BR112019027614B1 - SHORT DANGLING ARM BINDERS TO NUCLEOTIDES IN SEQUENCING APPLICATIONS - Google Patents

Abstract

The present disclosure relates to novel nucleotide and oligonucleotide compounds and their uses in nucleic acid sequencing applications.

Description

INCORPORATION BY REFERENCE TO ANY PRIORITY APPLICATION

[0001] This application claims the benefit of priority to United Kingdom (GB) Application No. 1711219.4, filed on 12 July 2017, which is incorporated by reference in its entirety.

BACKGROUND OF THE INVENTION

[0002] The present disclosure relates to novel nucleotide and oligonucleotide compounds and their uses in nucleic acid sequencing applications.

DESCRIPTION OF RELATED ARTWORK

[0003] As sequencing technology advances, a need has developed for improved sequencing reagents that are particularly amenable to high-throughput molecular methods such as solid-phase sequencing and the like.

[0004] In certain types of high-throughput sequencing, the nucleotides used contain fluorophores that specifically identify the incorporated base. The fluorophore can be attached to the nucleotide base via a cleavable linker. Therefore, once the incorporated base is identified, the linker can be cleaved, allowing the fluorophore to be removed, ready for the next base to be attached and identified. Such cleavage leaves a "scar" or "dangling arm" located at each of the detected nucleobases. Although it is possible to design reagents that leave no trace after cleavage, these tend to be slow to cleave and therefore are not particularly effective. A balance must be struck between efficient incorporation of the labeled nucleotides, efficient cleavage to remove all incorporated labels, and efficient incorporation of the next nucleotide. Optimized nucleotide structures that improve prior art nucleotide performance in Sequencing by Synthesis (SBS) cycles are described here.

[0005] Suitable nucleotide ligands are described, for example, in WO2004/018493. The compounds disclosed herein are shown as an example of formula (e): where B is a nucleoside base; and Fl is a fluorophore connected via an optional linker. This is cleaved to leave a pendant arm moiety of formula (ei):

[0006] The Applicant realized that changes in the hanging arm may provide improvements to the sequencing data obtained.

[0007] Improved nucleotide constructs and their uses in sequencing are described herein. The molecules described can be incorporated into nucleic acid strands. Also described are nucleic acid strands with certain modifications that allow for more efficient nucleotide incorporation. Also described are nucleic acid arrays and their uses in sequencing. Particular improvements can be observed in the efficiency of incorporation of labeled nucleotides, in the length and in the sequencing read accuracy obtained through the new constructs. The molecules described below are particularly advantageous in situations where long read lengths are required or where shorter nucleotide incorporation times are advantageous, and high-power excitation sources are used. SUMMARY

[0008] The present disclosure relates to improved reagents for nucleic acid sequencing. During repeated cycles of sequencing by synthesis (SBS), in which fluorescently labeled nucleotides are incorporated into an extended strand of nucleic acid, a "scarred" primer:DNA template is produced. The "scar" or "dangling arm" is a moiety located on each of the detected nucleobases, resulting from the chemical cleavage of the fluorophore from the nucleobase to which it is attached. These dangling arms accumulate on the primer:DNA template over repeated cycles of SBS, producing a primer:DNA template molecule with a molecular structure that differs from the natural primer:DNA template. These residual dangling arms slow the incorporation of the new incoming nucleotide and therefore increase the error rate and decrease data quality, especially in the later stages of a sequencing run and in long sequencing runs. The present disclosure aims to minimize the size of the overhanging arm left in the primer:DNA template during sequencing.

[0009] According to a first aspect, the present disclosure provides compounds of formula (I): where B is a nucleoside base; and Fl is a fluorophore connected through an optional linker.

[00010] Nucleosides are compounds that have a carbohydrate ribose attached to a nucleobase. The nucleobase may be a purine or a pyrimidine. Common naturally occurring purines are adenine (A) and guanine (G). Common naturally occurring pyrimidines are cytidine (C) and thymine (T) in DNA strands or uracil (U) in RNA strands. The ribose may be a 2'-deoxyribose (in DNA). A nucleoside with a phosphate moiety attached thereto is a nucleotide and therefore the compounds described herein may be in the form of a nucleotide. In any formulation as described herein, B represents a nucleotide base.

[00011] In any formulas described herein, B may represent a pyrimidine base. Base B may take the form of any of the four common natural bases, A, G, C, or T/U. The phosphate moiety may be a triphosphate moiety, which may be attached to the 5'-position of ribose. Compounds of the present disclosure may include compounds of formula (c), formula (t), or formula (a): where P is a triphosphate group; and Fl is a fluorophore connected through an optional linker.

[00012] The triphosphate moiety allows for incorporation of the nucleotide compound into a nucleic acid. Extension utilizing a nucleic acid polymerase allows for attachment of the compound to the 3'-OH of a nucleic acid primer. Thus, compounds of the present disclosure can be attached to an oligonucleic acid. The oligonucleotide can take the form of a nucleic acid primer that has been subjected to polymerase extension to incorporate a compound of the present disclosure. Thus, an oligonucleotide comprising a compound as disclosed herein is disclosed.

[00013] Disclosed is a nucleotide compound linked to an oligonucleotide. When the compound is fluorescently labeled, generally only a single modified compound would be linked to each oligonucleotide. Thus, disclosed is an oligonucleotide wherein the 3'-nucleotide is a compound of formula (I): where B is a nucleoside base; and Fl is a fluorophore connected through an optional linker.

[00014] For the avoidance of doubt, it is appreciated that upon incorporation, the triphosphate group is converted to a monophosphate diester. Thus, disclosed is an oligonucleotide wherein the 3'-nucleotide is a compound of formula (c), formula (t) or formula (a): where P is a monophosphate group; and Fl is a fluorophore connected through an optional linker.

[00015] The above compounds may alternatively be represented as an oligonucleotide, wherein the 3'-nucleotide is a compound of formula (c), formula (t) or formula (a): where Oln is an oligonucleotide; and Fl is a fluorophore connected via an optional linker.

[00016] After detection of the incorporated nucleobase, the fluorescent label and optional linker can be removed. Removal is accomplished by reduction of the azido group, leading to fragmentation of the O-CHNH2- moiety. After cleavage, a hydroxyl group is left attached to the nucleobase and the fluorescent moiety is highlighted.

[00017] Thus, a compound of formula (II) is disclosed: where B is a nucleotide base.

[00018] The nucleobase may be a purine or a pyrimidine. Common naturally occurring purines are adenine (A) and guanine (G). Common naturally occurring pyrimidines are cytidine (C) and thymine (T) in DNA strands or uracil (U) in RNA strands. The ribose may be a 2'-deoxyribose (in DNA).

[00019] The compound of formula II may be linked to an oligonucleotide. Thus, the B moiety may be a base linked to other bases. Thus, disclosed is an oligonucleotide wherein the 3'-nucleotide is a compound of formula (ci), formula (ti), or formula (ai): where Oln is an oligonucleotide.

[00020] The oligonucleotide having the hydroxyl pendant arm may have multiple pendant arms within the same oligonucleotide. The modified bases with the pendant arms would typically be contiguous in a sequence. An oligonucleotide comprising two or more copies of a compound according to formula (II) is disclosed. An oligonucleotide comprising ten or more copies of a composition according to formula (II) is disclosed. An oligonucleotide comprising one hundred or more copies of a compound according to formula (II) is disclosed.

[00021] Nucleotide incorporation can be accomplished using solutions with more than one type of nucleotide. Disclosed is a kit comprising two or more nucleotides, wherein at least one nucleotide is a labeled nucleotide as described herein. The kit may comprise two or more nucleotides, wherein at least two nucleotides are labeled nucleotides as described herein. The kit may comprise four nucleotides wherein at least two nucleotides are labeled nucleotides as described herein.

[00022] The nucleosides, nucleotides, oligonucleotides, and kits as described herein can be used in sequencing, expression analysis, hybridization analysis, genetic analysis, RNA analysis, or protein binding assays, or combinations thereof.

BRIEF DESCRIPTION OF THE FIGURES

[00023] Figure 1 illustrates the formation of the primer:template scarred or overhanging DNA during sequencing.

[00024] Figure 2 illustrates an example of the fully functionalized nucleotide (ffN) structure of the short overhanging arm used during sequencing.

[00025] Figure 3 illustrates examples of four short overhanging arm nucleotides for 2-channel SBS sequencing.

[00026] Figures 4A through 4C illustrate an example of the sequencing metrics observed with the standard and short overhanging arm nucleotides on a modified 2-channel Hiseq® with two different polymerases (PolA and PolB) and two different template libraries (PhiX and human).

[00027] Figures 5A and 5B demonstrate an example of the sequencing metrics observed with the standard nucleotide or the short overhanging arm nucleotide on a modified 2-channel Miseq®.

DETAILED DESCRIPTION

[00028] The present disclosure provides novel compounds particularly suitable for fluorescence detection and sequencing-by-synthesis methods. Compounds having a reduced residual overhanging arm improve certain nucleic acid sequencing applications.

[00029] According to a first aspect, the present disclosure provides compounds of formula (I): where B is a nucleoside base; and Fl is a fluorophore connected through an optional linker.

[00030] Nucleosides are compounds that have a carbohydrate ribose attached to a nucleobase. The nucleobase may be a purine or a pyrimidine. Common naturally occurring purines are adenine (A) and guanine (G). Common naturally occurring pyrimidines are cytidine (C) and thymine (T) in DNA strands or uracil (U) in RNA strands. The ribose may be a 2'-deoxyribose (in DNA). A nucleoside with a phosphate moiety attached thereto is a nucleotide, and therefore the compounds described herein may be in the form of a nucleotide. In any formulation as described herein, B represents a nucleotide base.

[00031] Fl is a fluorophore. Thus, the compounds of the present disclosure are fluorescently labeled. The nature of the fluorophore is not important and may include compounds selected from any known fluorescent species, for example, rhodamines or cyanines. The fluorophore may be linked to the nucleobase via an optional linker. The function of the linker is generally to aid in the chemical attachment of the fluorophore to the nucleotide. The linker may be, for example, an alkyl chain optionally having one or more heteroatom substitutions. The linker may contain amide or ester groups to facilitate chemical coupling reactions. The linker may be synthesized using click chemistry. The linker may contain triazole groups. The linker may contain other aryl groups.

[00032] Examples of ligands may include species such as the following: where n is an integer from 2 to 6 and Fl is a fluorophore.

[00033] The ribose moiety of the nucleoside or nucleotide may be a ribose or a deoxyribose. The ribose may have one or more 2' or 3' blocking groups intended to limit incorporation to a single nucleotide. The blocking group may be an azidomethyl group. The ribose may be a 2'-deoxyribose that has a 3'-azidomethyl blocking group. The blocking group may be an allyl group. The ribose may be a 2'-deoxyribose with a 3'-allyl blocking group.

[00034] In any formulas described herein, B may represent a pyrimidine base. Base B may take the form of any of the four common natural bases, A, G, C or T/U. The phosphate moiety may be a triphosphate moiety, which may be attached to the 5'-position of ribose. Compounds of the present disclosure may include compounds of formula (cl), formula (tl) or formula (al): where P is a triphosphate group; n is an integer from 2 to 6; and Fl is a fluorophore.

[00035] The triphosphate moiety allows for incorporation of the nucleotide compound into a nucleic acid. Extension using a nucleic acid polymerase allows for attachment of the compound to the 3'-OH of a nucleic acid primer. Thus, compounds of the present disclosure can be attached to an oligonucleic acid. The oligonucleotide can take the form of a nucleic acid primer that has been subjected to polymerase extension to incorporate a compound of the present disclosure. Thus, disclosed herein is an oligonucleotide comprising a compound as disclosed herein.

[00036] Disclosed is a nucleotide compound linked to an oligonucleotide. When the compound is fluorescently labeled, generally only a single modified compound would be linked to each oligonucleotide. Thus, disclosed is an oligonucleotide wherein the 3'-nucleotide is a compound of formula (III): where B is a nucleoside base; n is an integer from 2 to 6; and Fl is a fluorophore.

[00037] For the avoidance of doubt, it is appreciated that upon incorporation, the triphosphate group is converted to a monophosphate diester. Thus, disclosed is an oligonucleotide wherein the 3'-nucleotide is a compound of formula (cl), formula (tl) or formula (al): where P is a monophosphate group; n is an integer from 2 to 6; and Fl is a fluorophore.

[00038] The above compounds may alternatively be represented as an oligonucleotide wherein the 3'-nucleotide is a compound of formula (cl), formula (tl) or formula (al): where Oln is an oligonucleotide; n is an integer from 2 to 6; and Fl is a fluorophore.

[00039] In any of the examples provided n may be from 2 to 6. In any of the examples provided n may be equal to 2. In any of the examples provided n may be equal to 3. In any of the examples provided n may be equal to 4. In any of the examples provided n may be equal to 5. In any of the examples provided n may be equal to 6.

[00040] After detection of the incorporated nucleobase, the fluorescent label and optional linker can be removed. Removal is accomplished by reduction of the azido group, leading to fragmentation of the O-CHNH2- moiety. After cleavage, a hydroxyl group is left attached to the nucleobase and the fluorescent moiety is highlighted.

[00041] Thus, a compound of formula (II) is disclosed: where B is a nucleotide base.

[00042] The nucleobase may be a purine or a pyrimidine. Common naturally occurring purines are adenine (A) and guanine (G). Common naturally occurring pyrimidines are cytidine (C) and thymine (T) in DNA strands or uracil (U) in RNA strands. The ribose may be a 2'-deoxyribose (in DNA).

[00043] The compound of formula II may be linked to an oligonucleotide. Thus, the B moiety may be a base linked to other bases. Thus, disclosed is an oligonucleotide wherein the 3'-nucleotide is a compound of formula (ci), formula (ti), or formula (ai). where Oln is an oligonucleotide.

[00044] The oligonucleotide having the hydroxyl pendant arm may have multiple pendant arms on the same oligonucleotide. The modified bases with the pendant arms would typically be contiguous in a sequence. An oligonucleotide comprising two or more copies of a compound according to formula (II) is disclosed. An oligonucleotide comprising ten or more copies of a composition according to formula (II) is disclosed. An oligonucleotide comprising one hundred or more copies of a compound according to formula (II) is disclosed.

[00045] Nucleotide incorporation can be performed using solutions with more than one type of nucleotide. Thus, a kit comprising two or more nucleotides is disclosed, wherein at least one nucleotide is a labeled nucleotide as described herein. The kit may comprise two or more nucleotides, wherein at least two nucleotides are labeled nucleotides as described herein. The kit may comprise four nucleotides wherein at least two nucleotides are labeled nucleotides as described herein.

[00046] The nucleosides, nucleotides, oligonucleotides, and kits as described herein can be used in sequencing, expression analysis, hybridization analysis, genetic analysis, RNA analysis, or protein binding assays, or combinations thereof.

[00047] Provided herein are kits including two or more nucleotides, wherein at least one nucleotide is a nucleotide of the present disclosure. The kit may include two or more labeled nucleotides. The nucleotides may be labeled with two or more fluorescent labels. The two or more labels may be excited using a single excitation source, which may be a laser. For example, the excitation bands for the two or more labels may be at least partially overlapping, such that excitation in the overlapping region of the spectrum causes both labels to emit fluorescence. In particular embodiments, emission from the two or more labels will occur in different regions of the spectrum, such that the presence of at least one of the labels may be determined by optical distinction of the emission.

[00048] The kit may contain four labeled nucleotides, wherein the first of the four nucleotides is as disclosed herein. In such a kit, the second, third and fourth nucleotides may be labeled with a compound that is optionally spectrally different from the label on the first nucleotide and optionally spectrally different from the labels on each other. Thus, one or more of the compounds may have a distinct absorbance maximum and/or emission maximum, such that the compound(s) is/are distinguishable from the other compounds. For example, each compound may have a distinct absorbance maximum and/or emission maximum, such that each of the compounds is distinguishable from the other three compounds. It will be understood that some of the absorbance spectrum and/or emission spectrum other than the maxima may differ and these differences may be exploited to distinguish the compounds. The kit may be such that two or more of the compounds have a distinct absorbance maximum above 600 nm. The compounds of the invention typically absorb light in the region above 640 nm.

[00049] The compounds, nucleotides or kits presented herein can be used to detect, measure or identify a biological system (including, for example, processes or components thereof). Exemplary techniques that can employ the compounds, nucleotides or kits include sequencing, expression analysis, hybridization analysis, genetic analysis, RNA analysis, cellular assay (e.g., cell binding or cell function analysis), or protein assay (e.g., protein binding assay or protein activity assay). The use can be in an automated instrument to perform a specific technique, such as an automated sequencing instrument. The sequencing instrument can contain two lasers operating at different wavelengths.

[00050] The present disclosure provides fluorescently labeled nucleotide-nucleoside conjugates (modified nucleotides). The labeled nucleosides and nucleotides are useful for labeling polynucleotides formed by enzymatic synthesis, such as, by way of non-limiting example, in PCR amplification, isothermal amplification, solid phase amplification, polynucleotide sequencing (e.g., solid phase sequencing), nickel conversion reactions, and the like.

[00051] Nucleosides and nucleotides can be labeled at sites on the sugar or nucleobases. As known in the art, a "nucleotide" consists of a nitrogenous base, a sugar, and one or more phosphate groups. In RNA, the sugar is a ribose, and in DNA, it is a deoxyribose, that is, a sugar lacking a hydroxyl group that is present in ribose. The nitrogenous base is a derivative of a purine or pyrimidine. Purines can be adenine (A) or guanine (G), and pyrimidines can be cytosine (C), thymine (T), or, in the context of RNA, uracil (U). The C-1 atom of deoxyribose is bonded to N-1 of a pyrimidine or N-9 of a purine. A nucleotide is also a phosphate ester of a nucleoside, with esterification occurring at the hydroxyl group attached to C-3 or C-5 of the sugar. Nucleotides are usually mono-, di-, or triphosphates.

[00052] A "nucleoside" is structurally similar to a nucleotide, but without the presence of the phosphate moieties. An example of a nucleoside analogue would be one in which the label is attached to the base and there is no phosphate group attached to the sugar molecule.

[00053] Although the base is generally referred to as a purine or pyrimidine, one skilled in the art will appreciate that there are derivatives and analogues that do not alter the ability of the nucleotide or nucleoside to undergo Watson-Crick base pairing. "Derivative" or "analog" means a compound or molecule whose main structure is the same as or resembles that of an equivalent compound, but which has a chemical or physical modification, such as, for example, a different or additional side group, that allows the derivative nucleotide or nucleoside to be attached to another molecule. For example, the base may be a deazapurine. In particular embodiments, the derivatives are capable of undergoing Watson-Crick base pairing. "Derivative" and "analog" also include, for example, a synthetic nucleotide or derivative nucleoside with modified base moieties and/or modified sugar moieties. Such derivatives and analogues are discussed, for example, in Scheit, Nucleotide analogues (John Wiley & Son, 1980) and Uhlman et al., Chemical Reviews 90:543-584, 1990. Nucleotide analogues may also have modified phosphodiester linkages, including phosphorothioate, phosphorodithioate, alkylphosphonate, phosphoranilidate, phosphoramidate, and the like.

[00054] The fluorophore may be attached to any position on a nucleotide base, for example, via a linker. In particular embodiments, Watson-Crick basing may further be performed for the resulting analogue. Specific nucleobase labeling sites include the C5 position of a pyrimidine base or the C7 position of a 7-dezapurine base. As described above, a linker group may be used to covalently attach a dye to the nucleoside or nucleotide. In particular embodiments, the labeled nucleoside or nucleotide may be enzymatically incorporable and enzymatically extensible. Accordingly, a linker moiety may be of sufficient length to connect the nucleotide to the compound, such that the compound does not significantly interfere with the overall binding and recognition of the nucleotide by a nucleic acid replication enzyme. Thus, the linker may also comprise a spacer unit. The spacing unit distances, for example, the nucleotide base from a cleavage site or marker.

[00055] The labeled nucleosides or nucleotides according to the disclosure may have the formula: where Dye is a fluorescent compound, B is a nucleobase such as, for example, uracil, thymine, cytosine, adenine, guanine and the like, and L is an optional linking group which may or may not be present. R' can be H, monophosphate, diphosphate, triphosphate, thiophosphate, a phosphate ester analogue, -O- linked to a reactive phosphorus-containing group, or -O- protected by a blocking group. R'' can be H, OH, a phosphoramidite or a 3'OH blocking group and R''' is H or OH. R'' is phosphoramidite, R' is an acid-cleavable hydroxyl protecting group that allows subsequent monomer coupling under automated synthesis conditions.

[00056] In another alternative embodiment, there is no blocking group at the 3' carbon of the pentose sugar and the dye (or dye and linker construct) attached to the base, for example, may be of sufficient size or structure to act as a block for the incorporation of another nucleotide. Thus, the block may be due to steric hindrance or due to a combination of size, charge, and structure, regardless of whether or not the dye is attached to the 3' position of the sugar.

[00057] In another alternative embodiment, the blocking group is present on the 2' or 4' carbon of the pentose sugar and may be of sufficient size or structure to act as a block for the incorporation of an additional nucleotide.

[00058] The use of a blocking group allows polymerization to be controlled, such as by stopping extension when a modified nucleotide is incorporated. If the blocking effect is reversible, for example, by way of non-limiting example, by changing the chemical conditions or by removing a chemical block, extension can be stopped at certain points and then allowed to continue.

[00059] In another particular embodiment, a 3'OH blocking group will comprise moieties disclosed in WO2004/018497. For example, the blocking group may be azidomethyl (CH2N3) or allyl.

[00060] In a specific embodiment, a linker (between the dye and the nucleotide) and a blocking group are present and are separate moieties. In particular embodiments, the linker and the blocking group are cleavable under substantially similar conditions. Thus, the deprotection and deblocking processes may be more efficient, since only a single treatment will be required to remove the dye compound and the block. However, in some embodiments, a linker and a blocking group need not be cleavable under similar conditions, they may be individually cleavable under distinct conditions.

[00061] The present disclosure also encompasses polynucleotides incorporating fluorescent compounds. Such polynucleotides may be DNA or RNA comprised respectively of deoxyribonucleotides or ribonucleotides joined at the phosphodiester bond. Polynucleotides according to the disclosure may comprise naturally occurring nucleotides, non-naturally occurring (or modified) nucleotides other than the modified nucleotides of the present disclosure, or any combination thereof, in combination with at least one modified nucleotide (e.g., labeled with a dye compound) defined hereinafter. Polynucleotides according to the disclosure may also include non-natural backbone linkages and/or non-nucleotide chemical modifications. Chimeric structures comprised of mixtures of ribonucleotides and deoxyribonucleotides comprising at least one modified nucleotide according to the invention are also contemplated.

[00062] Modified nucleotides (or nucleosides) comprising a fluorescent compound according to the present disclosure may be used in any method of analysis, such as methods that include the detection of a fluorescent label attached to a nucleotide or nucleoside, either by itself, incorporated into, or associated with a larger molecular structure, or conjugated. In this context, the term "incorporated into a polynucleotide" may mean that the 5' phosphate is phosphodiester-linked to the 3' hydroxyl group of a second nucleotide (modified or unmodified), which may itself be part of a longer polynucleotide chain. The 3' end of a modified nucleotide disclosed herein may or may not be phosphodiester-linked to the 5' phosphate of another nucleotide (modified or unmodified). Thus, in a non-limiting embodiment, the disclosure provides a method for detecting a modified nucleotide incorporated into a polynucleotide comprising: (a) incorporating at least one modified nucleotide of the present disclosure into a polynucleotide and (b) detecting the modified nucleotide(s) incorporated into the polynucleotide by detecting the fluorescent signal of the dye compound bound to said modified nucleotide(s).

[00063] This method may include: a synthetic step (a) in which one or more modified nucleotides according to the disclosure are incorporated into a polynucleotide and a detection step (b) in which one or more modified nucleotides incorporated into the polynucleotide are detected, by detecting or quantitatively measuring their fluorescence(s).

[00064] In one embodiment of the present disclosure, at least one modified nucleotide is incorporated into a polynucleotide in a synthetic step by the action of a polymerase enzyme. However, other methods for attaching modified nucleotides to polynucleotides may be used, such as chemical synthesis of oligonucleotides or ligation of labeled oligonucleotides to unlabeled oligonucleotides. Therefore, the term "incorporating", when used in reference to a nucleotide and polynucleotide, may encompass the synthesis of polynucleotides by chemical methods as well as by enzymatic methods.

[00065] In a specific embodiment, a synthetic step is performed and may optionally comprise incubating a polynucleotide strand template with a reaction mixture comprising fluorescently labeled modified nucleotides of the present disclosure. A polymerase may also be provided under conditions that allow for the formation of a phosphodiester bond between a free 3' hydroxyl group on a polynucleotide strand annealed on the polynucleotide strand template and a 5' phosphate group on the modified nucleotide. Thus, a synthetic step may include forming a polynucleotide strand as directed by complementary base pairing of nucleotides with a strand template.

[00066] In all embodiments of the method, the detection step may be performed while the polynucleotide strand, into which the modified nucleotides are incorporated, is annealed to a strand template, or after a denaturation step in which the two strands are separated. Additional steps, e.g., chemical or enzymatic reaction steps or purification steps, may be included between a synthetic step and a detection step. In particular, the target strand incorporating the modified nucleotide(s) may be isolated or purified and then further processed, or used in a subsequent analysis. By way of example, target polynucleotides labeled with modified nucleotide(s) in a synthetic step may subsequently be used as labeled probes or primers. In other embodiments, the product of a synthetic step presented herein may be subjected to further reaction steps and, if desired, the product of these subsequent steps may be purified or isolated.

[00067] Suitable conditions for a synthetic step will be well known to those skilled in the art of molecular biology. In one embodiment, a synthetic step may be analogous to a standard primer extension reaction using nucleotide precursors, including modified nucleotides set forth herein, to form an extended target strand complementary to the template strand in the presence of a suitable polymerase enzyme. In other embodiments, a synthetic step may itself be part of an amplification reaction producing a labeled double-stranded amplification product composed of paired complementary strands derived from template copying of polynucleotide and target strands. Other exemplary synthetic steps include Nick translation, strand displacement polymerization, random labeling of primed DNA, etc. A particularly useful polymerase enzyme for a synthetic step is one that is capable of catalyzing the incorporation of one or more of the modified nucleotides set forth herein. A variety of naturally occurring or engineered polymerases may be used. By way of example, a thermostable polymerase may be used for a synthetic reaction carried out using thermocycling conditions, whereas a thermostable polymerase may also not be desired for isothermal primer extension reactions. Suitable thermostable polymerases that are capable of incorporating the modified nucleotides according to the disclosure include those described in WO 2005/024010 or WO06120433. In synthetic reactions carried out at lower temperatures, such as 37°C, the polymerase enzymes do not necessarily need to be thermostable polymerases, therefore the choice of polymerase will depend on several factors, such as reaction temperature, pH, strand unalignment activity and the like.

[00068] In specific non-limiting embodiments, the disclosure encompasses methods of nucleic acid sequencing, resequencing, whole genome sequencing, single nucleotide polymorphism scoring, or any other application involving detection of the modified nucleotide or nucleoside labeled with dyes set forth herein when incorporated into a polynucleotide. Any of a number of other applications that benefit from the use of polynucleotides labeled with the modified nucleotides comprising fluorophores may utilize modified nucleotides or nucleosides labeled with dyes set forth herein.

[00069] In a specific embodiment, the disclosure provides the use of modified nucleotides according to the disclosure in a polynucleotide sequencing by synthesis reaction. Sequencing by synthesis generally involves the sequential addition of one or more nucleotides or oligonucleotides to a growing polynucleotide chain in the 5' to 3' direction using a polymerase or ligase, in order to form an extended polynucleotide chain complementary to the nucleic acid template to be sequenced. The identity of the base present in the one or more added nucleotide(s) can be determined in a detection or "imaging" step. The identity of the added base can be determined after each nucleotide incorporation step. The sequence of the template can then be inferred using conventional Watson-Crick base pairing rules. The use of the dye-labeled modified nucleotides disclosed herein for determining the identity of a single base may be useful, for example, in classifying single nucleotide polymorphisms, and such single base extension reactions are within the scope of the present disclosure.

[00070] In one embodiment of the present disclosure, the sequence of a polynucleotide template is determined by detecting the incorporation of one or more nucleotides into a nascent strand complementary to the polynucleotide template to be sequenced by detecting fluorescent label(s) bound to the incorporated nucleotide(s). Sequencing of the polynucleotide template may be initiated with a suitable primer (or prepared as a harpin-like construct that will contain the primer as part of the harpin), and the nascent strand is extended in a stepwise manner by adding nucleotides to the 3' end of the primer in a polymerase-catalyzed reaction.

[00071] In particular embodiments, each of the different nucleotide triphosphates (A, T, G and C) may be labeled with a unique fluorophore and also comprise a blocking group at the 3' position to prevent runaway polymerization. Alternatively, one of the four nucleotides may be unlabeled (dark). The polymerase enzyme incorporates a nucleotide into the nascent strand complementary to the polynucleotide template, and the blocking group prevents further nucleotide incorporation. Any unincorporated nucleotides may be washed away and the fluorescent signal from each incorporated nucleotide may be "read" optically by suitable means, such as a charge-coupled device using laser excitation and suitable emission filters. The 3' blocking group and the fluorophore compounds may then be removed (deprotected), (simultaneously or sequentially) to expose the nascent strand for further nucleotide incorporation. Typically, the identity of the incorporated nucleotide will be determined after each incorporation step, but this is not strictly essential. Similarly, U.S. Patent No. 5,302,509 describes a method for sequencing polynucleotides immobilized on a solid support.

[00072] The method, as exemplified above, utilizes the incorporation of fluorescently labeled, 3'-blocked A, G, C, and T nucleotides into a growing strand complementary to the immobilized polynucleotide, in the presence of DNA polymerase. The polymerase incorporates a base complementary to the target polynucleotide, but is prevented from further addition by the 3'-blocking group. The label of the incorporated nucleotide can then be determined and the blocking group removed by chemical cleavage to allow further polymerization to occur. The nucleic acid template to be sequenced in a sequencing-by-synthesis reaction can be any polynucleotide that is desired to be sequenced. The nucleic acid template for a sequencing reaction will typically comprise a double-stranded region with a free 3' hydroxyl group that serves as a primer or initiation point for the addition of additional nucleotides in the sequencing reaction. The region of the template to be sequenced will extend beyond this free 3' hydroxyl group onto the complementary strand. The overhang region of the template to be sequenced may be single-stranded, but may be double-stranded, provided that a nick is present on the strand complementary to the template strand to be sequenced to provide a free 3' OH group for initiation of the sequencing reaction. In such embodiments, sequencing may be processed by strand displacement. In certain embodiments, a primer containing the free 3' hydroxyl group may be added as a separate component (e.g., a short oligonucleotide) that hybridizes to a single-stranded region of the template to be sequenced. Alternatively, the primer and the template strand to be sequenced may form part of a partially self-complementary nucleic acid strand capable of forming an intramolecular duplex, such as, for example, a harpin loop structure. Harpin polynucleotides and methods by which they may be attached to solid supports are disclosed in International Patent Publication Nos. WO0157248 and WO2005/047301. Nucleotides can be added successively to a growing primer, resulting in the synthesis of a polynucleotide chain in the 5' to 3' direction. The nature of the base that has been added can be determined, particularly, but not necessarily, after each nucleotide addition, thus providing sequence information for the nucleic acid template. Thus, a nucleotide is incorporated into a nucleic acid strand (or polynucleotide) by the attachment of the nucleotide to the free 3' hydroxyl group of the nucleic acid strand through the formation of a phosphodiester bond with the 5' phosphate group of the nucleotide.

[00073] The nucleic acid template to be sequenced may be DNA or RNA, or even a hybrid molecule composed of deoxynucleotides and ribonucleotides. The nucleic acid template may comprise naturally occurring and/or non-naturally occurring nucleotides and natural or non-natural backbone linkers, provided that they do not impede copying of the template in the sequencing reaction.

[00074] In certain embodiments, the nucleic acid template to be sequenced may be attached to a solid support via any suitable attachment method known in the art, e.g., via covalent attachment. In certain embodiments, the polynucleotide templates may be attached directly to a solid support (e.g., a silica-based support). However, in other embodiments of the present disclosure, the surface of the solid support may be modified in some manner to allow direct covalent attachment of the polynucleotide template or to immobilize the polynucleotide templates via a hydrogel or polyelectrolyte multilayer, which itself may be non-covalently attached to the solid support.

[00075] Arrangements in which the polynucleotides have been directly attached to silica-based supports are those, for example, described in WO00006770, in which the polynucleotides are immobilized on a glass support by reaction between an epoxide group pendant on the glass with an internal amino group on the polynucleotide. Furthermore, the polynucleotides may be attached to a solid support by reaction of a sulfur-based nucleophile with the solid support, for example, as described in WO2005/047301. Another example of a solid supported polynucleotide template is one in which the polynucleotide templates are attached to the hydrogel supported on silica-based supports or other solid supports, for example, as described in WO00/31148, WO001/01143, WO002/12566, WO003/014392, U.S. Patent No. 6,465,178 and WO000/53812.

[00076] A particular surface on which polynucleotide templates may be immobilized is a polyacrylamide hydrogel. Polyacrylamide hydrogels are described in the references cited above and in WO2005/065814.

[00077] Templates of DNA molecules can be attached to beads or microparticles. Attachment to beads or microparticles can be useful for sequencing applications. Libraries of beads can be prepared where each bead contains different DNA sequences. Exemplary libraries and methods for their creation are described in Nature. 437, 376-380 (2005); Science. 309, 5741, 1728-1732 (2005). Sequencing arrays of such beads using nucleotides presented herein is within the scope of the present disclosure.

[00078] The template(s) to be sequenced may be part of an "array" on a solid support; in this case, the array may take any convenient form. Thus, the method of the present disclosure is applicable to all types of high-density arrays, including single-molecule arrays, clustered arrays, and bead arrays. The modified nucleotides labeled with dye compounds of the present disclosure may be used to sequence templates on essentially any type of array, including, but not limited to, those formed by immobilizing nucleic acid molecules on a solid support.

[00079] However, the modified nucleotides of the present disclosure are particularly advantageous in the context of pooled array sequencing. In pooled arrays, distinct regions on the array (often referred to as sites or features) comprise multiple templates of polynucleotide molecules. Generally, the multiple polynucleotide molecules are not individually resolvable by optical means and are instead detected as a set. Depending on how the array is formed, each site on the array may comprise multiple copies of an individual polynucleotide molecule (e.g., the site is homogeneous for a given single- or double-stranded nucleic acid species) or even multiple copies of a small number of different polynucleotide molecules (e.g., multiple copies of two different nucleic acid species). Pooled arrays of nucleic acid molecules can be produced using techniques generally known in the art. By way of example, WO 98/44151 and WO00/18957, each of which is incorporated herein, describe methods of nucleic acid amplification in which the templates and amplification products are immobilized on a solid support to form arrays composed of clusters or "colonies" of immobilized nucleic acid molecules. The nucleic acid molecules present in the clustered arrays prepared according to these methods are suitable templates for sequencing using the modified nucleotides labeled with dye compounds of the present disclosure.

[00080] The modified nucleotides of the present disclosure are also useful in sequencing templates in single molecule arrays. The term "single molecule array" or "SMA" as used herein refers to a population of polynucleotide molecules, distributed (or arranged) on a solid support, wherein the spacing of any individual polynucleotide relative to the others in the population is such that it is possible to individually resolve the individual polynucleotide molecules. The target nucleic acid molecules immobilized on the surface of the solid support may thus be capable of being resolved by optical means in some embodiments. This means that one or more distinct signals, each representing a polynucleotide, will occur within the resolvable area of the imaging device used.

[00081] Single molecule detection may be achieved where the spacing between adjacent polynucleotide molecules in an array is at least 100 nm, more particularly at least 250 nm, even more particularly at least 300 nm, even more particularly at least 350 nm. Thus, each molecule is individually resolvable and detectable as a single molecule fluorescent spot, and the fluorescence from said single molecule fluorescent spot also exhibits photobleaching in a single step.

[00082] The terms "individually resolved" and "individual resolution" are used herein to specify that, when visualized, it is possible to distinguish a molecule in the array from its neighboring molecules. The separation between individual molecules in the array will be determined, in part, by the specific technique used to resolve the individual molecules. The general characteristics of single molecule arrays will be understood by reference to WO00/06770 and WO 01/57248. Although the use of the modified nucleotides of the present disclosure is applied in sequencing-by-synthesis reactions, the utility of the modified nucleotides is not limited to these methods. Indeed, the nucleotides may be advantageously utilized in any sequencing methodology that requires the detection of fluorescent labels attached to nucleotides incorporated into a polynucleotide.

[00083] In particular, the modified nucleotides of the present disclosure can be used in automated fluorescence sequencing protocols, particularly fluorophore-terminator cycle sequencing based on the Sanger chain termination sequencing method. Such methods generally use enzymes and cycle sequencing to incorporate fluorescently labeled dideoxynucleotides into a primer extension sequencing reaction. So-called Sanger sequencing methods and related (Sanger-like) protocols utilize random chain termination with labeled dideoxynucleotides.

[00084] The present disclosure also provides kits that include fluorophore-labeled modified nucleosides and/or nucleotides. Such kits generally include at least one modified nucleotide or labeled nucleoside as set forth herein along with at least one additional component. The additional component(s) may be one or more of the components identified in a method set forth above or in the Examples section below. Some non-limiting examples of components that may be combined in a kit of the present disclosure are set forth below.

[00085] In a particular embodiment, a kit may include at least one labeled modified nucleotide or nucleoside as set forth herein together with modified or unmodified nucleotides or nucleosides. For example, labeled modified nucleotides according to the present disclosure may be provided together with unlabeled or native nucleotides and/or with fluorescently labeled nucleotides or any combination thereof. Accordingly, kits may comprise modified nucleotides labeled with dyes according to the present disclosure and modified nucleotides labeled with other, e.g., dye compounds present in the prior art. The nucleotide combinations may be provided as separate individual components (e.g., one type of nucleotide per container or tube) or as mixtures of nucleotides (e.g., two or more nucleotides mixed in the same container or tube).

[00086] Where the kits comprise a plurality, particularly two, more particularly four, modified nucleotides labelled with a dye compound, the different nucleotides may be labelled with different dye compounds, or one may be dark, without dye compounds. Where the different nucleotides are labelled with different dye compounds, it is a feature of the kits that said dye compounds are spectrally distinguishable fluorophores. As used herein, the term "spectrally distinguishable fluorophores" refers to fluorophores that emit fluorescent energy at wavelengths that can be distinguished by fluorescence detection equipment (e.g., a commercial capillary-based DNA sequencing platform) when two or more such dyes are present in a sample. When two modified nucleotides labeled with fluorophoric compounds are provided in kit form, it is a feature of some embodiments that the spectrally distinguishable fluorophores can be excited at the same wavelength, such as, for example, by the same laser. When four modified nucleotides labeled with fluorophoric compounds are provided in kit form, it is a feature of some embodiments that two of the spectrally distinguishable fluorophores can be excited at one wavelength and the other two spectrally distinguishable dyes can be excited at another wavelength. Specific excitation wavelengths are 532 nm, 630 nm to 700 nm, particularly 660 nm.

[00087] In one embodiment, a kit includes a modified nucleotide labeled with a compound of the present disclosure and a second modified nucleotide labeled with a second dye, wherein the dyes have a difference in maximum absorbance of at least 10 nm, particularly from 20 nm to 50 nm. More particularly, the two dye compounds have Stokes shifts between 15 nm and 40 nm, where the "Stokes shift" is the distance between the absorption peak and the wavelength of the emission peak.

[00088] In a further embodiment, a kit may further include two further modified nucleotides labelled with fluorophores, wherein the dyes are excited by the same laser from 488 nm to 550 nm, particularly 532 nm. The dyes may have a difference in maximum absorbance of at least 10 nm, particularly 20 nm to 50 nm. More particularly, the two dye compounds may have Stokes shifts between 20 nm and 40 nm. Even more particularly, the two dye compounds may have a different maximum absorbance below 640 nm, particularly below 600 nm. Particular dyes that are spectrally distinguishable from the polymethine dyes of the present disclosure and that meet the above criteria are polymethine analogs such as described in U.S. Patent No. 5,268,486 (e.g. Cy3) or WO 0226891 (Alexa 532; Molecular Probes A20106) or asymmetric polymethines such as disclosed in U.S. Patent No. 6,924,372. Alternative dyes include rhodamine analogs, e.g. tetramethyl rhodamine and analogs thereof.

[00089] In an alternative embodiment, the kits of the present disclosure may contain nucleotides where the same base is labeled with two different compounds. A first nucleotide may be labeled with a first fluorescent compound, e.g., a 'red' fluorophore absorbing at greater than 650 nm. A second nucleotide may be labeled with a spectrally distinct compound, e.g., a 'green' fluorophore absorbing at less than 600 nm. A third nucleotide may be labeled as a mixture of the first fluorophore and the spectrally distinct compound, and the fourth nucleotide may be 'dark' and contain no label. In simple terms, nucleotides 1 through 4 may be labeled as 'green', 'red', 'red/green', and dark. To further simplify instrumentation, four nucleotides can be labeled with two dyes excited with a single laser, and thus the labeling of nucleotides 1 through 4 can be 'red 1,' 'red 2,' 'red 1/red 2' and dark or 'green 1,' 'green 2,' 'green 1/green 2' and dark.

[00090] Although the kits are exemplified above with respect to configurations with different nucleotides that are labeled with different dye compounds, it should be understood that the kits may include 2, 3, 4 or more different nucleotides that have the same dye compound.

[00091] In particular embodiments, a kit may include a polymerase enzyme capable of catalyzing the incorporation of the modified nucleotides into a polynucleotide. Other components to be included in such kits may include buffers and the like. The dye-labeled modified nucleotides according to the disclosure and other nucleotide components, including mixtures of different nucleotides, may be provided in the kit in a concentrated form to be diluted prior to use. In such embodiments, a suitable dilution buffer may also be included. Again, one or more of the components identified in a method set forth in this specification may be included in a kit of the present disclosure.

[00092] It is noted that, as used in this specification and the appended claims, the singular forms "a", "a", and "the" include plural referents unless expressly and unambiguously limited to one referent. It will be apparent to those skilled in the art that various modifications and variations may be made to various embodiments described herein without departing from the spirit or scope of the present teachings. Thus, it is intended that the various embodiments described herein encompass other modifications and variations within the scope of the appended claims and their equivalents.

EXAMPLES

[00093] Additional embodiments are disclosed in greater detail in the following examples, which are not intended to limit the scope of the claims. Example 1. Synthesis of Short Dangling Arm Nucleotide Triphosphate

Scheme 1: Example synthesis of two short pendant arm triphosphates (pppT-sPA-Fl and pppT-sPA2-Fl, where Fl is a fluorophore) Intermediate L2 synthesis

[00094] Starting material L1 (1.07 g, 4 mmol) was dissolved in anhydrous DMF (15 mL), then placed in nitrogen at 0 °C in an ice bath. N,N-Diisopropylethylamine (884 μL, 4.8 mmol) was added, followed by PyBOP (2.29 g, 4.4 mmol). The reaction was stirred in nitrogen at 0 °C for 20 min. Next, N-(5-aminopentyl)-2,2,2-trifluoroacetamide trifluoroacetate salt (1.5 g, 4.8 mmol) was added, followed by N,N-diisopropylethylamine (1 mL, 5.4 mmol). The reaction was removed from the ice bath and stirred at room temperature for 3 h. The solvent was removed under reduced pressure and the residue dissolved in ethyl acetate (100 mL). The solution was extracted with 3x 100 mL dilute aqueous KHSO4 (pH = 1), 1x 50 mL water, 2x 100 mL saturated aqueous NaHCO3. The organic phase was dried over anhydrous Na2SO4 and the solvent was removed under reduced pressure. The crude product was purified by flash chromatography on silica gel (linear gradient of ethyl acetate in DCM, 50% to 100%). L2 was isolated as a clear viscous oil (1.60 g, 3.59 mmol, 90%). 1H NMR (400 MHz,CDCl3): δ (ppm) 7.45 (m, 1H, Ar CH), 7.33 (m, 2H, Ar CH),7.07 (m, 2H, Ar CH, NH), 6.57 (t, J = 5.6 Hz, 1H, NH), 4.85 (t, J = 4.9 Hz, 1H, CH-N3), 4.22 (dd, J = 10.4, 5.1 Hz, 1H,CH2-OAr), 4.16 (dd, J = 10.5, 4.7 Hz, 1H, CH2-OAr), 4.00(ddd, J = 10.1, 4.9, 2.9 Hz, 1H, CH2-O), 3.83 (m, 2H, CH2-OH), 3.75 (ddd, J = 9.8, 6.6, 3.0 Hz, 1H, CH2-O), 3.45 (q, J = 6.7 Hz, 2H, CH2-NH), 3.36 (q, J = 6.6 Hz, 2H, CH2-NH), 2.78 (t, J = 6.2 Hz, 1H, OH), 1.65 (m, 4H, CH2-CH2-NH),1.41 (p, J = 7.4, 6.9 Hz, 2H, CH2-CH2-CH2-NH). 19F NMR(376.5 MHz, CDCl3): δ (ppm) -75.7. 13C NMR (100 MHz, CDCl3) (t), 69.4 (t), 61.6 (t), 39.7 (t), 39.4 (t), 29.0 (t),28.1 (t), 23.6 (t). LC-MS (ES and CI): (-ve) m/z 446 (M-H+); (+ve) m/z 448 (M+H+), 470 (M+Na+).

Synthesis of sPA ligand LN3 TFA

[00095] Alcohol L2 (500 mg, 1.12 mmol) was dissolved in acetonitrile (15 mL), then TEMPO (70 mg, 0.448 mmol) was added. NaH2PO4^2H2O (1.1 g, 7.2 mmol) and NaClO2 (405 mg, 4.48 mmol) were dissolved in 10 mL of water and added to the reaction. Then aqueous NaClO (14.5% available chlorine, 1.32 mL, 2.24 mmol) was added, and the solution immediately turned dark brown. The reaction was stirred at room temperature for 6 h. During this time, the brown color faded to orange. The reaction was quenched with concentrated aqueous Na2S2Oa until the reaction was colorless. Acetonitrile was evaporated under reduced pressure and the solution was diluted with 20 mL of water, and mixed with triethylamine (approx. 1 mL). The solution was extracted with 10 mL of ethyl acetate and the aqueous phase was concentrated under reduced pressure. The crude sPA LN3 TFA was purified by C18 reversed-phase chromatography (linear gradient of acetonitrile in water from 0% to 30%) and isolated as a clear oil (438 mg, 0.95 mmol, 85%). RP-HPLC: tR = 17.9 min (0.1 M TEAB/acetonitrile gradient from 5% to 50%, on a YMC-C18 analytical column). 1H NMR (400 MHz, CD3CN): δ (ppm) 8.03 (br s, 1H, NH), 7.63 (s, 1H, NH), 7.54 (s, 1H, Ar),7.42 (d, J = 7.7 Hz, 1H, Ar), 7.34 (t, J = 7.9 Hz, 1H, Ar),7.08 (d, J = 8.1 Hz, 1H, Ar), 5.10 (m, 1H, CH-N3), 4.35(dd, J = 10.9, 3.7 Hz, 1H, CH2O), 4.18 (dd, J = 10.8, 6.1Hz, 1H, CH2O), 4.09 (m, 2H, CH2O), 3.32 (m, 2H, CH2-NH) 19F NMR (376.5 MHz, CD3CN): δ (ppm) - 76.5. 13C NMR (100 MHz, CD3OD): δ (ppm) 173.1 (s), 170.0 (s), 159.9 (s), 137.5 (s), 131.0 (d), 125.7 (d), 121.4 (d), 119.2 (d), 114.5 (d), 90.8 (d), 70.5 (t), 66.7 (t), 41.0(t), 40μ.8 (t), 30.2 (t), 29.7 (t), 25.4 (t). LC-MS (ES eCI): (-ve) m/z 460 (M-H+). (+ve) m/z 484 (M+Na+), 561(M+Et3NH+).

Synthesis of intermediate L3

[00096] Starting material L1 (658 mg, 2.46 mmol) was dissolved in anhydrous DMF (10 mL), then placed under nitrogen at 0 °C in an ice bath. N,N-Diisopropylethylamine (544 μL, 2.95 mmol) was added, followed by PyBOP (1.41 g, 2.71 mmol). The reaction was stirred under nitrogen at 0 °C for 20 min, then N-(2-aminoethyl)-2,2,2-trifluoroacetamide trifluoroacetate salt (796 mg, 2.95 mmol) was added, followed by N,N-diisopropylethylamine (589 μL, 3.2 mmol). The reaction was removed from the ice bath and stirred at room temperature for 3 h. The solvent was removed under reduced pressure and the residue dissolved in ethyl acetate (100 mL). The solution was extracted with 3x 100 mL dilute aqueous KHSO4 (pH = 1), 1x 50 mL water, 2x 100 mL saturated aqueous NaHCO3 solution. The organic phase was dried over anhydrous Na2SO4 and the solvent was removed under reduced pressure. The crude product was purified by flash chromatography on silica gel (linear gradient of ethyl acetate in DCM, 50% to 100%) and isolated as a viscous oil (0.91 g, 2.24 mmol, 91%). 1H NMR (400 MHz, CDCl3): δ (ppm) 8.19 (br s,1H, NH), 7.39 (d, J = 1.7 Hz, 1H, Ar CH), 7.38 - 7.30 (m,2H, Ar), 7.24 (t, J = 5.5 Hz, 1H, Ar CH), 7.07 (dt, J =7.2, 2.1 Hz, 1H, Ar CH), 4.84 (t, J = 4.9 Hz, 1H, CH-N3), 4.20 (dd, J = 10.5, 5.1 Hz, 1H, CH2-OAr), 4.15 (dd, J =6.0, 4.5 Hz, 1H, CH2-OAr), 4.00 (ddd, J = 10.2, 4.8, 3.0Hz, 1H, CH2-O), 3.88 - 3.79 (m, 2H, CH2-OH), 3.74 (ddd, J =9.8, 6.4, 3.2 Hz, 1H, CH2-O), 3.65 (m, 2H, CH2-NH), 3.58(m, 2H, CH2-NH), 2.82 (t, J = 5.6 Hz 1H, OH). 19F NMR (376.5MHz, CDCl3): δ (ppm) -75.9. LC-MS (ES and CI): (-ve) m/z 404 (M-H+); (+ve) m/z 406 (M+H+), 428 (M+Na+).

Synthesis of sPA2 LN3 TFA

[00097] Alcohol L3 (230 mg, 0.567 mmol) was dissolved in acetonitrile (10 mL), then TEMPO (35 mg, 0.27 mmol) was added. NaH2PO4^2H2O (575 mg, 3.68 mmol) and NaClO2 (205 mg, 2.26 mmol) were dissolved in 10 mL of water and added to the reaction. Then aqueous NaClO (14.5% chlorine content, 0.671 mL, 1.13 mmol) was added, and the solution immediately turned dark brown. The reaction was kept at room temperature for 18 h. During this time, the brown color faded to orange. The reaction was quenched with concentrated aqueous Na2S2O3 until colorless. Acetonitrile was evaporated under reduced pressure and the solution was diluted with 10 mL of water, basified with triethylamine (approx. 0.6 mL) and extracted with 10 mL of ethyl acetate. The aqueous phase was concentrated under reduced pressure. Crude sPA2 LN3 TFA was purified by C18 reversed-phase chromatography (linear gradient of acetonitrile in water from 0% to 20%) and isolated as a clear oil (224 mg as triethylammonium salt, 0.43 mmol, 77%). RP-HPLC: tR = 17.9 min (0.1 M TEAB/acetonitrile gradient from 5% to 40%, on YMC-C18 analytical column). 1H NMR (400 MHz, CD3CN): δ (ppm) 8.52(br s, 1H, NH), 8.34 (br s, 1H, NH), 7.77 (s, 1H, Ar), 7.49(dt, J = 7.7, 1.1 Hz, 1H, Ar), 7.38 (t, J = 7.9 Hz, 1H,Ar) 6.7Hz, 1H, CH2O), 4.11 (d, J = 1.4 Hz, 2H, CH2O), 3. 66 — 3.42 (m, 4H, CH2-NH), 3.05 (q, J = 7.3 Hz, 5H, Et3N), 1.22 (t, J = 7.3 Hz, 8H, Et3N). 19F NMR(376.5 MHz, CD3CN): δ (ppm) -76.3. 13C NMR (101 MHz, CD3CN):δ (ppm) 173.1 (s), 166.9 (s), 157.6 (s), 135.7 (s), 129.3 (d), 120.0 (d), 118.0 (d), 117.0 (s), 112.1 (d), 88.4 (d), 68.8 (t), 67.6 (t), 45.1 (t), 39.2 (t), 38.4 (t), 7.5 (q).LC-MS (ES and CI): (-ve) m/z 418 (M-H+).

General synthesis of pppT-sPA and pppT-sPA2

[00098] The ligand (sPA-LN3-TFA or sPA2-LN3-TFA, 0.089 mmol) was coevaporated with 2x 2 mL of anhydrous N,N'-dimethylformamide (DMF), and then dissolved in 2 mL in anhydrous DMF. N,N-Diisopropylethylamine (33 μL, 0.178 mmol) was added, followed by O-(N-succinimidyl)-N,N,N',N'-tetramethyluronium tetrafluoroborate (TSTU, 32 mg, 0.106 mmol). The reaction was stirred with nitrogen at room temperature for 1 h. Meanwhile, an aqueous solution of PA-TTP triphosphate (0.1 mmol) was evaporated to dryness under reduced pressure and resuspended in 300 μL of 0.1 M triethylammonium bicarbonate (TEAB) solution in water. The ligand solution was added to the triphosphate, and the reaction was stirred at room temperature for 18 h. Then, the solvent was evaporated under reduced pressure, and the residue dissolved in 1 mL of methanol and 3 mL of 33% aqueous ammonium hydroxide. The solution was stirred at room temperature for 7 h, then evaporated to dryness. The crude product was purified by preparative-scale RP-HPLC using a YMC-Pack-Pro C18 column eluting with 0.1 M TEAB and acetonitrile. pppT-sPA: Yield: 67%. RP-HPLC: tR = 16.9 min (0.1 M TEAB/acetonitrile gradient from 5% to 35%, on YMC-C18 analytical column). LC-MS (ES and CI): (-ve) m/z 922 (M-H+), 461 (M-2H+). pppT- sPA2: Yield: 65%. RP-HPLC: tR = 17.5 min (0.1 M TEAB/acetonitrile gradient from 5% to 30%, on YMC-C18 analytical column). LC-MS (ES and CI): (-ve) m/z 880 (M-H+), 440 (M- 2H+).

General synthesis of pppT-sPA-Fl and pppT-sPA2-Fl

[00099] Fluorophore carboxylate (Fl, 0.0105 mmol) was coevaporated with 2x 2 mL of anhydrous N,N'-dimethylformamide (DMF) then dissolved in 2 mL of anhydrous N,N'-dimethylacetamide (DMA). N,N-Diisopropylethylamine (15 μL, 0.084 mmol) was added, followed by O-(N-succinimidyl)-N,N,N',N'-tetramethyluronium tetrafluoroborate (TSTU, 0.012 mmol). The reaction was stirred with nitrogen at room temperature for 45 min. Meanwhile, an aqueous solution of pppT-sPA or pppT-sPA2 triphosphate (0.084 mmol) was evaporated to dryness under reduced pressure and resuspended in 300 μL of 0.1 M triethylammonium bicarbonate (TEAB) solution in water. The NHS ester-fluorophore solution was added to the triphosphate, and the reaction was stirred at room temperature for 18 h. The crude product was purified by ion-exchange chromatography on DEAE-Sephadex (gradient 0.1 M TEAB to 1 M TEAB, with 20% acetonitrile) and by preparative scale RP-HPLC (YMC-Pack-Pro C18 column, eluting with 0.1 M TEAB and acetonitrile). General synthesis of N-(aminoalkyl)-2,2,2-trifluoroacetamide

Scheme 2: General synthesis of N-trifluoroacetamido-diamine.

[000100] tert-Butyl(aminoalkyl)carbamate (48 mmol) was dissolved in anhydrous DCM (60 mL) and placed in an ice-nitrogen bath. Triethylamine (103 mmol) was added, followed by trifluoroacetic anhydride (53 mmol) dropwise. The reaction was removed from the ice bath and stirred at room temperature for 1 h. The reaction was diluted with 200 mL of DCM and extracted with 2x 200 mL of saturated aqueous NaHCO3 solution. The organic phase was dried over anhydrous MgSO4 and concentrated under reduced pressure. The crude product was purified by flash chromatography on silica gel (petroleum ether/ethyl acetate 3:7).

[000101] tert-butyl(5-(2,2,2-trifluoroacetamido)pentyl) carbamate (n = 5): 1H NMR (400 MHz, CDCl3): δ (ppm) 6.40 (br s, 1H, NH), 4.49 (br s, 1H, NH), 3.30 (q, J = 6.8 Hz, 2H, CH2-NH), 3.06 (q, J = 6.6 Hz, 2H, CH2-NH), 1.56 (p, J = 7.2 Hz, 2H, CH2-CH2-NH), 1.45 (p,J = 7.0 Hz, 2H, CH2-CH2-NH), 1.37 (m, 9H, CH3), 1.31 (m, 2H,CH2-CH2- CH2-NH).19F NMR (376.5 MHz, CDCl3): δ (ppm) -75.8.

[000102] tert-butyl(2- (2,2,2-trifluoroacetamido)ethyl) carbamate (n = 2): 1H NMR (400MHz, CDCI3): δ (ppm) 7.72 (br s, 1H, NH), 4.86 (br s, 1H, NH), 3.39 (m, 2H, CH2-NH), 3.31 (m, 2H, CH2-NH), 1.38 (s,9H, CH3). 19F NMR (376.5 MHz, CDCl3): δ (ppm) -76.1.

[000103] tert-Butyl(2-(2,2,2-trifluoroacetamido)alkyl)carbamate (44 mmol) was dissolved in anhydrous DCM (40 mL) and trifluoroacetic acid (40 mL). The reaction was stirred at room temperature, open to air for 1 h. Volatiles were removed under reduced pressure. The residue was dissolved in water and extracted with 50 mL DCM, then the aqueous phase was evaporated to dryness. The residue was co-evaporated with 100 mL ethanol and 4x 100 mL acetonitrile.

[000104] N-(5-aminopentyl)-2,2,2-trifluoroacetamide (n = 5): 1H NMR (400 MHz, d6-DMSO): δ (ppm) 9.44 (br s, 1H,NH), 7.75 (br s, 3H, NH), 3.17 (q, J = 6.7 Hz, 2H, CH2-NH),2.77 (m, 2H, CH2-NH), 1.57-1.45 (m, 4H, CH2-CH2-NH), 1.29(m, 2H, CH2-CH2-CH2-NH).

[000105] N-(2-aminoethyl)-2,2,2-trifluoroacetamide (n = 2): 1H NMR (400 MHz, d6-DMSO): δ (ppm) 9.58 (br t, 1H,NHCO), 8.04 (br s, 3H, NH), 3.45 (m, 2H, CH2NHCO), 2.98 (m, 2H, CH2NH). 19F NMR (376.5 MHz, d6-DMSO): δ (ppm) -74.0, - 74.4.

Example 2. Use of short overhanging arm nucleotide triphosphates in SBS sequencing

[000106] The sequencing performance of short overhanging arm nucleotides was compared against the performance of standard nucleotides in SBS, the nucleotides containing the same fluorescent groups on a standard cleavable linker. Four short overhanging arm nucleotides (two As, C, T) labeled with fluorophores suitable for 2-channel SBS sequencing (Figure 3) and dark G were included in a solution comprising a DNA polymerase and an incorporation buffer, both used in a 2-channel modified Hiseq® or 2-channel modified Miseq® to sequence in two reads of 150 cycles each. Sequencing metrics (e.g., phasing, pre-phasing, % error rate, and Q30%) were compared between experiments performed using all four short overhang arm nucleotides or all standard nucleotides, under the same conditions (e.g., incorporation temperature, incorporation time, polymerase, template library). Results are shown in Figures 4A–4C and Figures 5A and 5B.

[000107] Figures 4A-4C demonstrate an example of the sequencing metrics (phasing, pre-phasing, % error rate, Q30%) observed with the standard nucleotide and the short overhang nucleotide on a modified 2-channel Hiseq®, using an embedding temperature of 50°C and an embedding time of 10 seconds, with two different polymerases (PolA and PolB) and two different template libraries (PhiX and human). When the short overhang nucleotides were used, a reduction in phasing and % error rate, and an increase in Q30%, were observed, regardless of the polymerase or template used.

[000108] Figures 5A and 5B demonstrate an example of sequencing metrics (phasing and error rate %) observed with standard nucleotide or short overhang nucleotide in a modified 2-channel Miseq® with embedding temperature equal to 60°C, in a PhiX template, allowing different embedding times.

[000109] The short overhanging arm nucleotides allowed an approximate 50% reduction in incorporation time without a significant impact on the percent error rate. At the shortest incorporation time tested (10 + 5 s), the percent error rate was still <1%, compared to an error rate of 2.5% with the standard nucleotides.FIGURESFigure 1 Figure 2 Figure 3 Figures 4A - 4C Figure 5A Figure 5B

Claims (25)

1. Compound characterized by the fact that it has the formula (I): where B is a nucleoside base or a nucleotide base; and Fl is a fluorophore; where when B is a nucleoside base or a purine nucleotide base, B is attached at position 8 and when B is a nucleoside base or a pyrimidine nucleotide base, B is attached at position 5. 2. Compound according to claim 1, characterized in that B is a nucleotide base. 3. Compound according to claim 2, characterized in that B is a pyrimidine base. 4. Compound according to claim 3, characterized in that the compound has formula (c) or formula (t): where P is a triphosphate group; and Fl is a fluorophore. 5. Compound according to claim 2, characterized in that B is a 7-deszapurine base. 6. Compound, according to claim 5, characterized by the fact that it has the formula (a): where P is a triphosphate group; and Fl is a fluorophore. 7. Compound according to any one of claims 1 to 6, characterized in that the fluorophore includes an alkyl chain ligand. 8. Compound according to claim 7, characterized in that the alkyl chain ligand has one or more heteroatom substitutions; and/or contains amide or ester groups; and/or contains aryl groups, optionally triazole groups. 9. Compound according to claim 2, characterized in that the compound is linked to an oligonucleotide. 10. Oligonucleotide characterized by the fact that it is labeled with a compound as defined in claim 2. 11. Compound characterized by the fact that it has the formula (II): where B is a nucleotide base; where when B is a purine nucleotide base, B is attached at position 8 and when B is a pyrimidine nucleotide base, B is attached at position 5. 12. Compound according to claim 11, characterized in that B is a pyrimidine base. 13. Compound, according to claim 12, characterized by the fact that it has the formula (ci) or the formula (ti): where in Oln is an oligonucleotide. 14. Compound according to claim 11, characterized in that B is a 7-deazapurine base. 15. Compound, according to claim 14, characterized by the fact that it has the formula (ai): where Oln is an oligonucleotide. 16. Oligonucleotide characterized by the fact that it comprises a compound as defined in any one of claims 11 to 15. 17. Oligonucleotide characterized by the fact that it comprises two or more copies of a compound as defined in any one of claims 11 to 15. 18. Oligonucleotide characterized by the fact that it comprises ten or more copies of a compound as defined in any one of claims 11 to 15. 19. Oligonucleotide characterized by the fact that it comprises one hundred or more copies of a compound as defined in any one of claims 11 to 15. 20. A kit comprising two or more nucleotides, wherein at least one nucleotide is a labeled nucleotide that is a compound as defined in any one of claims 2 to 8. 21. The kit of claim 20, comprising two or more nucleotides, wherein at least two nucleotides are labeled nucleotides that are compounds as defined in any one of claims 2 to 8. 22. The kit of claim 20, comprising four nucleotides, wherein at least two nucleotides are labeled nucleotides that are compounds as defined in any one of claims 2 to 8. 23. Use of a compound as defined in any one of claims 2 to 8 characterized in that it is for sequencing, expression analysis, hybridization analysis, genetic analysis, RNA analysis, or protein binding assays, or combinations thereof. 24. Use of an oligonucleotide as defined in claim 10 characterized in that it is for sequencing, expression analysis, hybridization analysis, genetic analysis, RNA analysis, or protein binding assays, or combinations thereof. 25. Use of an oligonucleotide as defined in claim 16 characterized in that it is for sequencing, expression analysis, hybridization analysis, genetic analysis, RNA analysis, or protein binding assays, or combinations thereof.