 mmol) was added dropwise. The bath was removed. The resulting cloudy reaction solution was stirred at room temperature for 1 hour. TLC and LCMS showed the reaction to be complete. The reaction was quenched with water (171 μL). Anhydrous MgSO ₄ (2.27 g) was added. The mixture was filtered through celite and the filtrate was concentrated to give the crude product as an off-white foam. The crude product was purified by normal phase column chromatography applying a gradient of 10-80% EtOAc in hexanes (each mobile phase contained 1% triethylamine) to afford the title compound as a white foam (8.37 g, 76.0% yield). was obtained as ^1H NMR (600 MHz, CDCl ₃ ) δ 13.34 (s, 1H), 8.25 (dd, J = 8.3, 1.4 Hz, 2H), 7.47 (ddt, J = 6.7, 2.6, 1.4 Hz, 4H), 7.46 - 7.42 (m, 3H), 7.36 - 7.32 (m, 6H), 7.31 - 7.24 (m, 6H), 7.22 (ddt, J = 9.3, 5.3, 1.8 Hz, 3H), 7.05 (d, J = 8.0 Hz, 1H), 6.83 - 6.76 (m, 4H), 5.56 (d, J = 7.9 Hz, 1H), 4.86 (dd, J = 13.6, 3.3 Hz, 1H), 4.70 - 4.60 (m, 2H), 3.75 (s) , 6H), 3.42 - 3.30 (m, 3H), 3.20 - 3.12 (m, 1H), 3.09 (dd, J = 9.6, 7.4 Hz, 1H), 2.95 - 2.86 (m, 1H), 1.72 (ddt, J = 12.7, 8.3, 4.1 Hz, 1H), 1.65 - 1.52 (m, 2H), 1.36 (dd, J = 14.6, 6.6 Hz, 1H), 1.31 - 1.24 (m, 1H), 1.14 (dq, J = 11.9 , 9.5 Hz, 1H), 0.53 (s, 3H); ³¹ P NMR (243 MHz, CDCl ₃ ) δ 156.70; MS (ESI), 931.17 [M + H] ⁺ .

3-((2R,4S,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-(((1S,3S,3aS)-3-((phenylsulfone) phonyl)methyl)tetrahydro-1H,3H-pyrrolo[1,2-c][1,3,2]oxazaphosphol-1-yl)oxy)tetrahydrofuran-2-yl)pyridin-2(1H ) -Synthesis of one

dry 3-[(2R,4R,5R)-5-[[bis(4-methoxyphenyl)-phenyl-methoxy]methyl]-4-hydroxy-tetrahydrofuran-2- in THF (31 mL) To a solution of yl]-1H-pyridin-2-one (4.17 g, 8.12 mmol) was added triethylamine (2.49 mL, 17.86 mmol). The reaction flask was set in a water bath. (3S,3aS)-3-(benzenesulfonylmethyl)-1-chloro-3a,4,5,6-tetrahydro-3H-pyrrolo[1,2-c][1,3,2]oxazapo Spall (0.89 M in THF, 13.68 mL, 12.18 mmol) was added dropwise. The bath was removed. The resulting cloudy reaction solution was stirred at room temperature for 2 hours and 45 minutes. The reaction was quenched with water (73 μL). Anhydrous MgSO ₄ (974 mg) was added. The mixture was filtered through celite and the filtrate was concentrated to give the crude product as an off-white foam. The crude product was purified by normal phase column chromatography applying a gradient of 20-100% EtOAc in hexanes (each mobile phase contained 1% triethylamine) to afford the title compound as a white foam (3.69 g, 57.0% yield). was obtained as ^1H NMR (600 MHz, CDCl ₃ ) δ 12.70 (s, 1H), 7.92 - 7.85 (m, 2H), 7.69 (ddd, J = 6.9, 2.1, 1.1 Hz, 1H), 7.59 - 7.54 (m, 1H) ), 7.54 - 7.44 (m, 4H), 7.38 - 7.31 (m, 4H), 7.28 (t, J = 7.7 Hz, 2H), 7.25 (dd, J = 6.5, 2.1 Hz, 1H), 7.22 - 7.18 ( m, 1H), 6.85 - 6.80 (m, 4H), 6.22 (t, J = 6.7 Hz, 1H), 5.23 (dd, J = 9.9, 5.7 Hz, 1H), 4.94 (q, J = 6.0 Hz, 1H) ), 4.66 (ddd, J = 9.1, 6.0, 2.6 Hz, 1H), 4.04 (q, J = 4.1 Hz, 1H), 3.78 (s, 6H), 3.59 (dq, J = 11.7, 5.9 Hz, 1H) , 3.52 - 3.43 (m, 2H), 3.36 (dd, J = 14.6, 5.5 Hz, 1H), 3.29 (dd, J = 10.0, 4.3 Hz, 1H), 3.20 (dd, J = 10.0, 4.2 Hz, 1H) ), 3.14 - 3.05 (m, 1H), 2.62 (ddd, J = 13.3, 5.8, 2.0 Hz, 1H), 1.86 (ddd, J = 13.0, 9.8, 6.2 Hz, 2H), 1.78 - 1.72 (m, 1H) ), 1.66 - 1.61 (m, 1H), 1.15 - 1.05 (m, 1H); ³¹ P NMR (243 MHz, CDCl ₃ ) δ 151.29; MS (ESI), 795.57 [M - H] ^- .

N-(9-((S)-3-(bis(4-methoxyphenyl)(phenyl)methoxy)-2-(((1S,3S,3aS)-3-((methyldiphenylsilyl)methyl )tetrahydro-1H,3H-pyrrolo[1,2-c][1,3,2]oxazaphosphol-1-yl)oxy)propyl)-6-oxo-6,9-dihydro-1H- Synthesis of purin-2-yl)isobutyramide

dry N-[9-[(2S)-3-[bis(4-methoxyphenyl)-phenyl-methoxy]-2-hydroxy-propyl]-6-oxo-1H-purine in THF (45 mL) To a solution of -2-yl]-2-methyl-propanamide (6.0 g, 10.04 mmol) was added triethylamine (5.04 mL, 36.14 mmol). The flask was set in a water bath. [(3S,3aS)-1-chloro-3a,4,5,6-tetrahydro-3H-pyrrolo[1,2-c][1,3,2]oxazaphosphol-3-yl]methyl- Methyl-diphenyl-silane (0.9574 M in THF, 18.87 mL, 18.07 mmol) was added dropwise. The bath was removed. The resulting cloudy reaction solution was stirred at room temperature for 1 hour. TLC and LCMS showed the reaction to be complete. The reaction was quenched with water (144 μL). Anhydrous MgSO ₄ (1.92 g) was added. The mixture was filtered through celite and the filtrate was concentrated to give the crude product as an off-white foam. The crude product was purified by normal phase column chromatography applying a gradient of 25-100% EtOAc in hexanes (each mobile phase contained 1% triethylamine) to afford the title compound as a white foam (8.18 g, 87.0% yield). was obtained as ¹ H NMR (600 MHz, CDCl ₃ ) δ 11.84 (s, 1H), 7.86 (s, 1H), 7.53 (s, 1H), 7.49 - 7.42 (m, 6H), 7.38 - 7.29 (m, 8H), 7.28 (q, J = 3.0, 2.2 Hz, 3H), 7.25 (d, J = 1.1 Hz, 1H), 7.23 - 7.19 (m, 1H), 6.83 - 6.78 (m, 4H), 4.73 (dt, J = 8.2, 6.2 Hz, 1H), 4.25 - 4.18 (m, 2H), 3.98 (dd, J = 14.2, 8.0 Hz, 1H), 3.76 (s, 6H), 3.31 - 3.26 (m, 1H), 3.23 (dd , J = 10.0, 5.3 Hz, 1H), 3.22 - 3.16 (m, 1H), 2.94 (dd, J = 9.9, 7.1 Hz, 1H), 2.92 - 2.87 (m, 1H), 2.52 (hept, J = 6.9 Hz, 1H), 1.71 (dtd, J = 12.8, 9.0, 8.4, 4.0 Hz, 1H), 1.61 - 1.52 (m, 2H), 1.37 (dd, J = 14.6, 6.6 Hz, 1H), 1.30 - 1.22 ( m, 7H), 1.10 (dq, J = 11.9, 9.7 Hz, 1H), 0.53 (s, 3H); ³¹ P NMR (243 MHz, CDCl ₃ ) δ 156.67; MS (ESI), 935.73 [M - H] ^- .

N-(9-((R)-3-(bis(4-methoxyphenyl)(phenyl)methoxy)-2-(((1S,3S,3aS)-3-((methyldiphenylsilyl)methyl )tetrahydro-1H,3H-pyrrolo[1,2-c][1,3,2]oxazaphosphol-1-yl)oxy)propyl)-6-oxo-6,9-dihydro-1H- Synthesis of purin-2-yl)isobutyramide

dry N-[9-[(2R)-3-[bis(4-methoxyphenyl)-phenyl-methoxy]-2-hydroxy-propyl]-6-oxo-1H-purine in THF (41 mL) To a solution of -2-yl]-2-methyl-propanamide (5.5 g, 9.2 mmol) was added triethylamine (4.62 mL, 33.13 mmol). The flask was set in a water bath. [(3S,3aS)-1-chloro-3a,4,5,6-tetrahydro-3H-pyrrolo[1,2-c][1,3,2]oxazaphosphol-3-yl]methyl- Methyl-diphenyl-silane (0.9574 M in THF, 17.3 mL, 16.56 mmol) was added dropwise. The bath was removed. The resulting cloudy reaction solution was stirred at room temperature for 1 hour. TLC and LCMS showed the reaction to be complete. The reaction was quenched with water (132 μL). Anhydrous MgSO ₄ (2.27 g) was added. The mixture was filtered through celite and the filtrate was concentrated to give the crude product as an off-white foam. The crude product was purified by normal phase column chromatography applying a gradient of 40-100% EtOAc in hexanes (each mobile phase contained 1% triethylamine) to afford the title compound as a white foam (6.265 g, 72.6% yield). was obtained as ¹ H NMR (600 MHz, CDCl ₃ ) δ 11.81 (s, 1H), 7.78 (s, 1H), 7.53 (ddd, J = 7.7, 3.8, 2.0 Hz, 4H), 7.42 - 7.38 (m, 3H), 7.36 - 7.31 (m, 3H), 7.31 - 7.27 (m, 3H), 7.27 - 7.24 (m, 6H), 7.21 - 7.17 (m, 1H), 6.81 - 6.74 (m, 4H), 4.74 (dt, J = 8.5, 6.2 Hz, 1H), 4.34 - 4.26 (m, 1H), 4.00 (dd, J = 14.2, 6.3 Hz, 1H), 3.87 (dd, J = 14.1, 4.4 Hz, 1H), 3.769 (s, 3H), 3.768 (s, 3H), 3.43 (ddt, J = 14.7, 10.7, 7.6 Hz, 1H), 3.31 (ddt, J = 9.6, 7.3, 5.6 Hz, 1H), 3.08 (dd, J = 9.9, 5.3 Hz, 1H), 3.00 (tdd, J = 10.9, 8.7, 4.5 Hz, 1H), 2.90 (dd, J = 9.9, 5.8 Hz, 1H), 2.47 (hept, J = 6.9 Hz, 1H), 1.78 ( ddt, J = 16.2, 8.0, 3.2 Hz, 1H), 1.67 - 1.56 (m, 2H), 1.40 (dd, J = 14.6, 6.5 Hz, 1H), 1.38 - 1.30 (m, 1H), 1.23 (d, J = 6.9 Hz, 3H), 1.21 (d, J = 6.9 Hz, 3H), 1.21 - 1.16 (m, 1H), 0.65 (s, 3H); ³¹ P NMR (243 MHz, CDCl ₃ ) δ 155.34; MS (ESI), 937.91 [M + H] ⁺ .

1-((S)-3-(bis(4-methoxyphenyl)(phenyl)methoxy)-2-(((1S,3S,3aS)-3-((methyldiphenylsilyl)methyl)tetrahydro -1H,3H-pyrrolo[1,2-c][1,3,2]oxazaphosphol-1-yl)oxy)propyl)-5-methylpyrimidine-2,4(1H,3H)-dione synthesis of

Dry 1-[(2S)-3-[bis(4-methoxyphenyl)-phenyl-methoxy]-2-hydroxy-propyl]-5-methyl-pyrimidine-2,4 in THF (45 mL) To a solution of -dione (6.0 g, 11.94 mmol) was added triethylamine (4.99 mL, 35.82 mmol). The flask was set in a water bath. [(3S,3aS)-1-chloro-3a,4,5,6-tetrahydro-3H-pyrrolo[1,2-c][1,3,2]oxazaphosphol-3-yl]methyl- Methyl-diphenyl-silane (0.9574 M in THF, 22.45 mL, 21.49 mmol) was added dropwise. The bath was removed. The resulting cloudy reaction solution was stirred at room temperature for 1 hour. TLC and LCMS showed the reaction to be complete. The reaction was quenched with water (172 μL). Anhydrous MgSO ₄ (2.29 g) was added. The mixture was filtered through celite and the filtrate was concentrated to give the crude product as an off-white foam. The crude product was purified by normal phase column chromatography applying a gradient of 10-80% EtOAc in hexanes (each mobile phase contained 1% triethylamine) to afford the title compound as a white foam (8.26 g, 82.2% yield). was obtained as ¹ H NMR (600 MHz, CDCl ₃ ) δ 8.17 (s, 1H), 7.48 (dt, J = 6.7, 1.4 Hz, 4H), 7.47 - 7.44 (m, 2H), 7.39 - 7.30 (m, 7H), 7.30 - 7.26 (m, 5H), 7.23 - 7.17 (m, 1H), 6.87 (d, J = 1.4 Hz, 1H), 6.85 - 6.79 (m, 4H), 4.77 (dt, J = 8.5, 5.9 Hz, 1H), 4.38 - 4.29 (m, 1H), 4.16 (dd, J = 14.1, 3.6 Hz, 1H), 3.76 (s, 6H), 3.41 (tdd, J = 14.5, 9.4, 7.2 Hz, 1H), 3.31 (dd, J = 14.0, 8.9 Hz, 1H), 3.26 - 3.18 (m, 1H), 3.15 (dd, J = 9.9, 4.7 Hz, 1H), 3.08 (dd, J = 9.9, 5.8 Hz, 1H), 3.00 - 2.92 (m, 1H), 1.81 - 1.73 (m, 1H), 1.76 (d, J = 1.2 Hz, 3H), 1.62 (qt, J = 11.0, 5.1 Hz, 1H), 1.56 (dd, J = 14.6, 8.6 Hz, 1H), 1.37 (dd, J = 14.6, 6.3 Hz, 1H), 1.32 (qd, J = 7.4, 3.0 Hz, 1H), 1.19 (dq, J = 12.1, 9.5 Hz, 1H), 0.56 (s, 3H); ³¹ P NMR (243 MHz, CDCl ₃ ) δ 155.31; MS (ESI), 840.68 [M - H] ^- .

1-((R)-3-(bis(4-methoxyphenyl)(phenyl)methoxy)-2-(((1S,3S,3aS)-3-((methyldiphenylsilyl)methyl)tetrahydro -1H,3H-pyrrolo[1,2-c][1,3,2]oxazaphosphol-1-yl)oxy)propyl)-5-methylpyrimidine-2,4(1H,3H)-dione synthesis of

dry 1-[(2R)-3-[bis(4-methoxyphenyl)-phenyl-methoxy]-2-hydroxy-propyl]-5-methyl-pyrimidine-2,4 in THF (41.6 mL) To a solution of -dione (5.54 g, 11.02 mmol) was added triethylamine (4.61 mL, 33.07 mmol). The flask was set in a water bath. [(3S,3aS)-1-chloro-3a,4,5,6-tetrahydro-3H-pyrrolo[1,2-c][1,3,2]oxazaphosphol-3-yl]methyl- Methyl-diphenyl-silane (0.9574 M in THF, 20.73 mL, 19.84 mmol) was added dropwise. The bath was removed. The resulting cloudy reaction solution was stirred at room temperature for 1 hour. TLC and LCMS showed the reaction to be complete. The reaction was quenched with water (159 μL). Anhydrous MgSO ₄ (2.115 g) was added. The mixture was filtered through celite and the filtrate was concentrated to give the crude product as an off-white foam. The crude product was purified by normal phase column chromatography applying a gradient of 20-100% EtOAc in hexanes (each mobile phase contained 1% triethylamine) to afford the title compound as a white foam (7.63 g, 82.2% yield). was obtained as ¹ H NMR (600 MHz, CDCl ₃ ) δ 8.17 (s, 1H), 7.48 (dt, J = 8.0, 1.6 Hz, 4H), 7.45 - 7.42 (m, 2H), 7.36 - 7.24 (m, 12H), 7.23 - 7.17 (m, 1H), 6.93 (q, J = 1.2 Hz, 1H), 6.84 - 6.77 (m, 4H), 4.74 (dt, J = 8.5, 6.1 Hz, 1H), 4.36 - 4.28 (m, 1H), 3.85 (dd, J = 14.0, 4.2 Hz, 1H), 3.78 (s, 3H), 3.77 (s, 3H), 3.54 (dd, J = 14.0, 8.0 Hz, 1H), 3.49 - 3.40 (m , 1H), 3.40 - 3.34 (m, 1H), 3.16 (dd, J = 10.1, 4.6 Hz, 1H), 3.03 (dd, J = 10.1, 4.4 Hz, 1H), 2.99 - 2.90 (m, 1H), 1.80 (d, J = 1.2 Hz, 3H), 1.78 - 1.72 (m, 1H), 1.68 - 1.58 (m, 1H), 1.54 (dd, J = 14.6, 8.5 Hz, 1H), 1.42 - 1.31 (m, 2H), 1.26 - 1.21 (m, 1H), 0.59 (s, 3H); ³¹ P NMR (243 MHz, CDCl ₃ ) δ 151.30; MS (ESI), 840.78 [M - H] ^- .

(2R,3S,4R,5R)-2-(4-acetamido-2-oxopyrimidin-1(2H)-yl)-5-((bis(4-methoxyphenyl)(phenyl)methyl Toxy)methyl)-4-(((1S,3S,3aS)-3-((phenylsulfonyl)methyl)tetrahydro-1H,3H-pyrrolo[1,2-c][1,3,2] Synthesis of oxazaphospholl-1-yl)oxy)tetrahydrofuran-3-yl)acetate

dry [(2R,3R,5R)-2-(4-acetamido-2-oxo-pyrimidin-1-yl)-5-[[bis(4-methoxyphenyl)- To a solution of phenyl-methoxy]methyl]-4-hydroxy-tetrahydrofuran-3-yl]acetate (10.0 g, 15.88 mmol) was added triethylamine (4.87 mL, 34.94 mmol). The reaction flask was set in a water bath. (3S,3aS)-3-(benzenesulfonylmethyl)-1-chloro-3a,4,5,6-tetrahydro-3H-pyrrolo[1,2-c][1,3,2]oxazapo Spall (0.43 M in THF, 55.4 mL, 23.82 mmol) was added dropwise. The bath was removed. The resulting cloudy reaction solution was stirred at room temperature for 1.5 hours. TLC and LCMS showed the reaction to be complete. The reaction was quenched with water (143 μL). Anhydrous MgSO ₄ (1.906 g) was added. The mixture was filtered through celite and the filtrate was concentrated to give the crude product as an off-white foam. The crude product was purified by normal phase column chromatography applying a gradient of 20-100% EtOAc in hexanes (each mobile phase contained 1% triethylamine) to afford the title compound as a white foam (9.35 g, 64.5% yield). was obtained as ^1H NMR (600 MHz, CDCl ₃ ) δ 9.69 (s, 1H), 7.93 - 7.89 (m, 2H), 7.87 (d, J = 7.6 Hz, 1H), 7.64 - 7.58 (m, 1H), 7.52 ( t, J = 7.8 Hz, 2H), 7.47 - 7.43 (m, 2H), 7.38 - 7.33 (m, 4H), 7.31 (dd, J = 8.4, 6.9 Hz, 2H), 7.28 (d, J = 7.5 Hz) , 1H), 7.26 - 7.22 (m, 1H), 6.88 - 6.83 (m, 4H), 6.31 (d, J = 4.1 Hz, 1H), 5.44 (dd, J = 4.2, 2.1 Hz, 1H), 5.04 ( q, J = 6.1 Hz, 1H), 4.58 (ddd, J = 9.2, 3.6, 2.2 Hz, 1H), 4.14 (dd, J = 7.2, 3.2 Hz, 1H), 3.79 (s, 6H), 3.65 (dq , J = 9.9, 6.0 Hz, 1H), 3.54 - 3.43 (m, 2H), 3.42 - 3.31 (m, 3H), 3.13 - 3.04 (m, 1H), 2.26 (s, 3H), 1.87 (dtd, J = 16.8, 8.1, 4.0 Hz, 1H), 1.81 (s, 3H), 1.80 - 1.71 (m, 1H), 1.64 (ddt, J = 12.0, 7.4, 4.2 Hz, 1H), 1.10 (dtd, J = 11.7 , 10.0, 8.5 Hz, 1H); ³¹ P NMR (243 MHz, CDCl ₃ ) δ 153.43; MS (ESI), 913.46 [M + H] ⁺ .

Synthesis of WV-NU-172 and amidite.

In some embodiments, WV-NU-172 was prepared as follows:

In some embodiments, WV-NU-172 was prepared at different scales as follows:

2 batches: To a solution of compound 1B (60 g, 137.52 mmol, 1 equiv) in DCM (1200 mL) was added chloro(isopropyl)magnesium (2 M, 103.14 mL, 1.5 equiv) at 20 °C for 1 hour Then tributyl(chloro)stannane (66.70 g, 204.91 mmol, 55.12 mL, 1.49 equiv) was slowly added and the mixture was stirred at 20° C. for 12 hours. TLC (petroleum ether: ethyl acetate = 3:1) showed compound 1B consumed. The two batches were combined for work-up. The reaction mixture was quenched by careful addition of water (500 mL) and the mixture was extracted with DCM (500 mL x 2). The organic phases were combined, washed with brine, and dried over Na ₂ SO ₄ . The solvent was removed under reduced pressure. The residue was purified by silica gel chromatography (petroleum ether/ethyl acetate = 10/1, 3/1) to give compound 1C (120 g, 200.19 mmol, 72.78% yield) as a white solid. TLC: (petroleum ether/ethyl acetate = 3:1), Rf = 0.25.

t-BuOK (79.09 g, 704.80 mmol, 1.05 equiv) was added to a solution of BnOH (145.18 g, 1.34 mol, 139.59 mL, 2 equiv) in THF (500 mL) and stirred until dissolved. This mixture was added dropwise to a solution of compound 1 (100 g, 671.24 mmol, 1 equiv) in DMF (500 mL) cooled to -78 °C under an inert atmosphere. The mixture was slowly warmed to 20 °C and stirred for 1 hour. TLC (petroleum ether: ethyl acetate = 3:1, Rf = 0.76) showed that compound 1 was completely consumed and one new major spot was formed. The reaction mixture was diluted with 1000 mL of H2O and extracted with EtOAc mL (500 mL * 2). The combined organic layers were washed with brine (100 mL), dried over Na ₂ SO ₄ , filtered, and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO ₂ , petroleum ether/ethyl acetate = 1/0 to 0/1) to give compound 2 (80 g, 362.56 mmol, 54.01% yield) as a white solid. TLC: (petroleum ether/ethyl acetate = 3:1), Rf = 0.76.

To a solution of compound 1C (85.57 g, 142.76 mmol, 1.26 equiv) in toluene (900 mL) was added 4-benzyloxy-2-chloro-pyrimidine (25 g, 113.30 mmol, 1 equiv), Pd(dppf)Cl ₂ . CH ₂ Cl ₂ (9.25 g, 11.33 mmol, 0.1 equiv) was added. The mixture was stirred at 120° C. under N ₂ for 3 hours. TLC (petroleum ether: ethyl acetate = 1:1) showed that reactant 1 was consumed and a new spot was found. The mixture was concentrated to give the crude product. The mixture was purified by MPLC (SiO ₂ , petroleum ether/ethyl acetate = 10:1, 5:1) to give compound 3 (45 g, 90.99 mmol, 80.31% yield) as a brown solid. TLC: (petroleum ether/ethyl acetate = 1:1), Rf = 0.24.

To a solution of compound 3 (45 g, 90.99 mmol, 1 equiv) in THF (400 mL) was added HCl (5 M, 90.99 mL, 5 equiv). The mixture was stirred at 15 °C for 2 h. TLC (petroleum ether: ethyl acetate = 0:1) showed that the desired material was detected. The reaction mixture was diluted with 50 mL of water and extracted with 90 mL of EtOAc (30 mL * 3). To the combined aqueous layers was added 2 N NaOH aq until pH>11, extracted with DCM (50 mL*3), and the combined organics were dried over Na ₂ SO ₄ , filtered and concentrated to give compound 4 (23 g, crude) was obtained as a yellow solid. TLC (petroleum ether/ethyl acetate = 0:1), Rf = 0.01.

To a solution of compound 4 (23 g, 91.17 mmol, 1 equiv) in MeCN (800 mL) was added NaH (7.29 g, 182.34 mmol, 60% purity, 2 equiv) and the mixture was stirred at 0 °C for 30 min. Then, compound 1E (47.01 g, 109.41 mmol, 1.2 eq) was added. The mixture was stirred at 15 °C for 12 hours. LCMS showed that compound 4 was consumed and the desired material was found. The reaction mixture was filtered, the cake was washed with DCM (100 mL), and the filtrate was concentrated to give the crude product. The mixture was purified by MPLC (SiO ₂ , DCM: MeOH = 20:1) to give compound 6 (30 g, crude) as a yellow oil. LCMS: (M+H+): 645.3. TLC (DCM: MeOH = 20:1), Rf = 0.24.

To a solution of compound 6 (30 g, 46.48 mmol, 1 equiv) in MeOH (600 mL) was added Pd/C (6, 46.48 mmol, 10% purity, 1 equiv) under N ₂ atmosphere. The suspension was degassed and purged with H ₂ three times. The mixture was stirred at 15° C. under H ₂ (15 Psi) for 12 hours. LCMS showed that compound 6 was consumed and the desired mass was found. The mixture was filtered and concentrated to give compound 7 (25 g, crude) as a yellow oil. LCMS: (M+H+): 555.2.

To a solution of compound 7 (2 g, 3.60 mmol, 1 equiv) in ammonia (200 mL), the mixture was stirred at 15 °C for 12 h. LCMS showed that compound 7 was consumed. The mixture was concentrated to give compound 8 (1 g, 3.59 mmol, 99.79% yield) as a yellow oil. To a solution of compound 7 (25 g, 45.02 mmol, 1 equiv) in ammonia (1000 mL), the mixture was stirred at 15 °C for 12 h. LCMS showed that compound 7 was consumed. The mixture was concentrated to give the crude product. The mixture was purified by MPLC (SiO2, dichloromethane:methanol = 20:1, 10:1, 5:1) to give compound 8 (11 g, 39.53 mmol, 87.82% yield) as a yellow oil. LCMS: (M+H+): 279.1. TLC: (dichloromethane:methanol = 10:1), Rf = 0.15.

A solution of compound 8 (5 g, 17.97 mmol, 1 equiv) and DMTC1 (6.39 g, 18.87 mmol, 1.05 equiv) in pyridine (60 mL) was added to the mixture and the solution was stirred at 20 °C for 1.5 h. LCMS showed that compound 8 was consumed and the desired material was found. MeOH (10 mL) was added to the mixture and concentrated to give the crude product. The mixture was purified by preparative HPLC (column: Phenomenex C18 250*70 mm 10u; mobile phase: [water(NH ₄ HCO ₃ )-ACN]; B%: 40% to 65%, 20 min) to obtain WV-NU-172 (2.5 g, 4.31 mmol, 23.96% yield) as a yellow solid. ¹ HNMR (400 MHz, DMSO-d6) δ = 11.74 (br s, 1H), 8.23 - 8.03 (m, 2H), 7.97 - 7.80 (m, 1H), 7.39 - 7.33 (m, 2H), 7.31 - 7.16 (m, 7H), 6.85 (br dd, J = 5.4, 8.5 Hz, 4H), 6.17 (br t, J = 6.0 Hz, 2H), 5.39 (br d, J = 4.1 Hz, 1H), 4.33 (br s, 1H), 3.96 (br d, J = 3.8 Hz, 1H), 3.71 (d, J = 3.8 Hz, 6H), 3.17 - 3.12 (m, 2H), 2.42 - 2.22 (m, 1H). LCMS: (M-H+): 579.3.

The amidite of WV-NU-172 can be prepared using various techniques according to the present invention. For example, in some embodiments the amidite is prepared as described below.

Nucleoside WV-NU-172 (1.9 g, 3.27 mmol, 1.0 equivalent) in a 250 mL three-neck flask was azeotroped with anhydrous toluene (30 mL) and dried in high vacuum for 48 hours. Anhydrous THF (10 mL) was added to the flask under argon and the solution was cooled to -10 °C. To the reaction mixture was added triethylamine (4.0 equiv.) followed by a solution of D-PSM-Cl (0.9 M) (2.0 equiv.) over 10 minutes. The reaction mixture was warmed to room temperature and reaction progress was monitored by HPLC. After the starting materials disappeared, water was added to quench the reaction and molecular sieves were added to dry. The reaction mixture was filtered through a glass tube. The reaction flask and precipitate were washed with dry THF (25 mL). The obtained filtrate was collected, and the solvent was removed under reduced pressure. The residue was purified by column chromatography (SiO2, 40-100% ethyl acetate in hexanes) to give D-PSM-WV-NU-172 amidite off-white solid (1.6 g, 57% yield). ³¹ P NMR (243 MHz, CDCl ₃ ) δ = 154.34. ¹ H NMR (600 MHz, CDCl ₃ ) δ 7.95 - 7.88 (m, 3H), 7.86 (d, J = 1.4 Hz, 1H), 7.71 (d, J = 1.4 Hz, 1H), 7.62 (tt, J = 7.3, 1.3 Hz, 1H), 7.54 - 7.48 (m, 2H), 7.43 - 7.38 (m, 2H), 7.34 - 7.27 (m, 4H), 7.26 - 7.20 (m, 1H), 6.85 (ddq, J = 8.4, 3.1, 1.8 Hz, 4H), 6.31 (dd, J = 6.6, 1.4 Hz, 1H), 6.04 (dd, J = 7.9, 5.5 Hz, 1H), 5.07 (dt, J = 7.4, 5.5 Hz, 1H) ), 4.79 (ddd, J = 8.2, 5.3, 2.5 Hz, 1H), 4.18 (td, J = 4.2, 2.2 Hz, 1H), 3.82 - 3.74 (m, 8H), 3.68 (ddd, J = 9.7, 5.5 , 2.7 Hz, 1H), 3.58 - 3.47 (m, 2H), 3.40 (dd, J = 14.4, 5.3 Hz, 1H), 3.30 (qd, J = 10.4, 4.2 Hz, 2H), 3.20 (ddd, J = 10.3, 4.0, 1.6 Hz, 1H), 2.56 (ddd, J = 13.5, 5.6, 2.3 Hz, 1H), 2.47 (ddd, J = 13.6, 8.0, 5.8 Hz, 1H), 1.96 - 1.81 (m, 4H) , 1.72 - 1.65 (m, 1H), 1.18 - 1.11 (m, 1H). ¹³ C NMR (151 MHz, CDCl ₃ ) δ 161.36, 158.64, 154.95, 152.50, 144.40, 139.41, 136.49, 135.47, 135.45, 135.06, 134.06, 130.09, 130. 01, 129.35, 128.10, 128.03, 127.99, 127.97, 126.99, 119.43 , 113.68, 113.28, 113.26, 86.71, 85.97, 85.95, 74.47, 74.41, 74.03, 73.94, 67.99, 66.33, 66.31, 63.12, 58.01, 57.99, 55.2 5, 46.79, 46.56, 41.15, 41.12, 27.37, 26.01, 25.99, 25.63 . LCMS: C ₄₅ H ₄₆ N ₅ O ₉ PS (MH ⁺ ): 865.04.

The nucleoside WV-NU-172 (0.9 g) was converted to L-PSM-WV-NU-172 amidite (510 mg, 45% yield) as an off-white solid. ³¹ P NMR (243 MHz, CDCl ₃ ) δ = 153.78. ¹ H NMR (600 MHz, CDCl ₃ ) δ 7.94 - 7.87 (m, 3H), 7.86 (d, J = 1.5 Hz, 1H), 7.70 (d, J = 1.4 Hz, 1H), 7.62 (tt, J = 7.3, 1.4 Hz, 1H), 7.53 - 7.47 (m, 2H), 7.42 - 7.36 (m, 2H), 7.34 - 7.27 (m, 4H), 7.26 - 7.20 (m, 1H), 6.85 (ddq, J = 8.4, 3.1, 1.8 Hz, 4H), 6.31 (dd, J = 6.6, 1.3 Hz, 1H), 6.03 (dd, J = 7.9, 5.4 Hz, 1H), 5.07 (dt, J = 7.4, 5.5 Hz, 1H) ), 4.79 (ddd, J = 8.2, 5.3, 2.5 Hz, 1H), 4.19 (td, J = 4.2, 2.2 Hz, 1H), 3.82 - 3.72 (m, 8H), 3.68 (ddd, J = 9.7, 5.5 , 2.7 Hz, 1H), 3.58 - 3.47 (m, 2H), 3.40 (dd, J = 14.4, 5.3 Hz, 1H), 3.30 (qd, J = 10.4, 4.3 Hz, 2H), 3.20 (ddd, J = 10.2, 4.0, 1.6 Hz, 1H), 2.56 (ddd, J = 13.5, 5.6, 2.3 Hz, 1H), 2.46 (ddd, J = 13.6, 8.0, 5.8 Hz, 1H), 1.95 - 1.80 (m, 4H) , 1.72 - 1.64 (m, 1H), 1.17 - 1.10 (m, 1H). ¹³ C NMR (151 MHz, CDCl ₃ ) δ 161.49, 158.77, 155.08, 152.63, 144.53, 139.54, 136.61, 135.60, 135.57, 135.18, 134.19, 130.22, 129. 48, 128.22, 128.12, 128.10, 127.12, 119.56, 113.81, 113.41 , 113.39, 86.84, 86.09, 86.08, 74.60, 74.54, 74.16, 74.07, 68.12, 66.46, 66.43, 63.25, 58.14, 58.12, 55.37, 46.92, 46.69, 41.28, 41.25, 27.50, 26.14, 26.12, 25.76. LCMS: C ₄₅ H ₄₆ N ₅ O ₉ PS (MH ⁺ ): 865.04.

N-(1-((S)-3-(bis(4-methoxyphenyl)(phenyl)methoxy)-2-(((1S,3S,3aS)-3-((phenylsulfonyl)methyl) Tetrahydro-1H,3H-pyrrolo[1,2-c][1,3,2]oxazaphosphol-1-yl)oxy)propyl)-2-oxo-1,2-dihydropyrimidine-4 -yl) synthesis of benzamide

dry N-[1-[(2S)-3-[bis(4-methoxyphenyl)-phenyl-methoxy]-2-hydroxy-propyl]-2-oxo-pyrimidine- in THF (48 mL) To a solution of 4-yl]benzamide (4.79 g, 8.1 mmol) was added triethylamine (6.1 mL, 43.73 mmol). (3S,3aS)-3-(benzenesulfonylmethyl)-1-chloro-3a,4,5,6-tetrahydro-3H-pyrrolo[1,2-c][1,3,2]oxazapo Spall (0.9 M in THF, 16.2 mL, 14.58 mmol) was added dropwise. The off-white slurry was stirred at room temperature for 7 hours. TLC and LCMS showed the reaction to be complete. The reaction was quenched with water (146 uL). Anhydrous MgSO ₄ (1.94 g) was added. The mixture was filtered through celite and the filtrate was concentrated to give the crude product as an off-white foam. The crude product was purified by normal phase column chromatography applying a gradient of 20-100% EtOAc in hexanes (each mobile phase contained 5% triethylamine) to afford the title compound as a light brown foam (5.63 g, 79.5% yield). was obtained as ^1H NMR (600 MHz, CDCl ₃ ) δ 8.62 (bs, 1H), 7.93 - 7.86 (m, 2H), 7.85 - 7.81 (m, 2H), 7.60 (t, J = 7.5 Hz, 1H), 7.56 ( tt, J = 7.6, 1.2 Hz, 1H), 7.51 (tt, J = 7.9, 1.6 Hz, 2H), 7.47 (dt, J = 7.1, 1.5 Hz, 2H), 7.42 (tt, J = 8.1, 1.6 Hz, 3H), 7.35 (dd, J = 8.9, 2.1 Hz, 4H), 7.30 (t, J = 7.7 Hz, 3H), 7.21 (tt, J = 7.4, 1.3 Hz, 1H), 6.85 (dd, J = 8.9, 1.5 Hz, 4H), 5.09 (q, J = 6.3 Hz, 1H), 4.59 - 4.52 (m, 1H), 4.41 (dd, J = 13.4, 3.3 Hz, 1H), 3.79 (s, 6H) , 3.71 - 3.62 (m, 1H), 3.57 (dd, J = 13.4, 9.1 Hz, 1H), 3.43 (dd, J = 14.3, 6.8 Hz, 1H), 3.39 - 3.33 (m, 1H), 3.30 (dd , J = 14.6, 6.1 Hz, 1H), 3.18 (qd, J = 9.9, 4.7 Hz, 2H), 3.01 (qd, J = 10.0, 4.4 Hz, 1H), 1.81 - 1.67 (m, 2H), 1.67 - 1.59 (m, 1H), 1.12 - 1.04 (m, 1H); ³¹ P NMR (243 MHz, CDCl ₃ ) δ 154.61; MS (ESI), 873.94 [M - H] ^- .

N-(1-((R)-3-(bis(4-methoxyphenyl)(phenyl)methoxy)-2-(((1S,3S,3aS)-3-((phenylsulfonyl)methyl) Tetrahydro-1H,3H-pyrrolo[1,2-c][1,3,2]oxazaphosphol-1-yl)oxy)propyl)-2-oxo-1,2-dihydropyrimidine-4 -yl) synthesis of benzamide

dry N-[1-[(2R)-3-[bis(4-methoxyphenyl)-phenyl-methoxy]-2-hydroxy-propyl]-2-oxo-pyrimidine- in THF (48 mL) To a solution of 4-yl]benzamide (4.81 g, 8.13 mmol) was added triethylamine (6.12 mL, 43.91 mmol). (3S,3aS)-3-(benzenesulfonylmethyl)-1-chloro-3a,4,5,6-tetrahydro-3H-pyrrolo[1,2-c][1,3,2]oxazapo Spall (0.9 M in THF, 16.3 mL, 14.64 mmol) was added dropwise. The off-white slurry was stirred at room temperature for 5 hours. TLC and LCMS showed the reaction to be complete. The reaction was quenched with water (146 uL). Anhydrous MgSO ₄ (1.94 g) was added. The mixture was filtered through celite and the filtrate was concentrated to give the crude product as a light brown foam. The crude product was purified by normal phase column chromatography applying a gradient of 20-100% EtOAc in hexanes (each mobile phase contained 5% triethylamine). Since the first half of the main peak fraction was still not pure, it was purified again by normal phase column chromatography applying a gradient of 30 to 100% DCM in hexanes (each mobile phase contained 2.5% triethylamine). The pure desired product fractions from the two columns were combined and concentrated to give the title compound as a brownish off-white foam (4.69 g, 65.9% yield). ¹ H NMR (600 MHz, CDCl ₃ ) δ 8.60 (bs, 1H), 7.96 (dt, J = 7.2, 1.3 Hz, 2H), 7.91 - 7.84 (m, 2H), 7.66 - 7.57 (m, 3H), 7.56 - 7.46 (m, 7H), 7.39 - 7.33 (m, 4H), 7.29 (t, J = 7.7 Hz, 2H), 7.21 (tt, J = 7.4, 1.3 Hz, 1H), 6.87 - 6.81 (m, 4H), 5.08 (q, J = 6.2 Hz, 1H), 4.59 (tdd, J = 12.1, 8.9, 4.1 Hz, 1H), 4.32 (dd, J = 13.4, 3.2 Hz, 1H), 3.791 (s, 3H) ), 3.789 (s, 3H), 3.78 - 3.73 (m, 1H), 3.58 (dd, J = 13.4, 8.9 Hz, 1H), 3.48 (dd, J = 14.3, 6.4 Hz, 1H), 3.46 - 3.39 ( m, 1H), 3.30 (dd, J = 14.2, 6.4 Hz, 1H), 3.23 (dd, J = 10.0, 3.7 Hz, 1H), 3.17 (dd, J = 10.0, 5.4 Hz, 1H), 3.07 - 2.98 (m, 1H), 1.85 - 1.70 (m, 2H), 1.69 - 1.63 (m, 1H), 1.08 (dq, J = 11.7, 9.5 Hz, 1H); ³¹ P NMR (243 MHz, CDCl ₃ ) δ 154.17; MS (ESI), 873.94 [M - H] ^- .

3-((2R,4S,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-hydroxytetrahydrofuran-2-yl)-6-methylpyrimidine -2,4(1H,3H)-dione (WV-NU-198) and 3-((2S,4S,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl) Synthesis of -4-hydroxytetrahydrofuran-2-yl) -6-methylpyrimidine-2,4 (1H, 3H) -dione (WV-NU-198A)

Step 1 . DMAP (4.55 g, 37.28 mol, 0.1 eq) was added, and Ac ₂ O (190.28 g, 1.86 mol, 174.57 mL, 5 eq) was added dropwise. The mixture was stirred at 15 °C for 12 hours. Pyridine was removed by rotary evaporator and the residue was co-evaporated with toluene (2*50 mL). The residue was diluted with DCM (300 mL), washed with 1 M HCl (100 mL), then sat.NaHCO ₃ (20 mL), dried over Na ₂ SO ₄ , filtered and concentrated to give crude product (2R, 4S,5R)-5-(acetoxymethyl)tetrahydrofuran-2,4-diyl diacetate (95 g, 365.05 mmol, 97.93% yield) was obtained as a white solid.

Step 2 . A solution of (2R,4S,5R)-5-(acetoxymethyl)tetrahydrofuran-2,4-diyl diacetate (14.54 g, 115.28 mmol, 1.5 equiv) stored under argon dissolved in DCE (300 mL). To the solution was added BSA (46.90 g, 230.56 mmol, 56.99 mL, 3 eq) and stirred at 80° C. for 0.5 h until the mixture became clear, stirring vigorously with 6-methylpyrimidine in DCE (150 mL). -2,4(1H,3H)-dione (20 g, 76.85 mmol, 1 equiv) followed by SnCl ₄ (22.02 g, 84.54 mmol, 9.88 mL, 1.1 equiv) was added dropwise to a slightly yellowish solution at 0 °C. did The mixture was stirred at 15 °C for 12 hours. The reaction mixture was quenched by adding 20 mL of NaHCO ₃ and extracted with 45 mL of DCM (15 mL x 3). The combined organic layers were dried over Na ₂ SO ₄ , filtered, and concentrated under reduced pressure to ((2R,3S,5R)-3-acetoxy-5-(4-methyl-2,6-dioxo-3,6- Dihydropyrimidin-1(2H)-yl)tetrahydrofuran-2-yl)methyl acetate (20 g, 61.29 mmol, 79.75% yield) was obtained as a white solid. LCMS (MH) ^- :325.1.

Step 3. ((2R,3S,5R)-3-acetoxy-5-(4-methyl-2,6-dioxo-3,6-dihydropyrimidine-1(2H) in MeOH (160 mL) To a solution of -yl)tetrahydrofuran-2-yl)methyl acetate (16 g, 49.03 mmol, 1 equiv) was added NaOMe (6.62 g, 122.59 mmol, 2.5 equiv). The mixture was stirred at 15 °C for 3 hours. The reaction mixture was quenched by addition of NH ₄ Cl (400 cmg) and then concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO ₂ , petroleum ether:ethyl acetate = 1:0 to 0:1) to obtain 3-((2R,4S,5R)-4-hydroxy-5-(hydroxymethyl) Tetrahydrofuran-2-yl)-6-methylpyrimidine-2,4(1H,3H)-dione (8 g, 33.03 mmol, 88.89% yield) was obtained as a white solid. LCMS : (MH ⁺ ):241.0.

Step 4. 3-((2R,4S,5R)-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-6-methylpyrimidine-2,4 in pyridine (90 mL) To a solution of (1H,3H)-dione (4 g, 16.51 mmol, 1 equiv) was added DMTC1 (6.71 g, 19.82 mmol, 1.2 equiv). The mixture was stirred at 15 °C for 2 hours. The reaction mixture was extracted with DCM (100 mL * 3). The combined organic layers were dried over Na ₂ SO ₄ , filtered and concentrated under reduced pressure to give a residue. The crude product was purified by column chromatography (SiO ₂ , petroleum ether:ethyl acetate = 1:0 to 0:1) and reverse phase HPLC (Column: Phenomenex Titank C18 bulk 250*70 mm 10u; mobile phase: [water (10 mM) NH ₄ HCO ₃ )-ACN]; B%: 46%-66%, 20 min @ 100 mL/min) to obtain 3-((2R,4S,5R)-5-((bis(4-meth) Koxyphenyl) (phenyl) methoxy) methyl) -4-hydroxytetrahydrofuran-2-yl) -6-methylpyrimidine-2,4 (1H, 3H) -dione (WV-NU-198) (0.83 g, 9.23% yield) as a white solid, and 3-((2S,4S,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-hydroxytetrahydro Obtained furan-2-yl)-6-methylpyrimidine-2,4(1H,3H)-dione (WV-NU-198A) (1.65 g, 18.35% yield) as a white solid. WV-NU-198: ¹ HNMR (400 MHz, DMSO-d ₆ ) δ = 11.15 - 10.94 (m, 1H), 7.53 - 7.34 (m, 2H), 7.31 - 7.18 (m, 6H), 6.96 - 6.79 ( m, 4H), 6.63 - 6.54 (m, 1H), 5.50 - 5.40 (m, 1H), 5.15 - 5.01 (m, 1H), 4.35 - 4.21 (m, 1H), 3.90 - 3.80 (m, 1H), 3.74 (d, J = 1.8 Hz, 6H), 3.40 - 3.29 (m, 1H), 3.27 - 3.12 (m, 1H), 3.10 - 2.96 (m, 1H), 2.14 - 1.89 (m, 4H); LCMS: (MH ⁺ ): 543.2. WV-NU-198A: ¹ H NMR (400 MHz, DMSO-d ₆ ) δ = 11.10 - 10.89 (m, 1H), 7.59 - 7.43 (m, 2H), 7.42 - 7.29 (m, 6H), 7.26 - 7.17 (m, 1H), 6.95 - 6.81 (m, 4H), 6.14 - 6.02 (m, 1H), 5.81 - 5.71 (m, 1H), 5.39 - 5.31 (m, 1H), 4.92 - 4.76 (m, 1H) , 3.79 - 3.68 (m, 6H), 3.65 (br s, 1H), 3.56 - 3.49 (m, 1H), 3.45 - 3.40 (m, 1H), 3.37 - 3.29 (m, 1H), 2.76 (br t, J = 11.9 Hz, 1H), 2.67 - 2.59 (m, 1H), 2.07 (s, 1H), 1.99 - 1.92 (m, 3H), 1.55 - 1.40 (m, 1H); LCMS: (MH ⁺ ):543.2.

9-((2R,4S,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-hydroxytetrahydrofuran-2-yl)-7,9-di Synthesis of hydro-1H-purine-6,8-dione (WV-NU-213)

Step 1 . Two batches: (2R,3S,5R)-5-(6-amino-9H-purin-9-yl in dioxane (400 mL) and AcONa (0.5 M, 1.87 L, 4.71 equiv) buffer (pH 4.3) To a solution of )-2-(hydroxymethyl)tetrahydrofuran-3-ol (50 g, 199.01 mmol, 1 equiv), a solution of Br ₂ (38.16 g, 238.81 mmol, 12.31 mL, 1.2 equiv) was stirred while stirring. it was added The mixture was stirred at 15 °C for 12 hours. The two batches were combined for work-up. Concentrated Na ₂ S ₂ O ₅ was added to the mixture until the red color disappeared. The mixture was neutralized to pH 7.0 with 0.5 m NaOH. The residue was evaporated when a white solid precipitated. The solid was filtered off, washed with cold 1,4-dioxane (50 mL), and dried under high vacuum to (2R,3S,5R)-5-(6-amino-8-bromo-9H-purine-9 Obtained -yl)-2-(hydroxymethyl)tetrahydrofuran-3-ol (110 g, 333.19 mmol, 83.71% yield) as a yellow solid. ^1HNMR (400MHz, DMSO-d6) δ = 8.22 - 7.98 (m, 1H), 7.53 (br s, 2H), 6.29 (dd, J = 6.5, 7.9 Hz, 1H), 5.35 (br d, J = 12.3 Hz, 2H), 4.58 - 4.38 ( m, 1H), 3.95 - 3.82 (m, 1H), 3.65 (dd, J = 4.5, 11.9 Hz, 1H), 3.48 (br dd, J = 4.5, 11.7 Hz, 1H), 3.36 (br s, 1H) , 3.24 (ddd, J = 6.1, 7.8, 13.4 Hz, 1H), 2.19 (ddd, J = 2.6, 6.4, 13.1 Hz, 1H); LCMS:(M+H+):330.14.

Step 2 . Two batches: (2R,3S,5R)-5-(6-amino-8-bromo-9H-purin-9-yl in 2-mercaptoethanol (39.22 g, 501.90 mmol, 35.01 mL, 3.01 equiv) A solution of )-2-(hydroxymethyl)tetrahydrofuran-3-ol (55 g, 166.60 mmol, 1 equiv) and TEA (168.58 g, 1.67 mol, 231.88 mL, 10 equiv) in water (1500 mL) Stirred at 110 °C for 4 hours. The solvent was removed under reduced pressure to give a residue which was purified by MPLC (dichloromethane:methanol = 5:1, 10:1) to give 6-amino-9-((2R,4S,5R)-4-hydroxy-5 Obtained -(hydroxymethyl)tetrahydrofuran-2-yl)-9H-purin-8-ol (65 g, 243.23 mmol, 73.00% yield) as a white solid. LCMS:(M+H+):267.24.

Step 3 . A solution of NaNO ₂ (15.49 g, 224.52 mmol, 2 equiv) in water (60 mL) was added to 6-amino-9-((2R,4S,5R)-4-hydroxy in HOAc (1500 mL, 95% purity). To a stirred solution of -5-(hydroxymethyl)tetrahydrofuran-2-yl)-9H-purin-8-ol (30 g, 112.26 mmol, 1 equiv) was added. The reaction mixture was stirred at 15 °C for 12 hours. The solvent was removed under reduced pressure. The crude product was triturated with DCM (500 ml) at 15 °C for 5 min to obtain 9-((2R,4S,5R)-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl )-7,9-dihydro-1H-purine-6,8-dione (22 g, 82.02 mmol, 73.06% yield) was obtained as a white solid. ¹ HNMR (400 MHz, DMSO-d ₆ ) δ = 7.98 (s, 1H), 6.12 (t, J = 7.3 Hz, 1H), 4.36 (td, J = 2.8, 5.8 Hz, 1H), 3.79 - 3.74 (m , 1H), 3.58 (dd, J = 5.0, 11.6 Hz, 1H), 3.44 (dd, J = 5.3, 11.6 Hz, 1H), 2.96 (ddd, J = 6.2, 7.6, 13.3 Hz, 1H), 2.01 ( ddd, J = 2.8, 6.7, 13.0 Hz, 1H), 1.90 (s, 1H); LCMS:(M+H+):268.23.

Step 4 . 9-((2R,4S,5R)-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-7,9-dihydro-1H-purine-6 in pyridine (400 mL) To a solution of ,8-dione (22 g, 82.02 mmol, 1 equiv) was added DMTC1 (22.23 g, 65.62 mmol, 0.8 equiv). The mixture was stirred at 15 °C for 12 hours. The reaction mixture was quenched by adding 400 mL of water at 15° C., then diluted with 200 mL of water and extracted with 900 mL of ethyl acetate (300 mL * 3). The combined organic layers were dried over Na ₂ SO ₄ , filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO ₂ , DCM: MeOH = 1:0 to 0:1). The crude product was triturated with DCM (300 ml) at 15° C. for 5 min to give WV-NU-213 (13.67 g, 30% yield) as a white solid. ¹ H NMR (400 MHz, DMSO-d ₆ ) δ = 11.37 (s, 1H), 7.78 (s, 1H), 7.35 (d, J = 7.4 Hz, 2H), 7.26 - 7.14 (m, 7H), 6.80 ( dd, J = 8.9, 14.5 Hz, 4H), 6.13 (t, J = 6.8 Hz, 1H), 5.21 (d, J = 4.8 Hz, 1H), 4.48 - 4.39 (m, 1H), 3.89 (td, J = 4.4, 6.4 Hz, 1H), 3.72 (d, J = 3.6 Hz, 6H), 3.33 (s, 1H), 3.20 - 3.03 (m, 2H), 2.96 (td, J = 6.5, 12.9 Hz, 1H) , 2.15 - 2.05 (m, 1H); LCMS :( M+H-):570.59, LCMS purity: 97.33%.

3-((2R,4S,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-(((1S,3S,3aS)-3-((phenylsulfone) phonyl)methyl)tetrahydro-1H,3H-pyrrolo[1,2-c][1,3,2]oxazaphosphol-1-yl)oxy)tetrahydrofuran-2-yl)-6-methylpyr Synthesis of midine-2,4(1H,3H)-dione

Dry 3-[(2R,4R,5R)-5-[[bis(4-methoxyphenyl)-phenyl-methoxy]methyl]-4-hydroxy-tetrahydrofuran-2- in THF (6.5 mL) To a solution of yl]-6-methyl-1H-pyrimidine-2,4-dione (0.83 g, 1.52 mmol) was added triethylamine (0.47 mL, 3.35 mmol). (3S,3aS)-3-(benzenesulfonylmethyl)-1-chloro-3a,4,5,6-tetrahydro-3H-pyrrolo[1,2-c][1,3,2]oxazapo Spall (0.43 M in THF, 5.32 mL, 2.29 mmol) was added dropwise rapidly. The resulting turbid reaction solution was stirred at room temperature for 5 hours. TLC showed the reaction to be complete. The reaction was quenched with water (14 μL). Anhydrous MgSO ₄ (183 mg) was added. The mixture was filtered through celite and the filtrate was concentrated to give the crude product as an off-white foam. The crude product was purified by normal phase column chromatography applying a gradient of 20-100% EtOAc in hexanes, each mobile phase containing 1% triethylamine, to afford the title compound as a white foam (0.549 g, 43.5% yield). was obtained as ¹ H NMR (600 MHz, chloroform-d) δ 9.50 (s, 1H), 7.89 - 7.84 (m, 2H), 7.60 (t, J = 7.4 Hz, 1H), 7.50 (t, J = 7.7 Hz, 2H ), 7.46 - 7.42 (m, 2H), 7.32 (ddd, J = 9.2, 5.6, 2.8 Hz, 4H), 7.22 (t, J = 7.6 Hz, 2H), 7.14 (t, J = 7.3 Hz, 1H) , 6.79 - 6.73 (m, 4H), 6.71 (dd, J = 8.9, 4.3 Hz, 1H), 5.46 (s, 1H), 4.93 (q, J = 6.1 Hz, 1H), 4.84 (dq, J = 8.8 , 6.2 Hz, 1H), 3.92 (td, J = 6.4, 3.9 Hz, 1H), 3.74 (s, 3H), 3.73 (s, 3H), 3.63 (dq, J = 11.8, 5.9 Hz, 1H), 3.43 - 3.27 (m, 5H), 2.94 (qd, J = 10.0, 4.1 Hz, 1H), 2.80 (ddd, J = 13.0, 8.2, 4.3 Hz, 1H), 2.26 (ddd, J = 13.6, 9.0, 6.1 Hz) , 1H), 1.99 (s, 3H), 1.83 (dtt, J = 11.9, 7.8, 3.2 Hz, 1H), 1.77 - 1.68 (m, 1H), 1.66 - 1.58 (m, 1H), 1.11 - 1.04 (m , 1H); ³¹ P NMR (243 MHz, chloroform-d) δ 149.82; MS (ESI), 826.14 [M - H] ^- .

3-((2S,4S,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-(((1S,3S,3aS)-3-((phenylsulfone) phonyl)methyl)tetrahydro-1H,3H-pyrrolo[1,2-c][1,3,2]oxazaphosphol-1-yl)oxy)tetrahydrofuran-2-yl)-6-methylpyr Synthesis of midine-2,4(1H,3H)-dione

Dry 3-[(2S,4R,5R)-5-[[bis(4-methoxyphenyl)-phenyl-methoxy]methyl]-4-hydroxy-tetrahydrofuran-2- in THF (12.5 mL) To a solution of yl]-6-methyl-1H-pyrimidine-2,4-dione (1.65 g, 3.03 mmol) was added triethylamine (0.93 mL, 6.67 mmol). (3S,3aS)-3-(benzenesulfonylmethyl)-1-chloro-3a,4,5,6-tetrahydro-3H-pyrrolo[1,2-c][1,3,2]oxazapo Spall (0.43 M in THF, 10.6 mL, 4.54 mmol) was added dropwise rapidly. The resulting turbid reaction solution was stirred at room temperature for 5 hours. TLC showed the reaction to be complete. The reaction was quenched with water (27 μL). Anhydrous MgSO ₄ (363 mg) was added. The mixture was filtered through celite and the filtrate was concentrated to give the crude product as an off-white foam. The crude product was purified by normal phase column chromatography applying a gradient of 20-100% EtOAc in hexanes (each mobile phase contained 1% triethylamine) to afford the title compound as a white foam (1.266 g, 50.5% yield). was obtained as ^1H NMR (600 MHz, chloroform-d) δ 9.07 (s, 1H), 7.93 (dd, J = 7.8, 1.6 Hz, 2H), 7.64 - 7.58 (m, 1H), 7.53 (t, J = 7.7 Hz , 2H), 7.51 - 7.47 (m, 2H), 7.41 - 7.36 (m, 4H), 7.25 (d, J = 7.6 Hz, 2H), 7.19 (t, J = 7.3 Hz, 1H), 6.79 (dd, J = 9.0, 2.2 Hz, 4H), 6.16 (d, J = 11.2 Hz, 1H), 5.45 (s, 1H), 5.04 (q, J = 6.0 Hz, 1H), 4.17 - 4.11 (m, 1H), 3.783 (s, 3H), 3.777 (s, 3H), 3.73 - 3.62 (m, 3H), 3.58 - 3.53 (m, 1H), 3.52 - 3.47 (m, 1H), 3.42 (dd, J = 14.6, 5.4 Hz, 1H), 3.06 - 2.97 (m, 1H), 2.95 - 2.88 (m, 1H), 2.86 (dd, J = 10.3, 4.2 Hz, 1H), 2.03 (s, 3H), 1.85 (dp, J = 12.2, 4.5 Hz, 1H), 1.78 - 1.70 (m, 1H), 1.66 (ddt, J = 7.8, 5.5, 2.5 Hz, 1H), 1.61 (dt, J = 13.6, 3.1 Hz, 1H), 1.21 - 1.11 (m, 1H); ³¹ P NMR (243 MHz, chloroform-d) δ 148.85; MS (ESI), 826.14 [M - H] ^- .

(Z)-N'-(9-((2R,3R,4R,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-3-((tert-butyldimethyl silyl)oxy)-4-(((1S,3S,3aS)-3-((phenylsulfonyl)methyl)tetrahydro-1H,3H-pyrrolo[1,2-c][1,3,2] Synthesis of oxazaphospholl-1-yl)oxy)tetrahydrofuran-2-yl)-8-oxo-8,9-dihydro-7H-purin-6-yl)-N,N-dimethylformimidamide

Dry N-[9-[(2R,3S,5R)-5-[[bis(4-methoxyphenyl)-phenyl-methoxy]methyl]-3-[tert-butyl(dimethyl) in THF (135 mL) A solution of )silyl]oxy-4-hydroxy-tetrahydrofuran-2-yl]-8-oxo-7H-purin-6-yl]-N,N-dimethyl-formamidine (18.0 g, 23.84 mmol) To this was added triethylamine (7.31 mL, 52.45 mmol). The reaction flask was set in a water bath. (3S,3aS)-3-(benzenesulfonylmethyl)-1-chloro-3a,4,5,6-tetrahydro-3H-pyrrolo[1,2-c][1,3,2]oxazapo Spall (0.43 M in THF, 83.17 mL, 35.76 mmol) was added dropwise rapidly. The bath was removed. The cloudy reaction solution was stirred at room temperature for 3 hours. TLC and LCMS showed the reaction to be incomplete. Additional TEA (1.46 mL, 10.47 mmol) was added. Added (3S,3aS)-3-(benzenesulfonylmethyl)-1-chloro-3a,4,5,6-tetrahydro-3H-pyrrolo[1,2-c][1,3,2]oxa Zappospol (0.43 M in THF, 16.6 mL, 7.14 mmol) was also added dropwise rapidly. It was stirred for an additional 1 hour. TLC showed the reaction to be complete. The reaction was quenched with water (343 μL). Anhydrous MgSO ₄ (4.577 g) was added. The mixture was filtered through celite and the filtrate was concentrated to give the crude product as a white foam. The crude product was purified by normal phase column chromatography applying a gradient of 20-100% EtOAc in hexanes, each mobile phase containing 1% triethylamine, to afford the title compound as a white foam (17.87 g, 72.2% yield). was obtained as ^1H NMR (600 MHz, chloroform- d ) δ 8.72 (s, 1H), 8.28 (s, 1H), 8.16 (s, 1H), 7.87 - 7.83 (m, 2H), 7.56 - 7.52 (m, 1H) , 7.49 - 7.42 (m, 4H), 7.38 - 7.31 (m, 4H), 7.20 (dd, J = 8.4, 6.8 Hz, 2H), 7.17 - 7.12 (m, 1H), 6.78 - 6.72 (m, 4H) , 5.94 (d, J = 5.5 Hz, 1H), 5.34 (t, J = 5.4 Hz, 1H), 4.96 (q, J = 6.2 Hz, 1H), 4.78 (dt, J = 10.8, 4.5 Hz, 1H) , 4.01 (q, J = 4.4 Hz, 1H), 3.75 (s, 6H), 3.67 (dq, J = 11.5, 6.0 Hz, 1H), 3.48 - 3.35 (m, 4H), 3.17 (dd, J = 10.2 , 4.9 Hz, 1H), 3.13 (s, 3H), 3.10 (s, 3H), 3.03 (qd, J = 9.5, 4.0 Hz, 1H), 1.89 - 1.81 (m, 1H), 1.78 - 1.72 (m, 1H), 1.69 - 1.62 (m, 1H), 1.15 - 1.06 (m, 1H), 0.81 (s, 9H), -0.02 (s, 3H), -0.14 (s, 3H); ³¹ P NMR (243 MHz, chloroform-d) δ 152.36; MS (ESI), 1036.85 [M - H] ^- .

9-((2R,4S,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-(((1S,3S,3aS)-3-((phenylsulfone) phonyl)methyl)tetrahydro-1H,3H-pyrrolo[1,2-c][1,3,2]oxazaphosphol-1-yl)oxy)tetrahydrofuran-2-yl)-7,9- Synthesis of dihydro-1H-purine-6,8-dione

dry 9-[(2R,4R,5R)-5-[[bis(4-methoxyphenyl)-phenyl-methoxy]methyl]-4-hydroxy-tetrahydrofuran-2- in THF (90 mL) To a solution of yl]-1,7-dihydropurine-6,8-dione (6.0 g, 10.52 mmol) was added triethylamine (3.08 mL, 22.08 mmol). (3S,3aS)-3-(benzenesulfonylmethyl)-1-chloro-3a,4,5,6-tetrahydro-3H-pyrrolo[1,2-c][1,3,2]oxazapo Spall (0.89 M in THF, 18.9 mL, 16.82 mmol) was added dropwise rapidly. Stir at room temperature for 2 hours. LCMS indicated a conversion of about 67%. Stirred for an additional 6 hours. TLC showed very little starting material. The reaction was quenched with water (113 μL). Anhydrous MgSO ₄ (1.51 g) was added. The mixture was filtered through celite and the filtrate was concentrated to give the crude product as an off-white foam. The crude product was purified by normal phase column chromatography applying a gradient of 0-100% ACN in EtOAc (each mobile phase contained 5% triethylamine) to afford the title compound as an off-white foam (5.32 g, 59.3% yield). was obtained as ^1H NMR (600 MHz, DMSO-d6) δ 11.42 (s, 2H), 7.88 - 7.81 (m, 3H), 7.60 (t, J = 7.4 Hz, 1H), 7.52 (t, J = 7.6 Hz, 2H) ), 7.33 (d, J = 7.8 Hz, 2H), 7.25 - 7.14 (m, 7H), 6.79 (dd, J = 18.2, 8.5 Hz, 4H), 6.11 (dd, J = 8.1, 4.6 Hz, 1H) , 5.10 - 5.00 (m, 2H), 3.86 - 3.79 (m, 2H), 3.73 - 3.69 (m, 6H), 3.69 - 3.65 (m, 1H), 3.58 (dt, J = 9.6, 5.3 Hz, 1H) , 3.24 (dd, J = 14.3, 7.6 Hz, 1H), 3.11 (dd, J = 10.4, 3.8 Hz, 1H), 3.08 - 3.03 (m, 1H), 2.85 (dt, J = 13.2, 6.1 Hz, 1H) ), 2.81 - 2.73 (m, 1H), 2.60 (qd, J = 9.8, 3.9 Hz, 1H), 2.27 (dt, J = 14.1, 7.4 Hz, 1H), 1.63 - 1.50 (m, 2H), 1.11 ( q, J = 10.2, 9.7 Hz, 1H); ³¹ P NMR (243 MHz, DMSO-d6) δ 144.02; MS (ESI), 852.62 [M - H] ^- .

The preparation of additional compounds useful for oligonucleotide preparation is described by way of example below.

General experimental procedure (A) for chloro reagent (2)

Dithiol (360 mmol) was dissolved in toluene (720 mL) under argon (3000 mL single neck flask) and then 4-methylmorpholine (35.4 mL, 792 mmol) was added. The mixture was added dropwise via cannula to an ice-cold solution of phosphorus trichloride (720 mL, 396 mmol) in toluene (720 mL) under an argon atmosphere over 30 minutes. After warming to room temperature for 1 hour, the mixture was carefully filtered under vacuum/argon. The resulting filtrate was concentrated by rotary evaporation (flushing with Ar) and then dried under high vacuum for 2 hours. The resulting crude compound was isolated as a thick oil and dissolved in THF to give a 1 M stock solution, which was used in the next step without further purification.

Data for 2: synthesized from compound 1 according to General Procedure A. ³¹ P NMR (243 MHz, THF-CDCl _3, 1:2) δ 168.77, 161.4

General experimental procedure for monomers (5 and 6) (B)

The 5'-ODMTr protected nucleoside 3 or 4 (6.9 mmol) was co-evaporated with anhydrous toluene (50 mL) in a 3-neck 250 mL round bottom flask and then dried under high vacuum for 18 h. The dried nucleoside was dissolved in dry THF (35 mL) under an argon atmosphere. Triethylamine (24.4 mmol, 3.5 eq) was then added to the reaction mixture and then cooled to about -10 °C. A THF solution of crude chloro reagent (1 M solution, 2.5 equiv, 17.4 mmol) was added to the above mixture via cannula over about 5 minutes and then slowly warmed to room temperature over about 1 hour. LCMS showed that the starting material was consumed. The reaction mixture was carefully filtered under vacuum/argon and the resulting filtrate was concentrated under reduced pressure to give a yellow foam which was further dried under high vacuum overnight. The crude mixture was purified by chromatography on a silica gel column [the column was pre-inactivated with acetonitrile followed by ethyl acetate (5% TEA) then equilibrated with ethyl acetate-hexanes] using ethyl acetate and hexanes as eluents. .

Stereorandom (Rp/Sp) monomer 5: 86% yield. The reaction was performed according to General Procedure B using nucleoside 3 and chloro reagent 2. ³¹ P NMR (243 MHz, CDCl ₃ ) δ 171.62, 155.50, 146.84, 146.17; m/z calcd for MS (ES) C ₃₅ H ₃₉ N ₂ O ₇ PS ₂ [M+K] ⁺ 733.16, found: 733.40 [M + K] ⁺ .

Stereorandom (Rp/Sp) monomer 6: 73% yield. The reaction was performed according to General Procedure B using nucleoside 4 and chloro reagent 2. ³¹ P NMR (243 MHz, CDCl ₃ ) δ 121.87, 106.20, 93.58, 92.99; m/z calcd for MS (ES) C ₃₅ H ₄₀ N ₃ O ₆ PS ₂ [M+K] ⁺ 773.28, found: 773.70 [M + K] ⁺ .

General experimental procedure for PS-PN dimers (7 and 8) (C):

To a stirred solution of monomer 5 or 6 (0.10 mmol, 2 eq, pre-dried by co-evaporation with dry acetonitrile and stored under vacuum for a minimum of 12 hours) in dry acetonitrile (0.5 mL) at room temperature under an argon atmosphere in acetonitrile. (0.2 mL) of 2-azido-1,3-dimethylimidazolinium hexafluorophosphate (0.11 mmol, 2.25 equiv) was added. After stirring the resulting reaction mixture for 10 min, DMTr protected alcohol (0.05 mmol, pre-dried by co-evaporation with dry acetonitrile and stored under vacuum for a minimum of 12 h) in dry acetonitrile (0.25 mL) and 1, 8-Diazabicyclo[5.4.0]undec-7-ene (0.23 mmol, 5 eq, 0.23 ml of a 1 M solution in dry acetonitrile) was added. The reaction was monitored and analyzed by LCMS. Approximate reaction completion time 10-20 minutes.

The reaction was carried out according to General Procedure C using stereorandom dimer 7:5. m/z calcd for MS (ES) C ₆₇ H ₇₂ N ₇ O ₁₄ PS [M+K] ⁺ 1300.42, found: 1300.70 [M + K] ⁺ .

The reaction was carried out according to General Procedure C using stereopure (Rp) dimer 8:6. m/z calcd for MS (ES) C ₆₇ H ₇₃ N ₈ O ₁₃ PS [M+K ^{] +} 1299.44, found: 1299.65 [M + K] ⁺ .

General experimental procedure for PS-PS dimers (9 and 10) (D):

To a stirred solution of monomer 5 or 6 (0.10 mmol, 2 eq, pre-dried by co-evaporation with dry acetonitrile and stored under vacuum for a minimum of 12 h) in dry acetonitrile (0.5 mL) at room temperature under an argon atmosphere in acetonitrile. A solution of 5-phenyl-3H-1,2,4-dithiazol-3-one (0.12 mmol, 2.5 equiv, 0.2 M) in The resulting reaction mixture was stirred for 10 min, then DMTr protected alcohol (0.05 mmol, 1 equiv., pre-dried by co-evaporation with dry acetonitrile and stored under vacuum for a minimum of 12 h) in dry acetonitrile (0.2 mL). and 1,8-diazabicyclo[5.4.0]undec-7-ene (0.23 mmol, 5 eq, 1 M solution in dry acetonitrile). Upon completion of the reaction (monitored by LCMS) the reaction mixture was analyzed by LCMS.

Dimer 9: The reaction was carried out according to General Procedure D using monomer 5. Reaction completion time is about 30 minutes. m/z calcd for MS (ES) ₆₂ H ₆₂ N ₄ O ₁₄ PS ₂ [M] ^- 1181.34, found: 1181.66 [M] ^- .

Dimer 10: The reaction was carried out according to General Procedure D using monomer 6. Reaction completion time is about 20 hours. m/z calcd for MS (ES) C ₆₂ H ₆₃ N ₅ O ₁₃ PS ₂ [M] ^- 1180.36, found: 1180.71 [M] ^- .

Additional useful compounds were prepared as examples:

MOE-G monomer 451: yield 81%. ³¹ P NMR (243 MHz, CDCl ₃ ) δ 175.14, 158.52, 150.30, 148.81; m/z calcd for MS (ES) C ₄₂ H ₅₀ N ₅ O ₉ PS ₂ [M+H] ⁺ 864.29, found: 864.56 [M + H] ⁺ .

OMe-A monomer 452: 92% yield. ³¹ P NMR (243 MHz, CDCl ₃ ) δ 175.65, 159.27, 151.04, 150.10; m/z calcd for MS (ES) C ₄₃ H ₄₄ N ₅ O ₇ PS ₂ [M+H] ⁺ 838.25, found: 838.05 [M + H] ⁺ .

OMe-U monomer 453: 94% yield. ³¹ P NMR (243 MHz, CDCl ₃ ) δ 175.09, 162.04, 154.12, 153.58; m/z calcd for MS (ES) C ₃₅ H ₃₉ N ₂ O ₈ PS ₂ [M+K] ⁺ 749.15, found: 749.06 [M + K] ⁺ .

MOE-5-Me-C monomer 454: yield 91%. ³¹ P NMR (243 MHz, CDCl ₃ ) δ 175.53, 162.04, 153.78, 153.61; m/z calcd for MS (ES) C ₄₅ H ₅₀ N ₃ O ₉ PS ₂ [M+H] ⁺ 872.28, found: 872.16 [M + H] ⁺ .

fG monomer 455: yield 97%. ³¹ P NMR (243 MHz, CDCl ₃ ) δ 176.88 (d), 161.94 (d), 154.16 (d), 152.48 (d); m/z calcd for MS (ES) C ₃₉ H ₄₃ FN ₅ O ₇ PS ₂ [M+H] ⁺ 808.24, found: 808.65 [M + H] ⁺ .

fA monomer 456: yield 99%. ³¹ P NMR (243 MHz, CDCl ₃ ) δ 177.43 (d), 159.63 (d), 149.76 (d), 149.55 (d); m/z calcd for MS (ES) C ₄₂ H ₄₁ FN ₅ O ₆ PS ₂ [M+H] ⁺ 826.23, found: 826.56 [M + H] ⁺ .

dA monomer 457: yield 98%. ³¹ P NMR (243 MHz, CDCl ₃ ) δ 171.85, 154.47, 146.19, 144.48; m/z calcd for MS (ES) C ₄₂ H ₄₂ N ₅ O ₆ PS ₂ [M+K] ⁺ 846.20, found: 846.56 [M + K] ⁺ .

Mor-G monomer 458: 72% yield. ³¹ P NMR (243 MHz, CDCl ₃ ) δ 121.26, 105.98, 93.48, 93.24; m/z calcd for MS (ES) C ₃₉ H ₄₅ N ₆ O ₆ PS ₂ [M+K ^{] +} 827.22, found: 827.60 [M + K] ⁺ .

Mor-A monomer 459: yield 37%. ³¹ P NMR (243 MHz, CDCl ₃ ) δ 121.87, 106.17, 93.23, 93.05; m/z calcd for MS (ES) C ₄₂ H ₄₃ N ₆ O ₅ PS ₂ [M+K] ⁺ 845.21, found: 845.32 [M + K] ⁺ .

Mor-C monomer 460: 68% yield. ³¹ P NMR (243 MHz, CDCl ₃ ) δ 122.34, 106.05, 93.33, 92.6116; m/z calcd for MS (ES) C ₄₁ H ₄₃ N ₄ O ₆ PS ₂ [M+K] ⁺ 821.20, found: 821.54 [M + K] ⁺ .

In some embodiments, the sugar is acyclic. In some embodiments, the present invention provides techniques, eg, reagents (eg, phosphoramidites), conditions, methods, etc., for preparing oligonucleotides comprising cyclic sugars. An example for sm18 is given below.

Certain acyclic morpholine monomers.

The 5′-ODMTr protected morpholino nucleoside (5.05 mmol) was co-evaporated with anhydrous toluene (50 mL) in a 3-neck 100 mL round bottom flask and then dried under high vacuum for 18 h. The dried nucleoside was dissolved in dry THF (25 mL) under an argon atmosphere. Triethylamine (17.6 mmol, 3.5 equiv) was then added to the reaction mixture and then cooled to about -10 °C. A THF solution of crude chloro reagent (1.4 M solution, 1.8 eq, 9.09 mmol) was added to the above mixture via cannula over about 3 minutes and then slowly warmed to room temperature over about 1 hour. LCMS showed that the starting material was consumed. It was then carefully filtered under vacuum/argon and the resulting filtrate was concentrated under reduced pressure to give a yellow foam which was further dried under high vacuum overnight. The crude mixture was purified by chromatography on a silica gel column [column was pre-inactivated with acetonitrile, then ethyl acetate (5% TEA) then equilibrated with ethyl acetate-hexanes] using ethyl acetate and hexanes as eluents. . Yield 66%. ³¹ P NMR (243 MHz, CDCl ₃ ) δ 154.93, 154.65, 154.58, 154.23, 150.54, 150.17, 145.69, 145.26; m/z calcd for MS (ES) C ₃₇ H ₄₆ N ₃ O ₇ PS [M+K ^{] +} 746.24, found: 746.38 [M + K] ⁺ .

To a solution of WV-SM-53a/50a (6 g, 10.70 mmol) in DCM (40 mL) was added Et ₃ N (3.25 g, 32.11 mmol) and MsCl (2.45 g, 21.40 mmol) in DCM (20 mL) to 0 was added at °C. The mixture was stirred at 0 °C for 4 hours. TLC showed that WV-SM-53a/50a was consumed and one new spot was detected. saturate the reaction mixture Quenched with NaHCO ₃ (aq., 50 mL) then extracted with EtOAc (50 mL * 3). The combined organic layers were washed with brine (50 mL), dried over Na ₂ SO ₄ , filtered, and concentrated under reduced pressure to give a residue. Compound 27 (8.0 g, crude) was obtained as a brown oil. TLC petroleum ether: ethyl acetate = 1:3, R _f = 0.50.

Two batches: To a solution of compound 27 (3.42 g, 5.35 mmol) in THF (20 mL) was added methanamine (10 g, 96.60 mmol, 30% purity). The mixture was stirred at 100 °C for 160 hours. LC-MS showed that compound 27 was consumed and one major peak with the desired MS was detected. TLC showed one major spot. The two batches were combined, and the reaction mixture was filtered and concentrated under reduced pressure to give a residue. The residue was purified by MPLC (SiO ₂ , petroleum ether/ethyl acetate = 5:1 to 0:1, 5% TEA). WV-SM-56a (2.9 g, 47.21% yield) was obtained as a yellow solid. ¹ H NMR (400 MHz, chloroform-d) δ = 7.29 - 7.24 (m, 2H), 7.20 - 7.06 (m, 8H), 6.72 (d, J=8.8 Hz, 4H), 6.08 - 5.87 (m, 1H) , 3.71 (s, 6H), 3.58 - 3.42 (m, 1H), 3.19 - 3.05 (m, 1H), 3.05 - 2.91 (m, 1H), 2.83 - 2.75 (m, 1H), 2.72 (d, J= 4.8 Hz, 2H), 2.31 (s, 3H), 1.61 (dd, J=0.9, 5.9 Hz, 3H), 1.36 (d, J=5.9 Hz, 3H), 0.96 - 0.77 (m, 3H). ¹³ C NMR (101 MHz, chloroform-d) δ = 163.71, 163.62, 158.47, 150.74, 150.58, 144.72, 135.94, 135.89, 135.86, 135.25, 135.15, 130.02, 129 .93, 129.89, 127.90 (dd, J=2.9, 22.0 Hz , 1C), 126.83, 126.81, 113.10, 113.08, 111.28, 111.24, 86.45, 86.39, 81.89, 81.82, 81.00, 80.58, 63.39, 63.15, 60.40, 56. 02, 55.23, 34.52, 34.17, 26.41, 23.11, 21.66, 21.59, 15.57, 15.09, 14.20, 12.46, 12.41. HPLC purity: 90.87%. LCMS (M+Na ⁺ ): 596.3. SFC: dr = 52.46: 47.54. TLC (ethyl acetate: methanol = 9:1), R _f = 0.19.

Preparation of compound 2. Two batches: To a solution of compound 1 (50 g, 137.99 mmol) in EtOH (1000 mL) was added NaIO ₄ (30.00 g, 140.26 mmol) in H ₂ O (500 mL). The mixture was stirred at 15° C. in the dark for 2 hours. TLC showed that compound 1 was consumed and one new spot was formed. Compound 2 (99.44 g, crude) was obtained as a white suspension, which was used in the next step. TLC (ethyl acetate: methanol = 9:1), R _f = 0.49.

Preparation of compound 3. Two batches: To a stirred solution of compound 2 (49.72 g, 137.99 mmol) in EtOH (1000 mL) and H ₂ O (500 mL) was added NaBH ₄ (10.44 g, 275.98 mmol) portionwise at 0 °C. The mixture was stirred at 15 °C for 1 hour. TLC showed that compound 2 was consumed and one new spot formed. 1 N HCl was added until pH = 7. Removal of the solvent gave a brown solid. To the solid was added saturated Na ₂ SO ₃ (aq., 500 mL), then extracted with EtOAc (500 mL * 8). The combined organic phases were dried over Na ₂ SO ₄ . Removal of the solvent under reduced pressure gave the product. Compound 3 (86.7 g, 86.22% yield) was obtained as a white solid. LCMS (M+Na ⁺ ) 386.9, purity 96.31%. TLC (ethyl acetate: methanol = 9:1), R _f = 0.38.

Preparation of compound 4. To a solution of compound 3 (86.7 g, 237.96 mmol) and TEA (120.40 g, 1.19 mmol) in DCM (700 mL) was added MsCl (59.97 g, 523.51 mmol) in DCM (300 mL). The mixture was stirred at 0 °C for 4 hours. TLC showed that compound 3 was consumed and two new spots formed. The reaction mixture was quenched by the addition of water (500 mL) and left for 36 hours. TLC showed that the intermediate was consumed and one spot remained. The aqueous layer was extracted with DCM (800 mL * 3). The combined organic layers were dried over Na ₂ SO ₄ , filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO ₂ , petroleum ether/ethyl acetate = 20/1 to 0:1, then MeOH/EtOAc = 0/1 to 1/10). Compound 4 (75 g, 74.26% yield) was obtained as a white solid. TLC (petroleum ether:ethyl acetate = 0: 1), R _f = 0.38; (ethyl acetate : methanol = 9 : 1), R _f = 0.13.

Preparation of compound 5. To a solution of compound 4 (75 g, 176.71 mmol) in DMF (650 mL) was added HI (100.46 g, 353.42 mmol, 59.09 mL, 45% purity). The mixture was stirred at 15 °C for 0.5 h. TLC showed that compound 4 was consumed and one major spot was detected. The reaction mixture was quenched with saturated NaHCO ₃ (aq.) to pH = 7. The residue was extracted with EtOAc (800 mL * 5). The combined organic layers were washed with brine (600 mL), dried over Na ₂ SO ₄ , filtered, and concentrated under reduced pressure to give a residue. Compound 5 (91.15 g, crude) was obtained as a brown oil. TLC (ethyl acetate: methanol = 9:1), R _f = 0.80.

Preparation of compound 6. A mixture of compound 5 (91 g, 164.75 mmol), Pd/C (28 g, 10% purity) and NaOAc (122.85 g, 1.50 mol) in EtOH (700 mL) was degassed and purged with H ₂ three times, The mixture was stirred at 15° C. under H ₂ atmosphere (15 psi) for 24 hours. TLC showed that compound 5 was consumed and one major spot was found. Pd/C was removed by filtration and the filtrate was evaporated. Water (500 mL) was added to the residue and extracted with EtOAc (500 mL*6). The organic layer was then washed with brine (500 mL), dried over Na ₂ SO ₄ , filtered, and concentrated under reduced pressure to give a residue. Compound 6 (76 g, crude) was obtained as a brown oil. TLC (petroleum ether: ethyl acetate = 1:3) R _f = 0.12.

Preparation of compound 7. To a solution of compound 6 (70 g, 164.15 mmol) in MeOH (1000 mL) was added NH ₃ .H ₂ O (1.15 kg, 8.21 mol, 1.26 L, 25% purity). The mixture was stirred at 15 °C for 16 hours. TLC showed that compound 6 was consumed and one new spot was formed. The reaction mixture was concentrated under reduced pressure to remove MeOH, and the aqueous phase was extracted with EtOAc (300 mL*8). The organic phase was dried over Na ₂ SO ₄ , filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO ₂ , petroleum ether/ethyl acetate = 20/1 to 0:1). Compound 7 (33 g, 62.37% yield) was obtained as a white solid. TLC (ethyl acetate: methanol = 9:1), R _f = 0.39.

Preparation of compound 8. To a solution of compound 7 (33 g, 102.38 mmol) in pyridine (120 mL) was added DMTC1 (41.63 g, 122.85 mmol). The mixture was stirred at 15 °C for 4 hours. TLC showed that compound 7 was consumed and one new spot was formed. saturate the reaction mixture Diluted with NaHCO ₃ (aq., 100 mL) and extracted with EtOAc (200 mL * 5). The combined organic layers were dried over Na ₂ SO ₄ , filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO ₂ , petroleum ether/ethyl acetate = 20/1 to 1/5, 5% TEA). Compound 8 (55 g, 86.00% yield) was obtained as a yellow solid. TLC (petroleum ether: ethyl acetate = 0:1) R _f = 0.65.

Preparation of WV-SM-47a. A mixture of compound 8 (55 g, 88.04 mmol), NaOH (42.26 g, 1.06 mol) in DMSO (300 mL) and water (300 mL) was degassed and purged with N ₂ three times, then the mixture was purged under N ₂ atmosphere. Stirred at 90° C. for 16 hours. LCMS and TLC showed compound 8 to be complete and one major peak was found with the desired MS 545 (NEG, MH ⁺ ). The reaction mixture was quenched by the addition of EtOAc (1000 mL), then diluted with H ₂ O (1000 mL) and extracted with EtOAc (1000 mL * 4). The combined organic layers were washed with brine (1000 mL), dried over Na ₂ SO ₄ , filtered, and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, petroleum ether/ethyl acetate = 20/1 to 1/3, 5% TEA). WV-SM-47a (37.5 g, 77.92% yield) was obtained as a white solid. LCMS (MH ⁺ ) 545.3. TLC (petroleum ether: ethyl acetate = 0:1, 5% TEA), R _f = 0.29.

Preparation of compound 9. To a solution of WV-SM-47a (37.5 g, 68.60 mmol) in DCM (400 mL) was added pyridine (81.40 g, 1.03 mmol, 83.06 mL) and Dess-Martin periodinane (34.92 g, 82.33 mmol). The mixture was stirred at 20 °C for 4 hours. LC-MS showed that WV-SM-47a was completely consumed and a new peak with the desired MS was detected. The reaction mixture was quenched by addition of saturated NaHCO ₃ (aq., 1000 mL) and 1000 mL of saturated Na ₂ SO ₃ (aq.), then extracted with EtOAc (100 mL * 5). The combined organic layers were washed with brine (500 mL), dried over Na ₂ SO ₄ , filtered, and concentrated under reduced pressure to give a residue. Compound 9 (43 g, crude) was obtained as a yellow solid. LCMS (MH ⁺ ) 543.3.

Preparation of WV-NU-53a and WV-NU-50a. To a solution of compound 9 (37.36 g, 68.60 mmol) in THF (300 mL) was added MeMgBr (3 M, 68.60 mL) at -40 °C. The mixture was stirred at -40-15 °C for 6 hours. LC-MS showed that compound 9 was completely consumed and a new peak with mass was detected. The reaction mixture was quenched by the addition of 0 °C water (20 mL) and extracted with EtOAc (300 mL* 3). The combined organic layers were washed with brine (200 mL), dried over Na ₂ SO ₄ , filtered, and concentrated under reduced pressure to give a residue. TLC showed one major spot. The residue was purified by column chromatography (SiO2, petroleum ether/ethyl acetate = 20/1 to 0/1, 5% TEA). 6 g of the residue was subjected to SFC (column: DAICEL CHIRALPAK AD-H (250 mm*30 mm, 5 um); mobile phase: [0.1% NH ₃ H ₂ O IPA]; B%: 39%-39%, 9.33 min. ) was purified. And crude WV-SM-50a was preparative HPLC (column: Agela Durashell 10u 250*50 mm; mobile phase: [water (0.04%NH ₃ H ₂ O)-ACN]; B%: 37%-56%, 20 min) was purified. WV-SM-53a (1.4 g, 23.33% yield) was obtained as a white solid. WV-SM-50a (1.8 g, 30.00% yield) was obtained as a white solid. 0.5 g of WV-SM-53a: ¹ H NMR (400 MHz, chloroform-d) δ = 7.37 - 7.30 (m, 2H), 7.28 - 7.18 (m, 8H), 7.12 (d, J = 1.1 Hz, 1H) , 6.80 (d, J=8.6 Hz, 4H), 6.08 (q, J=5.8 Hz, 1H), 4.09 - 3.99 (m, 1H), 3.79 (d, J=0.9 Hz, 6H), 3.51 (q, J=5.0 Hz, 1H), 3.20 - 3.05 (m, 2H), 2.70 (q, J=7.1 Hz, 2H), 1.71 (d, J=1.1 Hz, 3H), 1.46 (d, J=6.0 Hz, 3H), 1.14 - 1.10 (m, 3H). ¹³ C NMR (101 MHz, chloroform-d) δ = 163.19, 158.54, 150.48, 144.39, 135.53, 134.91, 129.86, 129.81, 127.90, 127.86, 126.93, 113.15, 111 .48, 86.73, 81.44, 81.24, 68.14, 63.45, 55.22, 45.74, 21.45, 18.01, 12.43. HPLC purity: 99.04%. LCMS (MH ⁺ ): 559.0. SFC dr = 99.83: 0.17. TLC (petroleum ether: ethyl acetate = 1:3), R _f = 0.28. 0.9 g of WV-SM-53a: ¹ H NMR (400 MHz, chloroform-d) δ = 7.36 - 7.30 (m, 2H), 7.29 - 7.15 (m, 9H), 7.13 (s, 1H), 6.80 (d, J=8.8 Hz, 4H), 6.08 (q, J=6.0 Hz, 1H), 4.11 - 3.97 (m, 1H), 3.79 (s, 6H), 3.51 (q, J=4.9 Hz, 1H), 3.13 ( dq, J=5.3, 10.1 Hz, 2H), 1.72 (s, 3H), 1.47 (d, J=6.2 Hz, 3H), 1.10 (d, J=6.4 Hz, 3H). ¹³ C NMR (101 MHz, chloroform-d) δ = 163.19, 158.54, 150.47, 144.39, 135.50, 134.92, 129.86, 129.81, 127.89, 127.87, 126.94, 113.15, 111 .48, 86.73, 81.44, 81.25, 68.14, 63.45, 55.22, 45.19, 21.46, 18.02, 12.44. HPLC purity: 97.56%. LCMS (MH ⁺ ): 559.1, purity 92.9%. SFC dr = 98.49: 1.51. 1.75 g of WV-SM-50a: ¹ H NMR (400 MHz, chloroform-d) δ = 8.41 (s, 1H), 7.35 - 7.31 (m, 2H), 7.26 - 7.19 (m, 7H), 7.11 (d, J=1.3 Hz, 1H), 6.82 - 6.77 (m, 4H), 6.00 (q, J=5.7 Hz, 1H), 4.09 - 4.00 (m, 1H), 3.79 (d, J=0.9 Hz, 6H), 3.51 - 3.44 (m, 1H), 3.22 (dd, J=5.3, 10.1 Hz, 1H), 3.02 (dd, J=5.3, 10.1 Hz, 1H), 2.20 (br s, 1H), 1.72 (d, J =0.9 Hz, 3H), 1.47 (d, J=6.1 Hz, 3H), 1.17 (d, J=6.6 Hz, 3H). ¹³ C NMR (101 MHz, chloroform-d) δ = 163.29, 158.50, 150.43, 144.40, 135.55, 135.45, 134.86, 129.88, 129.84, 127.93, 127.84, 126.94, 113 .12, 111.46, 86.55, 82.48, 82.43, 67.59, 63.24, 55.22, 21.40, 19.17, 12.43. HPLC purity: 96.51%. LCMS (MH ⁺ ): 559.2, purity 93.04%. SFC dr = 0.88: 99.12.

Example 3. Preparation of oligonucleotides and compositions.

For example, US 9982257, US 20170037399, US 20180216108, US 20180216107, US 9598458, WO 2017/062862, WO 2018/067973, WO 2017/160741, WO 2017/192679, WO 2017/210647, WO 2018/098264, WO 2018/223056, WO 2018/237194, WO 2019/032607, WO 2019/055951, WO 2019/075357, WO 2019/200185, WO 2019/217784, WO 2019/032612, WO 2020/1912 52, and/or WO 2021/ A variety of techniques for preparing oligonucleotides and oligonucleotide compositions (both stereorandom and chiral controlled) can be used in accordance with the present invention, including the methods and reagents described in 071858, each of which methods and reagents are disclosed herein. included by reference. Many oligonucleotides and compositions thereof, including the various oligonucleotides and compositions of Table 1, have been prepared and evaluated and found to provide various activities, such as adenosine editing.

Certain useful cycles are described below as examples for oligonucleotide preparation.

Each B is independently a nucleobase such as BA described herein (eg, A, C, G, T, U, etc.). Each B ^PRO is independently an optionally protected nucleobase such as a BA described herein (eg, ^Abz , C ^ac , G ^ibu , T, U, etc. suitable for oligonucleotide synthesis). As shown, a variety of linkages can be constructed to link monomers to nucleosides or oligonucleotides, including those on solid supports. As will be appreciated by those skilled in the art, these cycles can be used to couple monomers to the —OH of a variety of other types of sugars.

In some embodiments, the formulation comprises one or more DPSE and/or PSM cycles.

A number of oligonucleotide compositions have been synthesized and evaluated. The MS data of the oligonucleotides observed in some of the oligonucleotide compositions prepared are as follows (if multiple numbers are shown for the same oligonucleotide, the numbers may be MS data observed in different batches/experiments): WV-20666 :10167.1; WV-20689: 10183; WV-20690: 10198.4; WV-20691: 10215.3; WV-20692: 10230.3; WV-20693: 10246.5; WV-20694: 10262.7; WV-20695: 10278.9; WV-20696: 10294.3; WV-20697: 10311.3; WV-20698: 10327; WV-20699: 10342.9; WV-20700: 10358.5; WV-20701: 10376; WV-20702: 10391.1; WV-20703: 10407.5; WV-20704: 10423.6; WV-20706: 10199; WV-20707: 10215.3; WV-20708: 10230.6; WV-20709: 10246.5; WV-20710: 10262.6; WV-20711: 10279.3; WV-20712: 10294.2; WV-20713: 10310.8; WV-20714: 10327; WV-20715: 10342.9; WV-20716: 10358.7; WV-20717: 10246.3; WV-20718: 10262.7; WV-20719: 10278.3; WV-20720: 10294.2; WV-20721: 10311.4; WV-20722: 10327.1; WV-20723: 10342.8; WV-20724: 10358.7; WV-20725: 10374.8; WV-20726: 10391; WV-20727: 10182.9; WV-20728: 10182.7; WV-20729: 10182.7; WV-20730: 10182.9; WV-20731: 10230.8; WV-20732: 10199.1; WV-20733: 10663.7; WV-20734: 10194.7; WV-20735: 10222.7; WV-20736: 10250.5; WV-20737: 10278.3; WV-20738: 10306.7; WV-20739: 10334.8; WV-20740: 10362.9; WV-20741: 10194.8; WV-20742: 10208.5; WV-20743: 10236.8; WV-20744: 10263.9; WV-20745: 10293.1; WV-20746: 10320.4; WV-20747: 10093.9; WV-20748: 10098.1; WV-20749: 10101.9; WV-20750: 10106.4; WV-20751: 10110.5; WV-20752: 10113.5; WV-20753: 10118.3; WV-20754: 10122.6; WV-20755: 10098; WV-20756: 10100; WV-20757: 10104.3; WV-20758: 10107.7; WV-20759: 10111.8; WV-20760: 10116.7; WV-23388: 10098; WV-23395: 10612.3; WV-24111: 10046.8; WV-24112: 10047; WV-24113: 10047; WV-24114: 10046.8; WV-24115: 10047; WV-24116: 10046.8; WV-24117: 10046.9; WV-24118: 10046.8; WV-24119: 10046.9; WV-24120: 10047.1; WV-24121: 10047; WV-24122: 10047.1; WV-24123: 10047; WV-24124: 10047; WV-24125: 10046.9; WV-24126: 10046.9; WV-24127: 10047; WV-24128: 10046.5; WV-24129: 10047; WV-24130: 10046.8; WV-24131: 10046.8; WV-24132: 10047; WV-24133: 10047.1; WV-24134: 10047; WV-24135: 10047; WV-24136: 10046.9; WV-24137: 10047.1; WV-24138: 10047; WV-24139: 10046.8; WV-24140: 10046.4; WV-24141: 10046.9; WV-24142: 10047; WV-24143: 10047.1; WV-24144: 10047; WV-24145: 10047.1; WV-24146: 10046.9; WV-24147: 10046.7; WV-24148: 10047; WV-24149: 10047; WV-24150: 10047.1; WV-24151: 10047.1; WV-24152: 10047.1; WV-24153: 10047.1; WV-24154: 10047.1; WV-24155: 10047.1; WV-24156: 10046.7; WV-24157: 10047; WV-24158: 10047.1; WV-27457: 12613.1; WV-27458: 11954.6; WV-27459: 12631; WV-27460: 11972.7; WV-27521: 10064.1; WV-31133: 10737.8; WV-31134: 10869.1; WV-31135: 10790.3; WV-31137: 10779.4; WV-31138: 10788.2; WV-31139: 10039.1; WV-31140: 10168.8; WV-31141: 10091.0; WV-31143: 10079.0; WV-31144: 10089.6; WV-31632: 10772.7; WV-31633: 10786.6; WV-31634: 10072.7; WV-31635: 10087.2; WV-31748: 10762.5; WV-31749: 10064.4; WV-28788: 10169.1; WV-27458: 11954.6; WV-31940: 10285.5; WV-35741: 12352.0; WV-42028: 10252.9 (calculated 10254.9); WV-42680: 10293.4 (calculated 10294.9); WV-44278: 10326.5 (calculated 10329); WV-44279: 10331.2 (calculated 10333); WV-44280: 10346.3 (calculated 10348); WV-44281: 10266.5 (calculated 10268.9); WV-44282: 10195.8 (calculated 10197.9); WV-44283: 10200.1 (calculated 10201.8); WV-44284: 10135.4 (calculated 10137.8); WV-44285: 10368.2 (calculated 10369); WV-44286: 10307.1 (calculated 10308.9); WV-44287: 10235 (calculated 10237.9); WV-44288: 10175.9 (calculated 10177.8); WV-44327: 10398.4 (calculated 10399.1); WV-44328: 10357.7 (calculated 10359.1). Many others have also been fabricated, characterized and evaluated. See, for example, those in the drawings.

As described and identified herein, the techniques of the present invention are useful for preparing a variety of compositions of oligonucleotides comprising a variety of structural features. In some embodiments, as identified herein, a chiral auxiliary comprising an electron withdrawing group (eg, an electron withdrawing group (eg, -SO ₂ R ^C1 , -C(O) A technique using R ^C11 containing R ^C1 ) is the chiral control of oligonucleotides containing 2'-OH sugars (e.g., sugars where R ^2s = OH, such as those commonly found in natural RNA). It is particularly useful for preparing compositions (particularly when such sugars are linked to chirally controlled internucleotidic linkages). The preparation of WV-29874 is described below as an example.

Automated solid phase synthesis of a 25 umol scale chirally controlled oligonucleotide composition (WV-29874) was performed according to the cycle below.

IBN: isobutyronitrile; MeIm: N -methylimidazole; PhIMT: N -phenylimidazolium triflate; XH: xanthan hydride. The cycle was performed several times until the desired length was reached. PSM phosphoramidites were used for formation of chirally controlled internucleotidic linkages (protected with t-butyldimethylsilyl (TBS) in the case of 2'-OH).

After completion of the synthesis cycle, the PSM chiral auxiliary group was removed by anhydrous base treatment (DEA treatment). CPG was treated with 40% MeNH ₂ (5.0 mL) at 35° C. for 30 min, then cooled to room temperature and CPG was isolated by membrane filtration and washed with 8.0 mL of DMSO. TEA (triethylamine) -3HF (5.0 mL) was added to the filtrate and stirred at 45 °C for 1 hour, which can remove the TBS protecting group from 2'-OH. The reaction mixture was cooled to room temperature and diluted with 10 mL of 50 mM NaOAc (pH 5.2). The crude material was analyzed by LTQ and RP-UPLC. The crude material was purified by RP-HPLC using a linear gradient of MeCN in 50 mM TEAA (triethylammonium acetate) and desalted with a tC18 SepPak cartridge to obtain the target oligonucleotide.

Desalting was performed using the following procedure.

MeCN, if present, is evaporated from the sample.

Condition the column with 4 CV of 100% acetonitrile (HPLC grade).

The column is rinsed with 2 CV of 40% MeCN in endotoxin-free Millipore Bio-Pak water.

Rinse the column with 4 CV of water (Millipore Bio-Pak, endotoxin free).

The column is equilibrated with 2 CV of 50 mM TEAA in endotoxin-free Millipore Bio-Pak water.

The pure fraction is loaded onto the equilibration column. In some embodiments, loading by gravity provides the greatest amount of bonding, slow loading into vacuum provides adequate bonding, and fast loading into vacuum results in weak bonding.

Wash the column with 2 CV of BioPak water to wash out TEAA.

The column is washed with 2 CV of 100 mM NaOAc to exchange the ammonium on the backbone of the oligonucleotide for sodium instead.

Wash the column with BioPak water until the conductivity of the eluent is <20 uS/cm.

The product is eluted with 2 column volumes of 40% MeCN in endotoxin-free Millipore Bio-Pak water.

Place in a speed-vac overnight at 30°C to remove acetonitrile and concentrate.

Results from one preparation: Scale of synthesis: 25 umol; Crude ODs: 874 ODs; Crude UPLC purity: 32.17%; Crude LTQ purity: 62.45%; Final ODs: 59.8 OD; Final UPLC purity: 59.85%; Final MS purity: 74.51%; and final found MS: 10064.4 (calculated 10,063.68).

In accordance with the present invention, a number of techniques are available to those skilled in the art to prepare the oligonucleotides and compositions of the present invention.

For example, various chirally controlled oligonucleotide compositions were prepared. A specific useful procedure is described below as an example. In some embodiments, the oligonucleotide comprises a mixed PS (phosphorothioate)/PO (natural phosphate linkage)/PN (phosphorylguanidine internucleotide linkage, such as n001) backbone. Oligonucleotides with varying numbers of PS/PO/PN linkages (eg see Table 1) were prepared using techniques according to the present invention. For example, in some embodiments, phosphodiester (PO) linkages are formed using cyanoethyl amidite, and phosphorothioate (PS) linkages ( S p and R p; in some embodiments, both S p) was formed using DPSE chiral amidites, and phosphoroamidate linkages (PN; e.g., n001) ( S p and R p) linkages were formed using PSM amidites. Oligonucleotides typically contain a variety of sugar modifications such as 2'-modifications such as 2'-OMe, 2'-F and 2'-MOE, etc. (eg see Table 1). In some embodiments, the oligonucleotide comprises additional moieties such as, for example, a triantennary or GalNAc moiety at the 5'-end. To introduce a GalNAc moiety at the 5'-end, in some embodiments an oligonucleotide is synthesized by coupling with a C-6 amino modifier as a final coupling cycle and, after purification and desalting, conjugated with the tri-antennary GalNAc A conjugate was prepared.

Exemplary Procedure for Preparation of Oligonucleotide Compositions (25 μmol Scale)

For chiral controlled PS linkages, DPSE amidite was used, and for chiral controlled PN linkages such as n001, PSM amidite was used. Automated solid phase synthesis of oligonucleotides was performed according to the cycles presented below: regular amidite cycle for PO linkages, DPSE amidite cycle for chiral controlled PS linkages, PSM army for chiral controlled PN linkages such as n001 diet cycle.

regular amidite synthesis cycle

DPSE amidite synthesis cycle

PSM amidite synthesis cycle

In some embodiments, oligonucleotides were synthesized by coupling with a C-6 amino linker as a final coupling cycle to introduce a GalNAc moiety at the 5'-end.

Example procedure for cleavage and deprotection (25 μmol scale)

After completion of the cycle, the CPG support was subjected to a 5 column volume/20% diethylamine/acetonitrile wash step for 15 minutes, followed by an ACN wash cycle. The CPG solid support was dried, transferred to a 50 mL plastic tube, treated with 1X desilylation reagent (2.5 mL; 100 μL/umol) for 3 h at 28°C, and concentrated NH ₃ (5.0 mL; 200 μL/umol) at 37 °C. C for 24 hours. The reaction mixture was cooled to room temperature and CPG was separated by membrane filtration and washed with 15 mL of H ₂ O. The crude material (filtrate) was analyzed by LTQ and RP-UPLC. For certain oligonucleotides conjugated with other additional chemical moieties such as GalNac, oligonucleotides containing suitable reactive groups such as amino groups were purified by ion exchange chromatography on an AKTA pure system using a sodium chloride gradient. The desired product was desalted and further conjugated with a GalNAc containing acid. After the conjugation reaction is confirmed to be complete, the material is further purified by ion exchange chromatography and desalted using tangential flow filtration (TFF) to yield the desired product (e.g., WV-46312, WV-47606, WV-47608, WV-49085, WV-49086, WV-49087, WV-49088, WV-49089, WV-49090, WV-49092, WV-47603, WV-47604, WV-47605, WV-47607, WV-47609, WV- Various oligonucleotide compositions in Table 1 including 49091, WV-49093, WV-48453, WV-48454, etc.) were obtained.

For example, WV-47595 was prepared and then spliced to make WV-46312. A useful synthesis procedure is described below by way of example.

In preparation, synthesis of WV-47595 was performed on an AKTA OP100 synthesizer (GE healthcare) using a 3.5 cm diameter fineline column with a 1200 μmol scale using CPG support (loading 72 μmol/g). A specific synthesis cycle included five steps: detritylation, coupling, capping 1 (cap-1), oxidation/sulfation/imidation and capping 2 (cap-2).

Detritylation: Detritylation was performed using 3% DCA in toluene with the UV monitoring command set at 436 nm. After detritylation, the CPG scaffold was subjected to washing cycles using 2CV of acetonitrile.

Coupling: DPSE and PSM chiral amidites were prepared at 0.2 M concentration (either in ACN or at 20% IBN in ACN). The amidite was mixed in-line with the CMIMT activator (0.5 M in acetonitrile) at a ratio of 5.83 prior to addition to the column. After recycling the coupling mixture for 10 minutes to maximize the coupling efficiency, a column wash was performed with 2CV of ACN. Cyanoethyl amidite was prepared at a concentration of 0.2 M (either in ACN or at 20% IBN in ACN). The amidite was mixed in-line with the ETT activator (0.5 M in acetonitrile) at a ratio of 4.07 before being added to the column. After recycling the coupling mixture for 10 minutes to maximize the coupling efficiency, a column wash was performed with 2CV of ACN.

Capping 1: For steric coupling, the column was treated with a Capping 1 solution capable of acetylating chiral auxiliary amines (acetic anhydride, lutidine, ACN) at 1 CV for 2 minutes. After this step, the column was washed with 1.5 CV of acetonitrile. In the case of stereorandom coupling, capping 1 was not performed.

Sulfation/Imidation/Oxidation Step: Sulfation was performed with 0.1 M xanthan hydride in pyridine/acetonitrile (1.2 eq.) with a contact time of 6 min followed by a 2CV wash step. Imidation was performed with 0.3 M ADIH reagent in acetonitrile with 18 equivalents and a contact time of 15 minutes followed by a 2CV wash step. The oxidation step was performed using an oxidation reagent (50 mM I ₂ / pyridine-H ₂ O (9: 1, v/v)) at 3.5 equivalents for 2.5 minutes, followed by 2CV acetonitrile washing.

Capping 2: Capping 2 step was performed using in-line (1:1) mixed Capping A and Capping B reagents (eg see cap-2) followed by a 2 CV ACN wash.

After completion of the synthesis, the CPG support was subjected to a final 5 column volume/20% diethylamine/acetonitrile wash step for 15 minutes, followed by an ACN wash cycle. The CPG solid support was dried and transferred to a pressure vessel. DPSE was removed by treating the scaffold with desilylation reagent at a ratio of μmole support/100 μL desilylation reagent. A desilylation reagent was prepared by mixing DMSO:water:TEA:TEA 3HF in a ratio of 7.33:1.47:0.7:0.5. The CPG scaffold was incubated in the presence of desilylation reagent for 3 hours at 27° C. on an incubator shaker. Concentrated ammonia was then added at a ratio of μmole support/200 μL concentrated ammonia. The mixture was incubated at 37° C. for 24 hours and shaken. The mixture was cooled and filtered using a 0.2-0.45 micron filter and the CPG support was rinsed 3 times to collect all desired material as a filtrate. The filtrate containing the crude oligonucleotides was analyzed by RP-UPLC and quantified using a Nanodrop One spectrophotometer (Thermo Scientific) to give a yield of 110,000 OD/μmole.

Purification and Desalting: Crude oligonucleotides were loaded onto a Waters AP-2 glass column (2.0 cm x 20 cm) packed with Source 15Q (Cytiva). Purification was performed in AKTA150 Pure (GE Healthcare) using the following buffer: (Buffer A: 20 mM NaOH, 20% acetonitrile v/v) (Buffer B: 20 mM NaOH, 2.5 M NaCl, 20% acetonitrile v /v). The desired fractions containing the full length product in the range of 70-80% were pooled together. The pooled material was then desalted on a 2KD regenerated cellulose membrane followed by lyophilization to yield oligonucleotides as a fluffy white cake ready for conjugation.

Preparation of WV-46312: A variety of techniques for conjugating oligonucleotides with other moieties can be used in accordance with the present invention. A useful protocol for GalNAc conjugation is described below as an example. Pre-bonded material: WV-47595.01 (.01 indicates batch number). Produced substance: WV-46312.01.

Tri-antennae GalNAc acid (hydroxyl group protected with -OAc) and HATU were weighed in a 50 mL plastic tube and dissolved in anhydrous acetonitrile, then DIEA was added to the tube. The resulting mixture was stirred at 37° C. for 10 minutes. The lyophilized WV-47595 was reconstituted with water in a separate tube and the GalNAc mixture was added to the oligonucleotide solution and stirred at 37°C for 60 minutes. The reaction was monitored by RP-UPLC. The reaction was found to be complete within 1 hour. The reaction mixture was concentrated under vacuum to remove acetonitrile and the resulting GalNAc conjugated oligonucleotides were treated with concentrated ammonia at 37° C. for 2 hours. Formation of the final product was confirmed by mass spectrometry and RP-UPLC. The conjugated material was purified by anion exchange chromatography and desalted using tangential flow filtration (TFF) to obtain the final product (target mass: 121±0.65; observed mass: 12112.3). Various oligonucleotides and compositions were prepared using similar procedures.

Example 4. Provided technology may provide products with improved properties and/or activities.

As described herein, in some embodiments, provided techniques can correct mutations and provide improved or restored levels, properties, and/or activities of various products, such as proteins. For example, in some embodiments, provided technologies correct mutations and provide proteins, eg, wild-type proteins, with improved or restored levels, properties, and/or activities. In some embodiments, a provided technique provides an increased amount of a protein of interest, eg, a protein with improved properties and/or activity, compared to the corresponding protein prior to administration of the provided technique (eg, oligonucleotide, composition, etc.). level was provided. In some embodiments, provided technologies provide increased levels of wild-type protein. In some embodiments, provided technologies provide increased levels of properly folded proteins. Among other things, the present invention provides data confirming these various benefits using, for example, the compilation of SERPINA1's 1024 G>A.

In some embodiments, provided techniques are evaluated using cells, tissues, or animals comprising the 1024 G>A mutation in human SERPINA1. In some embodiments, the animal is a NOD.Cg-Prkdcscid Il2rgtm1Wjl Tg(SERPINA1*E342K)#Slcw/SzJ mouse (eg, The Jackson Laboratory Stock No: 028842; NSG-PiZ, also Borel F; Tang Q; Gernoux G; Greer C; Wang Z; Barzel A; Kay MA; Shultz LD; Greiner DL; Flotte TR; Brehm MA; Mueller C. 2017. Survival Advantage of Both Human Hepatocyte Xenografts and Genome-Edited Hepatocytes for Treatment of alpha-1 Antitrypsin Deficiency.Mol Ther 25(11):2477-2489PubMed:29032169MGI:J:243726] and Li S;Ling C;Zhong L;Li M;Su Q;He R;Tang Q;Greiner DL; Shultz LD; Brehm MA; Flotte TR; Mueller C; Srivastava A; Gao G. 2015. Efficient and Targeted Transduction of Nonhuman Primate Liver With Systemically Delivered Optimized AAV3B Vectors. Mol Ther 23(12):1867-76 PubMed: 26403887MGI: J: 230567]). In some embodiments, cells, tissues or organs from such animals are used to evaluate a given technique.

In some embodiments, primary murine hepatocytes are plated into wells of a 96-well plate, and one plate is irradiated for each time point. After an appropriate period of time, eg, 24 hours, the oligonucleotide composition is administered and, in some embodiments, the cells are cultured with 25 nM of the final oligo using a suitable technique, eg, RNAiMAX according to the manufacturer's instructions. Nucleotide concentrations were transfected with oligonucleotide compositions. Media was collected for protein analysis (e.g., using ELISA), and cells were collected at an appropriate time point, e.g., 120 hours, for RNA editing analysis (e.g., RNA lysis buffer for subsequent sequencing (Promega )at).

ELISA. In some embodiments, A1AT protein concentration is assessed using an A1AT ELISA assay, eg, the Abcam -ab108799 assay, according to the manufacturer's instructions. In some embodiments, standards were generated using recombinant A1AT protein diluted to 25 ng/ml in diluent and serially diluted 2-fold for 7 points. The cell culture medium was removed by centrifugation at 3000 g for 10 minutes before diluting 1-400 in diluent. Prepared standards and diluted culture medium were added to the wells of a SERPINA1 antibody coated and blocked 96-well plate and incubated for 2 hours at room temperature. After washing the plate 6 times (300 uL/well) with the provided ELISA wash buffer, the biotinylated SERPINA1 antibody was diluted to 1X in diluent and added to each well for 1 hour at room temperature. Wells were washed as described above and streptavidin-peroxidase complex diluted 1X in diluent was added to each well for 30 minutes at room temperature. The wells were washed one last time before 3,3′,5,5′-tetramethylbenzidine (TMB) was added to each well and the plates were allowed to develop for 20 minutes before stop solution was added. Plates were then read at 450 nm and 570 nm. Plates were quantified by subtracting the 570 nm reading from the 450 nm reading to account for optical defects. Specific data is presented in FIG. 1 .

As shown in FIG. 1 , the provided technology can provide for editing of mutations associated with a condition, disorder or disease (eg, the PiZ mutation in SERPINA1(SA1)). Among other things, provided techniques may not only provide editing at the RNA level, but may also provide proteins of improved levels, properties, and/or activity. For example, as shown in FIG. 1 , provided technologies, in addition to RNA editing, can provide increased levels of secreted proteins (e.g., WV-38621 compared to non-targeting (NT) control WV-37317, WV-38622 and WV-38630), which have improved folding and/or higher activity compared to proteins encoded by unedited RNA (e.g., proteins comprising the E342K mutation from the 1024G>A mutation). May contain protein. As will be appreciated by those skilled in the art, the level, nature and/or activity of comprising a sequence can also be assessed using other techniques such as mass spectrometry. In some embodiments, LC-MS based proteomics techniques are used to quantify A1AT protein (eg, wild-type and/or mutant proteins (eg, encoded by RNA with or without editing)).

Example 5. Various oligonucleotide compositions can provide editing.

A variety of oligonucleotides were designed and evaluated. Certain oligonucleotides target the PIZ target site. An oligonucleotide has a majority of 2'-F modified sugars in domain (5') and a majority of 2'-OMe modified sugars in another domain (3'), or a majority of 2'-OMe modified sugars in domain (5'). It was designed to have most of the 2'-F in the domain (5') different from the OMe modification. The oligonucleotide composition was then screened in 293T or ARPE19 cells stably expressing the SERPINA1-PIZ allele. As shown in (a) and (b) of Figure 2, the 5'- and / or 3'-side length of the nucleoside opposite to the specific sequence and / or target adenosine (eg, C) Certain oligonucleotide compositions provide higher levels of editing. In some embodiments, WV-42028 and WV-42029 provided higher editing levels than WV-42027 in all three cell lines 293T-SERPINA1-ADAR1-p110p110, 293T-SERPINA1-p150 and ARPE19-SERPINA1. In some embodiments, as shown in Figures 2(a) and (b), when an editing site moves from one domain to another, the surrounding 2' chemistry (eg, per 2'-OMe modification) is changed. Shifting can improve editing efficiency. In some embodiments, when the editing site is in domain (5'), a domain comprising multiple 2'-OMe modified sugars (and optionally another domain comprising multiple 2'-F modified sugars) It has been observed that this can be helpful.

Example 6. Various oligonucleotide compositions can provide editing.

A variety of oligonucleotides were designed and evaluated. Certain oligonucleotides target the PIZ target site. The oligonucleotide was designed to contain an 8-oxo-dA base modification in domain (3'). Oligonucleotides were then screened in 293T or SF8628 cells stably expressing the SERPINA1-PIZ allele. In some embodiments, WV-42680 and WV-42681 provided higher editing levels than WV-42679 in all three cell lines 293T-SERPINA1-ADAR1-p110, 293T-SERPINA1-p150 and SF8628-SERPINA1 (FIG. 3) .

Example 7. Various oligonucleotide compositions can provide editing.

Oligonucleotides containing various modified nucleobases (eg b008U) and/or various types of sugars (eg DNA sugars, RNA sugars, etc.) at or around the editing site were designed and evaluated around the PIZ target site. Oligonucleotide compositions were screened in 293T or SF8628 cells stably expressing the SERPINA1-PIZ allele. In some embodiments, WV-38621, WV-38622, WV-28923, WV-42328, WV-38629, WV-38630 and WV-42327 are 293T-SERPINA1-ADAR1-p110, 293T-SERPINA1-p150 and SF8628-SERPINA1 provided a higher editing level than WV-38620 (FIG. 4).

Example 8. Various oligonucleotide compositions can provide editing.

containing various base sequences, modified sugars (e.g. 2'-F, 2'-OMe, etc.) and/or modified internucleotide linkages (e.g. neutral internucleotide linkages such as n001, phosphorothioate internucleotide linkages, etc.) Oligonucleotides were designed and evaluated. Certain oligonucleotides target the PIZ target site. Oligonucleotides were administered via GalNAc-mediated uptake at multiple dose concentrations in primary mouse hepatocytes expressing human SERPINA1-PIZ. As shown in Figure 5, various oligonucleotides can provide editing activity. In some embodiments, as shown in Figure 5, the addition of one or more 2'F modified sugars, for example in domain-2 (3'), can increase editing efficiency.

Example 9. Various oligonucleotide compositions can provide editing.

Various types of sugars and modified internucleotide linkages, including modified sugars (e.g., 2'-F, 2'-OMe, etc.) (e.g., non-negatively charged internucleotidic linkages such as n001, phosphorothioates) oligonucleotides containing internucleotide linkages) were designed and evaluated. Certain oligonucleotides target the PIZ target site. Oligonucleotides were administered via zymotic uptake at multiple dose concentrations in primary mouse hepatocytes expressing human SERPINA1-PIZ. In some embodiments, as shown in Figure 6, various oligonucleotides can provide editing activity.

Example 10. Various oligonucleotide compositions can provide editing.

Various types of sugars, including modified bases (e.g., 8-oxo-dA), modified sugars (e.g., 2'-F, 2'-OMe, etc.), and/or modified internucleotide linkages (e.g., negative Oligonucleotides containing uncharged internucleotidic linkages, phosphorothioate internucleotidic linkages, etc.) were designed and evaluated. Certain oligonucleotides target the PIZ target site. Oligonucleotides were administered via zymotic uptake in primary mouse hepatocytes expressing human SERPINA1-PIZ. In some embodiments, WV-42680, WV-42935 and WV-42938 exhibited greater editing efficiency compared to WV-42028. In some embodiments, as shown in FIG. 7 , modified bases (eg, 8-oxo-dA), specific sugars (eg, arabino-cytidine), or combinations thereof increase editing efficiency. can represent

Example 11. Various oligonucleotide compositions can provide editing.

Various types of sugars, including modified bases (e.g., 8-oxo-dA), modified sugars (e.g., 2'-F, 2'-OMe, etc.), and/or modified internucleotide linkages (e.g., negative Oligonucleotides containing uncharged internucleotidic linkages, phosphorothioate internucleotidic linkages, etc.) were designed and evaluated. Certain oligonucleotides can target the PIZ target site. Oligonucleotides were administered in primary mouse hepatocytes expressing human SERPINA1-PIZ by zymnautic uptake. In some embodiments, WV-42680 and WV-42028 showed higher editing levels compared to WV-42679 and WV-42027. In some embodiments, shifting the target sequence by 1 nt, as shown in FIG. 8, can increase editing efficiency. In some embodiments, inclusion of a modified base (eg, 8-oxo-dA) can increase editing efficiency.

Example 12. Various oligonucleotide compositions can provide editing.

Various types of sugars, including modified bases (e.g., 8-oxo-dA), modified sugars (e.g., 2'-F, 2'-OMe, etc.), and/or modified internucleotide linkages (e.g., negative Oligonucleotides containing uncharged internucleotidic linkages, phosphorothioate internucleotidic linkages, etc.) were designed and evaluated. Certain oligonucleotides target the PIZ target site. Oligonucleotides were tested in primary mouse hepatocytes expressing human SERPINA1-PIZ by zymotic uptake. In some embodiments, WV-43112, WV-431113 and WV-43114 exhibited higher editing levels compared to WV-42680. See Figure 9. In some embodiments, adding a 2'-F modified sugar to an oligonucleotide, for example in domain-2(3'), can increase editing activity. In some embodiments, addition of a 2'-OMe modified sugar at the 5' end can improve editing efficiency.

Example 13. Various oligonucleotide compositions can provide editing.

Various types of sugars, including modified bases, modified sugars (e.g. 2'-F, 2'-OMe, etc.), and/or modified internucleotide linkages (e.g. non-negatively charged internucleotidic linkages such as n001, phospho Oligonucleotides containing porothioate internucleotidic linkages, etc.) were designed and evaluated. Certain oligonucleotides can target the PIZ target site. Oligonucleotides were tested in primary mouse hepatocytes expressing human SERPINA1-PIZ by zymotic uptake. As shown in FIG. 10 , in some embodiments, various oligonucleotides comprising modified internucleotide linkages at various positions can provide editing activity. In some embodiments, oligonucleotides with non-negatively charged internucleotidic linkages, such as n001, at certain positions provide higher activity than others.

Example 14. Various oligonucleotide compositions can provide editing.

Oligonucleotides containing various types of sugars, nucleobases, internucleotidic linkages and stereochemistry and their patterns were designed and evaluated. Certain oligonucleotides target the PIZ target site. Oligonucleotides were tested in primary mouse hepatocytes expressing human SERPINA1-PIZ by zymotic uptake. As shown in Figure 11, in some embodiments, Rp phosphorothioate internucleotidic linkages can be incorporated at various positions to provide oligonucleotides with editing activity, increasing the level of editing at specific sites .

Example 15. Various oligonucleotide compositions can provide editing.

Oligonucleotides containing various types of sugars, nucleobases, internucleotidic linkages and stereochemistry and their patterns were designed and evaluated. Certain oligonucleotides target the PIZ target site. Oligonucleotides were tested in primary mouse hepatocytes expressing human SERPINA1-PIZ by zymotic uptake. As shown in FIG. 12 , in some embodiments, multiple 2'-OR modified sugars such as, for example, domain-2(3') in certain oligonucleotides (R is an optionally substituted C _1-6 aliphatic (e.g., per 2'-OMe modification)), the addition of a 2'-F modified sugar to a specific site can increase or maintain the level of editing.

Example 16. Various oligonucleotide compositions can provide editing.

Oligonucleotides containing various types of sugars, nucleobases, internucleotidic linkages and stereochemistry and their patterns were designed and evaluated. Certain oligonucleotides target the PIZ target site. Oligonucleotides were tested in primary mouse hepatocytes expressing human SERPINA1-PIZ by zymotic uptake. As shown in Figure 13, in some embodiments, 2' to a specific site, eg, in a domain comprising multiple 2'-F modified sugars, such as domain-1 (5') in a specific oligonucleotide. Addition of an -OR modified sugar (R is an optionally substituted C _1-6 aliphatic (eg, 2′-OMe modified sugar)) may increase or maintain the level of editing. In some embodiments, a 2'-OR modified sugar (R is an optionally substituted C _1-6 aliphatic (eg, 2'-OMe modified sugar)) is at the 5'- and/or 3'-end of the oligonucleotide. used in

Example 17. Compositions of Oligonucleotides of Various Lengths Can Provide Editing.

Oligonucleotides containing various modifications and base sequences and having different lengths (eg, 28 nt, 29 nt, 30 nt, 31 nt, 32 nt) were designed and evaluated. Certain oligonucleotides target the PIZ target site. Oligonucleotides were tested in primary mouse hepatocytes expressing human SERPINA1-PIZ by zymotic uptake. As shown in Figure 14, oligonucleotides of various lengths, including those significantly shorter than those reported by others, can confer editing activity. In some embodiments, 31 nt and 32 nt oligonucleotides can provide improved levels of editing.

Example 18. Compositions of oligonucleotides containing various types of internucleotidic linkages can provide editing.

Oligonucleotides containing various types of sugars, nucleobases, internucleotidic linkages and stereochemistry and their patterns were designed and evaluated. Certain oligonucleotides target the PIZ target site. Oligonucleotides were tested in primary mouse hepatocytes expressing human SERPINA1-PIZ by zymotic uptake. As shown in FIG. 15 , in some embodiments, natural phosphate linkages, non-negatively charged internucleotide linkages such as n001 and phosphorothioate internucleotide linkages can be used at various locations to provide editing. In some embodiments, a natural phosphate linkage at a specific site, eg at a specific position in domain-2(3′), can increase the level of editing. In some embodiments, certain combinations of different internucleotidic linkages and/or stereochemistry at certain sites can increase the level of editing.

Example 19. Various oligonucleotide compositions can provide editing.

Oligonucleotides containing various types of sugars, nucleobases, internucleotidic linkages and stereochemistry and their patterns were designed and evaluated. Certain oligonucleotides target the PIZ target site. Oligonucleotides were tested in primary mouse hepatocytes expressing human SERPINA1-PIZ by GalNAc-mediated uptake. As shown in Figure 16, various oligonucleotides can provide editing activity. In some embodiments, at the 5'- and/or 3'-end of the oligonucleotide, a 2'-OR modification (R is an optionally substituted C1-6 aliphatic (e.g., 2'-OMe modification sugar)) The addition of additives can increase the level of editing.

Example 20. Various oligonucleotide compositions can provide editing.

Oligonucleotides containing various types of sugars, nucleobases, internucleotidic linkages and stereochemistry and their patterns were designed and evaluated. Certain oligonucleotides target the PIZ target site. GalNAc conjugated oligonucleotides were tested in primary mouse hepatocytes expressing human SERPINA1-PIZ. As shown in Figure 17, various oligonucleotides can provide editing activity. In some embodiments, oligonucleotides comprising increased levels of 2-'F modified sugars and/or specific bases/nucleobases (e.g., 8-oxo-dA, b001A, b008U, I, etc.) at specific positions are An increased level of editing can be provided.

Example 21. A provided editing area can improve editing.

A variety of editing regions were designed and evaluated, including oligonucleotides containing various types of sugars, nucleobases, internucleotidic linkages and stereochemistry and their patterns, and various sequences around the nucleoside opposite the target adenosine. Certain oligonucleotides target the PIZ target site. Oligonucleotides were tested in primary mouse hepatocytes expressing human SERPINA1-PIZ by zymnautic-mediated uptake. As shown in Figure 18 (a) to (c), various oligonucleotides can provide editing activity. In some embodiments, certain mismatches or wobble base pairs at positions 5' and/or 3' closest to the editing site may reduce the level of editing relative to fully complementary editing regions. In some embodiments, certain mismatches or wobble base pairs at positions 5' and/or 3' closest to the editing site in some embodiments maintain or increase the level of editing.

Example 22. Various oligonucleotide compositions can provide editing.

Oligonucleotides containing various types of sugars, nucleobases, internucleotidic linkages and stereochemistry and their patterns were designed and evaluated. Oligonucleotides were tested in primary mouse hepatocytes expressing human SERPINA1-PIZ by zymotic-mediated uptake. As shown in Figure 19, various oligonucleotides can provide editing activity. In some embodiments, introduction of a 2'-DNA nucleoside (eg, T instead of 2'-FU) adjacent to the editing site (eg, as N ₁ ) can increase the level of editing.

Example 23. Various oligonucleotide compositions can provide editing.

Oligonucleotides containing various types of sugars, nucleobases, internucleotidic linkages and stereochemistry and their patterns were designed and evaluated. Oligonucleotides were tested in primary mouse hepatocytes expressing human SERPINA1-PIZ by zymnautic-mediated uptake. These various oligonucleotides can provide editing activity. As shown in Figure 20, in some embodiments increasing the level of 2'-F modified sugars can increase the level of editing.

Example 24. Oligonucleotides of various designs provided can provide editing.

Oligonucleotides containing various types of sugars, nucleobases, internucleotidic linkages and stereochemistry and their patterns were designed and evaluated. Certain oligonucleotides target the PIZ target site. As shown in Figure 21, various types of sugars (e.g., DNA sugars, 2'-F modified sugars, 2'-OR (R is not hydrogen) modified sugars and their patterns), nucleobases (modified and unmodified bases and their patterns), internucleotidic linkages (eg, natural phosphate linkages, non-negatively charged internucleotide linkages, phosphorothioate internucleotide linkages and their patterns) and stereochemistry (eg, R p , S p and its pattern) and oligonucleotides comprising its pattern can provide editing activity. Oligonucleotides were tested in primary mouse hepatocytes expressing human SERPINA1-PIZ by zymotic uptake. In some embodiments, certain oligonucleotides provide higher levels of editing than others.

Example 25. Various oligonucleotide compositions can provide editing.

Oligonucleotides containing various types of sugars, nucleobases, internucleotidic linkages and stereochemistry and their patterns were designed and evaluated. Certain oligonucleotides target the PIZ target site. As shown in Figure 22, various types of sugars (e.g., DNA sugar, 2'-F modification sugar, 2'-OR (R is not hydrogen) modification sugar and their patterns), nucleobases (modification and unmodified bases and their patterns), internucleotidic linkages (eg, natural phosphate linkages, non-negatively charged internucleotide linkages, phosphorothioate internucleotide linkages and their patterns) and stereochemistry (eg, R p , S p and its pattern) and oligonucleotides comprising its pattern can provide editing activity. Oligonucleotides were tested in primary mouse hepatocytes expressing human SERPINA1-PIZ by zymotic uptake. In some embodiments, certain oligonucleotides provide higher levels of editing than others.

Example 26. Various oligonucleotide compositions can provide editing.

Oligonucleotides containing various types of sugars, nucleobases, internucleotidic linkages and stereochemistry and their patterns were designed and evaluated. Certain oligonucleotides target the PIZ target site. As shown in Figure 23, various types of sugars (e.g., DNA sugars, 2'-F modified sugars, 2'-OR (R is not hydrogen) modified sugars and their patterns), nucleobases (modified and unmodified bases and their patterns), internucleotidic linkages (eg, natural phosphate linkages, non-negatively charged internucleotide linkages, phosphorothioate internucleotide linkages and their patterns) and stereochemistry (eg, R p , S p and its pattern) and oligonucleotides comprising its pattern can provide editing activity. Oligonucleotides were tested in primary mouse hepatocytes expressing human SERPINA1-PIZ by zymotic uptake. In some embodiments, certain oligonucleotides provide higher levels of editing than others. In some embodiments, a 2'-OR modified sugar at the 5' and/or 3' end (R is not hydrogen) (eg, when R is optionally substituted C _1-6 aliphatic), such as 2'- per OMe strain. In some embodiments, the oligonucleotide comprises non-negatively charged internucleotidic linkages (eg, phosphoryl guanidine internucleotidic linkages such as n001) at both the 5'- and 3'-ends. In some embodiments, the oligonucleotide comprises a non-negatively charged internucleotidic linkage at the 5'-end (eg, a phosphoryl guanidine internucleotidic linkage such as n001). In some embodiments, the oligonucleotide comprises a non-negatively charged internucleotidic linkage at the 3'-end (eg, a phosphoryl guanidine internucleotidic linkage such as n001).

Example 27. Various oligonucleotide compositions can provide editing.

Oligonucleotides containing various types of sugars, nucleobases, internucleotidic linkages and stereochemistry and their patterns were designed and evaluated. Certain oligonucleotides target the PIZ target site. As shown in Figure 24, various types of sugars (e.g., DNA sugar, 2'-F modification sugar, 2'-OR (R is not hydrogen) modification sugar and their patterns), nucleobases (modification and unmodified bases and their patterns), internucleotidic linkages (eg, natural phosphate linkages, non-negatively charged internucleotide linkages, phosphorothioate internucleotide linkages and their patterns) and stereochemistry (eg, R p , S p and its pattern) and oligonucleotides comprising its pattern can provide editing activity. Oligonucleotides were tested in primary mouse hepatocytes expressing human SERPINA1-PIZ by zymotic uptake. In some embodiments, certain oligonucleotides provide higher levels of editing than others. In some embodiments, a 2'-OR modified sugar at the 5' and/or 3' end (R is not hydrogen) (eg, when R is optionally substituted C _1-6 aliphatic), such as 2'- per OMe or 2′-MOE variant. In some embodiments, the oligonucleotide comprises non-negatively charged internucleotidic linkages (eg, phosphoryl guanidine internucleotidic linkages such as n001) at the 5'- and/or 3'-terminus. In some embodiments, the natural DNA sugar is combined with modified internucleotide linkages, e.g., non-negatively charged internucleotide linkages (eg, phosphoryl guanidine internucleotide linkages, such as n001), phosphorothioate internucleotide linkages, and the like. terminal regions (eg, 5′-end regions as shown in FIG. 24).

Example 28. Various oligonucleotide compositions can provide editing.

Oligonucleotides containing various types of sugars, nucleobases, internucleotidic linkages and stereochemistry and their patterns were designed and evaluated. Certain oligonucleotides target the PIZ target site. Oligonucleotides were tested in primary mouse hepatocytes expressing human SERPINA1-PIZ by zymotic uptake. As shown in Figure 25, the nucleosides of various nucleobases, sugars, nucleosides opposite the target adenosine and/or nucleosides surrounding these nucleosides (e.g., b001A, b001rA, Csm15, I, A variety of oligonucleotides containing various types of sugars, nucleobases and internucleotidic linkages, including, etc.) can provide editing activity. In some embodiments, certain oligonucleotides provide higher levels of editing.

Example 29. Various oligonucleotide compositions can provide editing.

In some embodiments, an oligonucleotide comprises mismatches and/or wobble base pairs when aligned with a target nucleic acid. As demonstrated herein, these various oligonucleotides can provide editing activity. In some embodiments, oligonucleotides comprising G-U wobble base pairs at specific positions are designed to target PIZ target sites. Oligonucleotides were tested in primary mouse hepatocytes expressing human SERPINA1-PIZ by zymotic uptake. As shown in Figure 26, in various embodiments, oligonucleotides comprising G-U wobble base pairs provide editing activity.

Example 30. Various oligonucleotide compositions can provide editing.

sugars of various types (e.g., DNA sugars, 2'-F modified sugars, 2'-OR modified sugars (R is not hydrogen) and their patterns), nucleobases (modified and unmodified bases and their patterns), internucleotidic linkages (e.g., natural phosphate linkages, non-negatively charged internucleotide linkages, phosphorothioate internucleotide linkages and their patterns) and stereochemistry (e.g., R p, Sp and their patterns) and Various structural features in the editing region (e.g., 5MRm5dC, 5MSm5dC _{, 5MSm5fC} for various types of sugars, _nucleobases , _nucleosides , linkages, etc., e.g. oligonucleotides comprising patterns thereof, including fC, dC, m5dC, dA, 5MSdT, 5MRdT, etc.) can provide editing activity. In some embodiments, an oligonucleotide comprising a 5'-(R)-Me or 5'-(S)-Me modified sugar provides editing activity. Specific data is presented in FIG. 27 . Oligonucleotides were tested in primary human hepatocytes by GalNAc-mediated uptake at various concentrations.

Example 31. Various oligonucleotide compositions can provide editing.

Oligonucleotides containing various types of sugars, nucleobases, internucleotidic linkages and stereochemistry and their patterns were designed and evaluated. Certain oligonucleotides target the ACTB target site. Oligonucleotides were tested in primary human hepatocytes by GalNAc-mediated uptake at various concentrations. As shown in Figure 28, various oligonucleotides containing non-negatively charged internucleotidic linkages such as *n001 and/or Unlocked Nucleic Acids (UNA) sugars can provide editing activity.

Example 32. Various oligonucleotide compositions can provide editing.

In some embodiments, an oligonucleotide comprises a 5'-cap. In some embodiments, an oligonucleotide comprises a baseless 5'-cap. In some embodiments, an oligonucleotide comprises an additional chemical moiety, eg linked to the 5'-end of the oligonucleotide. A variety of these oligonucleotides were prepared and evaluated. In some embodiments, oligonucleotides are tested in primary human hepatocytes by GalNAc mediated uptake. As shown in Figure 29, these oligonucleotides can provide editing activity.

Example 33. A specific editing area provides a high level of editing.

In particular, the present invention provides an editing area that is particularly useful for editing. In some embodiments, the present invention provides a 5'-N ₁ N ₀ N _-1 -3' element that is particularly useful for editing. In some embodiments, they are fully complementary to the target adenosine and the nucleosides directly 5' and 3' to it. In some embodiments, they include one or more mismatched and/or wobbled base pairs. In some embodiments, those containing mismatches and/or wobble base pairs, including N ₋₁ and/or N ₁ , provide similar or higher levels of editing compared to absence of such mismatches and/or wobble base pairs. In some embodiments, oligonucleotides comprising various nucleosides at or directly 5' and 3' to the editing site have been designed and evaluated, which target, for example, the ACTB target. In some embodiments, plasmid reporters expressing full-length ACTB cDNA with various combinations of nucleosides directly 5' and 3' to the target adenosine were designed and tested with the corresponding oligonucleotides, each directly directed to the editing site. It is characterized by a unique combination of sugars and/or bases that are 5' and 3'. Plasmids and oligonucleotides were tested in 293T cells by transfection. As shown below, in some embodiments, oligonucleotides comprising specific mismatches and/or wobble base pairs that are directly 5' and/or 3' to the editing site can maintain or increase the level of editing. The combination of nearest neighbors for each target is displayed horizontally across the top of the chart (5' to 3' direction) and the combination of nearest neighbors to the editing site of the oligonucleotide is displayed vertically on the left side of the chart (3' to 5' direction). '). Endogenous ACTB transcripts are marked with *. Opposite the target adenosine in the oligonucleotide is dC. The average editing value for each reporter-oligonucleotide combination is plotted. From top to bottom, the oligonucleotides are WV-42331 to WV-42335, WV-37317, WV-42337 to WV-42349.

Example 34. Various oligonucleotide compositions can provide editing.

In some embodiments, an oligonucleotide comprises a 5'-cap. In some embodiments, an oligonucleotide comprises a baseless 5'-cap. In some embodiments, an oligonucleotide comprises an additional chemical moiety, eg linked to the 5'-end of the oligonucleotide. A variety of these oligonucleotides were prepared and evaluated. In some embodiments, the oligonucleotides are then tested in primary mouse hepatocytes expressing human ADAR-p110. As shown in Figure 30, these oligonucleotides can provide editing activity.

Example 35. Provided technology can provide in vivo editing.

Among other things, the present invention demonstrates that the provided oligonucleotides are capable of providing in vivo editing. In an example, a non-human primate (NHP) is administered a single subcutaneous (SC) dose of 50 mg/kg of WV-37317 (3X Macaca fascicularis) or PBS (1X Macaca fascicularis) as a control. received. Dosing details are provided below. Animals sacrificed on day 8 (administered on day 1, collected on day 8) and all tissues collected for PK/PD analysis. As shown in (a) of Figure 31, multiple tissues (kidney, liver, lung, heart, pancreas, pulmonary vein and artery, duodenum, ileum, jejunum, PBMC) showed ACTB editing, and WV-37317 showed all It was detected at a high level in tissues (see (b) of FIG. 31). Among other things, the oligonucleotides of the present invention can be delivered by SC administration, for example in NHP, allowing for a wide tissue distribution and efficient endogenous ADAR-mediated editing in a variety of tissue types.

Example 36. Provided technology can provide in vivo editing.

Among other things, the present invention demonstrates that the provided oligonucleotides are capable of providing in vivo editing. In the example, non-human primates (NHP) received a single intrathecal (IT) dose of 10 mg or 5 mg of WV-37317 (6X macaca fascicularis) or PBS (1X macaca fascicularis) as a control. Dosing details are provided below. Animals sacrificed on day 8 or 29 (administered on day 1, collected on days 8 and 29) and tissues collected for PK/PD analysis. As shown in (a) of FIG. 32, multiple tissues (eg, spinal cord, cortex, hippocampus, midbrain, cerebellum, corpus callosum, and optic nerve) showed ACTB editing. As shown in (b) of FIG. 32 , WV-37317 was detected in various CNS tissues. Among other things, the oligonucleotides of the present invention can be delivered by IT administration, for example in the NHP, and can provide broad distribution and efficient endogenous ADAR-mediated editing in various tissues, including CNS tissues.

Example 37. Various oligonucleotide compositions can provide editing.

In some embodiments, oligonucleotides include duplexed and targeting oligonucleotides. In some embodiments, such oligonucleotides can form duplexes with, for example, duplexed nucleic acids and oligonucleotides. In some embodiments, an oligonucleotide and a corresponding duplexed oligonucleotide. In some embodiments, oligonucleotides have been designed and evaluated to target a luciferase reporter target. In some embodiments, the design combines two oligonucleotide fragments that share 16 bp or 18 bp complementary sequences to allow association of both fragments within a cell. Certain oligonucleotides were tested in combination by transfection in 293T cells. Editing efficiency was calculated by determining the cLUC/gLUC ratio. As shown in Figure 33, in some embodiments certain combinations of oligonucleotide segments can provide editing. A specific duplex design is provided as an example in FIG. 35 . As will be appreciated by those skilled in the art, a variety of suitable lengths may be used for portions, regions, oligonucleotides, etc. in accordance with the present invention.

Example 38. Various oligonucleotide compositions can provide editing.

In some embodiments, oligonucleotides include double and single stranded regions as well as stem loops. In some embodiments, such oligonucleotides can be used as duplexed oligonucleotides to form complexes with oligonucleotides comprising duplexed and targeting regions. An exemplary design is presented in FIG. 35 . As will be appreciated by those skilled in the art, a variety of suitable lengths may be used for portions, regions, oligonucleotides, etc. in accordance with the present invention. For example, certain oligonucleotides have been designed to target a site on a luciferase reporter construct. The design combines two oligonucleotides that share complementary sequences (eg 15 bp), allowing the association of the two pieces and the formation of a stem-loop complex within the cell. Oligonucleotides were tested in combination by transfection in 293T cells. Editing efficiency was calculated by determining the cLUC/gLUC ratio. As shown in Figure 34, various combinations provide editing activity.

Example 39. Various oligonucleotide compositions can provide in vivo editing.

In particular, the provided technology may provide for in vivo editing. In some embodiments, oligonucleotides (e.g., WV-43120, WV-44464, WV-44465) are shown to confirm in vivo editing of the SERPINA1-Z allele in a human ADAR (huADAR) transgenic mouse described herein. appear. Thirty-two male mice from the JAX huADAR x SA1 mouse strain were used, all heterozygous for SA1-PiZ. Of these, 20 mice were also heterozygous for huADAR-p110 and 12 mice were wild type for mouse ADAR (no expression of huADAR-p110). UGP2 was used as a control for huADAR activity. Mice were administered subcutaneously (s.c.) with 10 mg/kg of the selected oligonucleotide or PBS control every other day for 3 days (Day 0, Day 2, Day 4). Serum was collected from mice before dosing and on day 7 after treatment, and liver biopsies were collected on day 7. Samples were subjected to PK and PD analysis and hybrid ELISA. Specific information is provided below:

In some embodiments, primary mouse hepatocytes from the transgenic model (expressing human ADARp110 and human SERPINA1-Z alleles) were treated with various GalNAc conjugated oligonucleotides for 48 hours. RNA editing was measured by Sanger sequencing. In some embodiments, as shown in Figure 36, various oligonucleotides provided in vitro editing of the SERPINA1-Z allele.

Liver biopsy samples collected from huADAR/SA1 transgenic mice on day 7 were subjected to Sanger sequencing to determine percent editing. In some embodiments, as shown in Figure 37, various oligonucleotide compositions provided about 20% or less, about 30% or less, or about 40% or less in vivo editing activity against the SERPINA1-Z allele.

From serum samples collected from mice before dosing and on day 7 after treatment, the concentration of total human AAT in serum was determined with a commercially available ELISA kit (AbCam). In some embodiments, as shown in FIG. 38 , in vivo editing from various oligonucleotide doses increased total human AAT concentrations in serum.

From serum samples collected from mice before dosing and on day 7 post treatment, the relative abundance of Z (mutant) to M (wild type) AAT isoforms was determined by mass spectrometry. Then, the absolute amount of each isoform was calculated by applying the relative abundance to the absolute concentration obtained from ELISA (see FIG. 38). In some embodiments, as shown in FIG. 39 , editing from treatment with WV-44464 resulted in a substantial reduction in secretion of wild-type AAT protein and mutant Z-AAT protein in serum. As identified herein, in some embodiments, provided technologies can increase wild-type SERPINA1 protein levels in the blood. In some embodiments, provided technologies can reduce mutant SERPINA1 protein levels in the blood. In some embodiments, as shown in FIG. 38 , about 75% of total AAT in blood is wild type.

Certain data are presented below as examples.

SERPINA1-Z allele editing in vivo in huADAR mice (eg, Figure 37):

Human AAT concentration in serum (ELISA) (eg, Figure 38):

AAI isoforms in serum (mass spectrometry; PBS and WV-44464) (eg, Figure 39):

Elastase inhibitory activity in serum (eg, FIG. 40)

It was confirmed that the provided technology can provide editing and functional proteins. From serum samples collected from mice before dosing and on day 7 after treatment, relative elastase inhibitory activity was determined using a commercially available kit (EnzChek® Elastase Assay Kit (E-12056)). Diluted serum was incubated with recombinant elastase enzyme and fluorescently labeled elastin substrate. The activity of the elastase enzyme can be detected by a fluorescence signal detected upon elastin cleavage. Relative inhibition was calculated for a control reaction in the absence of serum (100% elastase activity). Each sample was run in technical replicates. Among other things, the data presented in FIG. 40 confirmed that the wild-type AAT protein produced and secreted as a result of editing by the presented technique is functional, for example for elastase inhibition.

Among other things, the data presented herein confirms that transgenic mouse models expressing human ADARs are useful for evaluating ADAR editing agents, such as oligonucleotides. In some embodiments, as identified herein, editing of up to 40% or more of the SERPINA1 Z allele mRNA was provided (eg, in the liver at a specific time point). In some embodiments, the level of editing provided approximates correction for heterozygosity (MZ). In some embodiments, as identified herein, provided technologies provide a significant increase in circulating functional wild-type M-AAT protein in vivo. In some embodiments, provided techniques reduce the level of mutant Z-AAT protein, eg in liver, serum, etc.

Example 40. Provided technology can modulate protein-protein interactions .

As identified herein, among other things, the provided technology can modulate protein-protein interactions, for example, by adenosine editing in mRNA and altering the identity of amino acid residues in the polypeptide encoded thereby. In some embodiments, provided technologies modulate protein-protein interactions, activities and/or functions, for example by editing one or more amino acid residues of one or more proteins. As demonstrated herein, editing of various residues of Keap1 or Nrf2 can modulate their interactions, activities and/or functions. For example, in some embodiments, editing of residues of Keap1 or Nrf2 increases the level of Nrf2, the transcription of nucleic acids that can be activated by Nrf2, and/or the expression of Nrf2-regulated genes. Keap1 has been reported for NRF2 and mediates NRF2 proteasomal degradation. In some embodiments, disrupting the interaction between Keap1 and NRF2 allows post-transcriptional upregulation of NRF2 and translocation of NRF2 to the nucleus, which can activate transcription of NRF2-regulated genes. As demonstrated herein, various oligonucleotides were designed to target specific editing sites in the Keap1 or Nrf2 transcript. As shown in Figure 41(a), various oligonucleotides can provide editing at multiple sites in the Keap1 or NRF2 transcript. In some embodiments, editing of the Keap1 and/or Nrf2 transcript reduces the expression level of downstream genes regulated by NRF2 (eg, SRGN, HMOX1, SLC7a11, NQO1, etc., as shown in FIG. 41(b)). can be changed. In some embodiments, oligonucleotides provide for editing of Keap1 or NRF2 transcripts that change amino acid residues that could interfere with Keap1/NRF2 complex formation and stability, and of NRF2 levels, translocations, and/or nucleic acids regulated by NRF2. expression can be regulated. In some embodiments, certain oligonucleotides provide higher levels of editing than others. In some embodiments, the oligonucleotide comprises non-negatively charged internucleotidic linkages (eg, phosphoryl guanidine internucleotidic linkages such as n001) at the 5'- and/or 3'-terminus. In some embodiments, an oligonucleotide is a 2'-OR modified sugar at the 5' and/or 3' end (R is not hydrogen) (eg, R is optionally substituted C _1-6 aliphatic), such as 2 '-OMe contains modified sugars. In some embodiments, the oligonucleotide comprises a 2'-F modified sugar at the 5'- and/or 3'-end. One skilled in the art understands that the various oligonucleotide designs described herein can be applied to modulate interactions between polypeptides.

Example 41. Provided technology can provide robust, persistent in vivo editing.

In some embodiments, the present invention provides oligonucleotide compositions capable of conferring in vivo editing activity in a variety of systems, eg, a variety of cells, tissues, and/or organs, among others. Specific data confirming that the provided technology can provide persistent editing in various tissues in vivo, including the CNS, is presented in FIG. 42 . Human ADAR (hADAR) transgenic mice described herein were treated with a single 100 ug dose of the WV-40590 oligonucleotide composition via intraventricular (ICV) injection. Mice were sacrificed at 1 week, 2 weeks, 4 weeks, 8 weeks, 12 weeks and 16 weeks after administration, and multiple CNS tissues were collected and analyzed. As shown in Figure 42, UGP2 mRNA editing was achieved in all tissues analyzed. In some embodiments, the level of UGP2 editing was similar across the various time points analyzed. Among other things, these data demonstrate that the presented technique can provide effective editing of various tissues in vivo for at least 16 weeks.

Example 42. A provided technique may provide editing.

Oligonucleotides containing various types of sugars, nucleobases, internucleotidic linkages and stereochemistry and their patterns were designed and evaluated. Certain oligonucleotides target specific editing sites on the UGP2 transcript. 43 and 44, various types of sugars (e.g., DNA sugars, 2'-F modified sugars, 2'-OR (R is not hydrogen) modified sugars and their patterns), nucleobase (modified and unmodified bases and their patterns), internucleotide linkages (eg natural phosphate linkages, non-negatively charged internucleotidic linkages, phosphorothioate internucleotide linkages and their patterns) and stereochemistry (eg natural phosphate linkages, non-negatively charged internucleotide linkages) , Rp, Sp and their patterns) and oligonucleotides comprising their patterns can provide editing activity. In some embodiments, the 2'-F of the second domain (eg, in the region toward the 3' side of N ₀ ; in some embodiments, from N _-2 to the 3'-end of the oligonucleotide) and/or a natural phosphate linkage of a first domain (eg, in a region 5′ to N ₀ ; in some embodiments, from the 5′-end of an oligonucleotide to N ₂ ) and/or a second domain and/or 2 '-OR (R is an optionally substituted C _1-6 aliphatic (eg, 2'-OMe, 2'-MOE, etc.) provides improved editing efficiency. Oligonucleotides are transferred to human hepatocytes by zymotic uptake. (FIG. 43) and iPSC-derived neurons (FIG. 44). In some embodiments, certain oligonucleotides provide higher editing than others at certain concentrations.

Example 43. Provided technology can provide in vivo editing.

Oligonucleotides containing various types of sugars, nucleobases, internucleotidic linkages, stereochemistry, additional chemical moieties, etc. and their patterns were designed and evaluated. Certain oligonucleotides target specific editing sites on the UGP2 transcript. As shown in Figure 45, the provided oligonucleotide composition can provide editing activity in various tissues in vivo, including the liver. Oligonucleotides were tested in wild-type (Wt) and transgenic hADAR mice by subcutaneous administration of three doses of 10 mg/kg (day 0, day 2 and day 4, respectively). In some embodiments, certain oligonucleotide compositions provide higher editing than others. In some embodiments, certain oligonucleotide compositions provide significantly higher editing in hADAR mice compared to wt mice. In some embodiments, certain oligonucleotide compositions provide high levels of editing in both wt and hADAR mice.

Example 44. Provided technology can provide editing in various cell populations.

In some embodiments, the present invention provides oligonucleotide compositions capable of conferring editing activity in a variety of systems, eg, a variety of cells, tissues, and/or organs, among others. Certain data confirming that the provided technology is capable of providing editing in various immune cell populations, including PBMCs, is presented in FIG. 46 . Among other things, the provided technology can provide editing in cell populations such as CD4+, CD8+, CD14+, CD19+, NK, Treg cells, and the like. Cells were treated with 10 uM WV-37317 in activating (PHA addition) or inactivating conditions. RNA was isolated 4 days after treatment via a benchtop antibody/bead protocol. As shown in Figure 46, ACTB mRNA editing was achieved in multiple immune cell populations. In some embodiments, the level of ACTB editing was comparable for activated and inactive cell populations. In some embodiments, the level of ACTB editing is increased relative to an activated cell population.

Example 45. Provided technology can provide in vivo editing.

In some embodiments, the present invention provides oligonucleotide compositions capable of conferring in vivo editing activity in a variety of systems, eg, a variety of cells, tissues, and/or organs, among others. Certain data confirming that the provided technology can provide editing in vivo, including in the eye, is presented in FIG. 47 . A single 10 ug or 50 ug ICV injection of the WV-40590 oligonucleotide composition was administered to the posterior compartment of the eye of transgenic hADAR mice. RNA was isolated at 1 and 4 weeks post treatment. As shown in Figure 47, robust UGP2 mRNA editing was achieved in the eye at both doses.

Example 46. Provided technology can provide persistent in vivo editing.

In particular, the provided technology can provide persistent in vivo editing. Specific data confirming that the presented technology can provide persistent editing in a mouse model is presented in FIG. 48 . Wild-type and transgenic hADAR mice were treated with PBS or 10 mg/kg of the WV-44464 oligonucleotide composition on days 0, 2 and 4. Serum was collected via weekly blood draws and levels of total human AAT protein (total, wild-type (M-AAT) and mutant (Z-AAT)) were quantified by ELISA and mass spectrometry. As shown in FIG. 48 , provided technologies can increase total human AAT serum concentrations and produce or increase wild-type AAT protein (M-AAT). In some embodiments, AAT serum concentrations were observed to be >3-fold higher over 30 days after the last administration (FIG. 48(a)). In some embodiments, reconstituted wild-type M-AAT was detected over 30 days after the last administration (FIG. 48(b)).

Example 47. A provided technique may provide editing.

By designing and evaluating oligonucleotides comprising various types of sugars, nucleobases, internucleotidic linkages and stereochemistry and their patterns, alternating blocks comprising 2'-F and 2'-OR (R is C _1-6 aliphatic) (2'-OMe and/or 2'-MOE) blocks, natural phosphate linkages, phosphorothioate internucleotide linkages, internucleotide linkages that are not negatively charged (eg It was confirmed that oligonucleotides of various designs, including those with phosphoryl guanidine internucleotide linkages such as n001s), controlled stereochemistry, patterns thereof, and the like, can provide efficient editing. As shown in Figures 49 and 51, various types of sugars (e.g., DNA sugars, 2'-F modified sugars, 2'-OR (R is not hydrogen) modified sugars and their patterns), nucleobase (modified and unmodified bases and their patterns), internucleotide linkages (eg natural phosphate linkages, non-negatively charged internucleotidic linkages, phosphorothioate internucleotide linkages and their patterns) and stereochemistry (eg natural phosphate linkages, non-negatively charged internucleotide linkages) , Rp, Sp and their patterns) and oligonucleotides comprising their patterns can provide potent editing activity. Primary mouse hepatocytes transfected for the hADARp110 and SERPINA1-Z alleles were treated with GalNAc conjugated oligonucleotides via zymotic uptake. RNA was harvested 48 hours after treatment and RNA editing was measured by Sanger sequencing (n=2 biological replicates). Specific EC50 (nM) data are provided below (Figures 49 and 51):

Example 48. Provided technology can provide in vivo editing.

Blocks comprising alternating blocks comprising 2′-F and 2′-OR (R is C _1-6 aliphatic) (2′-OMe and/or 2′-MOE) blocks, natural phosphate linkages as described herein , phosphorothioate internucleotide linkages, non-negatively charged internucleotide linkages (e.g., phosphorylguanidine internucleotide linkages such as n001s), controlled stereochemistry, patterns thereof, and the like. Oligonucleotides containing types of sugars, nucleobases, internucleotidic linkages and stereochemistry and their patterns were designed and evaluated. Certain data confirming that the presented technique can provide robust editing in mouse models is presented in FIG. 50 . Male and female transgenic hADAR mice were treated with 5 mg/kg of the indicated oligonucleotide via subcutaneous administration on days 0, 2 and 4. Liver biopsies were collected on day 7 post treatment and RNA editing was measured by Sanger sequencing (n=3 mice per sex). As shown in Figure 50, provided oligonucleotide compositions can provide high levels of editing. In some embodiments, certain oligonucleotide compositions can provide higher levels of editing in male mice compared to female mice.

Example 49. Provided Technologies Can Provide Edited Polypeptides with Desired Properties and Functions In Vivo

In some embodiments, the present invention provides in vivo editing activity, particularly in a variety of systems, e.g., a variety of cells, tissues and/or organs, and has desired properties and activities, including polypeptides, e.g., in some embodiments, wild-type proteins. Provides an oligonucleotide composition capable of generating. Certain data confirming that, in some embodiments, provided technology can provide editing in mouse models and/or can produce increased levels of circulating proteins, including wild-type proteins, in serum are presented in FIG. 52 . Wild-type and transgenic hADAR mice were treated with PBS or 10 mg/kg of the WV-46312 oligonucleotide composition on days 0, 2 and 4. Serum was collected via weekly blood draws and levels of total human AAT protein (wild type (PiM) and mutant (PiZ)) were quantified by ELISA and mass spectrometry. As shown in FIG. 52 , provided technologies can increase AAT serum concentrations by about 4-fold or more and produce high levels of wild-type AAT in serum relative to baseline (eg, pre-administration levels).

Example 50. Provided technology can provide in vitro and in vivo editing.

Among other things, this embodiment provides data that further confirms that the provided technology can provide editing.

For example, Figure 53 confirms that oligonucleotides comprising various base modifications (e.g., s b001A, b001rA, CSM15, b008U, etc.), including various base modifications as described herein, can edit the target adenosine let it Primary mouse hepatocytes (huADAR/SA1 Tg) were treated with the indicated oligonucleotide composition targeting the SERPINA1-Z allele for 48 hours (Zymnautic). RNA editing was quantified by Sanger sequencing.

Figure 54 confirms that various modifications can be used in accordance with the present invention in oligonucleotides to provide editing. Primary mouse hepatocytes (huADAR/SA1 Tg) were treated with the indicated oligonucleotide composition targeting the SERPINA1-Z allele for 48 hours (Zymnautic). Modified nucleobases such as b008U at positions across the target adenosine editing site, linkages of various types (e.g., phosphorothioate (PS), natural phosphate linkages (PO)), and/or PNs (e.g., phosphoryl guanidines such as n001) oligonucleotides containing nucleotide linkages) and various types of sugars (eg, 2'-OMe modified sugars, 2'-F modified sugars, native DNA sugars, etc.) were evaluated and found to provide editing of the target adenosine. RNA editing was quantified by Sanger sequencing.

Figure 55 confirms that various modifications can be used in accordance with the present invention in oligonucleotides to provide editing. Primary mouse hepatocytes (huADAR/SA1 Tg) were treated with the indicated oligonucleotide composition targeting the SERPINA1-Z allele for 48 hours (Zymnautic). Modified nucleobases such as b001A at positions across the target adenosine editing site, linkages of various types (e.g., phosphorothioate (PS), natural phosphate linkages (PO)), and/or PNs (e.g., phosphoryl guanidines such as n001) oligonucleotides containing nucleotide linkages) and various types of sugars (eg, 2'-OMe modified sugars, 2'-F modified sugars, native DNA sugars, etc.) were evaluated and found to provide editing of the target adenosine. RNA editing was quantified by Sanger sequencing. As identified, non-negatively charged internucleotidic linkages such as phosphorylguanidine internucleotidic linkages such as n001 can be used in a variety of positions; R p phosphorothioate internucleotidic linkages and natural phosphate linkages can also be used. In some embodiments, the first domain comprises one or more negatively charged nucleotides, such as one or more R p phosphorothioate internucleotidic linkages, phosphorylguanidine internucleotide linkages such as n001, each optionally and independently in an R p configuration. liver linkages and one or more natural phosphate linkages. In some embodiments, as shown in the various figures, hypoxanthine is used in place of G when close to N ₀ , for example at position N _-1 .

Figure 56 confirms that various modifications can be used in accordance with the present invention in oligonucleotides to provide editing. Primary mouse hepatocytes (huADAR/SA1 Tg) were treated with the indicated oligonucleotide composition targeting the SERPINA1-Z allele for 48 hours (Zymnautic). Modified nucleobases such as b001A, b008U, b010U, b001C, b008C, b011U, b002G, b012U, etc. at positions across the target adenosine editing site, various types of modified linkages (e.g., phosphorothioate (PS), PN (e.g., phosphoryl guanidine linkages such as n001), etc.) and oligonucleotides containing various types of sugars (e.g., 2'-OMe modified sugars, 2'-F modified sugars, natural DNA sugars, etc.), and editing of target adenosines. It was confirmed that it can provide. RNA editing was quantified by Sanger sequencing. It was observed that certain base modifications can provide higher levels of editing under the conditions tested.

Figure 57 confirms that various modifications can be used in accordance with the present invention in oligonucleotides to provide editing. Primary mouse hepatocytes (huADAR/SA1 Tg) were treated with the indicated oligonucleotide composition targeting the SERPINA1-Z allele for 48 hours (Zymnautic). Modified nucleobases such as b008U, b010U, b001C, b008C, b011U and b012U (eg, N ₁ , N ₀ , etc.), various types of modified linkages (eg PS (phosphorothioate), PN (eg, phosphoryl guanidine linkages such as n001), etc.) and oligonucleotides containing various types of sugars (e.g., 2'-OMe modified sugars, 2'-F modified sugars, natural DNA sugars, etc.), and editing of target adenosines. It was confirmed that it can provide. RNA editing was quantified by Sanger sequencing. It was observed that certain base modifications can provide higher levels of editing under the conditions tested.

Figure 58 confirms that various modifications can be used in accordance with the present invention in oligonucleotides to provide editing. Primary mouse hepatocytes (huADAR/SA1 Tg) were treated with the indicated oligonucleotide composition targeting the SERPINA1-Z allele for 48 hours (Zymnautic). Oligonucleotides comprising modified nucleosides such as Usm04, Csm04 and rCsm13 of N ₀ and/or N ₁ were evaluated and found to provide editing of the target adenosine in certain cases. In some embodiments, certain modifications of N ₀ and/or N ₁ (eg, modifications containing UNA sugars such as sm04) have been observed to provide lower levels of editing compared to other modifications under the conditions tested. . RNA editing was quantified by Sanger sequencing.

Figure 59 confirms that various modifications can be used in accordance with the present invention in oligonucleotides to provide editing. Primary mouse hepatocytes (huADAR/SA1 Tg) were treated with the indicated oligonucleotide composition targeting the SERPINA1-Z allele for 48 hours (Zymnautic). Evaluate oligonucleotides containing various modifications such as Csm11, Csm12, b009Csm11, b009Csm12, Gsm11, Gsm12, Tsm11, Tsm12, L010, etc. (eg, at one or more of N ₁ , N ₀ and N _-1 positions); , confirmed to provide editing of the target adenosine. RNA editing was quantified by Sanger sequencing. In some embodiments, an oligonucleotide comprising a natural DNA sugar at N ₋₁ and/or N ₀ provides a higher level of editing compared to one comprising an acyclic sugar. In some embodiments, an acyclic sugar such as sm11, sm12, etc. may be used in N ₁ .

Figure 60 confirms that various modifications can be used in accordance with the present invention in oligonucleotides to provide editing. Primary mouse hepatocytes (huADAR/SA1 Tg) were treated with the indicated oligonucleotide composition targeting the SERPINA1-Z allele for 48 hours (Zymnautic). As described herein, oligonucleotides comprising various modifications and patterns thereof can provide robust editing. For example, in some embodiments, N ₀ sugars whose 2'-groups are independently selected from -H and -OH can provide robust editing (eg, natural DNA sugars, sm15, etc.). In some embodiments, the N ₁ sugar is a natural DNA sugar or a 2′-F modified sugar. In some embodiments, an oligonucleotide comprising a 2′-F modified or natural DNA sugar at the N ₁ position and a natural DNA sugar at the N ₀ and N ₋₁ positions can provide high levels of editing. RNA editing was quantified by Sanger sequencing.

Figure 61 confirms that various modifications can be used in accordance with the present invention in oligonucleotides to provide editing. Primary mouse hepatocytes (huADAR/SA1 Tg) were treated with the indicated oligonucleotide composition targeting the SERPINA1-Z allele for 48 hours (Zymnautic). different types of linkages (eg PS (phosphorothioate), PO (natural phosphate linkages) and/or PNs (eg phosphorylguanidine linkages such as n001) nucleotide linkages) and different types of sugars (eg 2'- OMe modified sugars, 2'-F modified sugars, natural DNA sugars, etc.) were evaluated and found to provide editing of the target adenosine. In some embodiments, oligonucleotides comprising increased levels of 2'-OMe modified sugars and PO linkages may provide similar or increased editing activity relative to reference at certain concentrations. RNA editing was quantified by Sanger sequencing. As demonstrated, per 2'-OR modification (R is not -H) (e.g., per 2'-OMe modification) is the first and last few nucleosides, first domain, first subdomain , It can be used in various locations including the third subdomain and the like. In some embodiments, about 30% to 80% of all sugars in the oligonucleotide (e.g., about 30% to 75%, 30% to 70%, 30% to 65%, 30% to 60%, 30% to 50%) %, 40% to 70%, 40% to 65%, 40% to 60%, 40% to 50%, or about 30%, 40%, 50%, 60%, 65%, or 70%) each independently per 2'-OR variant (R is not -H) (e.g. per 2'-OMe, 2'-MOE, 2'-OL ^B -4' variant). In some embodiments, about 30% to 80% of all sugars in the oligonucleotide (e.g., about 30% to 75%, 30% to 70%, 30% to 65%, 30% to 60%, 30% to 50%) %, 40% to 70%, 40% to 65%, 40% to 60%, 40% to 50%, or about 30%, 40%, 50%, 60%, 65%, or 70%) each independently as per 2'-OMe or 2'-MOE modifications. In some embodiments, about 30% to 80% of all sugars in the oligonucleotide (e.g., about 30% to 75%, 30% to 70%, 30% to 65%, 30% to 60%, 30% to 50%) %, 40% to 70%, 40% to 65%, 40% to 60%, 40% to 50%, or about 30%, 40%, 50%, 60%, 65%, or 70%) each independently as per the 2'-OMe variant. In some embodiments, an oligonucleotide is one or more (e.g., 1-10, 2-10, 3-9, 3-8, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) natural phosphate linkages. In some embodiments, natural phosphate linkages are used internally (eg, not linked to the first and last 1, 2 or 3 nucleosides). In some embodiments, at least about 50%, 60%, 70%, 75%, 80%, 85% or 90% of the natural phosphate linkages are each independently a 2'-OR variant (R is not -H) (e.g. eg, 2'-OMe, 2'-MOE, etc.). In some embodiments, each natural phosphate linkage is independently a 2'-OR variant (R is not -H). (eg, 2'-OMe, 2'-MOE, etc.).

Figure 62 confirms that various modifications can be used in accordance with the present invention in oligonucleotides to provide editing. Primary mouse hepatocytes (huADAR/SA1 Tg) were treated with the indicated oligonucleotide composition targeting the SERPINA1-Z allele for 48 hours (Zymnautic). Nucleobases of various types, linkages (eg, phosphorothioate (PS), natural phosphate linkages (PO), and/or PN (eg, phosphorylguanidine linkages, such as n001) nucleotide linkages) and sugars (eg, 2'- OMe modified sugars, 2'-F modified sugars, natural DNA sugars, etc.) were evaluated and found to provide editing of the target adenosine. As set forth herein, a 2'-OR modification (R is not -H) (e.g., 2'-OMe) is used at various locations in the first domain, first subdomain, and/or third subdomain It can be. RNA editing was quantified by Sanger sequencing.

In addition, sugar modifications, such as 2-OR modifications (R is not -H) (eg, 2'-OMe, 2'-MOE, etc.), 2'-F, etc. according to the present invention to provide editing. See, for example, FIGS. 63, 64, 65, 66, 67, 68, 69 and 70 for additional data confirming that it can be used with a variety of other structural elements. Primary mouse hepatocytes (huADAR/SA1 Tg) were treated with the indicated oligonucleotide composition targeting the SERPINA1-Z allele for 48 hours (Zymnautic). different types of linkages (eg PS (phosphorothioate), PO (natural phosphate linkages) and/or PNs (eg phosphorylguanidine linkages such as n001) nucleotide linkages) and different types of sugars (eg 2'- OMe modified sugars, 2'-MOE modified sugars, 2'-F modified sugars, native DNA sugars, etc.) were evaluated and found to provide editing of the target adenosine. In some embodiments, oligonucleotides comprising increased levels of 2'-OMe and/or 2'-MOE modified sugars and PO linkages provide similar or increased editing of target adenosines relative to reference under certain conditions. RNA editing was quantified by Sanger sequencing.

Figure 71 further confirms that various modifications can be used in accordance with the present invention in oligonucleotides to provide editing. Primary mouse hepatocytes (huADAR/SA1 Tg) were treated with the indicated oligonucleotide composition targeting the SERPINA1-Z allele for 48 hours (Zymnautic). different types of linkages (eg PS (phosphorothioate), PO (natural phosphate linkages) and/or PNs (eg phosphorylguanidine linkages such as n001) nucleotide linkages) and different types of sugars (eg 2'- OMe modified sugars, 2'-F modified sugars, natural DNA sugars, sm15, etc.) were evaluated and found to provide editing of the target adenosine. In some embodiments, oligonucleotides comprising sm15 or native RNA sugars at N _-2 can provide robust editing under certain conditions. RNA editing was quantified by Sanger sequencing.

As described herein, a variety of modified charged internucleotidic linkages can be used in accordance with the present invention. In some embodiments, the modified internucleotide linkage is a non-negatively charged internucleotide linkage. In some embodiments, the non-negatively charged internucleotidic linkages are neutral internucleotidic linkages. In some embodiments, the modified internucleotide linkage is a phosphoryl guanidine internucleotide linkage. In some embodiments, the modified internucleotide linkage is n001. In some embodiments, the modified internucleotide linkage has the structure -OP(O)(-N(R')SO ₂ R”)O- or a salt thereof, wherein each of R' and R" is independently as described herein In some embodiments, R' is R as described herein. In some embodiments, R' is -H or an optionally substituted C _1-6 aliphatic. In some embodiments, R' is -H In some embodiments, the modified internucleotide linkage has the structure -OP(O)(-NHSO ₂ R”)O- or a salt thereof, where R” is as described herein. In some embodiments, R" is R as described herein, wherein R is not -H. In some embodiments, R" is an optionally substituted group selected from C _1-6 aliphatic and phenyl. In some embodiments, R" is an optionally substituted phenyl. For example, in some embodiments, R" is 4-methylphenyl. In some embodiments, R″ is 4-(CH ₃ C(O)NH)C ₆ H ₄ . In some embodiments, R″ is optionally substituted C _1-6 aliphatic. In some embodiments, R" is an optionally substituted C _1-6 alkyl. In some embodiments, R" is methyl. In some embodiments, R" is ethyl. In some embodiments, R" is n-propyl. In some embodiments, R" is isopropyl. In some embodiments, R" is n-butyl. In some embodiments, the linkage is n002. In some embodiments, linkage is n006. In some embodiments, linkage is n020. In some embodiments, as shown in Figure 72, such internucleotide linkages can be used in place of phosphoryl guanidine internucleotide linkages such as n001. For example, in some embodiments, such internucleotide linkages are used at the 5'-end and/or the 3'-end. In some implementations, this connection is used internally. For example, in some embodiments, such an internucleotide linkage may be used between nucleosides N _-1 and N _-2 . For FIG. 72 , primary mouse hepatocytes (huADAR/SA1 Tg) were treated with the indicated oligonucleotide composition targeting the SERPINA1-Z allele for 48 hours (Zymnautic). RNA editing was quantified by Sanger sequencing.

In some embodiments, morpholine units can be used in place of natural sugars. Figure 73 confirms that such modifications can be used according to the present invention in oligonucleotides to provide editing. Primary mouse hepatocytes (huADAR/SA1 Tg) were treated with the indicated oligonucleotide composition targeting the SERPINA1-Z allele for 48 hours (Zymnautic). different types of linkages (eg PS (phosphorothioate), PO (natural phosphate linkages) and/or PNs (eg phosphorylguanidine linkages such as n001) nucleotide linkages) and different types of sugars (eg 2'- OMe modified sugars, 2'-F modified sugars, natural DNA sugars, morpholine sugars, etc.) were evaluated and found to provide editing of the target adenosine. In some embodiments, an oligonucleotide comprising a morpholine sugar and various modifications (eg, Gsm01, Tsm01, Tsm01n013, Gsm01n013, Tsm18) provides similar or reduced targeted adenosine editing relative to reference at particular concentrations. RNA editing was quantified by Sanger sequencing.

Figure 74 confirms that various modifications can be used in accordance with the present invention in oligonucleotides to provide editing. Primary mouse hepatocytes (huADAR/SA1 Tg) were treated with the indicated oligonucleotide composition targeting the SERPINA1-Z allele for 48 hours (Zymnautic). Different base modifications (e.g. b001A, b008U, etc.), type of linkage (e.g. PS (phosphorothioate), PO (natural phosphate linkage) and/or PN (e.g. phosphorylguanidine linkage such as n001) nucleotide linkage) And oligonucleotides containing various types of sugars (eg, 2'-OMe modified sugars, 2'-F modified sugars, natural DNA sugars, morpholine sugars, etc.) were evaluated and found to provide editing of the target adenosine. In some embodiments, an oligonucleotide comprising a morpholine sugar and various modifications (e.g., Gsm01, Tsm01, Csm01, Csm01n013, Tsm01n013, Gsm01n013, Tsm18) produces similar or reduced target adenosine editing relative to reference at particular concentrations. to provide. RNA editing was quantified by Sanger sequencing.

Dose response was evaluated for various oligonucleotide compositions. Specific results for specific compositions are given as examples below. Primary mouse hepatocytes (transformed for the human ADARp110 and SERPINA1-Z alleles) were treated for 48 hours with the indicated oligonucleotide composition targeting the SERPINA1-Z allele. RNA editing was quantified by Sanger sequencing. Oligonucleotides containing various modifications were evaluated and found to provide editing of the target adenosine. Serial dilution concentrations from about 1000 nM to about 0.5 nM. About 15% to 40% editing was observed at the lowest concentration and about 85% editing was observed at the highest concentration.

Among other things, the present invention provides a variety of nearest neighbor pairs that are not perfect matches at both N ₁ and N ₋₁ positions, but which in some implementations can provide robustness comparable to or better than perfect matches. 75 shows an example. Primary mouse hepatocytes (huADAR/SA1 Tg) were treated with the indicated oligonucleotide composition targeting the SERPINA1-Z allele for 48 hours (Zymnautic). RNA editing was quantified by Sanger sequencing.

Figure 76 confirms that various modifications can be used in accordance with the present invention in oligonucleotides to provide editing. Primary mouse hepatocytes (huADAR/SA1 Tg) were treated with the indicated oligonucleotide composition targeting the SERPINA1-Z allele for 48 hours (Zymnautic). Oligonucleotides containing various modifications (eg, b008U, b012U, b013U, b001A, b002A, b003A, b004I, b002G, b009U, etc.) were evaluated and found to provide editing of the target adenosine. In some embodiments, an oligonucleotide comprising a modified base (eg, b008U, b012U, b013U, b001A, b002A, b003A, b004I, b002G, b009U, etc.) across the editing site (position N ₀ ) is compared to a reference Provided similar or increased editing activity. RNA editing was quantified by Sanger sequencing.

As described herein, a variety of sugars and nucleobases can be utilized at positions comprising N ₁ . Figure 77 confirms that a variety of these sugars and/or nucleobases, including modified sugars and/or nucleobases, can be used in accordance with the present invention in oligonucleotides to provide editing. Primary mouse hepatocytes (huADAR/SA1 Tg) were treated with the indicated oligonucleotide composition targeting the SERPINA1-Z allele for 48 hours (Zymnautic). Oligonucleotides containing various nucleobases and sugars in N ₁ , such as dT, b002A, b003A, b008U, b001C, Tsm11, Tsm12, b004C, b007C, etc., were evaluated and confirmed to provide editing of the target adenosine. . In some embodiments, oligonucleotides comprising such sugars and/or nucleobases at the N ₁ position provided potent editing activity under certain conditions. RNA editing was quantified by Sanger sequencing. Confirmation that various sugars and nucleobases can be used in N ₁ along with other structural elements (eg, various sugars, nucleobases, internucleotide linkages, stereochemistry, etc.) in accordance with the present invention to provide editing Additional data is presented in FIG. 78 . For FIG. 78 , primary mouse hepatocytes (huADAR/SA1 Tg) were treated with the indicated oligonucleotide composition targeting the SERPINA1-Z allele for 48 hours (Zymnautic). For example, dT, b003A, b008U, b001C, b008C, Tsm11, Tsm12, b004C, Csm17, etc., oligonucleotides containing various sugars and nucleobases in N ₁ were evaluated and confirmed to provide editing of the target adenosine. . In some embodiments, oligonucleotides comprising such sugars and/or nucleobases at the N ₁ position provided potent editing activity under certain conditions. RNA editing was quantified by Sanger sequencing. As shown in the various figures, in many embodiments, natural and/or modified nucleobases (eg, C, b008U, etc.) and/or natural DNA sugars are utilized in N ₀ and/or natural and/or modified nuclei. A base (eg hypoxanthine) and/or natural DNA sugar is used in N _-1 .

Similarly, the present invention describes a variety of useful sugars and nucleobases for use at N _-1 , as well as useful internucleoside linkages that can be used to link N _-1 to its neighboring nucleoside. For example, Figure 79 confirms that a variety of sugars, nucleosides, internucleotide linkages, etc. can be used to provide editing. Primary hepatocytes were treated with the indicated oligonucleotide compositions targeting the SERPINA1-Z allele for 48 hours. Various sugars and nucleobases at N _-1 (e.g. in b001A, b003A, b008U, b001C, b008C, Tsm11, Tsm12, b004C, Csm17, etc.), various linkages (e.g. PS (phosphorus) between N _-1 and N _-2 polythioate) or PN (eg, phosphoryl guanidine linkages such as n001) linkages (eg, R p, Sp or sterically random), PS linkages between N ₀ and N _-1 , etc.) evaluated and confirmed to provide editing of the target adenosine. In some embodiments, certain nucleobases, sugars and/or internucleotidic linkages provide higher levels of editing than others. RNA editing was quantified by Sanger sequencing. Additional data are shown in Figure 80 (e.g., oligonucleotides comprising dI, b001A, b002A, b003A, b008U, b008C, Tsm11, Tsm12, b004C, Csm17, etc. in N _-1 ) and Figure 81 (e.g., dI, oligonucleotides including Csm11, Csm12, b009Csm11, b009Csm12, etc.). In some embodiments, certain sugars (eg, natural DNA sugars) and/or nucleobases (eg, hypoxanthine, b001A, b003A, etc.) of N _-1 provide higher levels of editing than others. In some embodiments, certain sugars (eg, DNA sugars) and/or nucleobases (eg, b008U) of N ₀ provide higher levels of editing than others.

Among other things, the present invention provides a variety of internucleotide linkages for use with other structural elements to provide oligonucleotides and compositions thereof. In some embodiments, an internucleotidic linkage is a non-negatively charged internucleotidic linkage. In some embodiments, the internucleotidic linkages are phosphoryl guanidine internucleotidic linkages. As shown in Figure 82, various internucleotide linkages, e.g., PN internucleotide linkages such as n001, n004, n008, n025, n026, etc., can be used in accordance with the present invention in oligonucleotides to provide editing. Primary mouse hepatocytes (huADAR/SA1 Tg) were treated with the indicated oligonucleotide composition targeting the SERPINA1-Z allele for 48 hours (Zymnautic). with various nucleobases (e.g. b008U, hypoxanthine, b014I, etc.), linkages (e.g. PS (phosphorothioate), PO (natural phosphate linkages) and/or PNs (e.g. n001, n004, n008, n025, n026, etc.) Evaluate oligonucleotides containing phosphoryl guanidine linkages) nucleotide linkages) and sugars (e.g., 2'-OMe modified sugars, 2'-F modified sugars, natural DNA sugars, 2'-MOE modified sugars, etc.) and target adenosine It was confirmed that the editing of RNA editing was quantified by Sanger sequencing. In some embodiments, oligonucleotides comprising various phosphorylguanidine internucleotidic linkages such as n001, n004, n008, n025, n026, etc. linked to N _-1 and N _-2 provide robust editing. In some embodiments, these internucleotidic linkages are chirally controlled and are Sp . In some embodiments, one or more non-n001 phosphoryl guanidine internucleotide linkages can independently be used in place of one or more n001 (and/or one or more other types of linkages).

As described herein, oligonucleotides may include duplex regions or may be used as duplexes. In some embodiments, the duplexed oligonucleotide forms a duplex with an oligonucleotide capable of targeting and editing a target adenosine. A specific example is given below by way of example. Serial dilution concentrations from about 1000 nM to about 0.5 nM. About 5% to 20% editing was observed at the lowest concentration and about 70% to 90% editing was observed at the highest concentration. Primary mouse hepatocytes (huADAR/SA1 Tg) were treated with the indicated oligonucleotide composition targeting the SERPINA1-Z allele for 48 hours (Zymnautic). Nucleobases of various types, linkages (eg, phosphorothioate (PS), natural phosphate linkages (PO), and/or PN (eg, phosphorylguanidine linkages, such as n001) nucleotide linkages) and sugars (eg, 2'- OMe modified sugars, 2'-F modified sugars, natural DNA sugars, 2'-MOE modified sugars, etc.) can form duplexes with corresponding duplexed oligonucleotides. Certain duplexes were evaluated as examples and confirmed to provide editing of the target adenosine. RNA editing was quantified by Sanger sequencing. In some embodiments, a particular duplex provided similar or increased editing activity relative to a reference. In some embodiments, the duplexed oligonucleotide has a 2'-OR modified sugar at both ends (where R is not -H, e.g., a 2'-OMe modified sugar, a 2'-MOE modified sugar, etc.) and/or Modified internucleotide linkages (eg, phosphorothioate internucleotide linkages). In some embodiments, the duplexed oligonucleotide is per 2'-F modification, per 2'-OR modification (where R is not -H, e.g., per 2'-OMe modification, per 2'-MOE modification, etc. ) and/or native RNA sugars. In some embodiments, it has been observed that duplexed oligonucleotides comprising an internal native RNA sugar can provide higher editing efficiencies when duplexed with a targeting oligonucleotide (eg, WV-46312).

As described herein, the provided technology can be used to edit target adenosines in a variety of nucleic acids. For example, as shown in FIG. 83 , various oligonucleotides comprising various modifications and patterns thereof can provide editing of the target adenosine in the UGP2 transcript. Primary human hepatocytes were treated with the indicated oligonucleotide compositions for 48 hours. RNA editing was quantified by Sanger sequencing. Additional data is presented as an example in FIG. 84 . Primary human hepatocytes were treated for 48 hours with the indicated oligonucleotide compositions targeting UGP2 at the indicated concentrations. RNA editing was quantified by Sanger sequencing. In some embodiments, per 2'-OR variant of a particular structural element, eg, a terminal region, multiple separate blocks (eg, one or more or each independently 2'-OR block) are separated. Oligonucleotides comprising a 2'-F block and/or a 2'-F modified sugar such as N _-3 may provide improved editing efficiency.

As described herein, provided technologies can provide for in vivo editing and provide a product, eg, a polypeptide, encoded by the edited nucleic acid. For example, FIG. 85 confirms in vivo editing of SERPINA1 and an increase in serum AAT levels. Mice transgenic for the human ADAR and SERPINA1-Z alleles were subcutaneously administered PBS or 10 mg/kg oligonucleotide on days 0, 2 and 4. Liver biopsies were collected on day 7 and serum AAT was collected before dosing and on day 7. As shown in Figure 85, provided oligonucleotide compositions delivered significant editing activity and increased levels of serum AAT relative to baseline (eg, PBS control, pre-administration levels). Serum AAT was quantified using ELISA. Certain additional results are presented in Figure 86, confirming that various modifications can be used in accordance with the present invention to provide oligonucleotides that are active in vivo. Mice transgenic for the human ADAR and SERPINA1-Z alleles were subcutaneously administered on day 0 with PBS or 10 mg/kg oligonucleotide. Liver biopsies were collected on day 10. Serum was collected prior to dosing, on days 7 and 10. Various oligonucleotide compositions were evaluated and found to provide targeted adenosine editing and increased serum AAT levels. RNA editing was quantified by Sanger sequencing. Serum AAT was quantified using ELISA.

Although various embodiments have been described and illustrated herein, those skilled in the art will readily envision various other means and/or structures for carrying out a function and/or obtaining a result and/or one or more advantages (described herein), and each such Changes and/or variations of are considered to be included. More generally, one skilled in the art should understand that all parameters, dimensions, materials and configurations described herein are exemplary and that the actual parameters, dimensions, materials and/or configurations may depend on the particular application or application for which the teachings of the present invention are being used. You will understand easily. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the embodiments of the present invention. Accordingly, it is to be understood that the foregoing implementations are presented by way of example only, and that within the scope of the appended claims and their equivalents, the claimed technology may be practiced otherwise than as specifically described and claimed. Also, any combination of two or more features, systems, articles, materials, kits, and/or methods is within the scope of the present invention, provided that such features, systems, articles, materials, kits, and/or methods do not contradict each other. do.

Claims (95)

As an oligonucleotide,
first domain; and
Including a second domain;
At this time,
the first domain comprises one or more 2'-F modifications;
the second domain comprises one or more sugars without a 2'-F modification;
About 30% to 70% (e.g., about 30% to 60%, 30% to 50%, or about 30%, 40%, 50%, 60%, or 70%) of the sugars in the first domain are independently contains a 2'-F variant;
30% to 70% (e.g., about 30% to 60%, 30% to 50%, or about 30%, 40%, 50%, 60%, or 70%) of the sugars in the first domain are 2'- OR, wherein R is an optionally substituted C _1-6 aliphatic. The oligonucleotide of claim 1 , wherein when the oligonucleotide is contacted with a target nucleic acid comprising the target adenosine in the system, the target adenosine in the target nucleic acid is modified, and the modification is or comprises conversion of the target adenosine to inosine. . The method of claim 2, wherein the first domain is one or more (eg, 1 to 20, 1 to 15, 1 to 14, 1 to 13, 1 to 12, 1 to 11, 1 to 10, 2 to 20, 3 ~15, 4~15, 5~15, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, etc.) of 2'-F blocks and one or more (e.g., 1-20, 1-15, 1-14, 1-13, 1-12, 1-11, 1-10, 2-20, 3-15, 4-15, 5-15, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 , 20, etc.), each sugar of each 2'-F block is independently a 2'-F modification sugar, and each sugar of each separation block is independently a 2'-F modification An oligonucleotide that is a sugar other than a sugar. 4. The oligonucleotide according to claim 3, wherein the first domain has three or more 2'-F blocks. 5. The oligonucleotide according to claim 4, wherein the first domain has two or more separating blocks. 6. The oligonucleotide of claim 5, wherein each sugar in the separating block is independently a 2'-OR modified sugar, wherein R is an optionally substituted C _1-6 aliphatic. 6. The oligonucleotide according to claim 5, wherein each block of the first domain linked to the 2'-F block of the first domain is a separate block. As an oligonucleotide,
first domain; and
Including a second domain;
At this time,
the first domain comprises one or more 2'-F modifications;
The oligonucleotide of claim 1 , wherein the second domain comprises one or more sugars without a 2'-F modification. An oligonucleotide comprising one or more modified sugars and/or one or more modified internucleotide linkages, wherein the oligonucleotide comprises a first domain and a second domain, each independently comprising one or more nucleobases. 10. The method of claim 8 or 9, wherein when the oligonucleotide is contacted with a target nucleic acid comprising the target adenosine in the system, the target adenosine in the target nucleic acid is modified, and the modification is or comprises conversion of the target adenosine to inosine. To, oligonucleotide. 8. The oligonucleotide of claim 7 having a length of about 26-35 nucleobases. 8. The oligonucleotide of claim 7, wherein each of the first and second domains independently has a length of about 10-50 nucleobases. 13. The oligonucleotide of claim 12, wherein about 50%-100% of the internucleotide linkages in the first domain are modified internucleotide linkages. 14. The oligonucleotide of claim 13, wherein the second domain comprises a nucleoside opposite to the target adenosine when the oligonucleotide is aligned with the target nucleic acid for complementarity. 15. The method of claim 14, wherein the opposite nucleobase is an optionally substituted or protected tautomer of U, an optionally substituted or protected tautomer of U, an optionally substituted or protected C, an optionally substituted or protected tautomer of C, or an optionally substituted tautomer of claim 14. is an optionally substituted or protected tautomer of A, an optionally substituted or protected nucleobase of pseudoisocytosine, an optionally substituted or protected tautomer of a nucleobase of pseudoisocytosine, or an optionally substituted or protected tautomer of nucleobase BA, and , BA is or comprises Ring BA or a tautomer thereof, and Ring BA is an optionally substituted 5-20 membered monocyclic, bicyclic or polycyclic ring having 0-10 heteroatoms. 16. The method of claim 15, wherein the nucleobase is BA, BA is or comprises ring BA or a tautomer thereof, and ring BA is an optionally substituted 5-20 membered monocyclic ring having 0-10 heteroatoms. , an oligonucleotide that is bicyclic or polycyclic. 17. The oligonucleotide according to claim 16, wherein BA has a weaker hydrogen bond with adenosine's target adenine than U. 17. The method of claim 16, wherein ring BA is X ² X ³ , X ² X ³ X ⁴ , -X ¹ ( ) X ² X ³ , -X ¹ ( ) X ² X ³ X ⁴ or a structure of formula BA-I, BA-Ia, BA-Ib, BA-II, BA-II-a, BA-II-b, BA-III, BA-III-a or BA-III-b Having, an oligonucleotide. 15. The method of claim 14, wherein the opposite nucleobase is Phosphorus, oligonucleotide. 15. The method of claim 14, wherein the opposite nucleobase is Phosphorus, oligonucleotide. 15. The method of claim 14, wherein the opposite nucleobase is , , , , , , , , , , , , , , , , , or Phosphorus, oligonucleotide. 15. The oligonucleotide of claim 14, wherein about 50% to 100% of the sugars in the second domain are independently modified sugars with modifications other than 2'-F. 23. The oligonucleotide of claim 22, wherein about 50%-100% of the internucleotide linkages in the second domain are modified internucleotide linkages. 24. The oligonucleotide of claim 23, wherein each modified internucleotide linkage is independently a phosphorothioate internucleotide linkage or a non-negatively charged internucleotide linkage. 25. The oligonucleotide of claim 24, wherein the second domain comprises one or more phosphorothioate internucleotidic linkages. 26. The oligonucleotide of claim 25, wherein the second domain comprises 1, 2, 3, 4, or 5 internucleotide linkages that are not negatively charged. 27. The oligonucleotide of claim 26, wherein the internucleotide linkage between the last nucleoside and the penultimate nucleoside of the second domain is a non-negatively charged internucleotide linkage. 26. The oligonucleotide of claim 25, wherein at least 50%-100% of the chiral internucleotidic linkages in the second domain are chirally controlled. 29. The oligonucleotide of claim 28, wherein the second domain comprises or consists of a first subdomain, a second subdomain, and a third subdomain in the 5' to 3' direction. 30. The oligonucleotide of claim 29, wherein the first subdomain is about 5-50 nucleobases in length. 31. The oligonucleotide of claim 30, wherein about 50%-100% of the sugars in the first subdomain are modified sugars with modifications other than 2'-F independently. 32. The oligonucleotide of claim 31, wherein the second subdomain is three nucleobases in length. 33. The oligonucleotide of claim 32, wherein the second subdomain comprises a nucleoside opposite to the target adenosine. 34. The oligonucleotide of claim 33, wherein the second subdomain comprises one or more natural DNA sugars. 35. The oligonucleotide of claim 34, wherein the second subdomain comprises one or more native RNA sugars. 35. The oligonucleotide of claim 34, wherein the second subdomain comprises about 2'-F modified sugars. 35. The oligonucleotide of claim 34, wherein the sugar of the opposite nucleoside comprises 2'-OH. 35. The oligonucleotide of claim 34, wherein the sugar of the opposing nucleoside is a natural DNA sugar. 35. The method of claim 34, wherein the sugar of the 5' adjacent nucleoside to the opposite nucleoside (5'-...N ₁ N ₀ ...3' to N ₁ where N ₀ is opposite to the target adenosine when aligned with the target The sugar of) is a natural DNA sugar, an oligonucleotide. 35. The method of claim 34, wherein the sugar of the 5' adjacent nucleoside to the opposite nucleoside (5'-...N ₁ N ₀ ...3' to N ₁ where N ₀ is opposite to the target adenosine when aligned with the target of) is an oligonucleotide comprising a 2'-F. 35. The method of claim 34, wherein the sugar of the adjacent nucleoside in the 3' direction to the opposite nucleoside (5'-...N ₀ N _-1 ...3' to N, where N ₀ is opposite to the target adenosine when aligned with the target _-1 sugar) is a natural DNA sugar, an oligonucleotide. 35. The method of claim 34, wherein each sugar of the opposite nucleoside, i.e., the sugar of the nucleoside adjacent in the 5' direction to the opposite nucleoside (when aligned with the target, N ₀ is opposite to the target adenosine, 5'-... N ₁ N ₀ ... sugar of N ₁ at 3'), and the sugar of the 3' adjacent nucleoside to the opposite nucleoside (5'-...N, where N ₀ is opposite the target adenosine when aligned with the target). ₀ N ₁ ...3' to N _-1 sugars) are independently natural DNA sugars. 35. The method of claim 34, wherein the sugar of the opposite nucleoside is a natural DNA sugar and the sugar of the adjacent nucleoside 5' to the opposite nucleoside (5' when aligned with the target, N ₀ is opposite to the target adenosine). -…N ₁ N ₀ …3' to N ₁ ) is the 2'-F modified sugar, and the sugar of the adjacent nucleoside in the 3' direction to the opposite nucleoside (N ₀ when aligned with the target is the target adenosine 5'-...N ₀ N _-1 ... 3' to N _-1 sugars) are natural DNA sugars, oligonucleotides. 35. The oligonucleotide of claim 34, wherein the nucleoside opposite the target nucleoside is linked to the adjacent nucleoside in the 3' direction via an R p phosphorothioate internucleoside linkage. 35. The method of claim 34, wherein the 3' adjacent nucleoside (position -1) to the opposite nucleoside (position 0) of the target nucleoside is 3' adjacent to the nucleoside (position -1) via a non-negatively charged internucleotide linkage. An oligonucleotide linked to a nucleoside (position -2). 35. The oligonucleotide of claim 34, wherein the 3' adjacent nucleoside comprises a base other than G. 35. The oligonucleotide of claim 34, wherein the 3' adjacent nucleoside comprises hypoxanthine. 35. The oligonucleotide of claim 34, wherein the third subdomain is about 1-10 nucleobases in length. 35. The oligonucleotide of claim 34, comprising a moiety that is or comprises GalNAc or a derivative thereof. An oligonucleotide comprising a modified nucleobase or modified linkage as described herein. An oligonucleotide identical to the oligonucleotide of any one of claims 1 to 50 except that there is a linkage having the structure -O ⁵ -P ^L (R ^CA )-O ³ - at the position of the linkage between the modified nucleotides.
(Wherein, P ^L is P or P(=W);
W is O, S, or W ^N ;
R ^CA is or contains an optionally substituted or capped chiral auxiliary moiety,
O ⁵ is an oxygen bonded to the 5'-carbon of the sugar;
O ³ is the oxygen attached to the 3'-carbon of the sugar). 52. The oligonucleotide according to claim 51, wherein each position of the modified internucleotide linkage independently has a linkage having the structure -O ⁵ -P ^L (W)(R ^CA )-O ³ -. 53. The method of claim 52, wherein each R ^CA is independently or , R ^C1 is R, -Si(R) ₃ or -SO ₂ R, and R ^C2 and R ^C3 are optionally substituted having 0 to 2 heteroatoms in addition to the nitrogen atom together with the atoms intervening therebetween. An oligonucleotide that forms a 3-7 membered saturated or partially unsaturated ring, and R ^C4 is -H or -C(O)R'. 53. The method of claim 52, wherein each R ^CA is independently or Phosphorus, oligonucleotide. 55. The oligonucleotide of claim 54, wherein R ^C1 is -SiPh ₂ Me or R ^C1 is -SO ₂ R, where R is optionally substituted phenyl. 56. The method of any one of claims 1 to 55, wherein the base sequence of the oligonucleotide is or comprises a sequence that differs from UUCAGUCCCUUUCTCIUCGA, CCCCAGCAGCUUCAGUCCCUUUCTCGUCGA, or CCCAGCAGCUUCAGUCCCUUUCTUIUCGAU in no more than 1, 2, 3, 4 or 5 positions, each An oligonucleotide wherein U may independently be replaced by T and vice versa. Mod001L001mCn001RmC*SmC*SfA*SfG*SmCmA*SfG*SfCmU*SfUn001RmCfA*SfGn001RfUmC*SfC*SfC*SfU*SmUmUfC*ST*Sb008U*SIn001SmUfC*SmG*SmAn001Rm As an oligonucleotide having a structure of U,
Mod001 is ego;
L001 is -NH-(CH ₂ ) ₆ -, wherein -NH- is linked to Mod001;
m represents the 2'-OMe modification to the nucleoside;
n001R represents R p n001 linkage, where n001 linkage has the structure of;
n001S represents an Sp n001 linkage;
*S represents an S p phosphorothioate linkage;
f represents a 2'-F modification to a nucleoside;
b008U is its base represents a phosphorus nucleoside;
I represents a nucleoside whose base is hypoxanthine. Mod001L001mCn001RmC*SmC*SfA*SfG*SmCmA*SfG*SfCmU*SfUn001RmCfA*SfGn001RfUmC*SfC*SfC*SfUn001RmUmUfC*ST*Sb008U*SIn001SmUfC*SmG*SmAn001 An oligonucleotide having the structure of RmU, wherein the modification is described in claim 57 or in the specification. As described above, oligonucleotides. Mod001L001mCn001RmC*SmC*SfA*SfG*SmCmAfG*SfCmU*SfUn001RmCfA*SfGn001RfUmC*SfC*SmCfUn001RmUmUfC*ST*Sb008U*SIn001SmUfC*SmG*SmAn001RmU An oligonucleotide having the structure, wherein the modifications are described in claim 57 (and/or specification). As described above, oligonucleotides. Mod001L001mCn001RmC*SmC*SfA*SfG*SfCmA*SfG*SmCmU*SfUn001RmCfA*SfGn001RfUmC*SfC*SfC*SfUn001RfU*SmUfC*ST*Sb008U*SIn001SmUfC*SmG*SmAn0 An oligonucleotide having the structure of 01RmU, wherein the modification is according to claim 57 (and / or oligonucleotides as described in the specification). Mod001L001mCn001RmC*SmC*SfA*SfG*SfCmA*SfG*SfCmU*SfUn001RmCfA*SfGn001RfUmC*SmCfC*SfUn001RfU*SmUfC*ST*Sb008U*SIn001SmUfC*SmG*SmAn001 An oligonucleotide having the structure of RmU, wherein the modification is as claimed in claim 57 (and/or oligonucleotide, as described in the specification). Mod001L001mCn001RmC*SmC*SfA*SfG*SmCmAfG*SfC*SfU*SfUn001RfC*SfAfGn001RfUmCmCfC*SfU*SmUmU*SfC*ST*Sb008U*SIn001SmUfC*SmG*SmAn001RmU An oligonucleotide having the structure, wherein the modification is defined in claim 57 (and/or oligonucleotide, as described in the specification). Mod001L001mCn001RmC*SmC*SfA*SfG*SfCmA*SfG*SfCmU*SfUn001RmCfA*SfGn001RfUmC*SfC*SfC*SfU*SfU*SmUfC*ST*Sb008U*SIn001SmUfC*SmG*SmAn001 An oligonucleotide having the structure of RmU, wherein the modification is as claimed in claim 57 An oligonucleotide, as described in (and/or specification). Mod001L001mCn001RmC*SmC*SfA*SfG*Sm5CeoAeofG*SfC*STeofUn001RmCfA*SfGn001RfUmC*SfC*SfC*SfUn001RTeoTeofC*ST*Sb008U*SIn001SmUfC*SmG*SmAn001R An oligonucleotide having the structure of mU, the modification of claim 57 (and/or specification) Oligonucleotides, as described in. Mod001L001mCn001RmC*SmC*SfA*SfG*SmCmAfG*SfC*SmUfUn001RmCfA*SmGn001RfUmC*SfC*SfC*SfUn001RmUmUfC*ST*Sb008U*SIn001SmUfC*SmG*SmAn001Rm An oligonucleotide having the structure of U, wherein the modification is described in claim 57 (and/or specification). Oligonucleotides, as described in. Mod001L001mCn001RmC*SmC*SfA*SfG*Sm5CeoAeofG*SfC*STeofUn001RmCfA*SmGn001RfUmC*SfC*SfC*SfUn001RTeoTeofC*ST*Sb008U*SIn001SmUfC*SmG*SmAn001R An oligonucleotide having the structure of mU, the modification of claim 57 (and/or specification) Oligonucleotides, as described in. Mod001L001mCn001RmC*SmC*SfA*SfG*Sm5CeoAeofG*SfC*STeofUn001RmCfA*SmGn001RfUm5Ceo*SfC*SfC*SfUn001RTeoTeofC*ST*Sb008U*SIn001SmUfC*SmG*SmAn00 An oligonucleotide having the structure of 1RmU, wherein the modification is described in claim 57 (and/or specification) Oligonucleotides, as described in. Structure of Mod001L001mCn001RmC*SmC*SfA*SfG*Sm5CeoAeofG*SmCTeo*SmUn001RmCfA*SfGn001RmUmCmC*SfC*SfU*STeoTeofC*ST*Sb008U*SIn001SmUfC*SmG*SmAn001RmU As an oligonucleotide having a, the modification of claim 57 (and / or specification) Oligonucleotides, as described in. Mod001L001mCn001RmC*SmC*SfA*SfG*Sm5CeoAeofG*SmCTeo*SmUn001RmCfA*SfGn001RmUm5CeomC*SfC*SfU*STeoTeofC*ST*Sb008U*SIn001SmUfC*SmG*SmAn001RmU An oligonucleotide having the structure of wherein the modification is described in claim 57 (and/or specification) Oligonucleotides, as described in. Mod001L001mCn001RmC*SmC*SfA*SfG*Sm5CeoAeofG*Sm5CeoTeo*SmUn001Rm5CeofA*SfGn001RmUm5Ceom5Ceo*SfC*SfU*STeoTeofC*ST*Sb008U*SIn001SmUfC*SmG*Sm An oligonucleotide having the structure of An001RmU, wherein the modification is described in claim 57 (and/or specification) Oligonucleotides, as described in. Mod001L001mCn001RmC*SmC*SfA*SfG*Sm5CeoAeofG*SfC*SmUmUn001RmCfA*SfGn001RfUm5Ceo*SfC*SmCmUn001RmUTeofC*ST*Sb008U*SIn001SmUfC*SmG*SmAn001 An oligonucleotide having the structure of RmU, wherein the modification is described in claim 57 (and/or specification) As described above, oligonucleotides. Mod001L001mCn001RmC*SmC*SfA*SfG*Sm5CeoAeofG*SfC*STeofUn001RmCfA*SfGn001RfUm5Ceo*SfC*SfC*SfUn001RTeoTeofC*ST*Sb008U*SIn001SmUfC*SmG*SmAn00 An oligonucleotide having the structure of 1RmU, wherein the modification is described in claim 57 (and/or specification) Oligonucleotides, as described in. 73. The oligonucleotide of any one of claims 1-72 in salt form. 74. The oligonucleotide of any one of claims 1-73 in the form of a pharmaceutically acceptable salt. 75. The method of any one of claims 1-74, wherein the diastereomeric excess of each chiral linking phosphorus is independently about or at least about 90%, 91%, 92%, 93%, 94%, 95%, 96% , 97%, 98% or 99%, an oligonucleotide. 76. The oligonucleotide of any one of claims 1-75 having a purity of about 10% to 100%. A pharmaceutical composition comprising or delivering an effective amount of the oligonucleotide of any one of claims 1 to 76 or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier. An oligonucleotide composition comprising a plurality of oligonucleotides, wherein the plurality of oligonucleotides
1) common base sequence and
2) share stereochemistry that is the same linkage independently in one or more chiral internucleotidic linkages ("chiral controlled internucleotidic linkages");
wherein each of the plurality of oligonucleotides is independently the oligonucleotide of any one of claims 1 to 76 or an acid, base or salt form thereof; or
An oligonucleotide composition comprising one or more of a plurality of oligonucleotides, wherein each of the plurality of oligonucleotides is independently
1) common base sequence and
2) share stereochemistry that is the same linkage independently in one or more chiral internucleotidic linkages ("chiral controlled internucleotidic linkages");
wherein each of the plurality of oligonucleotides is independently the oligonucleotide of any one of claims 1 to 76 or an acid, base or salt form thereof; or
A composition comprising a plurality of oligonucleotides of a particular oligonucleotide type, wherein the particular oligonucleotide type is
a) consensus sequence;
b) a common backbone connection pattern;
c) a common backbone chiral center pattern;
d) characterized by a deformation pattern that is a common backbone;
The composition is enriched in oligonucleotides of a particular oligonucleotide type relative to a substantially racemic preparation of oligonucleotides having the same common base sequence, backbone linkage pattern, and backbone phosphorus modification pattern, or within a composition that shares a common base sequence. chirally controlled in that all oligonucleotides at nonrandom levels are a plurality of oligonucleotides;
wherein each of the plurality of oligonucleotides is independently the oligonucleotide of any one of claims 1 to 76 or an acid, base or salt form thereof; or
An oligonucleotide composition comprising a plurality of oligonucleotides, wherein the plurality of oligonucleotides
1) common base sequence and
2) share stereochemistry that is the same linkage independently in one or more chiral internucleotidic linkages ("chiral controlled internucleotidic linkages");
wherein the consensus nucleotide sequence is complementary to a nucleotide sequence of a nucleic acid portion comprising a target adenosine; or
An oligonucleotide composition comprising one or more of a plurality of oligonucleotides, wherein each of the plurality of oligonucleotides is independently
1) common base sequence and
2) share stereochemistry that is the same linkage independently in one or more chiral internucleotidic linkages ("chiral controlled internucleotidic linkages");
a composition wherein each of the plurality of consensus nucleotide sequences is independently complementary to a nucleotide sequence of a nucleic acid portion comprising a target adenosine; or
A composition comprising a plurality of oligonucleotides of a particular oligonucleotide type, wherein the particular oligonucleotide type is
a) consensus sequence;
b) a common backbone connection pattern;
c) a common backbone chiral center pattern;
d) characterized by a deformation pattern that is a common backbone;
The composition is enriched in oligonucleotides of a particular oligonucleotide type relative to a substantially racemic preparation of oligonucleotides having the same common base sequence, backbone linkage pattern, and backbone phosphorus modification pattern, or within a composition that shares a common base sequence. chirally controlled in that all oligonucleotides at nonrandom levels are a plurality of oligonucleotides;
The consensus nucleotide sequence is complementary to the nucleotide sequence of the nucleic acid portion comprising the target adenosine, composition. 79. The composition of claim 78, wherein each of the plurality of oligonucleotides is an oligonucleotide of any one of claims 57-72 or a pharmaceutically acceptable salt thereof. 80. The method of claim 78 or 79, wherein the oligonucleotide level of the plurality of oligonucleotides in the composition that share a plurality of common base sequences is about or at least about (DS) ^nc (DS is about 85%-100% (e.g. , about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% or above) and nc is the number of chiral control internucleotide linkages), or the oligonucleotide level of a plurality of oligonucleotides in the composition that share the same configuration as the plurality of oligonucleotides or salts thereof is about or at least about (DS) ^nc (DS is About 85% to 100% (e.g., about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% , 98%, 99%, or 99.5% or more) and nc is the number of chiral control internucleotide linkages. A phosphoramidite wherein the nucleobase is a nucleobase or a tautomer thereof as described herein, wherein the nucleobase or tautomer thereof is optionally substituted or protected; or
A phosphoramidite wherein the nucleobase is or comprises Ring BA or a tautomer of Ring BA, wherein Ring BA is BA-I, BA-Ia, BA-Ib, BA-II, BA-II-a, BA-II-b, BA-III, BA-III-a, BA-III-b, BA-IV, BA-IV-a, BA-IV-b, BA-V, BA- A phosphoramidite having the structure Va, BA-Vb, or BA-VI, wherein the nucleobases are optionally substituted or protected. 82. The method of claim 81, having the structure R ^NS -P(OR)N(R) ₂ , R ^NS is an optionally protected nucleoside moiety, and each R is as described herein, preferably R A phosphoramidite having a structure of ^NS -P(OCH ₂ CH ₂ CN)N(i-Pr) ₂ . 82. The method of claim 81 comprising a chiral auxiliary moiety, wherein the phosphorus is bonded to the oxygen and nitrogen atoms of the chiral auxiliary moiety, preferably the phosphoramidite is Phosphoamidite, having a structure of. 84. The phosphoramidite of claim 83, wherein R ^C1 is -SiPh ₂ Me. 84. The phosphoramidite of claim 83, wherein R ^C1 is -SO ₂ R, wherein R is optionally substituted C _1-10 aliphatic or optionally substituted phenyl. 86. A method of preparing an oligonucleotide or composition comprising coupling the 5'-OH of an oligonucleotide or nucleoside with the phosphoramidite of any one of claims 81-85. As a method for characterizing oligonucleotides or compositions,
administering an oligonucleotide or composition to a cell or population comprising or expressing an ADAR1 polypeptide or a feature thereof, or a polynucleotide encoding an ADAR1 polypeptide or feature thereof; or
A method comprising administering an oligonucleotide or composition to a non-human animal or population comprising or expressing an ADAR1 polypeptide or feature thereof, or a polynucleotide encoding an ADAR1 polypeptide or feature thereof. 81. A method of modifying a target adenosine in a target nucleic acid comprising contacting the target nucleic acid with the oligonucleotide or composition of any one of claims 1-80; or
81. A method of deamination of a target adenosine in a target nucleic acid comprising contacting the target nucleic acid with an oligonucleotide or composition of any one of claims 1-80; or
81. A method of producing a product of a specific nucleic acid, or restoring or increasing the level of a product of a specific nucleic acid, comprising contacting a target nucleic acid with an oligonucleotide or composition of any one of claims 1-80, wherein the target nucleic acid is a method comprising a target adenosine, wherein a particular nucleic acid differs from a target nucleic acid in that it has I or G in place of the target adenosine; or
A method of reducing the product level of a target nucleic acid comprising contacting the target nucleic acid with an oligonucleotide or composition of any one of claims 1 - 80 , wherein the target nucleic acid comprises a target adenosine; or
A method comprising contacting the oligonucleotide or composition of any one of claims 1 to 80 with a sample comprising a target nucleic acid and an adenosine deaminase,
The base sequence of the oligonucleotide in the oligonucleotide or oligonucleotide composition is substantially complementary to the base sequence of the target nucleic acid,
the target nucleic acid comprises a target adenosine;
wherein the target adenosine is modified; or
As a method,
1) obtaining a modification of a first level of target adenosine in a target nucleic acid (the level observed when the first oligonucleotide composition is contacted with a sample comprising the target nucleic acid and adenosine deaminase), wherein the first oligonucleotide composition comprises: Comprising a first plurality of oligonucleotides sharing the same base sequence that is substantially complementary to the base sequence of the target nucleic acid, and
2) obtaining a modification of the reference level of target adenosine in the target nucleic acid (the level observed when the reference oligonucleotide composition is contacted with a sample comprising the target nucleic acid and adenosine deaminase), wherein the reference oligonucleotide composition is Comprising a plurality of reference oligonucleotides sharing the same base sequence that is substantially complementary to the base sequence,
At this time,
the first plurality of oligonucleotides comprises more sugars with 2'-F modifications, sugars with 2'-OR modifications (R is not -H), and/or chiral internucleotidic linkages than the reference plurality of oligonucleotides; do,
wherein the first oligonucleotide composition provides a higher level of modification relative to the oligonucleotides of the reference oligonucleotide composition; or
As a method,
obtaining a modification of a first level of target adenosine in the target nucleic acid (a level observed when the first oligonucleotide composition is contacted with a sample comprising the target nucleic acid and an adenosine deaminase), wherein the first oligonucleotide composition comprises: A first plurality of oligonucleotides sharing the same base sequence that is substantially complementary to the base sequence of the target nucleic acid,
The first level of modification of the target adenosine is higher than the reference level of modification of the target adenosine, the reference level observed when the reference oligonucleotide composition is contacted with a sample comprising a target nucleic acid and an adenosine deaminase, wherein the reference oligonucleotide composition is It includes a plurality of reference oligonucleotides that share the same base sequence that is substantially complementary to the base sequence of the target nucleic acid,
At this time,
the first plurality of oligonucleotides comprises more sugars with 2'-F modifications, sugars with 2'-OR modifications (R is not -H), and/or chiral internucleotidic linkages than the reference plurality of oligonucleotides; How to; or
As a method,
1) obtaining a modification of a first level of target adenosine in a target nucleic acid (the level observed when the first oligonucleotide composition is contacted with a sample comprising the target nucleic acid and adenosine deaminase), wherein the first oligonucleotide composition comprises: Comprising a first plurality of oligonucleotides sharing the same base sequence that is substantially complementary to the base sequence of the target nucleic acid, and
2) obtaining a modification of the reference level of target adenosine in the target nucleic acid (the level observed when the reference oligonucleotide composition is contacted with a sample comprising the target nucleic acid and adenosine deaminase), wherein the reference oligonucleotide composition is Comprising a plurality of reference oligonucleotides sharing the same base sequence that is substantially complementary to the base sequence,
At this time,
The first plurality of oligonucleotides may have more sugars with 2'-F modifications, sugars with 2'-OR modifications (R is not -H), and/or chiral controlled chiral internucleotidic linkages than the reference plurality of oligonucleotides. contains a lot,
wherein the first oligonucleotide composition provides a higher level of modification relative to the oligonucleotides of the reference oligonucleotide composition; or
As a method,
obtaining a modification of a first level of target adenosine in the target nucleic acid (a level observed when the first oligonucleotide composition is contacted with a sample comprising the target nucleic acid and an adenosine deaminase), wherein the first oligonucleotide composition comprises: A first plurality of oligonucleotides sharing the same base sequence that is substantially complementary to the base sequence of the target nucleic acid,
The first level of modification of the target adenosine is higher than the reference level of modification of the target adenosine, the reference level observed when the reference oligonucleotide composition is contacted with a sample comprising a target nucleic acid and an adenosine deaminase, wherein the reference oligonucleotide composition is It includes a plurality of reference oligonucleotides that share the same base sequence that is substantially complementary to the base sequence of the target nucleic acid,
At this time,
The first plurality of oligonucleotides may have more sugars with 2'-F modifications, sugars with 2'-OR modifications (R is not -H), and/or chiral controlled chiral internucleotidic linkages than the reference plurality of oligonucleotides. much inclusive, way; or
As a method,
1) obtaining a modification of a first level of target adenosine in a target nucleic acid (the level observed when the first oligonucleotide composition is contacted with a sample comprising the target nucleic acid and adenosine deaminase), wherein the first oligonucleotide composition comprises: Comprising a first plurality of oligonucleotides sharing the same base sequence that is substantially complementary to the base sequence of the target nucleic acid, and
2) obtaining a modification of the reference level of target adenosine in the target nucleic acid (the level observed when the reference oligonucleotide composition is contacted with a sample comprising the target nucleic acid and adenosine deaminase), wherein the reference oligonucleotide composition is Comprising a plurality of reference oligonucleotides sharing the same base sequence that is substantially complementary to the base sequence,
At this time,
the first plurality of oligonucleotides comprises one or more chiral controlled chiral internucleotidic linkages;
the reference plurality of oligonucleotides does not contain chiral controlled chiral internucleotide linkages (the reference oligonucleotide composition is a stereorandom composition);
wherein the first oligonucleotide composition provides a higher level of modification relative to the oligonucleotides of the reference oligonucleotide composition; or
As a method,
obtaining a modification of a first level of target adenosine in the target nucleic acid (a level observed when the first oligonucleotide composition is contacted with a sample comprising the target nucleic acid and an adenosine deaminase), wherein the first oligonucleotide composition comprises: It includes a first plurality of oligonucleotides sharing the same base sequence that is substantially complementary to the base sequence of the target nucleic acid,
The first level of modification of the target adenosine is higher than the reference level of modification of the target adenosine, the reference level observed when the reference oligonucleotide composition is contacted with a sample comprising a target nucleic acid and an adenosine deaminase, wherein the reference oligonucleotide composition is It includes a plurality of reference oligonucleotides that share the same base sequence that is substantially complementary to the base sequence of the target nucleic acid,
At this time,
the first plurality of oligonucleotides comprises one or more chiral controlled chiral internucleotidic linkages;
wherein the reference plurality of oligonucleotides does not contain chiral controlled chiral internucleotide linkages (the reference oligonucleotide composition is a stereorandom composition). 89. The method of claim 88, wherein the first oligonucleotide composition is the oligonucleotide composition of any one of claims 78-80. 89. The method of any one of claims 86-88, wherein the deaminase is an ADAR enzyme. 91. The method of any one of claims 87-90, wherein the target nucleic acid has an I or G at the position of the target adenosine instead of the target adenosine, compared to a nucleic acid that differs from the target nucleic acid, and is more related to the condition, disorder or disease. or is more related to a decrease in a desired property or function, or more related to an increase in an undesirable property or function. 92. The method of claim 91, wherein the target adenosine is a G to A mutation. 81. A method of preventing or treating a condition, disorder or disease comprising administering or delivering to a subject susceptible to or suffering from the condition, disorder or disease an effective amount of the oligonucleotide or composition of any one of claims 1-80; or
A method of preventing or treating a condition, disorder or disease associated with a G to A mutation, wherein an effective amount of the oligonucleotide or composition of any one of claims 1-80 is administered or delivered to a subject susceptible to or suffering from the condition, disorder or disease. How to include steps to do. 94. The method of claim 93, wherein the condition, disorder or disease is prone to A to G transformation or A to I transformation. A compound, oligonucleotide, composition or method of any one of the present specification or Exemplary Embodiments 1-1905.