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WO2026006774A1 - Altered cytidine deaminases and methods of use - Google Patents

Altered cytidine deaminases and methods of use

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Publication number
WO2026006774A1
WO2026006774A1 PCT/US2025/035754 US2025035754W WO2026006774A1 WO 2026006774 A1 WO2026006774 A1 WO 2026006774A1 US 2025035754 W US2025035754 W US 2025035754W WO 2026006774 A1 WO2026006774 A1 WO 2026006774A1
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WIPO (PCT)
Prior art keywords
acd
seq
functionally equivalent
amino acid
enhancing
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/US2025/035754
Other languages
French (fr)
Inventor
Anna TRACZYK
Lekha RAVICHANDRAPRABHU
Dewei Joel TOH
Shu Ting TAN
Saurabh Nirantar
Eric Brustad
June PAIS
Gaetano SPECIALE
Bridget DORSEY
Stephanie SILVA
Rebekah KARADEEMA
Rohit SUBRAMANIAN
Yvonne DEVADAS
Jing Liang
Jonathan Boutell
Vicki THOMSON
Hsu Myat NOE
Wei Wei
Zahra FAHMI
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Illumina Inc
Original Assignee
Illumina Inc
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Publication date
Application filed by Illumina Inc filed Critical Illumina Inc
Publication of WO2026006774A1 publication Critical patent/WO2026006774A1/en
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/78—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y305/00—Hydrolases acting on carbon-nitrogen bonds, other than peptide bonds (3.5)
    • C12Y305/04—Hydrolases acting on carbon-nitrogen bonds, other than peptide bonds (3.5) in cyclic amidines (3.5.4)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y305/00—Hydrolases acting on carbon-nitrogen bonds, other than peptide bonds (3.5)
    • C12Y305/04—Hydrolases acting on carbon-nitrogen bonds, other than peptide bonds (3.5) in cyclic amidines (3.5.4)
    • C12Y305/04005—Cytidine deaminase (3.5.4.5)

Definitions

  • Modified DNA cytosines including 5-methylcytosine (5mC) and 5 -hydroxymethyl cytosine (5hmC), are a well-studied epigenetic modification that play fundamental roles in human development and disease. Its genome-wide distribution differs between tissue types, and between healthy and diseased states. In recent years, 5mC has also gained prominence as a tool for clinical diagnostics: its distribution in cell-free DNA (cfDNA) - obtained from a liquid biopsy - can be used for the tissue-specific prediction of early-stage cancer or monitoring of cancer recurrence or remission after treatment. As a result, there has been an intense focus on developing methods for mapping modified DNA cytosines at single base resolution, with minimal loss of sample DNA quantity, quality, and complexity.
  • cfDNA cell-free DNA
  • mapping modified DNA cytosines exhibit limitations including (i) degradation of sample DNA due to prolonged chemical treatment at non-neutral pH and high temperatures, (ii) loss of sample DNA complexity due to conversion of unmethylated DNA bases to uracil, resulting in low complexity genome mapping, (iii) multi-step conversion, requiring both enzymes and chemical treatment, and (iv) for antibody-based 5mC detection, resolution of detection is limited to ⁇ 150bp, precluding the identification of its exact location in the genome.
  • the selectivity-enhancing alteration at amino acid 130, 131, 132, 133, 134, 135, or a combination thereof can be a substitution mutation to any amino acid, and in some embodiments the ACD includes two or more selectivity-enhancing alterations at a position functionally equivalent to amino acid 130, 131, 132, 133, 134, 135, or a combination thereof.
  • the amino acid sequence of amino acids 130, 131, 132, 133, and 134 can be selected from SEQ ID NO:25-34 of FIG. 4B, SEQ ID NO:35-50 of Table 2, or the amino acids at positions 130, 131, 132, 133, and 134 in the sequences found in Figure 8 or 9.
  • an ACD can further include at least one selectivity-enhancing alteration at a position functionally equivalent to amino acid 102, 103, 104, 105, or a combination thereof in a wild-type APOBEC3A (e.g., SEQ ID NO:3).
  • the amino acid sequence of amino acids 102, 103, 104, 105, 130, 131, 132, 133, 134, and 135 in an ACD is selected from SEQ ID NO: 100-478 of FIG. 8.
  • the amino acid sequence of amino acids 130, 131, 132, 133, and 134 is selected from SEQ ID NO: 23, SEQ ID NO:24, or the sequences described in FIG. 8 or FIG. 9 which demonstrate increased 5mC selectivity compared to wild-type (SEQ ID NO:3).
  • an ACD can include two or more stability mutations selected from I17T, T19Y, G25K, S45W, A59P, K60R, A61-68, R74L, G108A, G108C, C171A, G188P, and A104-105; a combination of stability mutations including T19Y, G25K, S45W, R74L, G108A, Cl 71 A, and G188R; or a combination of stability mutations including I17T, T19Y, G25K, S45W, A59P, K60R, A61-68, R74L, G108C, C171A, and G188R.
  • FIG. ID -IE shows the result of treating a DNA sample with a wild-type APOBEC3 A enzyme (FIG. ID), and an example of one-step detection of 5mC using an altered cytidine deaminase described herein (FIG. IE).
  • the top strand of FIG. 1D-E shows C and 5mC bases, and the bottom strand of FIG. 1D-E underlines the changed bases.
  • 5mC nucleobases are marked with CHB, 5 -hydroxymethyl uracil nucleobases are designated with small case "u,” and uracil nucleobases are designated with capital "U.”
  • FIG. 2 is a schematic showing alignment of cytidine deaminase amino acid sequences using the Clustal O algorithm.
  • An (asterisk) indicates positions which have a single, fully conserved residue between some cytidine deaminases.
  • a (colon) indicates conservation between groups of strongly similar properties as below - roughly equivalent to scoring > 0.5 in the Gonnet PAM 250 matrix.
  • the amino acids marked with " A " show the ZDD motif SEQ ID NO:1 (e.g., above amino acids 70 to 106 of sp
  • P3194111- 199 is a human APOBEC3A, SEQ ID NO:3;
  • XP_045219544.1 is an APOBEC3A from Macaca fascicularis, SEQ ID NO:4;
  • AER45717.1 is an APOBEC3A from Pongo pygmaeus, SEQ ID NO:5;
  • XP_003264816.1 is an AP0BEC3A from Nomascus leucogenys, SEQ ID NO:6;
  • PNI48846.1 is an APOBEC3A from Pan troglodytes, SEQ ID NO:7;
  • ADO85886.1 is an APOBEC3 A from Gorilla gorilla, SEQ ID NO:8.
  • FIG. 3 shows a bar graph representation of APOBEC3A(Y130X) deaminase activity
  • FIG. 4A-4B shows information related to the results of a high-throughput screen to test for mC specific mutants.
  • FIG. 4A The library diversity on positions Y130 to Pl 34 before screening and after 2 rounds of enrichment tested by NGS.
  • FIG. 4B The top 10 enriched mutants in high throughput assay from NGS analysis.
  • FIG. 5A-5B shows selectivity plot for (FIG. 5A) randomly picked mutants after 2 round of sorting, and (FIG. 5B) top enriched mutants based on NGS analysis of the library.
  • FIG. 6A-6C shows an exemplary selectivity plot for various mutations on positions S 103/ W104 in a ScF (SEQ ID NO:18) background (FIG. 6A); various mutations at positions S103/W104 combined with certain AxxxW mutants (FIG. 6B); and various mutations at positions F102/S103/W104/G105 combined with certain AxxxW mutations (FIG. 6C) (see, e.g., SEQ ID NO:955-968).
  • FIG. 7A-7C shows the activity of different exemplary ACD mutants on methylation reporting on pUC19 (CG methylated) and Lambda (fully unmethylated).
  • the parent backbone SEQ ID NO: 14 is shown in gray.
  • FIG. 8A-8M shows examples of sets of selectivity-enhancing alterations, e.g., substitution mutations, deletions, and/or insertions, of amino acids at positions functionally equivalent to amino acids 102, 103, 104, 105, 130, 131, 132, 133, 134, and/or 135 in SEQ ID NO: 14, SEQ ID NO: 17, SEQ ID NO: 18, or SEQ ID NO: 19.
  • selectivity-enhancing alterations e.g., substitution mutations, deletions, and/or insertions, of amino acids at positions functionally equivalent to amino acids 102, 103, 104, 105, 130, 131, 132, 133, 134, and/or 135 in SEQ ID NO: 14, SEQ ID NO: 17, SEQ ID NO: 18, or SEQ ID NO: 19.
  • FIG. 9A-9MM shows amino acid sequences of proteins described herein.
  • FIG. 10A-10C demonstrates expression and purification of ACD proteins in fusion with helicases.
  • First step of purification with Ni-NTA column (a) was followed by size exclusion column (b).
  • the purity of the proteins was confirmed by SDS gel (c).
  • FIG. 11 Testing ACD deamination activity in fusion constructs using Swal assay
  • a deaminase enzyme such as ACD
  • the enzyme If the enzyme is active on the substrate, it will deaminate methylcytosine (mC) to thymine (T), creating a mismatch with the complementary reverse oligonucleotide. If a mismatch occurs, the restriction enzyme Swal will cleave the double-stranded DNA oligonucleotide. The cleaved products can be then visualized by UREA-PAGE gel electrophoresis,
  • Swal assay results for ACD fusions with helicases For ACD fusions with helicases.
  • FIG. 12A-12B shows a schematic representation of NGS-based assay to assess ACD activity in helicase fusions.
  • FIG. 12A Substrate ssDNA oligo contains 17 unmethylated cytosines (C) and 16 methylated C sites (mC).
  • FIG. 12B Deamination by ACD results in conversion of unmethylated C residues to uracil, and conversion of mC to thymine. These deamination events are read out as C ⁇ >T mutations via Illumina sequencing.
  • FIG. 13A-13B demonstrates the testing helicase activity in fusion constructs using fluorescent oligos.
  • FIG. 13A Schematic of the fluorescent assay for helicase activity on dsDNA. The assay uses a 5'-F AM-labeled oligonucleotide, with the reverse complementary strand containing a quencher to suppress fluorescence. In the presence of helicase activity, the helicase unwinds the double-stranded DNA, replacing the quencher-containing strand with a competitor oligo that lacks the quencher. This replacement removes the quenching effect and results in an increase in FAM fluorescence, which can be detected.
  • FIG. 13B Helicase activity results for helicases in fusion with ACD.
  • FIG. 14A-14B demonstrates that the ACD in fusion with helicases can deaminate dsDNA.
  • FIG. 15 demonstrates that ACD in fusion with helicases can deaminate dsDNA in a complex substrate.
  • ACD protein or ACD-helicase fusions were added to mix of lambda DNA (non- methylated control) with fully methylated pUC19 DNA. DNA was either denatured with heating to 70°C in the presence of DMSO or not denatured at all. Only fusion constructs show activity if the denaturation step is skipped.
  • FIG. 16A-16C shows testing direct deamination of 5mC on-flow cell at NextSeq 2000 and NovaSeq X. Comparison of sequencing statistic (A) of 5mC conversion of ACD and Helicase-ACD. (B) Deamination with ACD using denaturation with NaOH without a wash on NextSeq 2000 (B) and NovaSeq X (C).
  • FIG. 17A-B shows testing direct deamination of 5mC on-flow cell with the Helicase- ACD fusion using (FIG. 17A) NextSeq 2000 and (FIG. 17B) Novaseq X.
  • FIG. 17A-B shows testing direct deamination of 5mC on-flow cell with the Helicase- ACD fusion using (FIG. 17A) NextSeq 2000 and (FIG. 17B) Novaseq X.
  • the schematic drawings are not necessarily to scale. Like numbers used in the figures refer to like components, steps and the like. However, it will be understood that the use of a number to refer to a component in a given figure is not intended to limit the component in another figure labeled with the same number. In addition, the use of different numbers to refer to components is not intended to indicate that the different numbered components cannot be the same or similar to other numbered components.
  • protein refers broadly to a polymer of two or more amino acids joined together by peptide bonds.
  • protein also includes molecules which contain more than one protein joined by disulfide bonds, ionic bonds, or hydrophobic interactions, or complexes of proteins that are joined together, covalently or noncovalently, as multimers (e.g., dimers, tetramers).
  • peptide, oligopeptide, and polypeptide are all included within the definition of protein and these terms are used interchangeably. It should be understood that these terms do not connote a specific length of a polymer of amino acids, nor are they intended to imply or distinguish whether the protein is produced using recombinant techniques, chemical or enzymatic synthesis, or is naturally occurring.
  • an “isolated” protein is one that has been removed from a cell.
  • an isolated protein is a protein that has been removed from the cytoplasm of a cell, and many of the proteins, nucleic acids, and other cellular material of its natural environment are no longer present. Proteins that are produced outside an organism, e.g., through chemical or recombinant means, are considered to be isolated and purified, since they were never present in a natural environment.
  • organism As used herein, the terms "organism,” “subject,” are used interchangeably and refer to microbes (e.g., prokaryotic or eukaryotic) animals and plants.
  • microbes e.g., prokaryotic or eukaryotic
  • An example of an animal is a mammal, such as a human.
  • target nucleic acid is intended as a semantic identifier for the nucleic acid in the context of a method or composition or kit set forth herein and does not necessarily limit the structure or function of the nucleic acid beyond what is otherwise explicitly indicated.
  • Reference to a nucleic acid such as a target nucleic acid includes both single-stranded and double-stranded nucleic acids, and both DNA and RNA, unless indicated otherwise.
  • the term library refers to the collection of target nucleic acids containing known common sequences, such as a universal sequence or adapter, at their 3' and 5' ends.
  • a target nucleic acid includes DNA.
  • a target nucleic acid includes single-stranded DNA.
  • a target nucleic acid includes double-stranded DNA.
  • a target nucleic acid includes RNA.
  • a target nucleic acid includes single-stranded RNA.
  • a target nucleic acid includes double- stranded RNA. which can be attached to a target nucleic acid.
  • An adapter can be single- stranded or double- stranded DNA, or can include both double-stranded and single-stranded regions.
  • An adapter can include a universal sequence that is substantially identical, or substantially complementary, to at least a portion of a primer, for example a universal primer; an index (also referred to herein as a barcode or tag) to assist with downstream error correction, identification, or sequencing; and/or a unique molecular identifier.
  • the adapter is substantially non-complementary to the 3' end or the 5' end of any target sequence present in the sample.
  • suitable adapter lengths are in the range of about 6-100 nucleotides, about 12-60 nucleotides, or about 15-50 nucleotides in length.
  • adaptor and “adapter” are used interchangeably.
  • universal when used to describe a nucleotide sequence, refers to a region of sequence that is common to two or more nucleic acid molecules where the molecules also have regions of sequence that differ from each other.
  • a universal sequence that is present in different members of a collection of nucleic acids can be used as, for instance, a "landing pad" in a subsequent step to anneal a nucleotide sequence that can be used as a primer for addition of another nucleotide sequence, such as an index, to a target nucleic acid.
  • a universal sequence that is present in different members of a collection of nucleic acids can allow capture of multiple different nucleic acids using a population of universal capture nucleic acids, e.g., capture oligonucleotides that are complementary to a portion of the universal sequence, e.g., a universal capture sequence.
  • Non-limiting examples of universal capture sequences include sequences that are identical to or complementary to P5 and P7 primers.
  • a universal sequence present in different members of a collection of molecules can allow the replication (e.g., sequencing) or amplification of multiple different nucleic acids using a population of universal primers that are complementary to a portion of the universal sequence, e g., a universal anchor sequence.
  • universal anchor sequences are used as a site to which a universal primer (e.g., a sequencing primer for read 1 or read 2) anneals for sequencing.
  • a capture oligonucleotide or a universal primer therefore includes a sequence that can hybridize specifically to a universal sequence.
  • P5 and P7 may be used when referring to a universal capture sequence or a capture oligonucleotide.
  • P5 1 (P5 prime)
  • P7 1 (P7 prime) refer to the complement of P5 and P7, respectively. It will be understood that any suitable universal capture sequence or a capture oligonucleotide can be used in the methods presented herein, and that the use of P5 and P7 are exemplary embodiments only.
  • capture oligonucleotides such as P5 and P7 or their complements onflow cells are known in the art, as exemplified by the disclosures of WO 2007/010251, WO 2006/064199, WO 2005/065814, WO 2015/106941, WO 1998/044151, and WO 2000/018957, which are incorporated by reference as to P5 and P7 and their uses.
  • any suitable forward amplification primer can be useful in the methods presented herein for hybridization to a complementary sequence and amplification of a sequence.
  • any suitable reverse amplification primer can be useful in the methods presented herein for hybridization to a complementary sequence and amplification of a sequence.
  • One of skill in the art will understand how to design and use primer sequences that are suitable for capture and/or amplification of nucleic acids as presented herein.
  • the term "primer” and its derivatives refer generally to any nucleic acid that can hybridize to a target sequence of interest.
  • the primer functions as a substrate onto which nucleotides can be polymerized by a polymerase or to which a polynucleotide can be ligated; in some embodiments, however, the primer can become incorporated into the synthesized nucleic acid strand and provide a site to which another primer can hybridize to prime synthesis of a new strand that is complementary to the synthesized nucleic acid molecule.
  • the primer can be used for hybridization to a predetermined sequence, for instance a predetermined sequence that includes one or more nucleotides that identify the location of a modified cytosine.
  • a “primer” includes a sequence present in a guide RNA used with a CRISPR-based system to hybridize to a predetermined sequence.
  • the primer can include any combination of nucleotides or analogs thereof.
  • the primer is a single-stranded oligonucleotide or polynucleotide.
  • polynucleotide and oligonucleotide and “nucleic acid” are used interchangeably herein to refer to a polymeric form of nucleotides of any length, and may include ribonucleotides, deoxyribonucleotides, analogs thereof, or mixtures thereof.
  • the terms should be understood to include, as equivalents, analogs of either DNA, RNA, cDNA, or antibody-oligo conjugates made from nucleotide analogs and to be applicable to single stranded (such as sense or antisense) and double stranded polynucleotides.
  • the term as used herein also encompasses cDNA, that is complementary or copy DNA produced from a RNA template, for example by the action of reverse transcriptase.
  • an “index” refers to a unique nucleic acid tag that can be used to identify a sample or source of the nucleic acid material, or a compartment in which a target nucleic acid was present.
  • the index can be present in solution or on a solid-support, or attached to or associated with a solidsupport and released in solution or compartment.
  • nucleic acid samples are derived from multiple sources, the nucleic acids in each nucleic acid sample can be tagged with different nucleic acid tags such that the source of the sample can be identified.
  • an index can include a six-base Index 1 (i7) sequence, an eight-base Index 1 (i7) sequence, an eight-base Index 2 (i5e) sequence, a ten-base Index 1 (i7) sequence, or a ten- base Index 2 (i5) sequence from Illumina, Inc. (San Diego, CA).
  • amplicon when used in reference to a nucleic acid, means the product of copying the nucleic acid, wherein the product has a nucleotide sequence that is the same as or complementary to at least a portion of the nucleotide sequence of the nucleic acid.
  • An amplicon can be produced by any of a variety of amplification methods that use the nucleic acid, or an amplicon thereof, as a template including, for example, polymerase extension, polymerase chain reaction (PCR), rolling circle amplification (RCA), ligation extension, or ligation chain reaction.
  • An amplicon can be a nucleic acid molecule having a single copy of a particular nucleotide sequence (e.g., a PCR product) or multiple copies of the nucleotide sequence (e.g., a conatemeric product of RCA).
  • a first amplicon of a target nucleic acid is typically a complementary copy.
  • Subsequent amplicons are copies that are created, after generation of the first amplicon, from the target nucleic acid or from the first amplicon.
  • a subsequent amplicon can have a sequence that is substantially complementary to the target nucleic acid or substantially identical to the target nucleic acid.
  • a primer refers to a single stranded nucleic acid molecule that can hybridize to a target sequence, such as an adapter attached to a fragment.
  • a flow cell surface bound primer can serve as a starting point for fragment amplification and cluster generation.
  • a flow cell surface bound primer can serve as a hybridization point for a spatial tag, and thus for targeting attachment of particular transposome complexes and DNA samples.
  • a primer e.g., a sequencing primer
  • Any primer can include any combination of nucleotides or analogs thereof.
  • the primer is a single-stranded oligonucleotide or polynucleotide.
  • the primer length can be any number of bases long.
  • each of the flow cell surface bound primer and the sequencing primer is a short strand, ranging from 10 to 60 bases, or from 20 to 40 bases.
  • amplify refer generally to any action or process whereby at least a portion of a nucleic acid molecule is replicated or copied into at least one additional nucleic acid molecule.
  • the additional nucleic acid molecule optionally includes sequence that is substantially identical or substantially complementary to at least some portion of the template nucleic acid molecule.
  • the template nucleic acid molecule can be single-stranded or double-stranded and the additional nucleic acid molecule can independently be single-stranded or double-stranded.
  • Amplification is typically the exponential replication of a nucleic acid molecule.
  • such amplification can be performed using isothermal conditions; in other embodiments, such amplification can include thermocycling.
  • the amplification is a multiplex amplification that includes the simultaneous amplification of a plurality of target sequences in a single amplification reaction.
  • "amplification” includes amplification of at least some portion of DNA and RNA based nucleic acids alone, or in combination.
  • the amplification reaction can include any of the amplification processes known to one of ordinary skill in the art.
  • the amplification reaction includes polymerase chain reaction (PCR). [00041] As used herein, the term "polymerase chain reaction” (“PCR”) refers to the method of Mullis U.S. Pat. Nos.
  • 4,683,195 and 4,683,202 which describe a method for increasing the concentration of a segment of a polynucleotide of interest in a mixture of genomic DNA without cloning or purification.
  • This process for amplifying the polynucleotide of interest consists of introducing a large excess of two oligonucleotide primers to the DNA mixture containing the desired polynucleotide of interest, followed by a series of thermal cycling in the presence of a DNA polymerase.
  • the two primers are complementary to their respective strands of the double stranded polynucleotide of interest.
  • the mixture is denatured at a higher temperature first and the primers are then annealed to complementary sequences within the polynucleotide of interest molecule. Following annealing, the primers are extended with a polymerase to form a new pair of complementary strands.
  • the steps of denaturation, primer annealing and polymerase extension can be repeated many times (referred to as thermocycling) to obtain a high concentration of an amplified segment of the desired polynucleotide of interest.
  • the length of the amplified segment of the desired polynucleotide of interest is determined by the relative positions of the primers with respect to each other, and therefore, this length is a controllable parameter.
  • the method is referred to as PCR.
  • the desired amplified segments of the polynucleotide of interest become the predominant nucleic acid sequences (in terms of concentration) in the mixture, they are said to be "PCR amplified”.
  • the target nucleic acid molecules can be PCR amplified using a plurality of different primer pairs, in some cases, one or more primer pairs per target nucleic acid molecule of interest, thereby forming a multiplex PCR reaction.
  • amplification conditions generally refers to conditions suitable for amplifying one or more nucleic acid sequences.
  • the amplification conditions can include isothermal conditions or alternatively can include thermocycling conditions, or a combination of isothermal and thermocycling conditions.
  • the conditions suitable for amplifying one or more nucleic acid sequences include polymerase chain reaction (PCR) conditions.
  • PCR polymerase chain reaction
  • the amplification conditions refer to a reaction mixture that is sufficient to amplify nucleic acids such as one or more target sequences flanked by a universal sequence, or target specific primers, or to amplify an amplified target sequence flanked by one or more adapters.
  • the amplification conditions include a catalyst for amplification or for nucleic acid synthesis, for example a polymerase; a primer that possesses some degree of complementarity to the nucleic acid to be amplified; and nucleotides, such as deoxyribonucleotide triphosphates (dNTPs) to promote extension of the primer once hybridized to the nucleic acid.
  • the amplification conditions can require hybridization or annealing of a primer to a nucleic acid, extension of the primer and a denaturing step in which the extended primer is separated from the nucleic acid sequence undergoing amplification.
  • amplification conditions can include thermocycling; in some embodiments, amplification conditions include a plurality of cycles where the steps of annealing, extending and separating are repeated.
  • the amplification conditions include cations such as Mg 2+ or Mn 2+ and can also include various modifiers of ionic strength.
  • multiplex amplification refers to selective and non-random amplification of two or more target sequences within a sample using at least one target-specific primer. In some embodiments, multiplex amplification is performed such that some or all of the target sequences are amplified within a single reaction vessel.
  • the "plexy” or “plex” of a given multiplex amplification refers generally to the number of different target-specific sequences that are amplified during that single multiplex amplification. In some embodiments, the plexy can be about 12-plex, 24-plex, 48-plex, 96-plex, 192-plex, 384-plex, 768-plex, 1536-plex, 3072-plex, 6144-plex or higher.
  • amplified target sequences by several different methodologies (e.g., gel electrophoresis followed by densitometry, quantitation with a bioanalyzer or quantitative PCR, hybridization with a labeled probe; incorporation of biotinylated primers followed by avidin-enzyme conjugate detection; incorporation of 32 P- labeled deoxynucleotide triphosphates into the amplified target sequence).
  • amplification site refers to a site in or on an array where one or more amplicons can be generated.
  • An amplification site can be further configured to contain, hold or attach at least one amplicon that is generated at the site.
  • an array As used herein, the term "array,” “analyte array,” and “microarray” are used interchangeably and refer to a population of sites that can be differentiated from each other according to relative location. Different molecules that are at different sites of an array can be differentiated from each other according to the locations of the sites in the array.
  • An individual site of an array can include one or more molecules of a particular type. For example, a site can include a single target nucleic acid molecule having a particular sequence or a site can include several nucleic acid molecules having the same sequence (and/or complementary sequence, thereof). The sites of an array can be different features located on the same substrate.
  • Exemplary features include without limitation, droplets, wells in a substrate, beads (or other particles) in or on a substrate, projections from a substrate, ridges on a substrate or channels in a substrate.
  • the sites of an array can be separate substrates each bearing a different molecule. Different molecules attached to separate substrates can be identified according to the locations of the substrates on a surface to which the substrates are associated or according to the locations of the substrates in a liquid or gel.
  • Exemplary arrays in which separate substrates are located on a surface include, without limitation, those having beads in wells.
  • compartment is intended to mean an area or volume that separates or isolates something from other things.
  • exemplary compartments include, but are not limited to, vials, tubes, wells, droplets, boluses, beads, vessels, surface features, flow cell, or areas or volumes separated by physical forces such as fluid flow, magnetism, electrical current or the like.
  • a compartment is a well of a multi-well plate, such as a 96- or 384- well plate.
  • a droplet may include a hydrogel bead, which is a bead for encapsulating one or more nuclei or cell, and includes a hydrogel composition.
  • the droplet is a homogeneous droplet of hydrogel material or is a hollow droplet having a polymer hydrogel shell. Whether homogenous or hollow, a droplet may be capable of encapsulating one or more nuclei or cells. In some embodiments, the droplet is a surfactant stabilized droplet. In some embodiments, a single cell or Nuclei is present per compartment. In some embodiments, two or more cells or Nuclei are present per compartment. In some embodiments, each compartment contains a compartment-specific index. In some embodiments, the index is in solution or attached or associated with a solid-phase in each compartment.
  • flow cell refers to a chamber comprising a solid surface across which one or more fluid reagents can be flowed.
  • a flow cell includes a vessel having an enclosed flow channel where a reaction can be carried out, or a vessel having a channel that is open to a surrounding environment and in which a reaction can be carried out.
  • the vessel with an open flow channel may be referred to herein as an open wafer flow cell.
  • Any example of the flow cell may include an inlet for delivering reagent(s) to the channel, and an outlet for removing reagent(s) from the channel.
  • the flow cell enables the detection of the reaction that occurs therein.
  • the flow cell can include one or more transparent surfaces allowing for the optical detection of arrays, optically labeled molecules, or the like.
  • Examples of flow cells and related fluidic systems and detection platforms that can be readily used in the methods of the present disclosure are described, for example, in Bentley et al., Nature 456:53-59 (2008), WO 04/018497; US 7,057,026; WO 91/06678; WO 07/123744; US 7,329,492; US 7,211,414; US 7,315,019; US 7,405,281, and US 2008/0108082.
  • clonal population refers to a population of nucleic acids that is homogeneous with respect to a particular nucleotide sequence.
  • the homogenous sequence is typically at least 10 nucleotides long, but can be even longer including for example, at least 50, 100, 250, 500 or 1000 nucleotides long.
  • a clonal population can be derived from a single target nucleic acid or template nucleic acid. Typically, all of the nucleic acids in a clonal population will have the same nucleotide sequence. It will be understood that a small number of mutations (e.g., due to amplification artifacts) can occur in a clonal population without departing from clonality.
  • a "pattern of cytosine modification,” also referred to as a "methylation profile,” refers to the pattern with which both methylation and unmethylation of cysteines is distributed in the genome of a cell or an organism.
  • a “pattern” is inclusive of both modified cytosines and non-modified cytosines.
  • the pattern can be defined in several distribution dimensions: by organ, by tissue, by status of disease or pathological condition (e.g., cancer, neurophysiological), by genome segment (e.g., chromosome or genetic coordinates on a chromosome), by gene, by CpG island, a group of cytosines, or by the site of a modified cytosine.
  • a pattern of cytosine modification can have a known correlation with a disease or pathological condition, or correlation of a pattern of cytosine modification with a disease or pathological condition can be identified using methods described herein.
  • a pattern of cytosine modification can be present at a specific locus (e.g., location) in a genome, and that specific location can be a single modified cytosine or a set of modified cytosines, e.g., a CpG island.
  • a pattern of cytosine modification can be identified by using a predetermined sequence, e.g., a method of using an altered cytidine deaminase can be designed and practiced with the intent of determining a pattern of cytosine modification, for instance, the methylation status of one of more specific cytosines, the methylation status of one or more specific cytosines present at a specific location of a genome, or the combination thereof.
  • a flow channel is an area that is defined between two bonded or otherwise attached components, or that is defined within a lane so that it is open to the surrounding environment.
  • the flow channel can selectively receive a liquid sample.
  • the flow channel may be defined between two patterned sequencing surfaces or a patterned sequencing surface and a lid, and thus may be in fluid communication with one or more components of the sequencing surface(s).
  • a fragment is a portion or piece of the DNA sample.
  • a “partially adapted fragment” is a portion or piece of the DNA sample that has been tagmented, and thus includes an adapter ligated to the 5’ end of the DNA fragment.
  • a “fully adapted fragment” is a portion or piece of the DNA sample that has adapters incorporated at both the 3’ and 5’ ends of the DNA fragment.
  • fragmentation is the breaking of nucleic acid into shorter lengths. Fragmentation methods include enzymatic methods, physical methods (including sonication, nebulization, needle shearing, microwave, etc ), and chemical methods (including depurination, hydrolysis, oxidation, etc.).
  • fragmenting enzymes or “enzyme-based fragmentation” or “enzyme fragmentation,” as used herein, refer to enzymes that fragment nucleic acids.
  • the enzymes can be a single enzyme or two or more enzymes that work together to fragment the nucleic acid.
  • fragmenting enzymes can cut the nucleic acid randomly or specifically.
  • fragmenting enzymes include transposase, restriction enzymes, Argonaute, CRISPR -associated nuclease (Cas), endonucleases, exonuclease, topoisomerase, FRAGMENTASETM (New England Biolabs, Ipswich, MA).
  • Preferred fragmentation examples include methods that fragment while retaining proximity information of the fragments.
  • each when used in reference to a collection of items, is intended to identify an individual item in the collection but does not necessarily refer to every item in the collection unless the context clearly dictates otherwise.
  • Conditions that are "suitable” for an event to occur such as converting 5 methylcytosine to thymidine by deamination, or “suitable” conditions are conditions that do not prevent such events from occurring. Thus, these conditions permit, enhance, facilitate, and/or are conducive to the event.
  • "providing" in the context of a protein, sample of DNA or RNA, or composition means making the protein, sample of DNA or RNA, or composition, purchasing the protein, sample of DNA or RNA, or composition, or otherwise obtaining the protein, sample of DNA or RNA, or composition.
  • polynucleotide sequences encoding an altered cytidine deaminase are described herein as DNA sequences, it is understood that the complements, reverse sequences, and reverse complements of the DNA sequences can be easily determined by the skilled person. It is also understood that the sequences described herein as DNA sequences can be converted from a DNA sequence to an RNA sequence by replacing each thymidine nucleotide with a uracil nucleotide.
  • Polynucleotide and/or polypeptides or protein sequences may include one or more forms of typographical emphasis (e.g., underlined text, bolded text, italicized text). It is understood that the typographical emphasis is non-limiting.
  • sequences stated with typographical emphasis include the stated sequence without the typographical emphasis. Additionally, polynucleotide sequences may be displayed in capital letters, lower case letters, or a combination thereof. The case of the letters in the polynucleotide sequences is non-limiting. Unless otherwise stated, lower case and upper-case letters simply indicate the identity of the nucleobase.
  • ACDs Altered cytidine deaminases
  • the present disclosure includes altered cytidine deaminases (ACDs, or the singular form ACD), compositions including ACDs, and methods for using ACDs.
  • ACDs altered cytidine deaminases
  • compositions including ACDs and methods for using ACDs.
  • Wild-type APOBEC3A deaminates cytosine (C), 5 methyl cytosine (5mC), and 5- hydroxymethyl cytosine (5hmC) efficiently in single-stranded DNA (FIG. 1A-C).
  • Treatment of DNA, such as genomic DNA, with wild-type APOBEC3A results in the conversion of C to uracil (U), 5mC to thymidine (T), and 5hmC to 5 -hydroxyuracil cytosine (5hmC) and reduces the complexity of the DNA sequencing (FIG. ID).
  • mutation of the tyrosine at position 130 in a human APOBEC3A protein to a different amino acid such as alanine resulted in preferential conversion of 5mC to thymidine (FIG. IE).
  • Analysis of the sample DNA after treatment with an altered cytidine deaminase described herein, for example, by sequencing of the sample DNA, and optional comparison to a reference (e g., reference sequence) permits easy identification of C to T point mutations, and these point mutations are inferred as 5mC positions.
  • a cytidine deaminase is considered to be an altered cytidine deaminase (ACD) if it has the activity of deaminating 5 methyl cytosine (5mC) and includes at least one of the substitution mutations described herein.
  • An ACD useful in the methods provided herein preferentially deaminates 5mC instead of C (i.e., converts 5mC to T at a greater rate than converting C to U) compared to the equivalent wild-type enzyme and is referred to herein as having “cytosinedefective deaminase activity” and “5mC-selective deaminase activity”, the terms being interchangeable.
  • ACDs include apolipoprotein B mRNA editing enzymes, catalytic polypeptide-like (APOBEC) and activation induced cytidine deaminase (AID). Wild-type APOBEC and AID cytidine deaminases have the activity of deaminating cytidine of DNA and/or RNA to form uridine (U).
  • An ACD of the present disclosure has an altered rate of deamination of 5mC when compared to the wild-type enzyme.
  • a cytidine deaminase of the present disclosure can be referred to herein as an "altered cytidine deaminase,” “recombinant cytidine deaminase,” “ACD,” “recombinant ACD,” “mutant cytidine deaminase,” or “modified cytidine deaminase” and refers to any of the engineered ACDs described herein that include one or more changes from a reference (i.e., wild-type) amino acid sequence that provide one or more of the activities described herein, including but not limited to an altered deamination profile, e.g., alters its ability to preferentially deaminate one form of cytosine over another, enhanced selectivity for 5mC or C, and/or enhanced stability.
  • a reference i.e., wild-type amino acid sequence
  • an ACD of the present disclosure is based on a member of the APOBEC protein family.
  • An ACD of the present disclosure that is "based on" a member of the APOBEC protein family means the ACD is an APOBEC protein that includes one or more of the substitution mutations described herein as compared to a reference APOBEC sequence.
  • An ACD of the present disclosure that is "based on” a member of the APOBEC protein family can also include conservative and/or nonconservative mutations as described herein. The positions of the alterations, substitutions or deletions will be at functionally equivalent amino acids from the reference sequence, as described herein.
  • An ACD of the present disclosure can be based on a member of the APOBEC protein family from a vertebrate, such as a mammal.
  • mammals include, but are not limited to, rodents, primates, rabbit, bovine (e.g., cow), porcine (e.g., pig), equine (e.g., horse), elephant, and aardvark.
  • An example of a primate is a human and a chimpanzee.
  • the APOBEC protein family is a member of the large cytidine deaminase superfamily that contains a canonical zinc-dependent deaminase (ZDD) signature motif embedded within a core cytidine deaminase fold.
  • ZDD canonical zinc-dependent deaminase
  • This fold includes a five-stranded mixed beta (b)-sheet surrounded by six alpha (a)-helices with the order al-bl-b2-a2-b3-a3-b4-a4-b5-a5-a6 (Salter et al., Trends Biochem Sci. 2016 41(7):578-594. Doi:10.1016/j.tibs.2016.05.001; Salter et al., Trends Biochem. Sci. 2018, 43(8):606-622 doi.org/10.1016/j .tibs.2018.04.013).
  • the H and two C residues coordinate a Zn atom
  • the E residue polarizes a water molecule near the Zn-atom for catalysis (Chen et al., 2021, Viruses, 13:497, doi.org/10.3390/vl3030497).
  • Some members of the APOBEC protein family include one copy of the ZDD motif.
  • Other members of the APOBEC protein family e.g., the APOBEC3B subfamily, the APOBEC3D subfamily, the APOBEC3F subfamily, and the APOBEC3G subfamily, include two copies of the ZDD motif, but often only the C-terminal copy is active (Salter et al., Trends Biochem Sci.
  • an ACD disclosed herein is a member of the APOBEC3 subfamily, e.g., APOBEC3A, APOBEC3B, APOBEC3C, APOBEC3D, APOBEC3F, or APOBEC3G, and can include one or more highly conserved sites that are part of the active site and within the ZDD motif SEQ ID NO: 1.
  • the sites include tryptophan at position 98 and serine or threonine at position 99 of SEQ ID NO:3 (Kouno et al., 2017, Nat. Comm, 8: 15024, DOI: 10.1038/ncomms 15024).
  • a member of the APOBEC protein family also includes other highly conserved residues that are part of the active site but not present as part of the ZDD motif SEQ ID NO:1.
  • a member the APOBEC3A subfamily, APOBEC3B subfamily, APOBEC3C subfamily, APOBEC3D subfamily, APOBEC3F subfamily, and APOBEC3G subfamily typically includes one or more of the following highly conserved sites that are part of the active site: arginine at position 28; histidine, asparagine, or arginine at position 29; serine or threonine, preferably threonine, at position 31; asparagine or aspartic acid at position 57; histidine at position 70; cysteine at position 101; cysteine at position 106; tyrosine or phenylalanine at position 130; asparagine or tyrosine at position 131; asparagine, tyrosine
  • An ACD of the present disclosure includes a substitution mutation, deletion, insertion, or a combination thereof, at one or more residues when compared to a reference cytidine deaminase.
  • a substitution mutation can be at the same position or a functionally equivalent position compared to the reference cytidine deaminase.
  • Reference cytidine deaminases and functionally equivalent positions are described in detail herein. As noted, positions of the altered amino acids described herein and in the tables are in reference to APOBECA3A (SEQ ID NO:3) but one skilled in the art is capable of deriving the functionally equivalent positions in the other referenced cytosine deaminases. The skilled person will readily appreciate that an altered cytidine deaminase described herein is not naturally occurring.
  • a reference cytidine deaminase can be a member of the APOBEC protein family. Essentially any known member of the APOBEC protein family can be a reference cytidine deaminase.
  • the skilled person can easily identify members of each of the subfamilies by using a publicly available database such as the Protein database available at the National Center for Biotechnology Information (ncbi.nlm.nih.gov/protein) and searching for APOBEC1, APOBEC2, APOBEC3A, APOBEC3B, APOBEC3C, APOBEC3D, APOBEC3F, APOBEC3G, APOBEC3H, APOBEC4, or, when identifying members of the AID family, Activation-induced cytidine deaminase.
  • cytidine deaminases include, but are not limited to, SEQ ID NO: 13, 14, 15, 16, 17, 18, 19, 20, 21, and 22.
  • any of the mutations discussed for selectivity e.g., AxxxW (SEQ ID NO:51) or AxxxWX (SEQ ID NO:52)
  • AxxxW SEQ ID NO:51
  • AxxxWX SEQ ID NO:52
  • an ACD has an amino acid sequence that is based on a reference sequence which is a member of the APOBEC protein family includes a ZDD motif H-[P/A/V]-E- X[23-28]-P-C-X[2-4]-C (SEQ ID NO:1) and at least one substitution mutation disclosed herein.
  • an altered cytidine deaminase includes a substitution mutation at one or more other active site residues disclosed herein.
  • Non-limiting examples of reference cytidine deaminase proteins for different APOBEC protein family members include UniProt Q9GZX7, UniProt G3QLD2, and UniProt Q9WVE0 (APOBEC protein family member AID); UniProt P41238, NCBI XP 030856728.1, and Uniprot P51908 (APOBEC protein family member APOBEC1); UniProt Q9Y235, Uniprot G3SGN8, and Uniprot Q9WV35 (APOBEC protein family member APOBEC2); UniProt P31941 , GenBank XP 045219544.1, GenBank AER45717.1, GenBank XP 003264816.1, GenBank PNI48846.1, and GenBank ADO85886.1 (APOBEC protein family member APOBEC3A); UniProt Q9UH17, Uniprot G3QV16, and Uniprot F6M3K5 (APOBEC protein family member APOBEC3B);
  • an ACD has an amino acid sequence that is based on a reference sequence that is a member of the APOBEC3A subfamily, and includes a ZDD motif HXEX24SW(S/T)PCX[2-4]CX6FX8LX5R(L/I) ⁇ X[8-11]LX 2 LX[1O]M (SEQ ID NO:2) (where X is any amino acid, and the subscript number or range of numbers after X refers to the number of amino acids) and at least one substitution mutation disclosed herein.
  • the substitution mutation is a substitution mutation at the underlined tyrosine, such as a substitution mutation to alanine (A).
  • the underlined tyrosine (Y) of SEQ ID NO:2 is the position functionally equivalent to the tyrosine amino acid 130 of the APOBEC3A protein SEQ ID NO:3.
  • the altered cytidine deaminase includes other active site residues disclosed herein.
  • the amino acid sequence of an ACD includes the amino acids of a member of the APOBEC3A subfamily: X[i 6 -26]-GRXXTXLCYXV-Xi 5 -GXXXN-Xi2-HAEXXF- X14-YXXTWXXSWSPC- X[2-4]-CA-X5-FL-X7-LXIXXXR(L/I)Y-Xs-GLXXLXXXG-X5-M-X4- FXXCWXXFV-X6-FXPW-X13-LXXI- X [2 -6] (SEQ ID NO:9) (where X is any amino acid, and the subscript number or range of numbers after X refers to the number of amino acids), or a subset thereof, and at least one substitution mutation disclosed herein.
  • the underlined tyrosine (Y) of SEQ ID NO:9 is the position functionally equivalent to the tyrosine amino acid 130 of the APOBEC3A protein SEQ ID NO :3.
  • the substitution mutation is a substitution mutation at the underlined tyrosine, such as a substitution mutation to alanine (A).
  • the altered cytidine deaminase includes other active site residues disclosed herein.
  • the underlined tyrosine (Y) of SEQ ID NO: 10 is the position functionally equivalent to the tyrosine amino acid 130 of the APOBEC3A protein SEQ ID NO:3.
  • the substitution mutation is a substitution mutation at the underlined tyrosine (Y), such as a substitution mutation to alanine (A).
  • the altered cytidine deaminase includes other active site residues disclosed herein.
  • a substitution mutation can be at the same position or a functionally equivalent position compared to a reference cytidine deaminase.
  • “functionally equivalent” it is meant that the altered cytidine deaminase has the amino acid substitution at the amino acid position in a reference cytidine deaminase that has the same functional role in both the reference cytidine deaminase and the altered cytidine deaminase.
  • FIG. 2 An example of a sequence alignment to identify positionally equivalent and/or functionally equivalent residues is set forth in FIG. 2.
  • the residues in the members of the APOBEC3A subfamily in FIG. 2 that are vertically aligned are considered positionally equivalent as well as functionally equivalent to the corresponding residue in the human APOBEC3A amino acid sequence.
  • FIG. 2 An example of a sequence alignment to identify positionally equivalent and/or functionally equivalent residues is set forth in FIG. 2.
  • the residues in the members of the APOBEC3A subfamily in FIG. 2 that are vertically aligned are considered positionally equivalent as well as functionally equivalent to the corresponding residue in the human APOBEC3A amino acid sequence.
  • an altered cytidine deaminase has an amino acid sequence that is structurally similar to a reference cytidine deaminase disclosed herein.
  • a reference cytidine deaminase is one that includes the amino acid sequence of SEQ ID NO:9 or SEQ ID NO:10.
  • Structural similarity of two amino acid sequences can be determined by aligning the residues of the two sequences (for example, a candidate ACD and a reference cytidine deaminase described herein) to optimize the number of identical amino acids along the lengths of their sequences; gaps in either or both sequences are permitted in making the alignment in order to optimize the number of identical amino acids, although the amino acids in each sequence must nonetheless remain in their proper order.
  • a candidate altered cytidine deaminase is the cytidine deaminase being compared to the reference cytidine deaminase.
  • a candidate ACD that has structural similarity with a reference cytidine deaminase and cytidine deaminase activity is an altered cytidine deaminase.
  • amino acid sequence of an altered cytidine deaminase protein having sequence similarity to a reference sequence may include conservative substitutions of amino acids present in that reference sequence.
  • a conservative substitution for an amino acid in a protein may be selected from other members of the class to which the amino acid belongs.
  • an amino acid belonging to a grouping of amino acids having a particular size or characteristic can be substituted for another amino acid without altering the activity of a protein, particularly in regions of the protein that are not directly associated with biological activity.
  • amino acids having a non-polar side chain include alanine, glycine, isoleucine, leucine, methionine, phenylalanine, proline, tryptophan, and valine; amino acids having a hydrophobic side chain include glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, and tryptophan; amino acids having a polar side chain include arginine, asparagine, aspartic acid, glutamine, glutamic acid, histidine, lysine, serine, cysteine, tyrosine, and threonine; and amino acids having an uncharged side chain include glycine, serine, cysteine, asparagine, glutamine, tyrosine, and threonine.
  • reference to a cytidine deaminase as described herein can include a protein having structural similarity to the reference cytidine deaminase, e g., at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence similarity to the reference cytidine deaminase.
  • a reference protein is SEQ ID NO:3. Examples of other reference proteins are described herein.
  • reference to a cytidine deaminase as described herein can include a protein having structural similarity to the reference cytidine deaminase, e.g., at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity to the reference cytidine deaminase.
  • a reference protein is SEQ ID NO:3. Examples of other reference proteins are described herein.
  • substitution mutations conferring increased selectivity or stability
  • the present disclosure provides new altered cytidine deaminases (ACDs) that preferentially deaminate 5mC instead of C (i.e., converts 5mC to T at a greater rate than converting C to U) compared to the equivalent wild-type enzyme and first and second generations of ACD that have been identified (International Application Publication NOs. WO 2023/196572, WO 2025/072793, and WO 2025/072800), and include a substitution mutation at a position functionally equivalent to a tyrosine at position 130 (Y130), for instance, Y130A in combination with the mutations described below.
  • ACDs cytidine deaminases
  • An optional substitution mutation is at a position functionally equivalent to a tyrosine at position 132 (Y132), for instance, Y130H.
  • Another optional mutation that increases the selectivity for 5mC is a substitution mutation at a position functionally equivalent to a aspartic acid at position 133 (Y133), for instance, D133W.
  • An example of an ACD with these three substitution mutations is ScA (SEQ ID NO: 13).
  • ACDs of the present disclosure include one or more selectivity-enhancing alterations that result in greater selectivity for 5mC than other ACDs, such as the ACD ScA (SEQ ID NO:13), as well a wild-type APOBEC3A cytidine deaminase (e.g., SEQ ID NO:3).
  • a selectivity-enhancing alteration is considered to enhance the selectivity of an ACD for 5mC if it has greater deamination of 5mC when compared to a reference ACD.
  • a suitable reference ACD is one having the same amino acid sequence (also referred to as the same backbone) but without the selectivity-enhancing alteration.
  • Examples of reference ACDs for use in determining whether an alteration is a selectivity-enhancing alteration include, but are not limited to, SEQ ID NO:3, SEQ ZD NO 13, SEQ ID NO: 14, SEQ ID NO:17, or SEQ ID NO 21.
  • the reference ACD is SEQ ID NO: 14.
  • a selectivity-enhancing alteration is a substitution mutation, a deletion, or an insertion.
  • an ACD disclosed herein having enhanced selectivity includes one or more selectivity-enhancing alterations that confer increased 5mC-selectivity compared to a reference ACD (an increase in 5mC deamination of at least 5%, at least 10%, at least 15%, least 20%, at least 25%, least 30%, least 40%, least 50%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95%).
  • Cytidine deaminase activity of a protein can be determined by in vitro assays.
  • One example of an in vitro assay is based on digestion with the restriction enzyme Swal as described in WO 2023/196572.
  • An ACD of the present disclosure can include a substitution mutation at a position functionally equivalent to tyrosine at position 130 (Y130) in a member of the APOBEC protein family, including a member of the APOBEC3A subfamily (for instance, SEQ ID NO:3).
  • the wild-type residue at a position functionally equivalent to Y130 is phenylalanine (F).
  • An ACD with a substitution mutation at a position functionally equivalent to Y130 preferentially acts on 5mC compared to cytosine (i.e., has 5mC-selective deaminase activity).
  • substitution mutation in an ACD is at a position functionally equivalent to position 130 in a member of the APOBEC protein family, including a member of the APOBEC3A subfamily (for instance, SEQ ID NO:3) and can be a mutation to alanine (A), glycine (G), phenylalanine (F), histidine (H), glutamine (Q), methionine (M), asparagine (N), lysine (K), valine (V), aspartic acid (D), glutamic acid I, serine (S), cysteine (C), proline (P), or threonine (T) (FIG. 2).
  • the substitution mutation at a position functionally equivalent to Y130 is Y130A, Y130S, or Y130H. In one embodiment, the substitution mutation at a position functionally equivalent to Y130 is Y130A. In one embodiment, the substitution mutation at a position functionally equivalent to Y130 is Y130S.
  • An ACD of the present disclosure can include a second substitution mutation at a position functionally equivalent to the proline at position 134 (Pl 34) in a member of the APOBEC protein family, including a member of the APOBEC3A subfamily (for instance, SEQ ID NO:3).
  • An ACD with a substitution mutation at a position functionally equivalent to Pl 34 preferentially acts on 5mC compared to cytosine (i.e., has 5mC-selective deaminase activity).
  • the substitution mutation in an ACD is at a position functionally equivalent to position Pl 34 is a mutation to tryptophan (P134W) or to tyrosine (P134Y).
  • the substitution mutation at a position functionally equivalent to Pl 34 is P134W.
  • the substitution mutation in an ACD is at a position functionally equivalent to position P134 is a mutation to threonine (P134T).
  • an ACD of the present disclosure comprises AxxxW (SEQ ID NO:51) at positions functionally equivalent to 130-134 of SEQ ID NO:3, and where X may be any amino acid (examples demonstrated in Tables 2 and 3 and Figures 8 and 9), and the ACD with AxxxW (SEQ ID NO:51) has an increase in 5mC selectivity as compared to wild-type APOBEC (e.g., SEQ ID NO:3), particularly, for example, has at least 75% increase 5mC specificity as compared to reference APOBEC sequence (e.g., an increase in 5mC deamination of at least 5%, at least 10%, at least 15%, least 20%, at least 25%, least 30%, least 40%, least 50%, at least 75%, at least 78%, at least 80%, at least 82%, at least 85%, at least 90%, at least 92%, or at least 95% as compared to WT APOBEC).
  • wild-type APOBEC e.g., SEQ ID NO
  • An ACD of the present disclosure can further include a substitution mutation at a position functionally equivalent to the aspartic acid at position 131 (DI 31), the tyrosine at position 132 (Y132), the aspartic acid at position 133 (D133), or any combination thereof.
  • a substitution mutation in an ACD at a position functionally equivalent to position Y130 is Y130A or Y130H, preferably Y130A
  • P134 is P134W or P134Y, preferably P134W
  • any amino acid can be present at DI 31 (D13 IX), Y132 (Y132X), and/or D133 (D133X).
  • substitution mutations in an ACD are at positions functionally equivalent to positions 131, 132, and/or 133 in a member of the APOBEC protein family, including a member of the APOBEC3A subfamily (for instance, SEQ ID NO:3).
  • a substitution mutation at D 131 can be histidine, arginine, lysine, proline, glycine, asparagine, alanine, or valine.
  • a substitution mutation at Y132 can be alanine, proline, aspartic acid, glutamic acid, glycine, threonine, valine, glutamine, or arginine.
  • a substitution mutation at D133 can be histidine, tyrosine, phenylalanine, threonine, isoleucine, or valine, [000105]
  • an ACD of the present disclosure can further include a substitution mutation at a position functionally equivalent to the leucine at position 135 (L135) in a member of the APOBEC protein family, including a member of the APOBEC3A subfamily (for instance, SEQ ID NO:3).
  • the substitution mutation at a position functionally equivalent to L135 preferentially acts on 5mC compared to cytosine (i.e., has 5mC-selective deaminase activity).
  • the substitution mutation in an ACD at a position functionally equivalent to position L135 is a mutation to tryptophan (L135W). In one embodiment, the substitution mutation in an ACD at a position functionally equivalent to position L 135 is a mutation to histidine (L135H), glutamine (L135Q), tyrosine (L135Y), or cysteine (L135C).
  • an ACD includes the substitution mutations at positions functionally equivalent to Y130A, Y132H, D133W, P134W, and L135X, where X is tryptophan (L135W), histidine (L135H), glutamine (L135Q), tyrosine (L135Y), or cysteine (L135C).
  • an ACD of the present disclosure includes a substitution mutation at positions functionally equivalent to Y130 (such as, but not limited to, Y130A) and Y134 (such as, but not limited to, P134W), and have any amino acid present at positions functionally equivalent to D131, Y132, D133, in a member of the APOBEC protein family, including a member of the APOBEC3A subfamily (for instance, SEQ ID NO:3), or an ACD having structural similarity to SEQ ID NO:3.
  • the structural similarity can be, but is not limited to, at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to SEQ ID NO:3.
  • an ACD of the present disclosure includes a substitution mutation at positions functionally equivalent to Y130 (such as, but not limited to, Y130A) and Y134 (such as, but not limited to, P134W), any amino acid present at positions functionally equivalent to D131, Y132, D133, and a substitution mutation at a position functionally equivalent to L135 in a member of the APOBEC protein family, including a member of the APOBEC3A subfamily (for instance, SEQ ID NO:3)
  • substitution mutations at positions functionally equivalent to 130, 131, 132, 133, and 134 that can be present in a wild-type APOBEC3A protein, such as SEQ ID NO:3, or present in an ACD and increase the selectivity for 5mC compared to cytosine include, but are not limited to, those shown in Table 2 (see Example 1).
  • examples of reference ACDs having these substitution mutations include, but are not limited to, SEQ ID NO: 13, 14, 15, 16, 17, 18, 19, 20, 21, and 22, and those having structural similarity to SEQ ID NO: 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22, where the amino acids at 130, 131, 132, 133 and 134 are those shown in Table 2.
  • the structural similarity can be, but is not limited to, at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22.
  • ACDs of SEQ ID NO: 13, 14, 15, 16, 17, 18, 19, 20, 21, and 22 include stability mutations, which are described herein.
  • the ACDs have increased 5mC specificity have mutations at position 103-105 in combination with AxxxW (SEQ ID NO:51) at positions 130- 134, as shown in FIG. 8.
  • Suitable ACD mutations for F102/S103/W104/G105 may include one or more mutations or deletions, two or more, or three or more mutaitons or deletions within F102/S103/W104/G105.
  • substitution mutations at positions functionally equivalent to 130, 131, 132, 133, and 134 that can be present in a wild-type APOBEC3A protein, such as SEQ ID NO:3, or present in an ACD and increase the selectivity for 5mC compared to cytosine include, but are not limited to, those shown in Table 2 (see Example 1).
  • examples of reference ACDs having these substitution mutations include, but are not limited to, SEQ ID NO:13, 14, 15, 16, 17, 18, 19, 20, 21, and 22, and those having structural similarity to SEQ ID NO: 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22, where the amino acids at 130, 131, 132, 133 and 134 are those shown in Table 2.
  • the structural similarity can be, but is not limited to, at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to SEQ ID NO:13, 14, 15, 16, 17, 18, 19, 20, 21, or 22.
  • ACDs of SEQ ID NO: 13, 14, 15, 16, 17, 18, 19, 20, 21, and 22 include stability mutations, which are described herein.
  • the ACDs have increased 5mC specificity have mutations at position 103-105 in combination with AxxxW (SEQ ID NO:51) at positions 130- 134, as shown in FIG. 8.
  • Suitable ACD mutations for F102/S103/W104/G105 may include one or more mutations or deletions, two or more, or three or more mutations or deletions within F102/S103/W104/G105.
  • substitution mutations at positions functionally equivalent to 130, 131, 132, 133, 134, and 135 that can be present in a wild-type APOBEC3A protein, such as SEQ ID NO:3, or present in an ACD and increase the selectivity for 5mC compared to cytosine include, but are not limited to, ADHWWX (SEQ ID NO: 52), where X any amino acid besides L, preferably wherein X is W, H, Q, Y or C.
  • examples of ACDs that can include these substitution mutations include, but are not limited to, SEQ ID NO:13, 14, 15, 16, 17, 18, 19, 20, 21, and 22, and those having structural similarity to SEQ ID NO: 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22, where the amino acids at 130, 131, 132, 133 134, and 135 is ADHWWX (SEQ ID NO:52), where X is W, H, Q, Y or C.
  • the structural similarity can be, but is not limited to, at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22.
  • these ACD may have an additional substitution or deletion at one or more positions functionally equivalent to 102, 103, 104 and 105, for example, one or more substitution mutations, one or more deletions, or one or more insertions, or a combination thereof, of the amino acids at positions functionally equivalent to a phenylalanine at position 102 (Fl 02), a serine at position 103 (SI 03), a tryptophan at position 104 (W104), and/or a glycine at position 105 (G105) in a member of the APOBEC protein family, including a member of the APOBEC3A subfamily (for instance, SEQ ID NO:3).
  • the substitution mutation at a position functionally equivalent to any one of Fl 02, SI 03, W104, and/or G105 can be any amino acid.
  • the deletion can be 1, 2, 3, or 4 of the amino acids at a position functionally equivalent to any one of F102, S103, W104, and/or G105. In one embodiment, deletion can be at a position functionally equivalent to W104, G105, or both W104 and G105.
  • Suitable sequences for the region from 102-105 can be selected from, e.g., SEQ ID NO:242-410, FIMA, QVP , YLPA, EMFA, FIIA, ELYA, YLPA, NIP A, VYNA, AFRA, YYIA, FSDD, FQAG, FSAG, AVPG, FVPA, AVPA, YLIA, RYAA, LFWA, TRY A, YLIA, VLYA, RHYA, FMHA, QFPA, RRPA, RIYA, YYIA, RMLA, VLYA, RMLA, AVQA, QVWA, FVPA, FVDA, FSAA, KRAA , RRAA, FPPA, KRRA, VP A A, TDHA, QTDA, YLAA, LPAA, EMAA, MFAA, YVRA, IKLA, YIKA, QTQA, TQ
  • a selectivity-enhancing alteration is one or more substitution mutations, one or more deletions, or one or more insertions, or a combination thereof, of the amino acids at positions functionally equivalent to a phenylalanine at position 102 (F102), a serine at position 103 (S103), a tryptophan at position 104 (W104), and/or a glycine at position 105 (G105) in a member of the APOBEC protein family, including a member of the APOBEC3A subfamily (for instance, SEQ ID NO:3).
  • the substitution mutation at a position functionally equivalent to any one of Fl 02, SI 03, W104, and/or G105 can be any amino acid.
  • the deletion can be 1, 2, 3, or 4 of the amino acids at a position functionally equivalent to any one of Fl 02, SI 03, W104, and/or G105.
  • deletion can be at a position functionally equivalent to W104, G105, or both W104 and G105.
  • the insertion can be the insertion of 1, 2, 3, or 4 amino acids between two amino acids at a position functionally equivalent to any one of F 102, S103, W104, or G105.
  • an insertion can be between Fl 02 and SI 03 and an insertion between amino acid SI 03 and W 104.
  • the one or more substitution mutations, one or more deletions, or one or more insertions can be at any combination of positions 102-105, e.g., at one, two, three, or all four positions. Examples of substitution mutations at positions functionally equivalent to SI 03 and W 104 include, but are not limited to, S103D/W104A, S103H/W104P, S103T/W104D, S103T/W104M, S103V/W104E, S103V/W104M, and S103V/W104P.
  • selectivity-enhancing alterations at positions functionally equivalent to S103, W104, and G105 include, but are not limited to, substitution mutations S103V/W104P and a deletion of G105.
  • these selectivity-enhancing alterations can be present in SEQ ID NO: 14 (see, for instance, SEQ ID NO:23 and SEQ ID NO:24.
  • a wild-type APOBEC3A protein such as SEQ ID NO:3, or present in an ACD and increase the selectivity for 5mC compared to cytosine include, but are not limited to, those shown in Table 3 (see Example 1).
  • examples of ACDs having these selectivity-enhancing alterations include, but are not limited to, SEQ ID NO: 13, 14, 15, 16, 17, 18, 19, 20, 21, and 22, and those having structural similarity to SEQ ID NO: 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22, where the amino acids at 102,
  • 103, 104, 105, 130, 131, 132, 133, and 134 are those shown in Table 3.
  • the structural similarity can be, but is not limited to, at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22.
  • Examples of selectivity-enhancing alterations at positions functionally equivalent to 102, 103, 104, 105, 130, 131, 132, 133, 134, and 135 that can be present in a wild-type APOBEC3A protein, such as SEQ ID NO:3, or present in an ACD and increase the selectivity for 5mC compared to cytosine include, but are not limited to, those shown in FIG. 8.
  • examples of ACDs having these selectivity-enhancing alterations include, but are not limited to, SEQ ID NO: 13, 14, 15, 16, 17, 18, 19, 20, 21, and 22, and those having structural similarity to SEQ ID NO:13, 14, 15, 16, 17, 18, 19, 20, 21, or 22, where the amino acids at 102, 103, 104, 105, 130, 131, 132, 133, 134, and 135 are those shown in FIG. 8.
  • the structural similarity can be, but is not limited to, at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22.
  • ACDs with additional mutations that increase selectivity for 5mC are described in International Application Publication Nos. WO 2025/072793 and WO 2025/072800.
  • An ACD described herein having enhanced 5mC-selective deaminase activity e.g., one or more selectivity-enhancing alterations at a position functionally equivalent to F102, 8013, W104, G105, Y130, D131, Y132, D133, P134, L135, or any combination thereof
  • a stability-enhancing alteration can be one or more substitution mutations, one or more deletions, one or more insertions, or a combination thereof, at a position functionally equivalent to an amino acid in a member of the APOBEC protein family, including a member of the APOBEC3A subfamily (for instance, SEQ ID NO:3).
  • a stability-enhancing alteration e.g., substitution mutation, deletion, or insertion
  • a stability-enhancing alteration is considered to be stability-enhancing if it can increase the melting temperature of an ACD by at least 1 °C, at least 2 °C, at least 3 °C, at least 4 °C, at least 5 °C, or at least 6 °C compared to the same ACD that does not have the stability-enhancing substitution mutation.
  • a suitable method of determining the melting temperature of an ACD described herein is by fluorimetry. However, it is well within one skilled in the art to determine the melting temperature of the ACDs described herein.
  • stability-enhancing alterations include, but are not limited to, substitution mutation at one or more positions functionally equivalent to I17X, T19X, G25X, S45X, A59X, K60X, R74X, G108X, A126X, C171X, G188X, a deletion of amino acids 61-68 (A61-68), a deletion of amino acids 104-105 (A 104- 105), or any combination thereof, in a member of the APOBEC protein family, including a member of the APOBEC3A subfamily (for instance, SEQ ID NO:3).
  • I17X is I17T.
  • T19X is T19Y.
  • Examples of G25X include G25K and G25R.
  • An example of S45X is S45W.
  • An example of A59X is A59P.
  • An example of K60X is K60R.
  • An example of R74X is R74L.
  • An example of A126X is A126C.
  • Examples of G108X include G108C and G108A.
  • An example of C171X is C171A.
  • An example of G188X is G188R.
  • Examples of ACDs having specific combinations of stability-enhancing alterations include, but are not limited to, those shown in Table 1.
  • Table 1 Examples of stability mutations in ACDs.
  • An ACD described herein having enhanced 5mC-selective deaminase activity can include one or more stability enhancing alterations selected from Hl IX, L12X, D14X, H16X, I17X, T19X, S20X, N21X, G25X, I26X, G27X, R28X, H29X, T31X, C34X, E38X, R39X, D41X, N42X, S45X, K47X, M48X, H51X, F54X, H56X, N57X, A59X, K60X, L62X, Y67X, L73X, R74X, D77X, L78X, V79X, P80X, S81X, D85X, A87
  • An ACD described herein having enhanced 5mC-selective deaminase activity can include one or more stability enhancing alterations selected from Hl IL, D14Y, H16D, I17T, T19Y, T19L, T19I, S20F, S20F, S20E, S20Y, S20I, G25R, G25V, G25K, I26R, T31I, T31W, S45W, S45R, K47P, A59P, K60R, K60G, L62P, R74L, L78H, P80R, P80T, P80S, P80G, I89R, I89T, I89H, I89R, S103R, S103Y, S103T, G108C,
  • an ACD having the one or more selectivity-enhancing alteration described herein e.g., a selectivity-enhancing alteration at a position functionally equivalent to F102, SO I 3, W104, G105, Y130, D131, Y132, D133, P134, L135, or any combination thereof
  • the stability-enhancing alterations T19Y/R74L/C171A e.g., T19Y/R74L/C171A
  • G105/G108C/C171A/G188R can further include one or more stability-enhancing substitution mutations at positions functionally equivalent to those selected from one or more ofH16D, S20F, S20E, S20Y, S20I, K60G, I89R, S103T, E109L, V110L, Q115K, Q115Y, E116L, A126C, E138R, E138Q, E138A, D145R, D180H, S183V, A185R, R189K, and A192E, or any combination thereof, where the position number designation is functionally equivalent to the position in a wild-type APOBEC3A (SEQ ID NO:3).
  • An ACD described herein can include a stability-enhancing alteration that is a deletion.
  • examples of other deletions are at positions functionally equivalent to AM1-L12 (the first 12 amino acids of human APOBECA3A (SEQ ID NO:3), AE36-G53, AN42, AN61-G68, AP86, AW104-G105, API 72, and AS187-N199, or any combination thereof, where the position number designation is functionally equivalent to the position in a wild-type APOBEC3 A (SEQ ID NO:3).
  • An ACD described herein can include the A61-68 deletion and the ancillary substitution mutation at (i) the position functionally equivalent to A59 where the mutation is to proline (A59P) or leucine (A59L), (ii) the position functionally equivalent to K60 where the mutation is to arginine (K60R), glutamic acid ((K60E), glutamine (K60Q), or glycine (K60G), or (iii) the position functionally equivalent to R69 (i.e., the position 69 prior to the deletion of 61-68) where the mutation is to tyrosine (R69Y), asparagine (R69N, histidine (R69H), aspartic acid (R69D), or leucine (R69L).
  • A61-68 deletion and ancillary substitution mutations are A61-68/A59P/K60R, A61-68/A59L/R69Y, A61- 68/A59L/K60E/R69N, A61-68/A59P/K60E/R69H, A61-68/A59P/K60Q/R69H, A61- 68/A59L/R69N, A61-68/R69D, A61-68/A59L/K60R, and A61-68/A59P/K60G/R69L.
  • An ACD described herein can include the Al 04- 105 deletion and the ancillary substitution mutation at the position functionally equivalent to Fl 02 where the mutation is to arginine (F102R), the substitution mutation at the position functionally equivalent to SI 03 where the mutation is to asparagine (S103N), or both substitution mutations F102R and S103N.
  • an ACD described herein can include both the A 104- 105 deletion and the Al-12 deletion.
  • Additional embodiments include: an ACD having the A104-105 deletion, the Al-12 deletion, and the substitution mutation F102R; an ACD having the A104-105 deletion, the Al-12 deletion, and the substitution mutation S103N; and an ACD having the A104-105 deletion, the Al-12 deletion, and the substitution mutations F102R and S103N.
  • selectivity-enhancing substitution mutations Y130A, D131X, Y132X, D133X, and Y134W e.g., the amino acids at 130, 131, 132, 133 and 134 are those shown in Table 2 (see Example 1), or
  • an ACD having enhanced 5mC-selective deaminase activity described herein can further include at least one of the following combinations of stabilizing mutations: R74L/C171A; R74L/T19Y; R74L/G25R; R74L/T19I/C171A;
  • the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 130-134 a sequence selected from: AVAHW (SEQ ID NO:25), AHIYW (SEQ ID NO:26), AHEYW (SEQ ID NO:27), ARAYW ((SEQ ID NO:28), ANGFW ((SEQ ID NO:29), AKSHW (SEQ ID NO:30), AHLYW (SEQ ID NO:31), AHDFW (SEQ ID NO:32), AKTHW (SEQ ID NO:33), AHMIW (SEQ ID NO:34), AKMVW (SEQ ID NO: 35), AQAFW (SEQ ID NO: 36, ARVKW (SEQ ID NO: 37), AHRVW (SEQ ID NO:38), AKTYW (SEQ ID NO:39), AVAKW (SEQ ID NO:40), AKNFW (SEQ ID NO:41), AHARW (SEQ ID NO:42), AGPYW (SEQ ID NO:25), A
  • the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 130-134, or functionally equivalent positions, a sequence selected from: AVAHW (SEQ ID NO:25) in combination with one or more combinations of stability enhancing mutations selected from Hl IX, L12X, D14X, H16X, I17X, T19X, S20X, N21X, G25X, I26X, G27X, R28X, H29X, T31X, C34X, E38X, R39X, D41X, N42X, S45X, K47X, M48X, H51X, F54X, H56X, N57X, A59X, K60X, L62X, Y67X, L73X, R74X, D77X, L78X, V79X, P80X, S81X, D85X, A87X, I89X, Y90X, R91X, T93X, W94X, I96X
  • the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 130-134, or functionally equivalent positions, a sequence selected from ARAYW ((SEQ ID NO:28) in combination with one or more combinations of stability enhancing mutations selected from Hl IX, L12X, D14X, H16X, I17X, T19X, S20X, N21X, G25X, I26X, G27X, R28X, H29X, T31X, C34X, E38X, R39X, D41X, N42X, S45X, K47X, M48X, H51X, F54X, H56X, N57X, A59X, K60X, L62X, Y67X, L73X, R74X, D77X, L78X, V79X, P80X, S81X, D85X, A87X, I89X, Y90X, R91X, T93X, W94X, I96X,
  • the ACD further comprises at least one selectivity-enhancing alteration at a position functionally equivalent to F102, S103, W104 and G105, for example, e.g., FVPG, FTDG, FVDG, FVPAG, FVPLG, FVPPG, FVPAAG, FVPLAG, FVPPAG, FVPASG, FVPLSG, FVPPSG, FVPAFG, FVPLFG, FVPPFG, FVPAQG, FVPLQG, FVPPQG, FVPADG, FVPLDG, FVPPDG, FVPAKG, FVPLKG, FVPPKG among others.
  • FVPG FTDG, FVDG, FVPAG, FVPLG, FVPPG, FVPAAG, FVPLAG, FVPPAG, FVPASG, FVPLSG, FVPPSG, FVPAFG, FVPLFG, FVPPFG, FVPAQG, FVPLQG, FVPPQG, FVPADG, F
  • the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 130-134, or functionally equivalent positions, a sequence selected from: ARAYW ((SEQ ID NO:28) in combination with one or more combinations of stability enhancing mutations selected from: at least one of the following combinations of stabilizing mutations: R74L/C 171 A/T 19 Y/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 88Q/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88Q/G25K/S45W; R74L/C 171 A/T 19 Y/Gl 88 A/S45 W;
  • the ACD further comprises at least one selectivity-enhancing alteration at a position functionally equivalent to Fl 02, S103, W104 and G105, for example, e g., FVPG, FTDG, FVDG, FVPAG, FVPLG, FVPPG, FVPAAG, FVPLAG, FVPPAG, FVPASG, FVPLSG, FVPPSG, FVPAFG, FVPLFG, FVPPFG, FVPAQG, FVPLQG, FVPPQG, FVPADG, FVPLDG, FVPPDG, FVPAKG, FVPLKG, FVPPKG among others.
  • FVPG FTDG, FVDG, FVPAG, FVPLG, FVPPG, FVPAAG, FVPLAG, FVPPAG, FVPASG, FVPLSG, FVPPSG, FVPAFG, FVPLFG, FVPPFG, FVPAQG, FVPLQG, FVPPQG, FVPADG, F
  • the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 130-134, or functionally equivalent positions, a sequence selected from ANGFW ((SEQ ID NO:29) in combination with one or more combinations of stability enhancing mutations selected from Hl IX, L12X, D14X, H16X, I17X, T19X, S20X, N21X, G25X, I26X, G27X, R28X, H29X, T31X, C34X, E38X, R39X, D41X, N42X, S45X, K47X, M48X, H51X, F54X, H56X, N57X, A59X, K60X, L62X, Y67X, L73X, R74X, D77X, L78X, V79X, P80X, S81X, D85X, A87X, I89X, Y90X, R91X, T93X, W94X, I96X, S
  • the ACD further comprises at least one selectivity-enhancing alteration at a position functionally equivalent to F102, S103, W104 and G105, for example, e.g., FVPG, FTDG, FVDG, FVPAG, FVPLG, FVPPG, FVPAAG, FVPLAG, FVPPAG, FVPASG, FVPLSG, FVPPSG, FVPAFG, FVPLFG, FVPPFG, FVPAQG, FVPLQG, FVPPQG, FVPADG, FVPLDG, FVPPDG, FVPAKG, FVPLKG, FVPPKG among others.
  • FVPG FTDG, FVDG, FVPAG, FVPLG, FVPPG, FVPAAG, FVPLAG, FVPPAG, FVPASG, FVPLSG, FVPPSG, FVPAFG, FVPLFG, FVPPFG, FVPAQG, FVPLQG, FVPPQG, FVPADG, F
  • the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 130-134, or functionally equivalent positions, a sequence selected from: ANGFW ((SEQ ID NO:29) in combination with one or more combinations of stability enhancing mutations selected from: at least one of the following combinations of stabilizing mutations: R74L/C 171 A/T 19 Y/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 88Q/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88Q/G25K/S45W; R74L/C 171 A/T 19 Y/Gl 88 A/S45 W;
  • R74L/C 171 A/T 19 Y/Gl 08C/G188R/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G188R/G25K/S45 W; T 19 Y/R74L/C 171 A;
  • the ACD further comprises at least one selectivity-enhancing alteration at a position functionally equivalent to Fl 02, S103, W104 and G105, for example, e g., FVPG, FTDG, FVDG, FVPAG, FVPLG, FVPPG, FVPAAG, FVPLAG, FVPPAG, FVPASG, FVPLSG, FVPPSG, FVPAFG, FVPLFG, FVPPFG, FVPAQG, FVPLQG, FVPPQG, FVPADG, FVPLDG, FVPPDG, FVPAKG, FVPLKG, FVPPKG among others.
  • FVPG FTDG, FVDG, FVPAG, FVPLG, FVPPG, FVPAAG, FVPLAG, FVPPAG, FVPASG, FVPLSG, FVPPSG, FVPAFG, FVPLFG, FVPPFG, FVPAQG, FVPLQG, FVPPQG, FVPADG, F
  • the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 130-134, or functionally equivalent positions, a sequence selected from: AKSHW (SEQ ID NO: 30) in combination with one or more combinations of stability enhancing mutations selected from Hl IX, L12X, D14X, H16X, I17X, T19X, S20X, N21X, G25X, I26X, G27X, R28X, H29X, T31X, C34X, E38X, R39X, D41X, N42X, S45X, K47X, M48X, H51X, F54X, H56X, N57X, A59X, K60X, L62X, Y67X, L73X, R74X, D77X, L78X, V79X, P80X, S81X, D85X, A87X, I89X, Y90X, R91X, T93X, W94X, I96X,
  • the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 130-134, or functionally equivalent positions, a sequence selected from: AKSHW (SEQ ID NO: 30) in combination with one or more combinations of stability enhancing mutations selected from: at least one of the following combinations of stabilizing mutations: R74L/C 171 A/T 19 Y/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 88Q/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88Q/G25K/S45W; R74L/C 171 A/T 19 Y/Gl 88 A/S45 W;
  • the ACD further comprises at least one selectivity-enhancing alteration at a position functionally equivalent to Fl 02, S103, W104 and G105, for example, e g., FVPG, FTDG, FVDG, FVPAG, FVPLG, FVPPG, FVPAAG, FVPLAG, FVPPAG, FVPASG, FVPLSG, FVPPSG, FVPAFG, FVPLFG, FVPPFG, FVPAQG, FVPLQG, FVPPQG, FVPADG, FVPLDG, FVPPDG, FVPAKG, FVPLKG, FVPPKG among others.
  • FVPG FTDG, FVDG, FVPAG, FVPLG, FVPPG, FVPAAG, FVPLAG, FVPPAG, FVPASG, FVPLSG, FVPPSG, FVPAFG, FVPLFG, FVPPFG, FVPAQG, FVPLQG, FVPPQG, FVPADG, F
  • the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 130-134, or functionally equivalent positions, a sequence selected from AHLYW (SEQ ID NO: 31) in combination with one or more combinations of stability enhancing mutations selected from Hl IX, L12X, D14X, H16X, I17X, T19X, S20X, N21X, G25X, I26X, G27X, R28X, H29X, T31X, C34X, E38X, R39X, D41X, N42X, S45X, K47X, M48X, H51X, F54X, H56X, N57X, A59X, K60X, L62X, Y67X, L73X, R74X, D77X, L78X, V79X, P80X, S81X, D85X, A87X, I89X, Y90X, R91X, T93X, W94X, I96X, S
  • the ACD further comprises at least one selectivity-enhancing alteration at a position functionally equivalent to F102, S103, W104 and G105, for example, e.g., FVPG, FTDG, FVDG, FVPAG, FVPLG, FVPPG, FVPAAG, FVPLAG, FVPPAG, FVPASG, FVPLSG, FVPPSG, FVPAFG, FVPLFG, FVPPFG, FVPAQG, FVPLQG, FVPPQG, FVPADG, FVPLDG, FVPPDG, FVPAKG, FVPLKG, FVPPKG among others.
  • FVPG FTDG, FVDG, FVPAG, FVPLG, FVPPG, FVPAAG, FVPLAG, FVPPAG, FVPASG, FVPLSG, FVPPSG, FVPAFG, FVPLFG, FVPPFG, FVPAQG, FVPLQG, FVPPQG, FVPADG, F
  • the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 130-134, or functionally equivalent positions, a sequence selected from: AHLYW (SEQ ID NO:31) in combination with one or more combinations of stability enhancing mutations selected from: at least one of the following combinations of stabilizing mutations: R74L/C 171 A/T 19 Y/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 88Q/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88Q/G25K/S45W; R74L/C 171 A/T 19 Y/Gl 88 A/S45 W;
  • R74L/C 171 A/T 19 Y/Gl 08C/G188R/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G188R/G25K/S45 W; T 19 Y/R74L/C 171 A;
  • the ACD further comprises at least one selectivity-enhancing alteration at a position functionally equivalent to Fl 02, S103, W104 and G105, for example, e g., FVPG, FTDG, FVDG, FVPAG, FVPLG, FVPPG, FVPAAG, FVPLAG, FVPPAG, FVPASG, FVPLSG, FVPPSG, FVPAFG, FVPLFG, FVPPFG, FVPAQG, FVPLQG, FVPPQG, FVPADG, FVPLDG, FVPPDG, FVPAKG, FVPLKG, FVPPKG among others.
  • FVPG FTDG, FVDG, FVPAG, FVPLG, FVPPG, FVPAAG, FVPLAG, FVPPAG, FVPASG, FVPLSG, FVPPSG, FVPAFG, FVPLFG, FVPPFG, FVPAQG, FVPLQG, FVPPQG, FVPADG, F
  • the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 130-134, or functionally equivalent positions, a sequence selected from: AHDFW (SEQ ID NO:32) in combination with one or more combinations of stability enhancing mutations selected from Hl IX, L12X, D14X, H16X, I17X, T19X, S20X, N21X, G25X, I26X, G27X, R28X, H29X, T31X, C34X, E38X, R39X, D41X, N42X, S45X, K47X, M48X, H51X, F54X, H56X, N57X, A59X, K60X, L62X, Y67X, L73X, R74X, D77X, L78X, V79X, P80X, S81X, D85X, A87X, I89X, Y90X, R91X, T93X, W94X, I96X, S
  • the ACD further comprises at least one selectivity-enhancing alteration at a position functionally equivalent to F102, S103, W104 and G105, for example, e.g., FVPG, FTDG, FVDG, FVPAG, FVPLG, FVPPG, FVPAAG, FVPLAG, FVPPAG, FVPASG, FVPLSG, FVPPSG, FVPAFG, FVPLFG, FVPPFG, FVPAQG, FVPLQG, FVPPQG, FVPADG, FVPLDG, FVPPDG, FVPAKG, FVPLKG, FVPPKG among others.
  • FVPG FTDG, FVDG, FVPAG, FVPLG, FVPPG, FVPAAG, FVPLAG, FVPPAG, FVPASG, FVPLSG, FVPPSG, FVPAFG, FVPLFG, FVPPFG, FVPAQG, FVPLQG, FVPPQG, FVPADG, F
  • the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 130-134, or functionally equivalent positions, a sequence selected from: AHDFW (SEQ ID NO:32), in combination with one or more combinations of stability enhancing mutations selected from: at least one of the following combinations of stabilizing mutations: R74L/C 171 A/T 19 Y/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 88Q/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88Q/G25K/S45W; R74L/C 171 A/T 19 Y/Gl 88 A/S45 W;
  • the ACD further comprises at least one selectivity-enhancing alteration at a position functionally equivalent to Fl 02, S103, W104 and G105, for example, e g., FVPG, FTDG, FVDG, FVPAG, FVPLG, FVPPG, FVPAAG, FVPLAG, FVPPAG, FVPASG, FVPLSG, FVPPSG, FVPAFG, FVPLFG, FVPPFG, FVPAQG, FVPLQG, FVPPQG, FVPADG, FVPLDG, FVPPDG, FVPAKG, FVPLKG, FVPPKG among others.
  • FVPG FTDG, FVDG, FVPAG, FVPLG, FVPPG, FVPAAG, FVPLAG, FVPPAG, FVPASG, FVPLSG, FVPPSG, FVPAFG, FVPLFG, FVPPFG, FVPAQG, FVPLQG, FVPPQG, FVPADG, F
  • the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 130-134, or functionally equivalent positions, a sequence selected from: AKTHW (SEQ ID NO:33), in combination with one or more combinations of stability enhancing mutations selected from Hl IX, L12X, D14X, H16X, I17X, T19X, S20X, N21X, G25X, I26X, G27X, R28X, H29X, T31X, C34X, E38X, R39X, D41X, N42X, S45X, K47X, M48X, H51X, F54X, H56X, N57X, A59X, K60X, L62X, Y67X, L73X, R74X, D77X, L78X, V79X, P80X, S81X, D85X, A87X, I89X, Y90X, R91X, T93X, W94X, I96X,
  • the ACD further comprises at least one selectivity-enhancing alteration at a position functionally equivalent to F102, S103, W104 and G105, for example, e.g., FVPG, FTDG, FVDG, FVPAG, FVPLG, FVPPG, FVPAAG, FVPLAG, FVPPAG, FVPASG, FVPLSG, FVPPSG, FVPAFG, FVPLFG, FVPPFG, FVPAQG, FVPLQG, FVPPQG, FVPADG, FVPLDG, FVPPDG, FVPAKG, FVPLKG, FVPPKG among others.
  • FVPG FTDG, FVDG, FVPAG, FVPLG, FVPPG, FVPAAG, FVPLAG, FVPPAG, FVPASG, FVPLSG, FVPPSG, FVPAFG, FVPLFG, FVPPFG, FVPAQG, FVPLQG, FVPPQG, FVPADG, F
  • the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 130-134, or functionally equivalent positions, a sequence selected from: AKTHW (SEQ ID NO:33), in combination with one or more combinations of stability enhancing mutations selected from: at least one of the following combinations of stabilizing mutations: R74L/C 171 A/T 19 Y/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 88Q/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88Q/G25K/S45W; R74L/C 171 A/T 19 Y/Gl 88 A/S45 W;
  • R74L/C 171 A/T 19 Y/Gl 08C/G188R/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G188R/G25K/S45 W; T 19 Y/R74L/C 171 A;
  • the ACD further comprises at least one selectivity-enhancing alteration at a position functionally equivalent to Fl 02, S103, W104 and G105, for example, e g., FVPG, FTDG, FVDG, FVPAG, FVPLG, FVPPG, FVPAAG, FVPLAG, FVPPAG, FVPASG, FVPLSG, FVPPSG, FVPAFG, FVPLFG, FVPPFG, FVPAQG, FVPLQG, FVPPQG, FVPADG, FVPLDG, FVPLDG, F
  • the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 130-134, or functionally equivalent positions, a sequence selected from: AHMIW (SEQ ID NO:34), in combination with one or more combinations of stability enhancing mutations selected from Hl IX, L12X, D14X, H16X, I17X, T19X, S20X, N21X, G25X, I26X, G27X, R28X, H29X, T31X, C34X, E38X, R39X, D41X, N42X, S45X, K47X, M48X, H51X, F54X, H56X, N57X, A59X, K60X, L62X, Y67X, L73X, R74X, D77X, L78X, V79X, P80X, S81X, D85X, A87X, I89X, Y90X, R91X, T93X, W94X, I96X,
  • the ACD further comprises at least one selectivity-enhancing alteration at a position functionally equivalent to F102, S103, W104 and G105, for example, e.g., FVPG, FTDG, FVDG, FVPAG, FVPLG, FVPPG, FVPAAG, FVPLAG, FVPPAG, FVPASG, FVPLSG, FVPPSG, FVPAFG, FVPLFG, FVPPFG, FVPAQG, FVPLQG, FVPPQG, FVPADG, FVPLDG, FVPPDG, FVPAKG, FVPLKG, FVPPKG among others.
  • FVPG FTDG, FVDG, FVPAG, FVPLG, FVPPG, FVPAAG, FVPLAG, FVPPAG, FVPASG, FVPLSG, FVPPSG, FVPAFG, FVPLFG, FVPPFG, FVPAQG, FVPLQG, FVPPQG, FVPADG, F
  • the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 130-134, or functionally equivalent positions, a sequence selected from: AHMIW (SEQ ID NO:34), in combination with one or more combinations of stability enhancing mutations selected from: at least one of the following combinations of stabilizing mutations: R74L/C 171 A/T 19 Y/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 88Q/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88Q/G25K/S45W; R74L/C 171 A/T 19 Y/Gl 88 A/S45 W;
  • the ACD further comprises at least one selectivity-enhancing alteration at a position functionally equivalent to Fl 02, S103, W104 and G105, for example, e g., FVPG, FTDG, FVDG, FVPAG, FVPLG, FVPPG, FVPAAG, FVPLAG, FVPPAG, FVPASG, FVPLSG, FVPPSG, FVPAFG, FVPLFG, FVPPFG, FVPAQG, FVPLQG, FVPPQG, FVPADG, FVPLDG, FVPPDG, FVPAKG, FVPLKG, FVPPKG among others.
  • FVPG FTDG, FVDG, FVPAG, FVPLG, FVPPG, FVPAAG, FVPLAG, FVPPAG, FVPASG, FVPLSG, FVPPSG, FVPAFG, FVPLFG, FVPPFG, FVPAQG, FVPLQG, FVPPQG, FVPADG, F
  • the ACD comprises a backbone sequence relative to AKMVW (SEQ ID NO: 35), in combination with one or more combinations of stability enhancing mutations selected from Hl IX, L12X, D14X, H16X, I17X, T19X, S20X, N21X, G25X, I26X, G27X, R28X, H29X, T31X, C34X, E38X, R39X, D41X, N42X, S45X, K47X, M48X, H51X, F54X, H56X, N57X, A59X, K60X, L62X, Y67X, L73X, R74X, D77X, L78X, V79X, P80X, S81X, D85X, A87X, I89X, Y90X, R91X, T93X, W94X, I96X, S97X, C101X, F102X, S103X, W104X, G105X, C
  • the ACD further comprises at least one selectivity-enhancing alteration at a position functionally equivalent to Fl 02, SI 03, W104 and G105, for example, e g., FVPG, FTDG, FVDG, FVPAG, FVPLG, FVPPG, FVPAAG, FVPLAG, FVPPAG, FVPASG, FVPLSG, FVPPSG, FVPAFG, FVPLFG, FVPPFG, FVPAQG, FVPLQG, FVPPQG, FVPADG, FVPLDG, FVPPDG, FVPAKG, FVPLKG, FVPPKG among others.
  • FVPG selectivity-enhancing alteration at a position functionally equivalent to Fl 02, SI 03, W104 and G105
  • FVPG FTDG, FVDG, FVPAG, FVPLG, FVPPG, FVPAAG, FVPLAG, FVPPAG, FVPASG, FVPLSG, FVPPSG, FVPAFG, FVPL
  • the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 130-134, or functionally equivalent positions, a sequence selected from: AKMVW (SEQ ID NO: 35), in combination with one or more combinations of stability enhancing mutations selected from: at least one of the following combinations of stabilizing mutations: R74L/C 171 A/T 19 Y/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 88Q/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88Q/G25K/S45W; R74L/C 171 A/T 19 Y/Gl 88 A/S45 W;
  • the ACD further comprises at least one selectivity-enhancing alteration at a position functionally equivalent to Fl 02, S103, W104 and G105, for example, e g., FVPG, FTDG, FVDG, FVPAG, FVPLG, FVPPG, FVPAAG, FVPLAG, FVPPAG, FVPASG, FVPLSG, FVPPSG, FVPAFG, FVPLFG, FVPPFG, FVPAQG, FVPLQG, FVPPQG, FVPADG, FVPLDG, FVPPDG, FVPAKG, FVPLKG, FVPPKG among others.
  • FVPG FTDG, FVDG, FVPAG, FVPLG, FVPPG, FVPAAG, FVPLAG, FVPPAG, FVPASG, FVPLSG, FVPPSG, FVPAFG, FVPLFG, FVPPFG, FVPAQG, FVPLQG, FVPPQG, FVPADG, F
  • the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 130-134, or functionally equivalent positions, a sequence selected from: AQAFW (SEQ ID NO: 36), in combination with one or more combinations of stability enhancing mutations selected from Hl IX, L12X, D14X, H16X, I17X, T19X, S20X, N21X, G25X, I26X, G27X, R28X, H29X, T31X, C34X, E38X, R39X, D41X, N42X, S45X, K47X, M48X, H51X, F54X, H56X, N57X, A59X, K60X, L62X, Y67X, L73X, R74X, D77X, L78X, V79X, P80X, S81X, D85X, A87X, I89X, Y90X, R91X, T93X, W94X, I96X, S
  • the ACD further comprises at least one selectivity-enhancing alteration at a position functionally equivalent to F102, S103, W104 and G105, for example, e.g., FVPG, FTDG, FVDG, FVPAG, FVPLG, FVPPG, FVPAAG, FVPLAG, FVPPAG, FVPASG, FVPLSG, FVPPSG, FVPAFG, FVPLFG, FVPPFG, FVPAQG, FVPLQG, FVPPQG, FVPADG, FVPLDG, FVPPDG, FVPAKG, FVPLKG, FVPPKG among others.
  • FVPG FTDG, FVDG, FVPAG, FVPLG, FVPPG, FVPAAG, FVPLAG, FVPPAG, FVPASG, FVPLSG, FVPPSG, FVPAFG, FVPLFG, FVPPFG, FVPAQG, FVPLQG, FVPPQG, FVPADG, F
  • the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 130-134, or functionally equivalent positions, a sequence selected from: AQAFW (SEQ ID NO: 36), in combination with one or more combinations of stability enhancing mutations selected from: at least one of the following combinations of stabilizing mutations: R74L/C 171 A/T 19 Y/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 88Q/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88Q/G25K/S45W; R74L/C 171 A/T 19 Y/Gl 88 A/S45 W;
  • the ACD further comprises at least one selectivity-enhancing alteration at a position functionally equivalent to Fl 02, S103, W104 and G105, for example, e g., FVPG, FTDG, FVDG, FVPAG, FVPLG, FVPPG, FVPAAG, FVPLAG, FVPPAG, FVPASG, FVPLSG, FVPPSG, FVPAFG, FVPLFG, FVPPFG, FVPAQG, FVPLQG, FVPPQG, FVPADG, FVPLDG, FVPPDG, FVPAKG, FVPLKG, FVPPKG among others.
  • FVPG FTDG, FVDG, FVPAG, FVPLG, FVPPG, FVPAAG, FVPLAG, FVPPAG, FVPASG, FVPLSG, FVPPSG, FVPAFG, FVPLFG, FVPPFG, FVPAQG, FVPLQG, FVPPQG, FVPADG, F
  • the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 130-134, or functionally equivalent positions, a sequence selected from: ARVKW (SEQ ID NO: 37), in combination with one or more combinations of stability enhancing mutations selected from Hl IX, L12X, D14X, H16X, I17X, T19X, S20X, N21X, G25X, I26X, G27X, R28X, H29X, T31X, C34X, E38X, R39X, D41X, N42X, S45X, K47X, M48X, H51X, F54X, H56X, N57X, A59X, K60X, L62X, Y67X, L73X, R74X, D77X, L78X, V79X, P80X, S81X, D85X, A87X, I89X, Y90X, R91X, T93X, W94X, I96X, S
  • the ACD further comprises at least one selectivity-enhancing alteration at a position functionally equivalent to F102, S103, W104 and G105, for example, e.g., FVPG, FTDG, FVDG, FVPAG, FVPLG, FVPPG, FVPAAG, FVPLAG, FVPPAG, FVPASG, FVPLSG, FVPPSG, FVPAFG, FVPLFG, FVPPFG, FVPAQG, FVPLQG, FVPPQG, FVPADG, FVPLDG, FVPPDG, FVPAKG, FVPLKG, FVPPKG among others.
  • FVPG FTDG, FVDG, FVPAG, FVPLG, FVPPG, FVPAAG, FVPLAG, FVPPAG, FVPASG, FVPLSG, FVPPSG, FVPAFG, FVPLFG, FVPPFG, FVPAQG, FVPLQG, FVPPQG, FVPADG, F
  • the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 130-134, or functionally equivalent positions, a sequence selected from: ARVKW (SEQ ID NO: 37), in combination with one or more combinations of stability enhancing mutations selected from: at least one of the following combinations of stabilizing mutations: R74L/C 171 A/T 19 Y/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 88Q/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88Q/G25K/S45W; R74L/C 171 A/T 19 Y/Gl 88 A/S45 W;
  • R74L/C 171 A/T 19 Y/Gl 08C/G188R/G25K/S45 W; T 19 Y/R74L/C 171 A; 117T/T 19 Y/G25R/R74L/C 171 A; T 19 Y/G25K/S45 WR74L/G108 A/C 171 A/Gl 88R;
  • the ACD further comprises at least one selectivity-enhancing alteration at a position functionally equivalent to Fl 02, S103, W104 and G105, for example, e g., FVPG, FTDG, FVDG, FVPAG, FVPLG, FVPPG, FVPAAG, FVPLAG, FVPPAG, FVPASG, FVPLSG, FVPPSG, FVPAFG, FVPLFG, FVPPFG, FVPAQG, FVPLQG, FVPPQG, FVPADG, FVPLDG, FVPPDG, FVPAKG, FVPLKG, FVPPKG among others.
  • FVPG FTDG, FVDG, FVPAG, FVPLG, FVPPG, FVPAAG, FVPLAG, FVPPAG, FVPASG, FVPLSG, FVPPSG, FVPAFG, FVPLFG, FVPPFG, FVPAQG, FVPLQG, FVPPQG, FVPADG, F
  • the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 130-134, or functionally equivalent positions, a sequence selected from: AHRVW (SEQ ID NO: 38), in combination with one or more combinations of stability enhancing mutations selected from Hl IX, L12X, D14X, H16X, I17X, T19X, S20X, N21X, G25X, I26X, G27X, R28X, H29X, T31X, C34X, E38X, R39X, D41X, N42X, S45X, K47X, M48X, H51X, F54X, H56X, N57X, A59X, K60X, L62X, Y67X, L73X, R74X, D77X, L78X, V79X, P80X, S81X, D85X, A87X, I89X, Y90X, R91X, T93X, W94X, I96X,
  • the ACD further comprises at least one selectivity-enhancing alteration at a position functionally equivalent to F102, S103, W104 and G105, for example, e.g., FVPG, FTDG, FVDG, FVPAG, FVPLG, FVPPG, FVPAAG, FVPLAG, FVPPAG, FVPASG, FVPLSG, FVPPSG, FVPAFG, FVPLFG, FVPPFG, FVPAQG, FVPLQG, FVPPQG, FVPADG, FVPLDG, FVPPDG, FVPAKG, FVPLKG, FVPPKG among others.
  • FVPG FTDG, FVDG, FVPAG, FVPLG, FVPPG, FVPAAG, FVPLAG, FVPPAG, FVPASG, FVPLSG, FVPPSG, FVPAFG, FVPLFG, FVPPFG, FVPAQG, FVPLQG, FVPPQG, FVPADG, F
  • the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 130-134, or functionally equivalent positions, a sequence selected from: AHRVW (SEQ ID NO: 38), in combination with one or more combinations of stability enhancing mutations selected from: at least one of the following combinations of stabilizing mutations: R74L/C 171 A/T 19 Y/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 88Q/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88Q/G25K/S45W; R74L/C 171 A/T 19 Y/Gl 88 A/S45 W;
  • the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 130-134, or functionally equivalent positions, a sequence selected from: AKTYW (SEQ ID NO: 39), in combination with one or more combinations of stability enhancing mutations selected from Hl IX, L12X, D14X, H16X, I17X, T19X, S20X, N21X, G25X, I26X, G27X, R28X, H29X, T31X, C34X, E38X, R39X, D41X, N42X, S45X, K47X, M48X, H51X, F54X, H56X, N57X, A59X, K60X, L62X, Y67X, L73X, R74X, D77X, L78X, V79X, P80X, S81X, D85X, A87X, I89X, Y90X, R91X, T93X, W94X, I96X,
  • the ACD further comprises at least one selectivity-enhancing alteration at a position functionally equivalent to F102, S103, W104 and G105, for example, e.g., FVPG, FTDG, FVDG, FVPAG, FVPLG, FVPPG, FVPAAG, FVPLAG, FVPPAG, FVPASG, FVPLSG, FVPPSG, FVPAFG, FVPLFG, FVPPFG, FVPAQG, FVPLQG, FVPPQG, FVPADG, FVPLDG, FVPPDG, FVPAKG, FVPLKG, FVPPKG among others.
  • FVPG FTDG, FVDG, FVPAG, FVPLG, FVPPG, FVPAAG, FVPLAG, FVPPAG, FVPASG, FVPLSG, FVPPSG, FVPAFG, FVPLFG, FVPPFG, FVPAQG, FVPLQG, FVPPQG, FVPADG, F
  • R74L/C 171 A/T 19 Y/Gl 08C/G188R/G25K/S45 W; T 19 Y/R74L/C 171 A; 117T/T 19 Y/G25R/R74L/C 171 A; T 19 Y/G25K/S45 WR74L/G108 A/C 171 A/Gl 88R;
  • the ACD further comprises at least one selectivity-enhancing alteration at a position functionally equivalent to Fl 02, S103, W104 and G105, for example, e g., FVPG, FTDG, FVDG, FVPAG, FVPLG, FVPPG, FVPAAG, FVPLAG, FVPPAG, FVPASG, FVPLSG, FVPPSG, FVPAFG, FVPLFG, FVPPFG, FVPAQG, FVPLQG, FVPPQG, FVPADG, FVPLDG, FVPLDG, F
  • the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 130-134, or functionally equivalent positions, a sequence selected from: AVAKW (SEQ ID NO:40), in combination with one or more combinations of stability enhancing mutations selected from Hl IX, L12X, D14X, H16X, I17X, T19X, S20X, N21X, G25X, I26X, G27X, R28X, H29X, T31X, C34X, E38X, R39X, D41X, N42X, S45X, K47X, M48X, H51X, F54X, H56X, N57X, A59X, K60X, L62X, Y67X, L73X, R74X, D77X, L78X, V79X, P80X, S81X, D85X, A87X, I89X, Y90X, R91X, T93X, W94X, I96X, S
  • the ACD further comprises at least one selectivity-enhancing alteration at a position functionally equivalent to F102, S103, W104 and G105, for example, e.g., FVPG, FTDG, FVDG, FVPAG, FVPLG, FVPPG, FVPAAG, FVPLAG, FVPPAG, FVPASG, FVPLSG, FVPPSG, FVPAFG, FVPLFG, FVPPFG, FVPAQG, FVPLQG, FVPPQG, FVPADG, FVPLDG, FVPPDG, FVPAKG, FVPLKG, FVPPKG among others.
  • FVPG FTDG, FVDG, FVPAG, FVPLG, FVPPG, FVPAAG, FVPLAG, FVPPAG, FVPASG, FVPLSG, FVPPSG, FVPAFG, FVPLFG, FVPPFG, FVPAQG, FVPLQG, FVPPQG, FVPADG, F
  • the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 130-134, or functionally equivalent positions, a sequence selected from: AVAKW (SEQ ID NO:40), in combination with one or more combinations of stability enhancing mutations selected from: at least one of the following combinations of stabilizing mutations: R74L/C 171 A/T 19 Y/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 88Q/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88Q/G25K/S45W; R74L/C 171 A/T 19 Y/Gl 88 A/S45 W;
  • the ACD further comprises at least one selectivity-enhancing alteration at a position functionally equivalent to Fl 02, S103, W104 and G105, for example, e g., FVPG, FTDG, FVDG, FVPAG, FVPLG, FVPPG, FVPAAG, FVPLAG, FVPPAG, FVPASG, FVPLSG, FVPPSG, FVPAFG, FVPLFG, FVPPFG, FVPAQG, FVPLQG, FVPPQG, FVPADG, FVPLDG, FVPPDG, FVPAKG, FVPLKG, FVPPKG among others.
  • FVPG FTDG, FVDG, FVPAG, FVPLG, FVPPG, FVPAAG, FVPLAG, FVPPAG, FVPASG, FVPLSG, FVPPSG, FVPAFG, FVPLFG, FVPPFG, FVPAQG, FVPLQG, FVPPQG, FVPADG, F
  • the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 130-134, or functionally equivalent positions, a sequence selected from: AKNFW (SEQ ID NO:41), in combination with one or more combinations of stability enhancing mutations selected from Hl IX, L12X, D14X, H16X, I17X, T19X, S20X, N21X, G25X, I26X, G27X, R28X, H29X, T31X, C34X, E38X, R39X, D41X, N42X, S45X, K47X, M48X, H51X, F54X, H56X, N57X, A59X, K60X, L62X, Y67X, L73X, R74X, D77X, L78X, V79X, P80X, S81X, D85X, A87X, I89X, Y90X, R91X, T93X, W94X, I96X,
  • the ACD further comprises at least one selectivity-enhancing alteration at a position functionally equivalent to F102, S103, W104 and G105, for example, e.g., FVPG, FTDG, FVDG, FVPAG, FVPLG, FVPPG, FVPAAG, FVPLAG, FVPPAG, FVPASG, FVPLSG, FVPPSG, FVPAFG, FVPLFG, FVPPFG, FVPAQG, FVPLQG, FVPPQG, FVPADG, FVPLDG, FVPPDG, FVPAKG, FVPLKG, FVPPKG among others.
  • FVPG FTDG, FVDG, FVPAG, FVPLG, FVPPG, FVPAAG, FVPLAG, FVPPAG, FVPASG, FVPLSG, FVPPSG, FVPAFG, FVPLFG, FVPPFG, FVPAQG, FVPLQG, FVPPQG, FVPADG, F
  • the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 130-134, or functionally equivalent positions, a sequence selected from: AKNFW (SEQ ID NO:41), in combination with one or more combinations of stability enhancing mutations selected from: at least one of the following combinations of stabilizing mutations: R74L/C 171 A/T 19 Y/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 88Q/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88Q/G25K/S45W; R74L/C 171 A/T 19 Y/Gl 88 A/S45 W;
  • R74L/C 171 A/T 19 Y/Gl 08C/G188R/G25K/S45 W; T 19 Y/R74L/C 171 A; 117T/T 19 Y/G25R/R74L/C 171 A; T 19 Y/G25K/S45 WR74L/G108 A/C 171 A/Gl 88R;
  • the ACD further comprises at least one selectivity-enhancing alteration at a position functionally equivalent to Fl 02, S103, W104 and G105, for example, e g., FVPG, FTDG, FVDG, FVPAG, FVPLG, FVPPG, FVPAAG, FVPLAG, FVPPAG, FVPASG, FVPLSG, FVPPSG, FVPAFG, FVPLFG, FVPPFG, FVPAQG, FVPLQG, FVPPQG, FVPADG, FVPLDG, FVPPDG, FVPAKG, FVPLKG, FVPPKG among others.
  • FVPG FTDG, FVDG, FVPAG, FVPLG, FVPPG, FVPAAG, FVPLAG, FVPPAG, FVPASG, FVPLSG, FVPPSG, FVPAFG, FVPLFG, FVPPFG, FVPAQG, FVPLQG, FVPPQG, FVPADG, F
  • the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 130-134, or functionally equivalent positions, a sequence selected from: AHARW (SEQ ID NO:42), in combination with one or more combinations of stability enhancing mutations selected from Hl IX, L12X, D14X, H16X, I17X, T19X, S20X, N21X, G25X, I26X, G27X, R28X, H29X, T31X, C34X, E38X, R39X, D41X, N42X, S45X, K47X, M48X, H51X, F54X, H56X, N57X, A59X, K60X, L62X, Y67X, L73X, R74X, D77X, L78X, V79X, P80X, S81X, D85X, A87X, I89X, Y90X, R91X, T93X, W94X, I96X,
  • the ACD further comprises at least one selectivity-enhancing alteration at a position functionally equivalent to F102, S103, W104 and G105, for example, e.g., FVPG, FTDG, FVDG, FVPAG, FVPLG, FVPPG, FVPAAG, FVPLAG, FVPPAG, FVPASG, FVPLSG, FVPPSG, FVPAFG, FVPLFG, FVPPFG, FVPAQG, FVPLQG, FVPPQG, FVPADG, FVPLDG, FVPPDG, FVPAKG, FVPLKG, FVPPKG among others.
  • FVPG FTDG, FVDG, FVPAG, FVPLG, FVPPG, FVPAAG, FVPLAG, FVPPAG, FVPASG, FVPLSG, FVPPSG, FVPAFG, FVPLFG, FVPPFG, FVPAQG, FVPLQG, FVPPQG, FVPADG, F
  • the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 130-134, or functionally equivalent positions, a sequence selected from: AHARW (SEQ ID NO:42), in combination with one or more combinations of stability enhancing mutations selected from: at least one of the following combinations of stabilizing mutations: R74L/C 171 A/T 19 Y/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 88Q/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88Q/G25K/S45W; R74L/C 171 A/T 19 Y/Gl 88 A/S45 W;
  • the ACD further comprises at least one selectivity-enhancing alteration at a position functionally equivalent to Fl 02, S103, W104 and G105, for example, e g., FVPG, FTDG, FVDG, FVPAG, FVPLG, FVPPG, FVPAAG, FVPLAG, FVPPAG, FVPASG, FVPLSG, FVPPSG, FVPAFG, FVPLFG, FVPPFG, FVPAQG, FVPLQG, FVPPQG, FVPADG, FVPLDG, FVPPDG, FVPAKG, FVPLKG, FVPPKG among others.
  • FVPG FTDG, FVDG, FVPAG, FVPLG, FVPPG, FVPAAG, FVPLAG, FVPPAG, FVPASG, FVPLSG, FVPPSG, FVPAFG, FVPLFG, FVPPFG, FVPAQG, FVPLQG, FVPPQG, FVPADG, F
  • the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 130-134, or functionally equivalent positions, a sequence selected from: AGPYW (SEQ ID NO:43), in combination with one or more combinations of stability enhancing mutations selected from Hl IX, L12X, D14X, H16X, I17X, T19X, S20X, N21X, G25X, I26X, G27X, R28X, H29X, T31X, C34X, E38X, R39X, D41X, N42X, S45X, K47X, M48X, H51X, F54X, H56X, N57X, A59X, K60X, L62X, Y67X, L73X, R74X, D77X, L78X, V79X, P80X, S81X, D85X, A87X, I89X, Y90X, R91X, T93X, W94X, I96X,
  • the ACD further comprises at least one selectivity-enhancing alteration at a position functionally equivalent to F102, S103, W104 and G105, for example, e.g., FVPG, FTDG, FVDG, FVPAG, FVPLG, FVPPG, FVPAAG, FVPLAG, FVPPAG, FVPASG, FVPLSG, FVPPSG, FVPAFG, FVPLFG, FVPPFG, FVPAQG, FVPLQG, FVPPQG, FVPADG, FVPLDG, FVPPDG, FVPAKG, FVPLKG, FVPPKG among others.
  • FVPG FTDG, FVDG, FVPAG, FVPLG, FVPPG, FVPAAG, FVPLAG, FVPPAG, FVPASG, FVPLSG, FVPPSG, FVPAFG, FVPLFG, FVPPFG, FVPAQG, FVPLQG, FVPPQG, FVPADG, F
  • the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 130-134, or functionally equivalent positions, a sequence selected from: AGPYW (SEQ ID NO:43), in combination with one or more combinations of stability enhancing mutations selected from: at least one of the following combinations of stabilizing mutations: R74L/C 171 A/T 19 Y/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 88Q/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88Q/G25K/S45W; R74L/C 171 A/T 19 Y/Gl 88 A/S45 W;
  • R74L/C 171 A/T 19 Y/Gl 08C/G188R/G25K/S45 W; T 19 Y/R74L/C 171 A; 117T/T 19 Y/G25R/R74L/C 171 A; T 19 Y/G25K/S45 WR74L/G108 A/C 171 A/Gl 88R;
  • the ACD further comprises at least one selectivity-enhancing alteration at a position functionally equivalent to Fl 02, S103, W104 and G105, for example, e g., FVPG, FTDG, FVDG, FVPAG, FVPLG, FVPPG, FVPAAG, FVPLAG, FVPPAG, FVPASG, FVPLSG, FVPPSG, FVPAFG, FVPLFG, FVPPFG, FVPAQG, FVPLQG, FVPPQG, FVPADG, FVPLDG, FVPPDG, FVPAKG, FVPLKG, FVPPKG among others.
  • FVPG FTDG, FVDG, FVPAG, FVPLG, FVPPG, FVPAAG, FVPLAG, FVPPAG, FVPASG, FVPLSG, FVPPSG, FVPAFG, FVPLFG, FVPPFG, FVPAQG, FVPLQG, FVPPQG, FVPADG, F
  • the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 130-134, or functionally equivalent positions, a sequence selected from: AKYPW (SEQ ID NO:44), in combination with one or more combinations of stability enhancing mutations selected from Hl IX, L12X, D14X, H16X, I17X, T19X, S20X, N21X, G25X, I26X, G27X, R28X, H29X, T31X, C34X, E38X, R39X, D41X, N42X, S45X, K47X, M48X, H51X, F54X, H56X, N57X, A59X, K60X, L62X, Y67X, L73X, R74X, D77X, L78X, V79X, P80X, S81X, D85X, A87X, I89X, Y90X, R91X, T93X, W94X, I96X
  • the ACD further comprises at least one selectivity-enhancing alteration at a position functionally equivalent to F102, S103, W104 and G105, for example, e.g., FVPG, FTDG, FVDG, FVPAG, FVPLG, FVPPG, FVPAAG, FVPLAG, FVPPAG, FVPASG, FVPLSG, FVPPSG, FVPAFG, FVPLFG, FVPPFG, FVPAQG, FVPLQG, FVPPQG, FVPADG, FVPLDG, FVPPDG, FVPAKG, FVPLKG, FVPPKG among others.
  • FVPG FTDG, FVDG, FVPAG, FVPLG, FVPPG, FVPAAG, FVPLAG, FVPPAG, FVPASG, FVPLSG, FVPPSG, FVPAFG, FVPLFG, FVPPFG, FVPAQG, FVPLQG, FVPPQG, FVPADG, F
  • the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 130-134, or functionally equivalent positions, a sequence selected from: AKYPW (SEQ ID NO:44), in combination with one or more combinations of stability enhancing mutations selected from: at least one of the following combinations of stabilizing mutations: R74L/C 171 A/T 19 Y/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 88Q/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88Q/G25K/S45W; R74L/C 171 A/T 19 Y/Gl 88 A/S45 W;
  • the ACD further comprises at least one selectivity-enhancing alteration at a position functionally equivalent to Fl 02, S103, W104 and G105, for example, e g., FVPG, FTDG, FVDG, FVPAG, FVPLG, FVPPG, FVPAAG, FVPLAG, FVPPAG, FVPASG, FVPLSG, FVPPSG, FVPAFG, FVPLFG, FVPPFG, FVPAQG, FVPLQG, FVPPQG, FVPADG, FVPLDG, FVPPDG, FVPAKG, FVPLKG, FVPPKG among others.
  • FVPG FTDG, FVDG, FVPAG, FVPLG, FVPPG, FVPAAG, FVPLAG, FVPPAG, FVPASG, FVPLSG, FVPPSG, FVPAFG, FVPLFG, FVPPFG, FVPAQG, FVPLQG, FVPPQG, FVPADG, F
  • the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 130-134, or functionally equivalent positions, a sequence selected from: AKPFW (SEQ ID NO:45), in combination with one or more combinations of stability enhancing mutations selected from Hl IX, L12X, D14X, H16X, I17X, T19X, S20X, N21X, G25X, I26X, G27X, R28X, H29X, T31X, C34X, E38X, R39X, D41X, N42X, S45X, K47X, M48X, H51X, F54X, H56X, N57X, A59X, K60X, L62X, Y67X, L73X, R74X, D77X, L78X, V79X, P80X, S81X, D85X, A87X, I89X, Y90X, R91X, T93X, W94X, I96X,
  • AKPFW SEQ
  • the ACD further comprises at least one selectivity-enhancing alteration at a position functionally equivalent to F102, S103, W104 and G105, for example, e.g., FVPG, FTDG, FVDG, FVPAG, FVPLG, FVPPG, FVPAAG, FVPLAG, FVPPAG, FVPASG, FVPLSG, FVPPSG, FVPAFG, FVPLFG, FVPPFG, FVPAQG, FVPLQG, FVPPQG, FVPADG, FVPLDG, FVPPDG, FVPAKG, FVPLKG, FVPPKG among others.
  • FVPG FTDG, FVDG, FVPAG, FVPLG, FVPPG, FVPAAG, FVPLAG, FVPPAG, FVPASG, FVPLSG, FVPPSG, FVPAFG, FVPLFG, FVPPFG, FVPAQG, FVPLQG, FVPPQG, FVPADG, F
  • the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 130-134, or functionally equivalent positions, a sequence selected from AKPFW (SEQ ID NO:45), in combination with one or more combinations of stability enhancing mutations selected from: at least one of the following combinations of stabilizing mutations: R74L/C 171 A/T 19 Y/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 88Q/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88Q/G25K/S45W; R74L/C 171 A/T 19 Y/Gl 88 A/S45 W;
  • the ACD further comprises at least one selectivity-enhancing alteration at a position functionally equivalent to Fl 02, S103, W104 and G105, for example, e g., FVPG, FTDG, FVDG, FVPAG, FVPLG, FVPPG, FVPAAG, FVPLAG, FVPPAG, FVPASG, FVPLSG, FVPPSG, FVPAFG, FVPLFG, FVPPFG, FVPAQG, FVPLQG, FVPPQG, FVPADG, FVPLDG, FVPPDG, FVPAKG, FVPLKG, FVPPKG among others.
  • FVPG FTDG, FVDG, FVPAG, FVPLG, FVPPG, FVPAAG, FVPLAG, FVPPAG, FVPASG, FVPLSG, FVPPSG, FVPAFG, FVPLFG, FVPPFG, FVPAQG, FVPLQG, FVPPQG, FVPADG, F
  • the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 130-134, or functionally equivalent positions, a sequence selected from: AKLIW (SEQ ID NO:46), in combination with one or more combinations of stability enhancing mutations selected from Hl IX, L12X, D14X, H16X, I17X, T19X, S20X, N21X, G25X, I26X, G27X, R28X, H29X, T31X, C34X, E38X, R39X, D41X, N42X, S45X, K47X, M48X, H51X, F54X, H56X, N57X, A59X, K60X, L62X, Y67X, L73X, R74X, D77X, L78X, V79X, P80X, S81X, D85X, A87X, I89X, Y90X, R91X, T93X, W94X, I96X
  • the ACD further comprises at least one selectivity-enhancing alteration at a position functionally equivalent to F102, S103, W104 and G105, for example, e.g., FVPG, FTDG, FVDG, FVPAG, FVPLG, FVPPG, FVPAAG, FVPLAG, FVPPAG, FVPASG, FVPLSG, FVPPSG, FVPAFG, FVPLFG, FVPPFG, FVPAQG, FVPLQG, FVPPQG, FVPADG, FVPLDG, FVPPDG, FVPAKG, FVPLKG, FVPPKG among others.
  • FVPG FTDG, FVDG, FVPAG, FVPLG, FVPPG, FVPAAG, FVPLAG, FVPPAG, FVPASG, FVPLSG, FVPPSG, FVPAFG, FVPLFG, FVPPFG, FVPAQG, FVPLQG, FVPPQG, FVPADG, F
  • the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 130-134, or functionally equivalent positions, a sequence selected from: AKLIW (SEQ ID NO:46), in combination with one or more combinations of stability enhancing mutations selected from: at least one of the following combinations of stabilizing mutations: R74L/C 171 A/T 19 Y/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 88Q/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88Q/G25K/S45W; R74L/C 171 A/T 19 Y/Gl 88 A/S45 W;
  • the ACD further comprises at least one selectivity-enhancing alteration at a position functionally equivalent to Fl 02, S103, W104 and G105, for example, e g., FVPG, FTDG, FVDG, FVPAG, FVPLG, FVPPG, FVPAAG, FVPLAG, FVPPAG, FVPASG, FVPLSG, FVPPSG, FVPAFG, FVPLFG, FVPPFG, FVPAQG, FVPLQG, FVPPQG, FVPADG, FVPLDG, FVPPDG, FVPAKG, FVPLKG, FVPPKG among others.
  • FVPG FTDG, FVDG, FVPAG, FVPLG, FVPPG, FVPAAG, FVPLAG, FVPPAG, FVPASG, FVPLSG, FVPPSG, FVPAFG, FVPLFG, FVPPFG, FVPAQG, FVPLQG, FVPPQG, FVPADG, F
  • the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 130-134, or functionally equivalent positions, a sequence selected from AHVVW (SEQ ID NO:47), in combination with one or more combinations of stability enhancing mutations selected from Hl IX, L12X, D14X, H16X, I17X, T19X, S20X, N21X, G25X, I26X, G27X, R28X, H29X, T31X, C34X, E38X, R39X, D41X, N42X, S45X, K47X, M48X, H51X, F54X, H56X, N57X, A59X, K60X, L62X, Y67X, L73X, R74X, D77X, L78X, V79X, P80X, S81X, D85X, A87X, I89X, Y90X, R91X, T93X, W94X, I96X,
  • the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 130-134, or functionally equivalent positions, a sequence selected from: AHVVW (SEQ ID NO:47), in combination with one or more combinations of stability enhancing mutations selected from: at least one of the following combinations of stabilizing mutations: R74L/C 171 A/T 19 Y/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 88Q/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88Q/G25K/S45W; R74L/C 171 A/T 19 Y/Gl 88 A/S45 W;
  • the ACD further comprises at least one selectivity-enhancing alteration at a position functionally equivalent to Fl 02, S103, W104 and G105, for example, e g., FVPG, FTDG, FVDG, FVPAG, FVPLG, FVPPG, FVPAAG, FVPLAG, FVPPAG, FVPASG, FVPLSG, FVPPSG, FVPAFG, FVPLFG, FVPPFG, FVPAQG, FVPLQG, FVPPQG, FVPADG, FVPLDG, FVPPDG, FVPAKG, FVPLKG, FVPPKG among others.
  • FVPG FTDG, FVDG, FVPAG, FVPLG, FVPPG, FVPAAG, FVPLAG, FVPPAG, FVPASG, FVPLSG, FVPPSG, FVPAFG, FVPLFG, FVPPFG, FVPAQG, FVPLQG, FVPPQG, FVPADG, F
  • the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 130-134, or functionally equivalent positions, a sequence selected from: AGRFW (SEQ ID NO:48), in combination with one or more combinations of stability enhancing mutations selected from Hl IX, L12X, D14X, H16X, I17X, T19X, S20X, N21X, G25X, I26X, G27X, R28X, H29X, T31X, C34X, E38X, R39X, D41X, N42X, S45X, K47X, M48X, H51X, F54X, H56X, N57X, A59X, K60X, L62X, Y67X, L73X, R74X, D77X, L78X, V79X, P80X, S81X, D85X, A87X, I89X, Y90X, R91X, T93X, W94X, I96X, S
  • the ACD further comprises at least one selectivity-enhancing alteration at a position functionally equivalent to F102, S103, W104 and G105, for example, e.g., FVPG, FTDG, FVDG, FVPAG, FVPLG, FVPPG, FVPAAG, FVPLAG, FVPPAG, FVPASG, FVPLSG, FVPPSG, FVPAFG, FVPLFG, FVPPFG, FVPAQG, FVPLQG, FVPPQG, FVPADG, FVPLDG, FVPPDG, FVPAKG, FVPLKG, FVPPKG among others.
  • FVPG FTDG, FVDG, FVPAG, FVPLG, FVPPG, FVPAAG, FVPLAG, FVPPAG, FVPASG, FVPLSG, FVPPSG, FVPAFG, FVPLFG, FVPPFG, FVPAQG, FVPLQG, FVPPQG, FVPADG, F
  • the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 130-134, or functionally equivalent positions, a sequence selected from: AGRFW (SEQ ID NO:48), in combination with one or more combinations of stability enhancing mutations selected from: at least one of the following combinations of stabilizing mutations: R74L/C 171 A/T 19 Y/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 88Q/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88Q/G25K/S45W; R74L/C 171 A/T 19 Y/Gl 88 A/S45 W;
  • the ACD further comprises at least one selectivity-enhancing alteration at a position functionally equivalent to Fl 02, S103, W104 and G105, for example, e g., FVPG, FTDG, FVDG, FVPAG, FVPLG, FVPPG, FVPAAG, FVPLAG, FVPPAG, FVPASG, FVPLSG, FVPPSG, FVPAFG, FVPLFG, FVPPFG, FVPAQG, FVPLQG, FVPPQG, FVPADG, FVPLDG, FVPPDG, FVPAKG, FVPLKG, FVPPKG among others.
  • FVPG FTDG, FVDG, FVPAG, FVPLG, FVPPG, FVPAAG, FVPLAG, FVPPAG, FVPASG, FVPLSG, FVPPSG, FVPAFG, FVPLFG, FVPPFG, FVPAQG, FVPLQG, FVPPQG, FVPADG, F
  • the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 130-134, or functionally equivalent positions, a sequence selected from: AIAHW (SEQ ID NO:49), in combination with one or more combinations of stability enhancing mutations selected from Hl IX, L12X, D14X, H16X, I17X, T19X, S20X, N21X, G25X, I26X, G27X, R28X, H29X, T31X, C34X, E38X, R39X, D41X, N42X, S45X, K47X, M48X, H51X, F54X, H56X, N57X, A59X, K60X, L62X, Y67X, L73X, R74X, D77X, L78X, V79X, P80X, S81X, D85X, A87X, I89X, Y90X, R91X, T93X, W94X, I96X, S
  • the ACD further comprises at least one selectivity-enhancing alteration at a position functionally equivalent to F102, S103, W104 and G105, for example, e.g., FVPG, FTDG, FVDG, FVPAG, FVPLG, FVPPG, FVPAAG, FVPLAG, FVPPAG, FVPASG, FVPLSG, FVPPSG, FVPAFG, FVPLFG, FVPPFG, FVPAQG, FVPLQG, FVPPQG, FVPADG, FVPLDG, FVPPDG, FVPAKG, FVPLKG, FVPPKG among others.
  • FVPG FTDG, FVDG, FVPAG, FVPLG, FVPPG, FVPAAG, FVPLAG, FVPPAG, FVPASG, FVPLSG, FVPPSG, FVPAFG, FVPLFG, FVPPFG, FVPAQG, FVPLQG, FVPPQG, FVPADG, F
  • the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 130-134, or functionally equivalent positions, a sequence selected from: AIAHW (SEQ ID NO:49), in combination with one or more combinations of stability enhancing mutations selected from: at least one of the following combinations of stabilizing mutations: R74L/C 171 A/T 19 Y/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 88Q/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88Q/G25K/S45W; R74L/C 171 A/T 19 Y/Gl 88 A/S45 W;
  • the ACD further comprises at least one selectivity-enhancing alteration at a position functionally equivalent to Fl 02, S103, W104 and G105, for example, e g., FVPG, FTDG, FVDG, FVPAG, FVPLG, FVPPG, FVPAAG, FVPLAG, FVPPAG, FVPASG, FVPLSG, FVPPSG, FVPAFG, FVPLFG, FVPPFG, FVPAQG, FVPLQG, FVPPQG, FVPADG, FVPLDG, FVPPDG, FVPAKG, FVPLKG, FVPPKG among others.
  • FVPG FTDG, FVDG, FVPAG, FVPLG, FVPPG, FVPAAG, FVPLAG, FVPPAG, FVPASG, FVPLSG, FVPPSG, FVPAFG, FVPLFG, FVPPFG, FVPAQG, FVPLQG, FVPPQG, FVPADG, F
  • the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 130-134, or functionally equivalent positions, a sequence selected from: AKMYW (SEQ ID NO:50), in combination with one or more combinations of stability enhancing mutations selected from Hl IX, L12X, D14X, H16X, I17X, T19X, S20X, N21X, G25X, I26X, G27X, R28X, H29X, T31X, C34X, E38X, R39X, D41X, N42X, S45X, K47X, M48X, H51X, F54X, H56X, N57X, A59X, K60X, L62X, Y67X, L73X, R74X, D77X, L78X, V79X, P80X, S81X, D85X, A87X, I89X, Y90X, R91X, T93X, W94X, I96X,
  • the ACD further comprises at least one selectivity-enhancing alteration at a position functionally equivalent to F102, S103, W104 and G105, for example, e.g., FVPG, FTDG, FVDG, FVPAG, FVPLG, FVPPG, FVPAAG, FVPLAG, FVPPAG, FVPASG, FVPLSG, FVPPSG, FVPAFG, FVPLFG, FVPPFG, FVPAQG, FVPLQG, FVPPQG, FVPADG, FVPLDG, FVPPDG, FVPAKG, FVPLKG, FVPPKG among others.
  • FVPG FTDG, FVDG, FVPAG, FVPLG, FVPPG, FVPAAG, FVPLAG, FVPPAG, FVPASG, FVPLSG, FVPPSG, FVPAFG, FVPLFG, FVPPFG, FVPAQG, FVPLQG, FVPPQG, FVPADG, F
  • the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 130-134, or functionally equivalent positions, a sequence selected from: AKMYW (SEQ ID NO:50), among others in combination with one or more combinations of stability enhancing mutations selected from: at least one of the following combinations of stabilizing mutations: R74L/C171A/T19Y/G25K/S45W;
  • the ACD further comprises at least one selectivity-enhancing alteration at a position functionally equivalent to Fl 02, S103, W104 and G105, for example, e g., FVPG, FTDG, FVDG, FVPAG, FVPLG, FVPPG, FVPAAG, FVPLAG, FVPPAG, FVPASG, FVPLSG, FVPPSG, FVPAFG, FVPLFG, FVPPFG, FVPAQG, FVPLQG, FVPPQG, FVPADG, FVPLDG, FVPPDG, FVPAKG, FVPLKG, FVPPKG among others.
  • FVPG FTDG, FVDG, FVPAG, FVPLG, FVPPG, FVPAAG, FVPLAG, FVPPAG, FVPASG, FVPLSG, FVPPSG, FVPAFG, FVPLFG, FVPPFG, FVPAQG, FVPLQG, FVPPQG, FVPADG, F
  • the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 102-105, or functionally equivalent positions, the sequence FVPG, and from 130-134, or functionally equivalent positions, the sequence AKYPW, in combination with one or more combinations of stability enhancing mutations selected from: at least one of the following combinations of stabilizing mutations:
  • the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 102-105 the sequence FVPG, and from 130-134, or functionally equivalent positions, the sequence AKYPW (SEQ ID NO:44), in combination with one or more combinations of stability enhancing mutations selected from Hl IX, L12X, D14X, H16X, I17X, T19X, S20X, N21X, G25X, I26X, G27X, R28X, H29X, T31X, C34X, E38X, R39X, D41X, N42X, S45X, K47X, M48X, H51X, F54X, H56X, N57X, A59X, K60X, L62X, Y67X, L73X, R74X, D77X, L78X, V79X, P80X, S81X, D85X, A87X, I89X,
  • the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 102-105 the sequence FTDG, and from 130-134, or functionally equivalent positions, the sequence AKYPW, in combination with one or more combinations of stability enhancing mutations selected from: at least one of the following combinations of stabilizing mutations: R74L/C171A/T19Y/G25K/S45W;
  • the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 102-105 the sequence FTDG, and from 130-134, or functionally equivalent positions, the sequence AKYPW (SEQ ID NO:44), in combination with one or more combinations of stability enhancing mutations selected from Hl IX, L12X, D14X, H16X, I17X, T19X, S20X, N21X, G25X, I26X, G27X, R28X, H29X, T31X, C34X, E38X, R39X, D41X, N42X, S45X, K47X, M48X, H51X, F54X, H56X, N57X, A59X, K60X, L62X, Y67X, L73X, R74X, D77X, L78X, V79X, P80X, S81X, D85X, A87X, I89X, Y90X, R91X, T93
  • the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 102-105 the sequence FVDG, and from 130-134, or functionally equivalent positions, the sequence AKYPW, in combination with one or more combinations of stability enhancing mutations selected from: at least one of the following combinations of stabilizing mutations: R74L/C171A/T19Y/G25K/S45W;
  • the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 102-105 the sequence FVDG, and from 130-134, or functionally equivalent positions, the sequence AKYPW (SEQ ID NO:44), in combination with one or more combinations of stability enhancing mutations selected from Hl IX, L12X, D14X, H16X, I17X, T19X, S20X, N21X, G25X, I26X, G27X, R28X, H29X, T31X, C34X, E38X, R39X, D41X, N42X, S45X, K47X, M48X, H51X, F54X, H56X, N57X, A59X, K60X, L62X, Y67X, L73X, R74X, D77X, L78X, V79X, P80X, S81X, D85X, A87X, I89X, Y90X, R91X, T93X
  • the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 102-105 the sequence FVPAG, and from 130-134, or functionally equivalent positions, the sequence AKYPW, in combination with one or more combinations of stability enhancing mutations selected from: at least one of the following combinations of stabilizing mutations: R74L/C171A/T19Y/G25K/S45W;
  • the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 102-105 the sequence FVPAG, and from 130-134, or functionally equivalent positions, the sequence AKYPW (SEQ ID NO:44), in combination with one or more combinations of stability enhancing mutations selected from Hl IX, L12X, D14X, H16X, I17X, T19X, S20X, N21X, G25X, I26X, G27X, R28X, H29X, T31X, C34X, E38X, R39X, D41X, N42X, S45X, K47X, M48X, H51X, F54X, H56X, N57X, A59X, K60X, L62X, Y67X, L73X, R74X, D77X, L78X, V79X, P80X, S81X, D85X, A87X, I89X, Y90X, R91X, T93X
  • the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 102-105 the sequence FVPLG, and from 130-134, or functionally equivalent positions, the sequence AKYPW, in combination with one or more combinations of stability enhancing mutations selected from: at least one of the following combinations of stabilizing mutations: R74L/C171A/T19Y/G25K/S45W;
  • the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 102-105 the sequence FVPLG, and from 130-134, or functionally equivalent positions, the sequence AKYPW (SEQ ID NO:44), in combination with one or more combinations of stability enhancing mutations selected from Hl IX, L12X, D14X, H16X, I17X, T19X, S20X, N21X, G25X, I26X, G27X, R28X, H29X, T31X, C34X, E38X, R39X, D41X, N42X, S45X, K47X, M48X, H51X, F54X, H56X, N57X, A59X, K60X, L62X, Y67X, L73X, R74X, D77X, L78X, V79X, P80X, S81X, D85X, A87X, I89X, Y90X, R91X, T93X
  • the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 102-105 the sequence FVPPG, and from 130-134, or functionally equivalent positions, the sequence AKYPW, in combination with one or more combinations of stability enhancing mutations selected from: at least one of the following combinations of stabilizing mutations: R74L/C171A/T19Y/G25K/S45W;
  • the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 102-105 the sequence FVPPG, and from 130-134, or functionally equivalent positions, the sequence AKYPW (SEQ ID NO:44), in combination with one or more combinations of stability enhancing mutations selected from Hl IX, L12X, D14X, H16X, I17X, T19X, S20X, N21X, G25X, I26X, G27X, R28X, H29X, T31X, C34X, E38X, R39X, D41X, N42X, S45X, K47X, M48X, H51X, F54X, H56X, N57X, A59X, K60X, L62X, Y67X, L73X, R74X, D77X, L78X, V79X, P80X, S81X, D85X, A87X, I89X, Y90X, R91X, T93X
  • the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 102-105 the sequence FVPAAG, and from 130-134, or functionally equivalent positions, the sequence AKYPW, in combination with one or more combinations of stability enhancing mutations selected from: at least one of the following combinations of stabilizing mutations: R74L/C171A/T19Y/G25K/S45W;
  • the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 102-105 the sequence FVPAAG, and from 130-134, or functionally equivalent positions, the sequence AKYPW (SEQ ID NO:44), in combination with one or more combinations of stability enhancing mutations selected from Hl IX, L12X, D14X, H16X, I17X, T19X, S20X, N21X, G25X, I26X, G27X, R28X, H29X, T31X, C34X, E38X, R39X, D41X, N42X, S45X, K47X, M48X, H51X, F54X, H56X, N57X, A59X, K60X, L62X, Y67X, L73X, R74X, D77X, L78X, V79X, P80X, S81X, D85X, A87X, I89X, Y90X, R91X, T93
  • the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 102-105 the sequence FVPLAG, and from 130-134, or functionally equivalent positions, the sequence AKYPW, in combination with one or more combinations of stability enhancing mutations selected from: at least one of the following combinations of stabilizing mutations: R74L/C171A/T19Y/G25K/S45W;
  • the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 102-105 the sequence FVPLAG, and from 130-134, or functionally equivalent positions, the sequence AKYPW (SEQ ID NO:44), in combination with one or more combinations of stability enhancing mutations selected from Hl IX, L12X, D14X, H16X, I17X, T19X, S20X, N21X, G25X, I26X, G27X, R28X, H29X, T31X, C34X, E38X, R39X, D41X, N42X, S45X, K47X, M48X, H51X, F54X, H56X, N57X, A59X, K60X, L62X, Y67X, L73X, R74X, D77X, L78X, V79X, P80X, S81X, D85X, A87X, I89X, Y90X, R91X, T
  • the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 102-105 the sequence FVPPAG, and from 130-134, or functionally equivalent positions, the sequence AKYPW, in combination with one or more combinations of stability enhancing mutations selected from: at least one of the following combinations of stabilizing mutations: R74L/C171A/T19Y/G25K/S45W;
  • the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 102-105 the sequence FVPPAG, and from 130-134, or functionally equivalent positions, the sequence AKYPW (SEQ ID NO:44), in combination with one or more combinations of stability enhancing mutations selected from Hl IX, L12X, D14X, H16X, I17X, T19X, S20X, N21X, G25X, I26X, G27X, R28X, H29X, T31X, C34X, E38X, R39X, D41X, N42X, S45X, K47X, M48X, H51X, F54X, H56X, N57X, A59X, K60X, L62X, Y67X, L73X, R74X, D77X, L78X, V79X, P80X, S81X, D85X, A87X, I89X, Y90X, R91X, T93
  • the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 102-105 the sequence FVPASG, and from 130-134, or functionally equivalent positions, the sequence AKYPW, in combination with one or more combinations of stability enhancing mutations selected from: at least one of the following combinations of stabilizing mutations: R74L/C171A/T19Y/G25K/S45W;
  • the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 102-105 130-134, or functionally equivalent positions, the sequence FIMA AHIYW, in combination with one or more combinations of stability enhancing mutations selected from: at least one of the following combinations of stabilizing mutations: R74L/C 171 A/T 19 Y/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 88Q/G25R/S45 W;
  • the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 102-105 130-134, or functionally equivalent positions, the sequence FIMA AHIYW, in combination with one or more combinations of stability enhancing mutations selected from Hl IX, L12X, D14X, H16X, I17X, T19X, S20X, N21X, G25X, I26X, G27X, R28X, H29X, T31X, C34X, E38X, R39X, D41X, N42X, S45X, K47X, M48X, H51X, F54X, H56X, N57X, A59X, K60X, L62X, Y67X, L73X, R74X, D77X, L78X, V79X, P80X, S81X, D85X, A87X, I89X, Y90X, R91X, T93X, W94X, I96X, S97X, C
  • the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 102- 105 130-134, or functionally equivalent positions, the sequence HLTG AHEYW , in combination with one or more combinations of stability enhancing mutations selected from: at least one of the following combinations of stabilizing mutations:
  • the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 102-105 130-134, or functionally equivalent positions, the sequence HLTG AHEYW, in combination with one or more combinations of stability enhancing mutations selected from Hl IX, L12X, D14X, H16X, I17X, T19X, S20X, N21X, G25X, I26X, G27X, R28X, H29X, T31X, C34X, E38X, R39X, D41X, N42X, S45X, K47X, M48X, H51X, F54X, H56X, N57X, A59X, K60X, L62X, Y67X, L73X, R74X, D77X, L78X, V79X, P80X, S81X, D85X, A87X, I89X, Y90X, R91X, T93X, W94X, I96X, S97X,
  • the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 102-105 130-134, or functionally equivalent positions, the sequence QVPA AHDFW , in combination with one or more combinations of stability enhancing mutations selected from: at least one of the following combinations of stabilizing mutations: R74L/C 171 A/T 19 Y/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 88Q/G25R/S45 W;
  • the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 102-105 130-134, or functionally equivalent positions, the sequence QVPA AHDFW, in combination with one or more combinations of stability enhancing mutations selected from Hl IX, L12X, D14X, H16X, I17X, T19X, S20X, N21X, G25X, I26X, G27X, R28X, H29X, T31X, C34X, E38X, R39X, D41X, N42X, S45X, K47X, M48X, H51X, F54X, H56X, N57X, A59X, K60X, L62X, Y67X, L73X, R74X, D77X, L78X, V79X, P80X, S81X, D85X, A87X, I89X, Y90X, R91X, T93X, W94X, I96X, S97X, C
  • the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 102-105 130-134, or functionally equivalent positions, the sequence EFNQ ANVHW, in combination with one or more combinations of stability enhancing mutations selected from: at least one of the following combinations of stabilizing mutations: R74L/C 171 A/T 19 Y/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 88Q/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88Q/G25K/S45W; R74L/C 171 A/T 19 Y/Gl 88 A/S45 W;
  • the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 102-105 130-134, or functionally equivalent positions, the sequence EFNQ ANVHW, in combination with one or more combinations of stability enhancing mutations selected from Hl IX, L12X, D14X, H16X, I17X, T19X, S20X, N21X, G25X, I26X, G27X, R28X, H29X, T31X, C34X, E38X, R39X, D41X, N42X, S45X, K47X, M48X, H51X, F54X, H56X, N57X, A59X, K60X, L62X, Y67X, L73X, R74X, D77X, L78X, V79X, P80X, S81X, D85X, A87X, I89X, Y90X, R91X, T93X, W94X, I96X, S97X,
  • the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 102-105 130-134, or functionally equivalent positions, the sequence QTDH AAEHW, in combination with one or more combinations of stability enhancing mutations selected from: at least one of the following combinations of stabilizing mutations: R74L/C 171 A/T 19 Y/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 88Q/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88Q/G25K/S45W; R74L/C 171 A/T 19 Y/Gl 88 A/S45 W;
  • the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 102-105 130-134, or functionally equivalent positions, the sequence QTDH AAEHW, in combination with one or more combinations of stability enhancing mutations selected from Hl IX, L12X, D14X, H16X, I17X, T19X, S20X, N21X, G25X, I26X, G27X, R28X, H29X, T31X, C34X, E38X, R39X, D41X, N42X, S45X, K47X, M48X, H51X, F54X, H56X, N57X, A59X, K60X, L62X, Y67X, L73X, R74X, D77X, L78X, V79X, P80X, S81X, D85X, A87X, I89X, Y90X, R91X, T93X, W94X, I96X, S97X,
  • the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 102-105 130-134, or functionally equivalent positions, the sequence GLNG AHEYW, in combination with one or more combinations of stability enhancing mutations selected from: at least one of the following combinations of stabilizing mutations: R74L/C 171 A/T 19 Y/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 88Q/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88Q/G25K/S45W; R74L/C 171 A/T 19 Y/Gl 88 A/S45 W; R74L/C 171 A/T 19 Y/Gl 88 A/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88 A/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88 A/G25R
  • the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 102-105 130-134, or functionally equivalent positions, the sequence GLNG AHEYW, in combination with one or more combinations of stability enhancing mutations selected from Hl IX, L12X, D14X, H16X, I17X, T19X, S20X, N21X, G25X, I26X, G27X, R28X, H29X, T31X, C34X, E38X, R39X, D41X, N42X, S45X, K47X, M48X, H51X, F54X, H56X, N57X, A59X, K60X, L62X, Y67X, L73X, R74X, D77X, L78X, V79X, P80X, S81X, D85X, A87X, I89X, Y90X, R91X, T93X, W94X, I96X, S97X,
  • the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 102-105 130-134, or functionally equivalent positions, the sequence YLPA ANGFW, in combination with one or more combinations of stability enhancing mutations selected from: at least one of the following combinations of stabilizing mutations: R74L/C 171 A/T 19 Y/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 88Q/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88Q/G25K/S45W; R74L/C 171 A/T 19 Y/Gl 88 A/S45 W;
  • the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 102-105 130-134, or functionally equivalent positions, the sequence YLPA ANGFW, in combination with one or more combinations of stability enhancing mutations selected from Hl IX, L12X, D14X, H16X, I17X, T19X, S20X, N21X, G25X, I26X, G27X, R28X, H29X, T31X, C34X, E38X, R39X, D41X, N42X, S45X, K47X, M48X, H51X, F54X, H56X, N57X, A59X, K60X, L62X, Y67X, L73X, R74X, D77X, L78X, V79X, P80X, S81X, D85X, A87X, I89X, Y90X, R91X, T93X, W94X, I96X, S97X,
  • the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 102-105 130-134, or functionally equivalent positions, the sequence EMFA ANDFW , in combination with one or more combinations of stability enhancing mutations selected from: at least one of the following combinations of stabilizing mutations: R74L/C 171 A/T 19 Y/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 88Q/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88Q/G25K/S45W; R74L/C 171 A/T 19 Y/Gl 88 A/S45 W;
  • the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 102-105 130-134, or functionally equivalent positions, the sequence EMFA ANDFW, in combination with one or more combinations of stability enhancing mutations selected from Hl IX, L12X, D14X, H16X, I17X, T19X, S20X, N21X, G25X, I26X, G27X, R28X, H29X, T31X, C34X, E38X, R39X, D41X, N42X, S45X, K47X, M48X, H51X, F54X, H56X, N57X, A59X, K60X, L62X, Y67X, L73X, R74X, D77X, L78X, V79X, P80X, S81X, D85X, A87X, I89X, Y90X, R91X,
  • the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 102-105 130-134, or functionally equivalent positions, the sequence YKQY AGEYW, in combination with one or more combinations of stability enhancing mutations selected from: at least one of the following combinations of stabilizing mutations: R74L/C 171 A/T 19 Y/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 88Q/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88Q/G25K/S45W; R74L/C 171 A/T 19 Y/Gl 88 A/S45 W;
  • the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 102-105 130-134, or functionally equivalent positions, the sequence YKQY AGEYW, in combination with one or more combinations of stability enhancing mutations selected from Hl IX, L12X, D14X, H16X, I17X, T19X, S20X, N21X, G25X, I26X, G27X, R28X, H29X, T31X, C34X, E38X, R39X, D41X, N42X, S45X, K47X, M48X, H51X, F54X, H56X, N57X, A59X, K60X, L62X, Y67X, L73X, R74X, D77X, L78X, V79X, P80X, S81X, D85X, A87X, I89X, Y90X, R91X, T93X, W94X, I96X, S97
  • the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 102-105 130-134, or functionally equivalent positions, the sequence YVRL AVPFW, in combination with one or more combinations of stability enhancing mutations selected from: at least one of the following combinations of stabilizing mutations: R74L/C 171 A/T 19 Y/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 88Q/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88Q/G25K/S45W; R74L/C 171 A/T 19 Y/Gl 88 A/S45 W;
  • the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 102-105 130-134, or functionally equivalent positions, the sequence YVRL AVPFW, in combination with one or more combinations of stability enhancing mutations selected from Hl IX, L12X, D14X, H16X, I17X, T19X, S20X, N21X, G25X, I26X, G27X, R28X, H29X, T31X, C34X, E38X, R39X, D41X, N42X, S45X, K47X, M48X, H51X, F54X, H56X, N57X, A59X, K60X, L62X, Y67X, L73X, R74X, D77X, L78X, V79X, P80X, S81X, D85X, A87X, I89X, Y90X, R91X, T93X, W94X, I96X, S97X,
  • the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 102-105 130-134, or functionally equivalent positions, the sequence YIKL AVAHW, in combination with one or more combinations of stability enhancing mutations selected from: at least one of the following combinations of stabilizing mutations: R74L/C 171 A/T 19 Y/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 88Q/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88Q/G25K/S45W; R74L/C 171 A/T 19 Y/Gl 88 A/S45 W;
  • the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 102-105 130-134, or functionally equivalent positions, the sequence YIKL AVAHW, in combination with one or more combinations of stability enhancing mutations selected from Hl IX, L12X, D14X, H16X, I17X, T19X, S20X, N21X, G25X, I26X, G27X, R28X, H29X, T31X, C34X, E38X, R39X, D41X, N42X, S45X, K47X, M48X, H51X, F54X, H56X, N57X, A59X, K60X, L62X, Y67X, L73X, R74X, D77X, L78X, V79X, P80X, S81X, D85X, A87X, I89X, Y90X, R91X, T93X, W94X, I96X, S
  • the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 102-105 130-134, or functionally equivalent positions, the sequence FILA ALAHW, in combination with one or more combinations of stability enhancing mutations selected from: at least one of the following combinations of stabilizing mutations: R74L/C 171 A/T 19 Y/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 88Q/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88Q/G25K/S45W; R74L/C 171 A/T 19 Y/Gl 88 A/S45 W;

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Abstract

The present disclosure is concerned with altered cytidine deaminases, methods, compositions, and kits for mapping of methylation status of nucleic acids. In one embodiment, altered cytidine deaminases are provided that selectively act on certain modified cytosines of target nucleic acids and converts them to thymidine. Also provided are compositions and kits that include one or more of the proteins and methods for using one or more of the proteins.

Description

ALTERED CYTIDINE DEAMINASES AND METHODS OF USE
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of U.S. Provisional Application Serial No. 63/665,656, filed June 28, 2024, which is incorporated by reference herein in its entirety.
SEQUENCE LISTING
[0002] This application contains a Sequence Listing electronically submitted to the United States Patent and Trademark Office as an XML file entitled "IP-2811-PCT.xml" having a size of 960,863 bytes and created on June 27, 2025. The information contained in the Sequence Listing is incorporated by reference herein.
FIELD
[0003] Embodiments of the present disclosure relate to identifying the methylation status of nucleotides. In particular, embodiments of the proteins, methods, compositions, and kits provided herein relate to mapping of methylation status of nucleotides in a genome. BACKGROUND
[0004] Modified DNA cytosines, including 5-methylcytosine (5mC) and 5 -hydroxymethyl cytosine (5hmC), are a well-studied epigenetic modification that play fundamental roles in human development and disease. Its genome-wide distribution differs between tissue types, and between healthy and diseased states. In recent years, 5mC has also gained prominence as a tool for clinical diagnostics: its distribution in cell-free DNA (cfDNA) - obtained from a liquid biopsy - can be used for the tissue-specific prediction of early-stage cancer or monitoring of cancer recurrence or remission after treatment. As a result, there has been an intense focus on developing methods for mapping modified DNA cytosines at single base resolution, with minimal loss of sample DNA quantity, quality, and complexity. Current methods for mapping modified DNA cytosines, however, exhibit limitations including (i) degradation of sample DNA due to prolonged chemical treatment at non-neutral pH and high temperatures, (ii) loss of sample DNA complexity due to conversion of unmethylated DNA bases to uracil, resulting in low complexity genome mapping, (iii) multi-step conversion, requiring both enzymes and chemical treatment, and (iv) for antibody-based 5mC detection, resolution of detection is limited to ~150bp, precluding the identification of its exact location in the genome. SUMMARY OF THE APPLICATION
[0005] The present disclosure provides altered cytidine deaminases (ACDs) that include 5mC- selective deaminase activity. An ACD includes at least one, in some embodiments at least two, selectivity-enhancing alteration at a position functionally equivalent to amino acid 130, 131, 132, 133, 134, 135, or a combination thereof, in a wild-type APOBEC3A (e.g., SEQ ID NO:3). The selectivity-enhancing alteration at amino acid 130, 131, 132, 133, 134, 135, or a combination thereof can be a substitution mutation to any amino acid, and in some embodiments the ACD includes two or more selectivity-enhancing alterations at a position functionally equivalent to amino acid 130, 131, 132, 133, 134, 135, or a combination thereof. The amino acid sequence of amino acids 130, 131, 132, 133, and 134 can be selected from SEQ ID NO:25-34 of FIG. 4B, SEQ ID NO:35-50 of Table 2, or the amino acids at positions 130, 131, 132, 133, and 134 in the sequences found in Figure 8 or 9.
[0006] In some embodiments, an ACD can further include at least one selectivity-enhancing alteration at a position functionally equivalent to amino acid 102, 103, 104, 105, or a combination thereof in a wild-type APOBEC3A (e.g., SEQ ID NO:3). In some embodiments, the amino acid sequence of amino acids 102, 103, 104, 105, 130, 131, 132, 133, 134, and 135 in an ACD is selected from SEQ ID NO: 100-478 of FIG. 8. In some embodiments, the amino acid sequence of amino acids 130, 131, 132, 133, and 134 is selected from SEQ ID NO: 23, SEQ ID NO:24, or the sequences described in FIG. 8 or FIG. 9 which demonstrate increased 5mC selectivity compared to wild-type (SEQ ID NO:3).
[0007] In some embodiments, an ACD can further include one or more stability-enhancing alterations, two or more stability-enhancing alteration, or three or more stability-enhancing alterations. Examples of stability-enhancing alterations include, but are not limited to, substitution mutations at a position functionally equivalent to Hl IX, L12X, D14X, H16X, H7X, T19X, S20X, N21X, G25X, I26X, G27X, R28X, H29X, T31X, C34X, E38X, R39X, D41X, N42X, S45X, K47X, M48X, H51X, F54X, H56X, N57X, A59X, K60X, L62X, Y67X, L73X, R74X, D77X, L78X, V79X, P80X, S81X, D85X, A87X, I89X, Y90X, R91X, T93X, W94X, I96X, S97X, C101X, F102X, S103X, W104X, G105X, C106X, A107X, G108X, E109X, VI 10X, R11 IX, Al 12X, LI 14X, QI 15X, El 16X, N117X, T118X, Hl 19X, V120X, L122X, R123X, R128X, L135X, Y136X, E138X, A139X, Q141X, M142X, D145X, A146X, A148X, D156X, E157X, K159X, C161X, D163X, T164X, D167X, Q169X, C171X, D177X, D180X, H182X, S183X, Q184X, A185X, L186X, S187X, G188X, R189X, A192X, or N196X a deletion selected from AM1-L12, AE36-G53, AN61-G68, AW104-G105 , AQ195-N199 , or AI26-G27, or combinations thereof, wherein the position number designation is functionally equivalent to the position in a wild-type AP0BEC3A (e.g., SEQ ID NO:3) and X is an amino acid substitution different from the wild-type amino acid at that position. For instance, an ACD can include two or more stability mutations selected from I17T, T19Y, G25K, S45W, A59P, K60R, A61-68, R74L, G108A, G108C, C171A, G188P, and A104-105; a combination of stability mutations including T19Y, G25K, S45W, R74L, G108A, Cl 71 A, and G188R; or a combination of stability mutations including I17T, T19Y, G25K, S45W, A59P, K60R, A61-68, R74L, G108C, C171A, and G188R. Specific examples of ACDs provided herein include SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:479-517, 520-605, 607-615, 623, 635-907, and 915-921. BRIEF DESCRIPTION OF THE FIGURES
[0008] The following detailed description of illustrative embodiments of the present disclosure may be best understood when read in conjunction with the following drawings.
[0009] FIG. ID -IE shows the result of treating a DNA sample with a wild-type APOBEC3 A enzyme (FIG. ID), and an example of one-step detection of 5mC using an altered cytidine deaminase described herein (FIG. IE). The top strand of FIG. 1D-E shows C and 5mC bases, and the bottom strand of FIG. 1D-E underlines the changed bases. 5mC nucleobases are marked with CHB, 5 -hydroxymethyl uracil nucleobases are designated with small case "u," and uracil nucleobases are designated with capital "U."
[00010] FIG. 2 is a schematic showing alignment of cytidine deaminase amino acid sequences using the Clustal O algorithm. An (asterisk) indicates positions which have a single, fully conserved residue between some cytidine deaminases. A (colon) indicates conservation between groups of strongly similar properties as below - roughly equivalent to scoring > 0.5 in the Gonnet PAM 250 matrix. A (period) indicates conservation between groups of weakly similar properties as below - roughly equivalent to scoring =< 0.5 and > 0 in the Gonnet PAM 250 matrix. The amino acids marked with "A" show the ZDD motif SEQ ID NO:1 (e.g., above amino acids 70 to 106 of sp|P3194111-199). The amino acids marked with "A" and "#" show the ZDD motif SEQ ID NO:2 (e.g., above amino acids 70 to 153 of sp|P3194111-199). sp|P3194111- 199 is a human APOBEC3A, SEQ ID NO:3; XP_045219544.1 is an APOBEC3A from Macaca fascicularis, SEQ ID NO:4; AER45717.1 is an APOBEC3A from Pongo pygmaeus, SEQ ID NO:5; XP_003264816.1 is an AP0BEC3A from Nomascus leucogenys, SEQ ID NO:6; PNI48846.1 is an APOBEC3A from Pan troglodytes, SEQ ID NO:7; and ADO85886.1 is an APOBEC3 A from Gorilla gorilla, SEQ ID NO:8.
[00011] FIG. 3 shows a bar graph representation of APOBEC3A(Y130X) deaminase activity [00012] FIG. 4A-4B shows information related to the results of a high-throughput screen to test for mC specific mutants. (FIG. 4A) The library diversity on positions Y130 to Pl 34 before screening and after 2 rounds of enrichment tested by NGS. (FIG. 4B) The top 10 enriched mutants in high throughput assay from NGS analysis.
[00013] FIG. 5A-5B shows selectivity plot for (FIG. 5A) randomly picked mutants after 2 round of sorting, and (FIG. 5B) top enriched mutants based on NGS analysis of the library. [00014] FIG. 6A-6C shows an exemplary selectivity plot for various mutations on positions S 103/ W104 in a ScF (SEQ ID NO:18) background (FIG. 6A); various mutations at positions S103/W104 combined with certain AxxxW mutants (FIG. 6B); and various mutations at positions F102/S103/W104/G105 combined with certain AxxxW mutations (FIG. 6C) (see, e.g., SEQ ID NO:955-968).
[00015] FIG. 7A-7C shows the activity of different exemplary ACD mutants on methylation reporting on pUC19 (CG methylated) and Lambda (fully unmethylated). The parent backbone SEQ ID NO: 14 is shown in gray.
[00016] FIG. 8A-8M shows examples of sets of selectivity-enhancing alterations, e.g., substitution mutations, deletions, and/or insertions, of amino acids at positions functionally equivalent to amino acids 102, 103, 104, 105, 130, 131, 132, 133, 134, and/or 135 in SEQ ID NO: 14, SEQ ID NO: 17, SEQ ID NO: 18, or SEQ ID NO: 19.
[00017] FIG. 9A-9MM shows amino acid sequences of proteins described herein.
[00018] FIG. 10A-10C demonstrates expression and purification of ACD proteins in fusion with helicases. First step of purification with Ni-NTA column (a) was followed by size exclusion column (b). The purity of the proteins was confirmed by SDS gel (c).
[00019] FIG. 11 Testing ACD deamination activity in fusion constructs using Swal assay, (a) Schematic of restriction enzyme (Swal) -based assay for deamination. In this assay, the oligonucleotide is incubated with a deaminase enzyme (such as ACD). If the enzyme is active on the substrate, it will deaminate methylcytosine (mC) to thymine (T), creating a mismatch with the complementary reverse oligonucleotide. If a mismatch occurs, the restriction enzyme Swal will cleave the double-stranded DNA oligonucleotide. The cleaved products can be then visualized by UREA-PAGE gel electrophoresis, (b) Swal assay results for ACD fusions with helicases.
[00020] FIG. 12A-12B shows a schematic representation of NGS-based assay to assess ACD activity in helicase fusions. FIG. 12A, Substrate ssDNA oligo contains 17 unmethylated cytosines (C) and 16 methylated C sites (mC). FIG. 12B, Deamination by ACD results in conversion of unmethylated C residues to uracil, and conversion of mC to thymine. These deamination events are read out as C~>T mutations via Illumina sequencing.
[00021] FIG. 13A-13B demonstrates the testing helicase activity in fusion constructs using fluorescent oligos. (FIG. 13A) Schematic of the fluorescent assay for helicase activity on dsDNA. The assay uses a 5'-F AM-labeled oligonucleotide, with the reverse complementary strand containing a quencher to suppress fluorescence. In the presence of helicase activity, the helicase unwinds the double-stranded DNA, replacing the quencher-containing strand with a competitor oligo that lacks the quencher. This replacement removes the quenching effect and results in an increase in FAM fluorescence, which can be detected. (FIG. 13B) Helicase activity results for helicases in fusion with ACD.
[00022] FIG. 14A-14B demonstrates that the ACD in fusion with helicases can deaminate dsDNA. (a) ACD protein deamination with either ds or ssDNA. (b) Deamination of double stranded DNA by ACD-helicases fusion proteins in different buffer conditions.
[00023] FIG. 15 demonstrates that ACD in fusion with helicases can deaminate dsDNA in a complex substrate. ACD protein or ACD-helicase fusions were added to mix of lambda DNA (non- methylated control) with fully methylated pUC19 DNA. DNA was either denatured with heating to 70°C in the presence of DMSO or not denatured at all. Only fusion constructs show activity if the denaturation step is skipped.
[00024] FIG. 16A-16C shows testing direct deamination of 5mC on-flow cell at NextSeq 2000 and NovaSeq X. Comparison of sequencing statistic (A) of 5mC conversion of ACD and Helicase-ACD. (B) Deamination with ACD using denaturation with NaOH without a wash on NextSeq 2000 (B) and NovaSeq X (C).
[00025] FIG. 17A-B shows testing direct deamination of 5mC on-flow cell with the Helicase- ACD fusion using (FIG. 17A) NextSeq 2000 and (FIG. 17B) Novaseq X. [00026] The schematic drawings are not necessarily to scale. Like numbers used in the figures refer to like components, steps and the like. However, it will be understood that the use of a number to refer to a component in a given figure is not intended to limit the component in another figure labeled with the same number. In addition, the use of different numbers to refer to components is not intended to indicate that the different numbered components cannot be the same or similar to other numbered components.
DETAILED DESCRIPTION
[00027] Terms used herein will be understood to take on their ordinary meaning in the relevant art unless specified otherwise. Several terms used herein and their meanings are set forth below. [00028] As used herein, the term "protein" refers broadly to a polymer of two or more amino acids joined together by peptide bonds. The term "protein" also includes molecules which contain more than one protein joined by disulfide bonds, ionic bonds, or hydrophobic interactions, or complexes of proteins that are joined together, covalently or noncovalently, as multimers (e.g., dimers, tetramers). Thus, the terms peptide, oligopeptide, and polypeptide are all included within the definition of protein and these terms are used interchangeably. It should be understood that these terms do not connote a specific length of a polymer of amino acids, nor are they intended to imply or distinguish whether the protein is produced using recombinant techniques, chemical or enzymatic synthesis, or is naturally occurring.
[00029] An “isolated” protein is one that has been removed from a cell. For instance, an isolated protein is a protein that has been removed from the cytoplasm of a cell, and many of the proteins, nucleic acids, and other cellular material of its natural environment are no longer present. Proteins that are produced outside an organism, e.g., through chemical or recombinant means, are considered to be isolated and purified, since they were never present in a natural environment.
[00030] As used herein, the terms "organism," "subject," are used interchangeably and refer to microbes (e.g., prokaryotic or eukaryotic) animals and plants. An example of an animal is a mammal, such as a human.
[00031] As used herein, the term "target nucleic acid," is intended as a semantic identifier for the nucleic acid in the context of a method or composition or kit set forth herein and does not necessarily limit the structure or function of the nucleic acid beyond what is otherwise explicitly indicated. Reference to a nucleic acid such as a target nucleic acid includes both single-stranded and double-stranded nucleic acids, and both DNA and RNA, unless indicated otherwise. The term library refers to the collection of target nucleic acids containing known common sequences, such as a universal sequence or adapter, at their 3' and 5' ends.
[00032] As used herein, the term "adapter" and its derivatives, e.g., universal adapter, refers generally to any linear oligonucleotide. In one embodiment, a target nucleic acid includes DNA. In one embodiment, a target nucleic acid includes single-stranded DNA. In one embodiment, a target nucleic acid includes double-stranded DNA. In one embodiment, a target nucleic acid includes RNA. In one embodiment, a target nucleic acid includes single-stranded RNA. In one embodiment, a target nucleic acid includes double- stranded RNA. which can be attached to a target nucleic acid. An adapter can be single- stranded or double- stranded DNA, or can include both double-stranded and single-stranded regions. An adapter can include a universal sequence that is substantially identical, or substantially complementary, to at least a portion of a primer, for example a universal primer; an index (also referred to herein as a barcode or tag) to assist with downstream error correction, identification, or sequencing; and/or a unique molecular identifier. In some embodiments, the adapter is substantially non-complementary to the 3' end or the 5' end of any target sequence present in the sample. In some embodiments, suitable adapter lengths are in the range of about 6-100 nucleotides, about 12-60 nucleotides, or about 15-50 nucleotides in length. For instance, the terms "adaptor" and "adapter" are used interchangeably. [00033] As used herein, the term "universal," when used to describe a nucleotide sequence, refers to a region of sequence that is common to two or more nucleic acid molecules where the molecules also have regions of sequence that differ from each other. A universal sequence that is present in different members of a collection of nucleic acids can be used as, for instance, a "landing pad" in a subsequent step to anneal a nucleotide sequence that can be used as a primer for addition of another nucleotide sequence, such as an index, to a target nucleic acid. A universal sequence that is present in different members of a collection of nucleic acids can allow capture of multiple different nucleic acids using a population of universal capture nucleic acids, e.g., capture oligonucleotides that are complementary to a portion of the universal sequence, e.g., a universal capture sequence. Non-limiting examples of universal capture sequences include sequences that are identical to or complementary to P5 and P7 primers. Similarly, a universal sequence present in different members of a collection of molecules can allow the replication (e.g., sequencing) or amplification of multiple different nucleic acids using a population of universal primers that are complementary to a portion of the universal sequence, e g., a universal anchor sequence. In one embodiment universal anchor sequences are used as a site to which a universal primer (e.g., a sequencing primer for read 1 or read 2) anneals for sequencing. A capture oligonucleotide or a universal primer therefore includes a sequence that can hybridize specifically to a universal sequence.
[00034] The terms "P5" and "P7" may be used when referring to a universal capture sequence or a capture oligonucleotide. The terms "P51 " (P5 prime) and "P71 " (P7 prime) refer to the complement of P5 and P7, respectively. It will be understood that any suitable universal capture sequence or a capture oligonucleotide can be used in the methods presented herein, and that the use of P5 and P7 are exemplary embodiments only. Uses of capture oligonucleotides such as P5 and P7 or their complements onflow cells are known in the art, as exemplified by the disclosures of WO 2007/010251, WO 2006/064199, WO 2005/065814, WO 2015/106941, WO 1998/044151, and WO 2000/018957, which are incorporated by reference as to P5 and P7 and their uses. For example, any suitable forward amplification primer, whether immobilized or in solution, can be useful in the methods presented herein for hybridization to a complementary sequence and amplification of a sequence. Similarly, any suitable reverse amplification primer, whether immobilized or in solution, can be useful in the methods presented herein for hybridization to a complementary sequence and amplification of a sequence. One of skill in the art will understand how to design and use primer sequences that are suitable for capture and/or amplification of nucleic acids as presented herein.
[00035] As used herein, the term "primer" and its derivatives refer generally to any nucleic acid that can hybridize to a target sequence of interest. Typically, the primer functions as a substrate onto which nucleotides can be polymerized by a polymerase or to which a polynucleotide can be ligated; in some embodiments, however, the primer can become incorporated into the synthesized nucleic acid strand and provide a site to which another primer can hybridize to prime synthesis of a new strand that is complementary to the synthesized nucleic acid molecule. In some embodiments, the primer can be used for hybridization to a predetermined sequence, for instance a predetermined sequence that includes one or more nucleotides that identify the location of a modified cytosine. In one embodiment, a “primer” includes a sequence present in a guide RNA used with a CRISPR-based system to hybridize to a predetermined sequence. The primer can include any combination of nucleotides or analogs thereof. In some embodiments, the primer is a single-stranded oligonucleotide or polynucleotide.
[00036] The terms "polynucleotide" and "oligonucleotide" and “nucleic acid” are used interchangeably herein to refer to a polymeric form of nucleotides of any length, and may include ribonucleotides, deoxyribonucleotides, analogs thereof, or mixtures thereof. The terms should be understood to include, as equivalents, analogs of either DNA, RNA, cDNA, or antibody-oligo conjugates made from nucleotide analogs and to be applicable to single stranded (such as sense or antisense) and double stranded polynucleotides. The term as used herein also encompasses cDNA, that is complementary or copy DNA produced from a RNA template, for example by the action of reverse transcriptase.
[00037] As used herein, an "index" (also referred to as an "index region," "index adaptor," "tag," or a "barcode") refers to a unique nucleic acid tag that can be used to identify a sample or source of the nucleic acid material, or a compartment in which a target nucleic acid was present. The index can be present in solution or on a solid-support, or attached to or associated with a solidsupport and released in solution or compartment. When nucleic acid samples are derived from multiple sources, the nucleic acids in each nucleic acid sample can be tagged with different nucleic acid tags such that the source of the sample can be identified. Any suitable index or set of indexes can be used, as known in the art and as exemplified by the disclosures of U.S. Pat. No. 8,053,192, PCT Publication No. WO 05/068656, and U.S. Pat. Publication No. 2013/0274117. In some embodiments, an index can include a six-base Index 1 (i7) sequence, an eight-base Index 1 (i7) sequence, an eight-base Index 2 (i5e) sequence, a ten-base Index 1 (i7) sequence, or a ten- base Index 2 (i5) sequence from Illumina, Inc. (San Diego, CA).
[00038] As used herein, the term "amplicon," when used in reference to a nucleic acid, means the product of copying the nucleic acid, wherein the product has a nucleotide sequence that is the same as or complementary to at least a portion of the nucleotide sequence of the nucleic acid. An amplicon can be produced by any of a variety of amplification methods that use the nucleic acid, or an amplicon thereof, as a template including, for example, polymerase extension, polymerase chain reaction (PCR), rolling circle amplification (RCA), ligation extension, or ligation chain reaction. An amplicon can be a nucleic acid molecule having a single copy of a particular nucleotide sequence (e.g., a PCR product) or multiple copies of the nucleotide sequence (e.g., a conatemeric product of RCA). A first amplicon of a target nucleic acid is typically a complementary copy. Subsequent amplicons are copies that are created, after generation of the first amplicon, from the target nucleic acid or from the first amplicon. A subsequent amplicon can have a sequence that is substantially complementary to the target nucleic acid or substantially identical to the target nucleic acid.
[00039] As used herein the term “primer” refers to a single stranded nucleic acid molecule that can hybridize to a target sequence, such as an adapter attached to a fragment. As one example, a flow cell surface bound primer can serve as a starting point for fragment amplification and cluster generation. As another example, a flow cell surface bound primer can serve as a hybridization point for a spatial tag, and thus for targeting attachment of particular transposome complexes and DNA samples. As still another example, a primer (e.g., a sequencing primer) may be introduced that can hybridize to DNA fragments in order to prime synthesis of a new strand that is complementary to the fragments. Any primer can include any combination of nucleotides or analogs thereof. In some examples, the primer is a single-stranded oligonucleotide or polynucleotide. The primer length can be any number of bases long. In an example, each of the flow cell surface bound primer and the sequencing primer is a short strand, ranging from 10 to 60 bases, or from 20 to 40 bases.
[00040] As used herein, "amplify," "amplifying," or "amplification reaction" and their derivatives, refer generally to any action or process whereby at least a portion of a nucleic acid molecule is replicated or copied into at least one additional nucleic acid molecule. The additional nucleic acid molecule optionally includes sequence that is substantially identical or substantially complementary to at least some portion of the template nucleic acid molecule. The template nucleic acid molecule can be single-stranded or double-stranded and the additional nucleic acid molecule can independently be single-stranded or double-stranded. Amplification is typically the exponential replication of a nucleic acid molecule. In some embodiments, such amplification can be performed using isothermal conditions; in other embodiments, such amplification can include thermocycling. In some embodiments, the amplification is a multiplex amplification that includes the simultaneous amplification of a plurality of target sequences in a single amplification reaction. In some embodiments, "amplification" includes amplification of at least some portion of DNA and RNA based nucleic acids alone, or in combination. The amplification reaction can include any of the amplification processes known to one of ordinary skill in the art. In some embodiments, the amplification reaction includes polymerase chain reaction (PCR). [00041] As used herein, the term "polymerase chain reaction" ("PCR") refers to the method of Mullis U.S. Pat. Nos. 4,683,195 and 4,683,202, which describe a method for increasing the concentration of a segment of a polynucleotide of interest in a mixture of genomic DNA without cloning or purification. This process for amplifying the polynucleotide of interest consists of introducing a large excess of two oligonucleotide primers to the DNA mixture containing the desired polynucleotide of interest, followed by a series of thermal cycling in the presence of a DNA polymerase. The two primers are complementary to their respective strands of the double stranded polynucleotide of interest. The mixture is denatured at a higher temperature first and the primers are then annealed to complementary sequences within the polynucleotide of interest molecule. Following annealing, the primers are extended with a polymerase to form a new pair of complementary strands. The steps of denaturation, primer annealing and polymerase extension can be repeated many times (referred to as thermocycling) to obtain a high concentration of an amplified segment of the desired polynucleotide of interest. The length of the amplified segment of the desired polynucleotide of interest (amplicon) is determined by the relative positions of the primers with respect to each other, and therefore, this length is a controllable parameter. By virtue of repeating the process, the method is referred to as PCR. Because the desired amplified segments of the polynucleotide of interest become the predominant nucleic acid sequences (in terms of concentration) in the mixture, they are said to be "PCR amplified". In a modification to the method discussed above, the target nucleic acid molecules can be PCR amplified using a plurality of different primer pairs, in some cases, one or more primer pairs per target nucleic acid molecule of interest, thereby forming a multiplex PCR reaction.
[00042] As used herein, "amplification conditions" and its derivatives, generally refers to conditions suitable for amplifying one or more nucleic acid sequences. In some embodiments, the amplification conditions can include isothermal conditions or alternatively can include thermocycling conditions, or a combination of isothermal and thermocycling conditions. In some embodiments, the conditions suitable for amplifying one or more nucleic acid sequences include polymerase chain reaction (PCR) conditions. Typically, the amplification conditions refer to a reaction mixture that is sufficient to amplify nucleic acids such as one or more target sequences flanked by a universal sequence, or target specific primers, or to amplify an amplified target sequence flanked by one or more adapters. Generally, the amplification conditions include a catalyst for amplification or for nucleic acid synthesis, for example a polymerase; a primer that possesses some degree of complementarity to the nucleic acid to be amplified; and nucleotides, such as deoxyribonucleotide triphosphates (dNTPs) to promote extension of the primer once hybridized to the nucleic acid. The amplification conditions can require hybridization or annealing of a primer to a nucleic acid, extension of the primer and a denaturing step in which the extended primer is separated from the nucleic acid sequence undergoing amplification. Typically, but not necessarily, amplification conditions can include thermocycling; in some embodiments, amplification conditions include a plurality of cycles where the steps of annealing, extending and separating are repeated. Typically, the amplification conditions include cations such as Mg2+ or Mn2+ and can also include various modifiers of ionic strength.
[00043] As defined herein "multiplex amplification" refers to selective and non-random amplification of two or more target sequences within a sample using at least one target-specific primer. In some embodiments, multiplex amplification is performed such that some or all of the target sequences are amplified within a single reaction vessel. The "plexy" or "plex" of a given multiplex amplification refers generally to the number of different target-specific sequences that are amplified during that single multiplex amplification. In some embodiments, the plexy can be about 12-plex, 24-plex, 48-plex, 96-plex, 192-plex, 384-plex, 768-plex, 1536-plex, 3072-plex, 6144-plex or higher. It is also possible to detect the amplified target sequences by several different methodologies (e.g., gel electrophoresis followed by densitometry, quantitation with a bioanalyzer or quantitative PCR, hybridization with a labeled probe; incorporation of biotinylated primers followed by avidin-enzyme conjugate detection; incorporation of 32P- labeled deoxynucleotide triphosphates into the amplified target sequence).
[00044] As used herein, the term "amplification site" refers to a site in or on an array where one or more amplicons can be generated. An amplification site can be further configured to contain, hold or attach at least one amplicon that is generated at the site.
[00045] As used herein, the term "array," "analyte array," and "microarray" are used interchangeably and refer to a population of sites that can be differentiated from each other according to relative location. Different molecules that are at different sites of an array can be differentiated from each other according to the locations of the sites in the array. An individual site of an array can include one or more molecules of a particular type. For example, a site can include a single target nucleic acid molecule having a particular sequence or a site can include several nucleic acid molecules having the same sequence (and/or complementary sequence, thereof). The sites of an array can be different features located on the same substrate. Exemplary features include without limitation, droplets, wells in a substrate, beads (or other particles) in or on a substrate, projections from a substrate, ridges on a substrate or channels in a substrate. The sites of an array can be separate substrates each bearing a different molecule. Different molecules attached to separate substrates can be identified according to the locations of the substrates on a surface to which the substrates are associated or according to the locations of the substrates in a liquid or gel. Exemplary arrays in which separate substrates are located on a surface include, without limitation, those having beads in wells.
[00046] As used herein, the term "compartment" is intended to mean an area or volume that separates or isolates something from other things. Exemplary compartments include, but are not limited to, vials, tubes, wells, droplets, boluses, beads, vessels, surface features, flow cell, or areas or volumes separated by physical forces such as fluid flow, magnetism, electrical current or the like. In one embodiment, a compartment is a well of a multi-well plate, such as a 96- or 384- well plate. As used herein, a droplet may include a hydrogel bead, which is a bead for encapsulating one or more nuclei or cell, and includes a hydrogel composition. In some embodiments, the droplet is a homogeneous droplet of hydrogel material or is a hollow droplet having a polymer hydrogel shell. Whether homogenous or hollow, a droplet may be capable of encapsulating one or more nuclei or cells. In some embodiments, the droplet is a surfactant stabilized droplet. In some embodiments, a single cell or Nuclei is present per compartment. In some embodiments, two or more cells or Nuclei are present per compartment. In some embodiments, each compartment contains a compartment-specific index. In some embodiments, the index is in solution or attached or associated with a solid-phase in each compartment.
[00047] The term "flow cell" as used herein refers to a chamber comprising a solid surface across which one or more fluid reagents can be flowed. A flow cell includes a vessel having an enclosed flow channel where a reaction can be carried out, or a vessel having a channel that is open to a surrounding environment and in which a reaction can be carried out. The vessel with an open flow channel may be referred to herein as an open wafer flow cell. Any example of the flow cell may include an inlet for delivering reagent(s) to the channel, and an outlet for removing reagent(s) from the channel. In some examples, the flow cell enables the detection of the reaction that occurs therein. For example, the flow cell can include one or more transparent surfaces allowing for the optical detection of arrays, optically labeled molecules, or the like. [00048] Examples of flow cells and related fluidic systems and detection platforms that can be readily used in the methods of the present disclosure are described, for example, in Bentley et al., Nature 456:53-59 (2008), WO 04/018497; US 7,057,026; WO 91/06678; WO 07/123744; US 7,329,492; US 7,211,414; US 7,315,019; US 7,405,281, and US 2008/0108082.
[00049] As used herein, the term "clonal population" refers to a population of nucleic acids that is homogeneous with respect to a particular nucleotide sequence. The homogenous sequence is typically at least 10 nucleotides long, but can be even longer including for example, at least 50, 100, 250, 500 or 1000 nucleotides long. A clonal population can be derived from a single target nucleic acid or template nucleic acid. Typically, all of the nucleic acids in a clonal population will have the same nucleotide sequence. It will be understood that a small number of mutations (e.g., due to amplification artifacts) can occur in a clonal population without departing from clonality.
[00050] As used herein, a "pattern of cytosine modification," also referred to as a "methylation profile," refers to the pattern with which both methylation and unmethylation of cysteines is distributed in the genome of a cell or an organism. A “pattern” is inclusive of both modified cytosines and non-modified cytosines. The pattern can be defined in several distribution dimensions: by organ, by tissue, by status of disease or pathological condition (e.g., cancer, neurophysiological), by genome segment (e.g., chromosome or genetic coordinates on a chromosome), by gene, by CpG island, a group of cytosines, or by the site of a modified cytosine. A pattern of cytosine modification can have a known correlation with a disease or pathological condition, or correlation of a pattern of cytosine modification with a disease or pathological condition can be identified using methods described herein. A pattern of cytosine modification can be present at a specific locus (e.g., location) in a genome, and that specific location can be a single modified cytosine or a set of modified cytosines, e.g., a CpG island. A pattern of cytosine modification can be identified by using a predetermined sequence, e.g., a method of using an altered cytidine deaminase can be designed and practiced with the intent of determining a pattern of cytosine modification, for instance, the methylation status of one of more specific cytosines, the methylation status of one or more specific cytosines present at a specific location of a genome, or the combination thereof.
[00051] As used herein, a flow channel is an area that is defined between two bonded or otherwise attached components, or that is defined within a lane so that it is open to the surrounding environment. The flow channel can selectively receive a liquid sample. In some examples, the flow channel may be defined between two patterned sequencing surfaces or a patterned sequencing surface and a lid, and thus may be in fluid communication with one or more components of the sequencing surface(s).
[00052] As used herein, a fragment is a portion or piece of the DNA sample. A “partially adapted fragment” is a portion or piece of the DNA sample that has been tagmented, and thus includes an adapter ligated to the 5’ end of the DNA fragment. A “fully adapted fragment” is a portion or piece of the DNA sample that has adapters incorporated at both the 3’ and 5’ ends of the DNA fragment.
[00053] As used herein, the term fragmentation is the breaking of nucleic acid into shorter lengths. Fragmentation methods include enzymatic methods, physical methods (including sonication, nebulization, needle shearing, microwave, etc ), and chemical methods (including depurination, hydrolysis, oxidation, etc.). The terms “fragmenting enzymes” or “enzyme-based fragmentation” or “enzyme fragmentation,” as used herein, refer to enzymes that fragment nucleic acids. The enzymes can be a single enzyme or two or more enzymes that work together to fragment the nucleic acid. Some enzymes work on single stranded nucleic acid whereas others work on double stranded nucleic acid and yet others work on one strand of a double stranded nucleic acid. Fragmenting enzymes can cut the nucleic acid randomly or specifically. Examples of fragmenting enzymes include transposase, restriction enzymes, Argonaute, CRISPR -associated nuclease (Cas), endonucleases, exonuclease, topoisomerase, FRAGMENTASE™ (New England Biolabs, Ipswich, MA). Preferred fragmentation examples include methods that fragment while retaining proximity information of the fragments.
[00054] As used herein, the term "each," when used in reference to a collection of items, is intended to identify an individual item in the collection but does not necessarily refer to every item in the collection unless the context clearly dictates otherwise.
[00055] As used in this specification and the appended claims, the term "or" is generally employed in its sense including "and/or" unless the content clearly dictates otherwise. The term "and/or" means one or all of the listed elements or a combination of any two or more of the listed elements. The use of "and/or" in some instances does not imply that the use of "or" in other instances may not mean "and/or." [00056] Unless otherwise specified, "a," "an," "the," and "at least one" are used interchangeably and mean one or more than one.
[00057] As used in this specification and the appended claims, the term "or" is generally employed in its sense including "and/or" unless the content clearly dictates otherwise. The term "and/or" means one or all of the listed elements or a combination of any two or more of the listed elements. The use of "and/or" in some instances does not imply that the use of "or" in other instances may not mean "and/or."
[00058] The words "preferred" and "preferably" refer to embodiments of the disclosure that may afford certain benefits, under certain circumstances. However, other embodiments may also be preferred, under the same or other circumstances. Furthermore, the recitation of one or more preferred embodiments does not imply that other embodiments are not useful, and is not intended to exclude other embodiments from the scope of the disclosure.
[00059] As used herein, "have," "has," "having," "include," "includes," "including," "comprise," "comprises," "comprising" or the like are used in their open ended inclusive sense, and generally mean "include, but not limited to," "includes, but not limited to," or "including, but not limited to."
[00060] It is understood that wherever embodiments are described herein with the language "have," "has," "having," "include," "includes," "including," "comprise," "comprises," "comprising" and the like, otherwise analogous embodiments described in terms of "consisting of' and/or "consisting essentially of' are also provided. The term "consisting of' means including, and limited to, whatever follows the phrase "consisting of." That is, "consisting of' indicates that the listed elements are required or mandatory, and that no other elements may be present. The term "consisting essentially of' indicates that any elements listed after the phrase are included, and that other elements than those listed may be included provided that those elements do not interfere with or contribute to the activity or action specified in the disclosure for the listed elements.
[00061] Conditions that are "suitable" for an event to occur, such as converting 5 methylcytosine to thymidine by deamination, or "suitable" conditions are conditions that do not prevent such events from occurring. Thus, these conditions permit, enhance, facilitate, and/or are conducive to the event. [00062] As used herein, "providing" in the context of a protein, sample of DNA or RNA, or composition means making the protein, sample of DNA or RNA, or composition, purchasing the protein, sample of DNA or RNA, or composition, or otherwise obtaining the protein, sample of DNA or RNA, or composition.
[00063] Reference throughout this specification to "one embodiment," "an embodiment," "certain embodiments," or "some embodiments," etc., means that a particular feature, configuration, composition, or characteristic described in connection with the embodiment is included in at least one embodiment of the disclosure. Thus, the appearances of such phrases in various places throughout this specification are not necessarily referring to the same embodiment of the disclosure. Furthermore, the particular features, configurations, compositions, or characteristics may be combined in any suitable manner in one or more embodiments.
[00064] While polynucleotide sequences encoding an altered cytidine deaminase are described herein as DNA sequences, it is understood that the complements, reverse sequences, and reverse complements of the DNA sequences can be easily determined by the skilled person. It is also understood that the sequences described herein as DNA sequences can be converted from a DNA sequence to an RNA sequence by replacing each thymidine nucleotide with a uracil nucleotide. [00065] Polynucleotide and/or polypeptides or protein sequences may include one or more forms of typographical emphasis (e.g., underlined text, bolded text, italicized text). It is understood that the typographical emphasis is non-limiting. Sequences stated with typographical emphasis include the stated sequence without the typographical emphasis. Additionally, polynucleotide sequences may be displayed in capital letters, lower case letters, or a combination thereof. The case of the letters in the polynucleotide sequences is non-limiting. Unless otherwise stated, lower case and upper-case letters simply indicate the identity of the nucleobase.
[00066] Throughout this disclosure, various aspects of the disclosure can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the disclosure. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 2.7, 3, 4, 4.5, 5, 5.3, and 6. This applies regardless of the breadth of the range.
[00067] In the description herein particular embodiments may be described in isolation for clarity. Unless otherwise expressly specified that the features of a particular embodiment are incompatible with the features of another embodiment, certain embodiments can include a combination of compatible features described herein in connection with one or more embodiments.
[00068] Altered cytidine deaminases (ACDs)
[00069] The present disclosure includes altered cytidine deaminases (ACDs, or the singular form ACD), compositions including ACDs, and methods for using ACDs.
[00070] Wild-type APOBEC3A deaminates cytosine (C), 5 methyl cytosine (5mC), and 5- hydroxymethyl cytosine (5hmC) efficiently in single-stranded DNA (FIG. 1A-C). Treatment of DNA, such as genomic DNA, with wild-type APOBEC3A results in the conversion of C to uracil (U), 5mC to thymidine (T), and 5hmC to 5 -hydroxyuracil cytosine (5hmC) and reduces the complexity of the DNA sequencing (FIG. ID).
[00071] As described herein, mutation of the tyrosine at position 130 in a human APOBEC3A protein to a different amino acid such as alanine resulted in preferential conversion of 5mC to thymidine (FIG. IE). Analysis of the sample DNA after treatment with an altered cytidine deaminase described herein, for example, by sequencing of the sample DNA, and optional comparison to a reference (e g., reference sequence) permits easy identification of C to T point mutations, and these point mutations are inferred as 5mC positions.
[00072] A cytidine deaminase is considered to be an altered cytidine deaminase (ACD) if it has the activity of deaminating 5 methyl cytosine (5mC) and includes at least one of the substitution mutations described herein. An ACD useful in the methods provided herein preferentially deaminates 5mC instead of C (i.e., converts 5mC to T at a greater rate than converting C to U) compared to the equivalent wild-type enzyme and is referred to herein as having “cytosinedefective deaminase activity” and “5mC-selective deaminase activity”, the terms being interchangeable.
[00073] ACDs include apolipoprotein B mRNA editing enzymes, catalytic polypeptide-like (APOBEC) and activation induced cytidine deaminase (AID). Wild-type APOBEC and AID cytidine deaminases have the activity of deaminating cytidine of DNA and/or RNA to form uridine (U). An ACD of the present disclosure has an altered rate of deamination of 5mC when compared to the wild-type enzyme. A cytidine deaminase of the present disclosure can be referred to herein as an "altered cytidine deaminase," "recombinant cytidine deaminase," “ACD,” “recombinant ACD,” “mutant cytidine deaminase,” or “modified cytidine deaminase” and refers to any of the engineered ACDs described herein that include one or more changes from a reference (i.e., wild-type) amino acid sequence that provide one or more of the activities described herein, including but not limited to an altered deamination profile, e.g., alters its ability to preferentially deaminate one form of cytosine over another, enhanced selectivity for 5mC or C, and/or enhanced stability.
[00074] Whether a protein has cytidine deaminase activity may be determined by in vitro assays. One example of an in vitro assay is based on digestion with the restriction enzyme Awal as described in WO 2023/196572. A protein that can deaminate 5mC to thymidine has cytidine deaminase activity.
[00075] In certain embodiments, an ACD of the present disclosure is based on a member of the APOBEC protein family. An ACD of the present disclosure that is "based on" a member of the APOBEC protein family means the ACD is an APOBEC protein that includes one or more of the substitution mutations described herein as compared to a reference APOBEC sequence. An ACD of the present disclosure that is "based on" a member of the APOBEC protein family can also include conservative and/or nonconservative mutations as described herein. The positions of the alterations, substitutions or deletions will be at functionally equivalent amino acids from the reference sequence, as described herein.
[00076] The APOBEC protein family includes subfamilies AID, APOBEC 1, APOBEC2, APOBEC3 (including 3 A, 3B, 3C, 3D, 3F, 3G, 3H), and APOBEC4. An ACD of the present disclosure can be based on a member of the AID subfamily, the APOBEC 1 subfamily, the APOBEC2 subfamily, the APOBEC3 subfamily (e.g., the 3A subfamily, the 3B subfamily, the 3C subfamily, the 3D subfamily, the 3F subfamily, the 3G subfamily, or the 3H subfamily), or the APOBEC4 subfamily. An ACD of the present disclosure can be based on a member of the APOBEC protein family from a vertebrate, such as a mammal. Examples of mammals include, but are not limited to, rodents, primates, rabbit, bovine (e.g., cow), porcine (e.g., pig), equine (e.g., horse), elephant, and aardvark. An example of a primate is a human and a chimpanzee. [00077] The APOBEC protein family is a member of the large cytidine deaminase superfamily that contains a canonical zinc-dependent deaminase (ZDD) signature motif embedded within a core cytidine deaminase fold. This fold includes a five-stranded mixed beta (b)-sheet surrounded by six alpha (a)-helices with the order al-bl-b2-a2-b3-a3-b4-a4-b5-a5-a6 (Salter et al., Trends Biochem Sci. 2016 41(7):578-594. Doi:10.1016/j.tibs.2016.05.001; Salter et al., Trends Biochem. Sci. 2018, 43(8):606-622 doi.org/10.1016/j .tibs.2018.04.013). Each cytidine deaminase domain core structure of APOBEC proteins contains a highly conserved spatial arrangement of the catalytic center residues of a zinc-binding motif H-[P/A/V]-E-X[23-28]-P-C-X[2- 4]-C (SEQ ID NO:1) (referred to herein as the ZDD motif, where X is any amino acid, and the subscript range of numbers after X refers to the number of amino acids) (Salter et al., Trends Biochem Sci. 2016 41(7):578— 594. Doi : 10.1016/j .tibs.2016.05.001). Without intending to be limited by theory, the H and two C residues coordinate a Zn atom, and the E residue polarizes a water molecule near the Zn-atom for catalysis (Chen et al., 2021, Viruses, 13:497, doi.org/10.3390/vl3030497).
[00078] Some members of the APOBEC protein family, e.g., the AID subfamily, the APOBEC 1 subfamily, the APOBEC2 subfamily, the APOBEC3A subfamily, the APOBEC3C subfamily, the APOBEC3H subfamily, and the APOBEC4 subfamily, include one copy of the ZDD motif. Other members of the APOBEC protein family, e.g., the APOBEC3B subfamily, the APOBEC3D subfamily, the APOBEC3F subfamily, and the APOBEC3G subfamily, include two copies of the ZDD motif, but often only the C-terminal copy is active (Salter et al., Trends Biochem Sci. 2016 41(7)578-594. Doi: 10.1016/j .tibs.2016.05.001). Thus, an altered cytidine deaminase disclosed herein includes one or two ZDD motifs. In one embodiment, an altered cytidine deaminase based on a member of the APOBEC3A subfamily that includes the following ZDD motif: HXEX24SW(S/T)PCX[2-4]CX6FX8LX5R(L/I)YX[8-n]LX2LX[io]M (SEQ ID NO:2) (where X is any amino acid, and the subscript number or range of numbers after X refers to the number of amino acids) (Salter et al., Trends Biochem Sci. 2016 41(7)578-594. Doi: 10.1016/j. tibs.2016.05.001).
[00079] In one embodiment, an ACD disclosed herein is a member of the APOBEC3 subfamily, e.g., APOBEC3A, APOBEC3B, APOBEC3C, APOBEC3D, APOBEC3F, or APOBEC3G, and can include one or more highly conserved sites that are part of the active site and within the ZDD motif SEQ ID NO: 1. The sites include tryptophan at position 98 and serine or threonine at position 99 of SEQ ID NO:3 (Kouno et al., 2017, Nat. Comm, 8: 15024, DOI: 10.1038/ncomms 15024).
[000801 In addition to the ZDD motif, a member of the APOBEC protein family also includes other highly conserved residues that are part of the active site but not present as part of the ZDD motif SEQ ID NO:1. A member the APOBEC3A subfamily, APOBEC3B subfamily, APOBEC3C subfamily, APOBEC3D subfamily, APOBEC3F subfamily, and APOBEC3G subfamily typically includes one or more of the following highly conserved sites that are part of the active site: arginine at position 28; histidine, asparagine, or arginine at position 29; serine or threonine, preferably threonine, at position 31; asparagine or aspartic acid at position 57; histidine at position 70; cysteine at position 101; cysteine at position 106; tyrosine or phenylalanine at position 130; asparagine or tyrosine at position 131; asparagine, tyrosine, or phenylalanine, preferably tyrosine, at position 132; and arginine or lysine at position 189 of SEQ ID NO:3 (Kouno et al., 2017, Nat. Comm, 8: 15024, DOI: 10.1038/ncommsl5024).
[00081] An ACD of the present disclosure includes a substitution mutation, deletion, insertion, or a combination thereof, at one or more residues when compared to a reference cytidine deaminase. A substitution mutation can be at the same position or a functionally equivalent position compared to the reference cytidine deaminase. Reference cytidine deaminases and functionally equivalent positions are described in detail herein. As noted, positions of the altered amino acids described herein and in the tables are in reference to APOBECA3A (SEQ ID NO:3) but one skilled in the art is capable of deriving the functionally equivalent positions in the other referenced cytosine deaminases. The skilled person will readily appreciate that an altered cytidine deaminase described herein is not naturally occurring.
[00082] A reference cytidine deaminase can be a member of the APOBEC protein family. Essentially any known member of the APOBEC protein family can be a reference cytidine deaminase. The skilled person can easily identify members of each of the subfamilies by using a publicly available database such as the Protein database available at the National Center for Biotechnology Information (ncbi.nlm.nih.gov/protein) and searching for APOBEC1, APOBEC2, APOBEC3A, APOBEC3B, APOBEC3C, APOBEC3D, APOBEC3F, APOBEC3G, APOBEC3H, APOBEC4, or, when identifying members of the AID family, Activation-induced cytidine deaminase. A wild-type reference cytidine deaminase has the activity of binding singlestranded DNA (ssDNA) and deaminating a cytosine present on the ssDNA to convert it to uracil. In one embodiment, a wild-type reference cytidine deaminase has the activity of binding singlestranded RNA (ssRNA) and deaminating a cytosine present on the ssRNA to convert it to uracil. Methods for determining whether a protein binds ssDNA or ssRNA and deaminates a cytosine present are known to the skilled person. Other reference cytidine deaminases include, but are not limited to, SEQ ID NO: 13, 14, 15, 16, 17, 18, 19, 20, 21, and 22. Thus, any of the mutations discussed for selectivity (e.g., AxxxW (SEQ ID NO:51) or AxxxWX (SEQ ID NO:52)) may be substituted into the backbone of any one of SEQ ID NO: 13-22 instead of the SEQ ID NO:3 wildtype reference backbone and are contemplated as aspects of the present invention.
[00083] In one embodiment, an ACD has an amino acid sequence that is based on a reference sequence which is a member of the APOBEC protein family includes a ZDD motif H-[P/A/V]-E- X[23-28]-P-C-X[2-4]-C (SEQ ID NO:1) and at least one substitution mutation disclosed herein. Optionally, an altered cytidine deaminase includes a substitution mutation at one or more other active site residues disclosed herein. Non-limiting examples of reference cytidine deaminase proteins for different APOBEC protein family members include UniProt Q9GZX7, UniProt G3QLD2, and UniProt Q9WVE0 (APOBEC protein family member AID); UniProt P41238, NCBI XP 030856728.1, and Uniprot P51908 (APOBEC protein family member APOBEC1); UniProt Q9Y235, Uniprot G3SGN8, and Uniprot Q9WV35 (APOBEC protein family member APOBEC2); UniProt P31941 , GenBank XP 045219544.1, GenBank AER45717.1, GenBank XP 003264816.1, GenBank PNI48846.1, and GenBank ADO85886.1 (APOBEC protein family member APOBEC3A); UniProt Q9UH17, Uniprot G3QV16, and Uniprot F6M3K5 (APOBEC protein family member APOBEC3B); UniProt Q9NRW3, Uniprot Q694B5, and Uniprot B0LW74 (APOBEC protein family member APOBEC3C); UniProt Q96AK3, NCBI NP_001332895.1, and NCBI NP_001332931.1 (APOBEC protein family member APOBEC3D); UniProt Q8IUX4, Uniprot G3RD21, and Uniprot Q1G0Z6 (APOBEC protein family member APOBEC3F); UniProt Q9HC16, Uniprot Q694C1, and Uniprot U5NDB3 (APOBEC protein family member APOBEC3G); UniProt Q6NTF7, Uniprot B7T0U7, and Uniprot Q19Q52 (APOBEC protein family memberAPOBEC3H); and UniProt Q8WW27, NCBI
XP 004028087.1, and Uniprot Q497M3 (APOBEC protein family member APOBEC4). [00084] In one embodiment, an ACD has an amino acid sequence that is based on a reference sequence that is a member of the APOBEC3A subfamily, and includes a ZDD motif HXEX24SW(S/T)PCX[2-4]CX6FX8LX5R(L/I)¥X[8-11]LX2LX[1O]M (SEQ ID NO:2) (where X is any amino acid, and the subscript number or range of numbers after X refers to the number of amino acids) and at least one substitution mutation disclosed herein. In one embodiment, the substitution mutation is a substitution mutation at the underlined tyrosine, such as a substitution mutation to alanine (A). The underlined tyrosine (Y) of SEQ ID NO:2 is the position functionally equivalent to the tyrosine amino acid 130 of the APOBEC3A protein SEQ ID NO:3. Optionally, the altered cytidine deaminase includes other active site residues disclosed herein.
[00085] In one embodiment, the amino acid sequence of an ACD includes the amino acids of a member of the APOBEC3A subfamily: X[i6-26]-GRXXTXLCYXV-Xi5-GXXXN-Xi2-HAEXXF- X14-YXXTWXXSWSPC- X[2-4]-CA-X5-FL-X7-LXIXXXR(L/I)Y-Xs-GLXXLXXXG-X5-M-X4- FXXCWXXFV-X6-FXPW-X13-LXXI- X[2-6] (SEQ ID NO:9) (where X is any amino acid, and the subscript number or range of numbers after X refers to the number of amino acids), or a subset thereof, and at least one substitution mutation disclosed herein. The underlined tyrosine (Y) of SEQ ID NO:9 is the position functionally equivalent to the tyrosine amino acid 130 of the APOBEC3A protein SEQ ID NO :3. In one embodiment, the substitution mutation is a substitution mutation at the underlined tyrosine, such as a substitution mutation to alanine (A). Optionally, the altered cytidine deaminase includes other active site residues disclosed herein. [00086] In one embodiment, the amino acid sequence of an ACD includes the amino acids of a member of the APOBEC3A subfamily: X26-GRXXTXLCYXV-X15-G-X16-HAEXXF-X14- YXXTWXXSWSPC-X4-CA-X5-FL-X7-LXIFXXR(L/I)Y-X8-GLXXLXXXG-X5-M-X4- FXXCWXXFV-X6-FXPW-X13-LXXI-X6 (SEQ ID NO: 10) (where X is any amino acid, and the subscript number after X refers to the number of amino acids present), or a subset thereof, and at least one substitution mutation disclosed herein. The underlined tyrosine (Y) of SEQ ID NO: 10 is the position functionally equivalent to the tyrosine amino acid 130 of the APOBEC3A protein SEQ ID NO:3. In one embodiment, the substitution mutation is a substitution mutation at the underlined tyrosine (Y), such as a substitution mutation to alanine (A). Optionally, the altered cytidine deaminase includes other active site residues disclosed herein.
[00087] A substitution mutation can be at the same position or a functionally equivalent position compared to a reference cytidine deaminase. By "functionally equivalent" it is meant that the altered cytidine deaminase has the amino acid substitution at the amino acid position in a reference cytidine deaminase that has the same functional role in both the reference cytidine deaminase and the altered cytidine deaminase.
[00088] In general, functionally equivalent substitution mutations in two or more different cytidine deaminases occur at homologous amino acid positions in the amino acid sequences of the cytidine deaminases. Hence, use herein of the term "functionally equivalent" also encompasses mutations that are "positionally equivalent" or "homologous" to a given mutation, regardless of whether or not the particular function of the mutated amino acid is known. It is possible to identify the locations of functionally equivalent and positionally equivalent amino acid residues in the amino acid sequences of two or more different cytidine deaminases on the basis of sequence alignment and/or molecular modelling. An example of a sequence alignment to identify positionally equivalent and/or functionally equivalent residues is set forth in FIG. 2. For example, the residues in the members of the APOBEC3A subfamily in FIG. 2 that are vertically aligned are considered positionally equivalent as well as functionally equivalent to the corresponding residue in the human APOBEC3A amino acid sequence. Thus, for example, as shown in FIG. 2, the tyrosine at residue 130 of the APOBEC3A proteins of Homo sapiens, Pongo pygmaeus, Nomascus leucogenys, Pan troglodytes, and Gorilla gorilla and the tyrosine at residue 133 of the APOBEC3A protein from Macaca fascicularis are functionally equivalent and positionally equivalent. The skilled person can easily identify functionally equivalent residues in cytidine deaminases.
[00089] In one embodiment, an altered cytidine deaminase has an amino acid sequence that is structurally similar to a reference cytidine deaminase disclosed herein. In one embodiment, a reference cytidine deaminase is one that includes the amino acid sequence of SEQ ID NO:9 or SEQ ID NO:10. Other examples of reference sequences include, but are not limited to, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:479-517, SEQ ID N0:520-605, SEQ ID NO:607-615, SEQ ID NO:635-954, and SEQ ID NO:962-968
[00090] As used herein, an ACD may be "structurally similar" or have “structural similarity” to a reference cytidine deaminase if the amino acid sequence of the ACD possesses a specified amount of sequence similarity and/or sequence identity compared to the reference cytidine deaminase. [00091] Structural similarity of two amino acid sequences can be determined by aligning the residues of the two sequences (for example, a candidate ACD and a reference cytidine deaminase described herein) to optimize the number of identical amino acids along the lengths of their sequences; gaps in either or both sequences are permitted in making the alignment in order to optimize the number of identical amino acids, although the amino acids in each sequence must nonetheless remain in their proper order. A candidate altered cytidine deaminase is the cytidine deaminase being compared to the reference cytidine deaminase. A candidate ACD that has structural similarity with a reference cytidine deaminase and cytidine deaminase activity is an altered cytidine deaminase.
[00092] Unless modified as otherwise described herein, a pair-wise comparison analysis of amino acid sequences can be conducted, for instance, by the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson & Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by visual inspection (see generally Current Protocols in Molecular Biology, Ausubel et al., eds., Current Protocols, a joint venture between Greene Publishing Associates, Inc. and John Wiley & Sons, Inc., supplemented through 2004). One example of an algorithm that is suitable for determining structural similarity is the BLAST® algorithm, which is described in Altschul et al., J. Mol. Biol. 215:403-410 (1990). The BLAST® algorithm can be used to calculate percent sequence identity and percent sequence similarity between two sequences. Software for performing BLAST® analyses is publicly available through the National Center for Biotechnology Information.
[00093] In the comparison of two amino acid sequences, structural similarity may be referred to by percent "identity" or may be referred to by percent "similarity." "Identity" refers to the presence of identical amino acids. "Similarity" refers to the presence of not only identical amino acids but also the presence of conservative substitutions. Thus, in one embodiment the amino acid sequence of an altered cytidine deaminase protein having sequence similarity to a reference sequence may include conservative substitutions of amino acids present in that reference sequence. [00094] A conservative substitution for an amino acid in a protein may be selected from other members of the class to which the amino acid belongs. For example, it is well-known in the art of protein biochemistry that an amino acid belonging to a grouping of amino acids having a particular size or characteristic (such as charge, hydrophobicity, or hydrophilicity) can be substituted for another amino acid without altering the activity of a protein, particularly in regions of the protein that are not directly associated with biological activity. For example, amino acids having a non-polar side chain include alanine, glycine, isoleucine, leucine, methionine, phenylalanine, proline, tryptophan, and valine; amino acids having a hydrophobic side chain include glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, and tryptophan; amino acids having a polar side chain include arginine, asparagine, aspartic acid, glutamine, glutamic acid, histidine, lysine, serine, cysteine, tyrosine, and threonine; and amino acids having an uncharged side chain include glycine, serine, cysteine, asparagine, glutamine, tyrosine, and threonine.
[00095] Thus, as used herein, reference to a cytidine deaminase as described herein, such as reference to the amino acid sequence of one or more SEQ ID NOs described herein can include a protein having structural similarity to the reference cytidine deaminase, e g., at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence similarity to the reference cytidine deaminase. In one embodiment, a reference protein is SEQ ID NO:3. Examples of other reference proteins are described herein.
[00096] Alternatively, as used herein, reference to a cytidine deaminase as described herein, such as reference to the amino acid sequence of one or more SEQ ID NOs described herein can include a protein having structural similarity to the reference cytidine deaminase, e.g., at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity to the reference cytidine deaminase. In one embodiment, a reference protein is SEQ ID NO:3. Examples of other reference proteins are described herein.
[00097] Substitution mutations conferring increased selectivity or stability [00098] In one aspect, the present disclosure provides new altered cytidine deaminases (ACDs) that preferentially deaminate 5mC instead of C (i.e., converts 5mC to T at a greater rate than converting C to U) compared to the equivalent wild-type enzyme and first and second generations of ACD that have been identified (International Application Publication NOs. WO 2023/196572, WO 2025/072793, and WO 2025/072800), and include a substitution mutation at a position functionally equivalent to a tyrosine at position 130 (Y130), for instance, Y130A in combination with the mutations described below. An optional substitution mutation is at a position functionally equivalent to a tyrosine at position 132 (Y132), for instance, Y130H. Another optional mutation that increases the selectivity for 5mC is a substitution mutation at a position functionally equivalent to a aspartic acid at position 133 (Y133), for instance, D133W. An example of an ACD with these three substitution mutations is ScA (SEQ ID NO: 13). ACDs of the present disclosure include one or more selectivity-enhancing alterations that result in greater selectivity for 5mC than other ACDs, such as the ACD ScA (SEQ ID NO:13), as well a wild-type APOBEC3A cytidine deaminase (e.g., SEQ ID NO:3).
[00099] A selectivity-enhancing alteration is considered to enhance the selectivity of an ACD for 5mC if it has greater deamination of 5mC when compared to a reference ACD. A suitable reference ACD is one having the same amino acid sequence (also referred to as the same backbone) but without the selectivity-enhancing alteration. Examples of reference ACDs for use in determining whether an alteration is a selectivity-enhancing alteration include, but are not limited to, SEQ ID NO:3, SEQ ZD NO 13, SEQ ID NO: 14, SEQ ID NO:17, or SEQ ID NO 21. In one embodiment, the reference ACD is SEQ ID NO: 14. A selectivity-enhancing alteration is a substitution mutation, a deletion, or an insertion. In one embodiment, an ACD disclosed herein having enhanced selectivity includes one or more selectivity-enhancing alterations that confer increased 5mC-selectivity compared to a reference ACD (an increase in 5mC deamination of at least 5%, at least 10%, at least 15%, least 20%, at least 25%, least 30%, least 40%, least 50%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95%). Cytidine deaminase activity of a protein can be determined by in vitro assays. One example of an in vitro assay is based on digestion with the restriction enzyme Swal as described in WO 2023/196572.
[000100] An ACD of the present disclosure can include a substitution mutation at a position functionally equivalent to tyrosine at position 130 (Y130) in a member of the APOBEC protein family, including a member of the APOBEC3A subfamily (for instance, SEQ ID NO:3). In some APOBEC family proteins, the wild-type residue at a position functionally equivalent to Y130 is phenylalanine (F). An ACD with a substitution mutation at a position functionally equivalent to Y130 preferentially acts on 5mC compared to cytosine (i.e., has 5mC-selective deaminase activity). The substitution mutation in an ACD is at a position functionally equivalent to position 130 in a member of the APOBEC protein family, including a member of the APOBEC3A subfamily (for instance, SEQ ID NO:3) and can be a mutation to alanine (A), glycine (G), phenylalanine (F), histidine (H), glutamine (Q), methionine (M), asparagine (N), lysine (K), valine (V), aspartic acid (D), glutamic acid I, serine (S), cysteine (C), proline (P), or threonine (T) (FIG. 2). In one embodiment, the substitution mutation at a position functionally equivalent to Y130 is Y130A, Y130S, or Y130H. In one embodiment, the substitution mutation at a position functionally equivalent to Y130 is Y130A. In one embodiment, the substitution mutation at a position functionally equivalent to Y130 is Y130S.
[000101] An ACD of the present disclosure can include a second substitution mutation at a position functionally equivalent to the proline at position 134 (Pl 34) in a member of the APOBEC protein family, including a member of the APOBEC3A subfamily (for instance, SEQ ID NO:3). An ACD with a substitution mutation at a position functionally equivalent to Pl 34 preferentially acts on 5mC compared to cytosine (i.e., has 5mC-selective deaminase activity). . In one embodiment, the substitution mutation in an ACD is at a position functionally equivalent to position Pl 34 is a mutation to tryptophan (P134W) or to tyrosine (P134Y). one embodiment, the substitution mutation at a position functionally equivalent to Pl 34 is P134W. In one embodiment, the substitution mutation in an ACD is at a position functionally equivalent to position P134 is a mutation to threonine (P134T).
[000102] In one embodiment, an ACD of the present disclosure comprises AxxxW (SEQ ID NO:51) at positions functionally equivalent to 130-134 of SEQ ID NO:3, and where X may be any amino acid (examples demonstrated in Tables 2 and 3 and Figures 8 and 9), and the ACD with AxxxW (SEQ ID NO:51) has an increase in 5mC selectivity as compared to wild-type APOBEC (e.g., SEQ ID NO:3), particularly, for example, has at least 75% increase 5mC specificity as compared to reference APOBEC sequence (e.g., an increase in 5mC deamination of at least 5%, at least 10%, at least 15%, least 20%, at least 25%, least 30%, least 40%, least 50%, at least 75%, at least 78%, at least 80%, at least 82%, at least 85%, at least 90%, at least 92%, or at least 95% as compared to WT APOBEC). [000103] An ACD of the present disclosure can further include a substitution mutation at a position functionally equivalent to the aspartic acid at position 131 (DI 31), the tyrosine at position 132 (Y132), the aspartic acid at position 133 (D133), or any combination thereof. [000104] In one embodiment, when the substitution mutation in an ACD at a position functionally equivalent to position Y130 is Y130A or Y130H, preferably Y130A, and at a position functionally equivalent to position P134 is P134W or P134Y, preferably P134W, then any amino acid can be present at DI 31 (D13 IX), Y132 (Y132X), and/or D133 (D133X). The substitution mutations in an ACD are at positions functionally equivalent to positions 131, 132, and/or 133 in a member of the APOBEC protein family, including a member of the APOBEC3A subfamily (for instance, SEQ ID NO:3). In one embodiment, a substitution mutation at D 131 can be histidine, arginine, lysine, proline, glycine, asparagine, alanine, or valine. In one embodiment, a substitution mutation at Y132 can be alanine, proline, aspartic acid, glutamic acid, glycine, threonine, valine, glutamine, or arginine. In one embodiment, a substitution mutation at D133 can be histidine, tyrosine, phenylalanine, threonine, isoleucine, or valine, [000105] In some embodiments, an ACD of the present disclosure can further include a substitution mutation at a position functionally equivalent to the leucine at position 135 (L135) in a member of the APOBEC protein family, including a member of the APOBEC3A subfamily (for instance, SEQ ID NO:3). The substitution mutation at a position functionally equivalent to L135 preferentially acts on 5mC compared to cytosine (i.e., has 5mC-selective deaminase activity). In one embodiment, the substitution mutation in an ACD at a position functionally equivalent to position L135 is a mutation to tryptophan (L135W). In one embodiment, the substitution mutation in an ACD at a position functionally equivalent to position L 135 is a mutation to histidine (L135H), glutamine (L135Q), tyrosine (L135Y), or cysteine (L135C). In one embodiment, an ACD includes the substitution mutations at positions functionally equivalent to Y130A, Y132H, D133W, P134W, and L135X, where X is tryptophan (L135W), histidine (L135H), glutamine (L135Q), tyrosine (L135Y), or cysteine (L135C).
[000106] In some embodiments, an ACD of the present disclosure includes a substitution mutation at positions functionally equivalent to Y130 (such as, but not limited to, Y130A) and Y134 (such as, but not limited to, P134W), and have any amino acid present at positions functionally equivalent to D131, Y132, D133, in a member of the APOBEC protein family, including a member of the APOBEC3A subfamily (for instance, SEQ ID NO:3), or an ACD having structural similarity to SEQ ID NO:3. The structural similarity can be, but is not limited to, at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to SEQ ID NO:3. In some embodiments, an ACD of the present disclosure includes a substitution mutation at positions functionally equivalent to Y130 (such as, but not limited to, Y130A) and Y134 (such as, but not limited to, P134W), any amino acid present at positions functionally equivalent to D131, Y132, D133, and a substitution mutation at a position functionally equivalent to L135 in a member of the APOBEC protein family, including a member of the APOBEC3A subfamily (for instance, SEQ ID NO:3)
[000107] Examples of substitution mutations at positions functionally equivalent to 130, 131, 132, 133, and 134 that can be present in a wild-type APOBEC3A protein, such as SEQ ID NO:3, or present in an ACD and increase the selectivity for 5mC compared to cytosine include, but are not limited to, those shown in Table 2 (see Example 1). Thus, examples of reference ACDs having these substitution mutations include, but are not limited to, SEQ ID NO: 13, 14, 15, 16, 17, 18, 19, 20, 21, and 22, and those having structural similarity to SEQ ID NO: 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22, where the amino acids at 130, 131, 132, 133 and 134 are those shown in Table 2. The structural similarity can be, but is not limited to, at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22. ACDs of SEQ ID NO: 13, 14, 15, 16, 17, 18, 19, 20, 21, and 22 include stability mutations, which are described herein. In some aspects, the ACDs have increased 5mC specificity have mutations at position 103-105 in combination with AxxxW (SEQ ID NO:51) at positions 130- 134, as shown in FIG. 8. Suitable ACD mutations for F102/S103/W104/G105 may include one or more mutations or deletions, two or more, or three or more mutaitons or deletions within F102/S103/W104/G105.
[000108] Examples of substitution mutations at positions functionally equivalent to 130, 131, 132, 133, and 134 that can be present in a wild-type APOBEC3A protein, such as SEQ ID NO:3, or present in an ACD and increase the selectivity for 5mC compared to cytosine include, but are not limited to, those shown in Table 2 (see Example 1). Thus, examples of reference ACDs having these substitution mutations include, but are not limited to, SEQ ID NO:13, 14, 15, 16, 17, 18, 19, 20, 21, and 22, and those having structural similarity to SEQ ID NO: 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22, where the amino acids at 130, 131, 132, 133 and 134 are those shown in Table 2. The structural similarity can be, but is not limited to, at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to SEQ ID NO:13, 14, 15, 16, 17, 18, 19, 20, 21, or 22. ACDs of SEQ ID NO: 13, 14, 15, 16, 17, 18, 19, 20, 21, and 22 include stability mutations, which are described herein. In some aspects, the ACDs have increased 5mC specificity have mutations at position 103-105 in combination with AxxxW (SEQ ID NO:51) at positions 130- 134, as shown in FIG. 8. Suitable ACD mutations for F102/S103/W104/G105 may include one or more mutations or deletions, two or more, or three or more mutations or deletions within F102/S103/W104/G105.
[000109] Examples of substitution mutations at positions functionally equivalent to 130, 131, 132, 133, 134, and 135 that can be present in a wild-type APOBEC3A protein, such as SEQ ID NO:3, or present in an ACD and increase the selectivity for 5mC compared to cytosine include, but are not limited to, ADHWWX (SEQ ID NO: 52), where X any amino acid besides L, preferably wherein X is W, H, Q, Y or C. Thus, examples of ACDs that can include these substitution mutations include, but are not limited to, SEQ ID NO:13, 14, 15, 16, 17, 18, 19, 20, 21, and 22, and those having structural similarity to SEQ ID NO: 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22, where the amino acids at 130, 131, 132, 133 134, and 135 is ADHWWX (SEQ ID NO:52), where X is W, H, Q, Y or C. The structural similarity can be, but is not limited to, at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22.
[000110] Additionally, these ACD may have an additional substitution or deletion at one or more positions functionally equivalent to 102, 103, 104 and 105, for example, one or more substitution mutations, one or more deletions, or one or more insertions, or a combination thereof, of the amino acids at positions functionally equivalent to a phenylalanine at position 102 (Fl 02), a serine at position 103 (SI 03), a tryptophan at position 104 (W104), and/or a glycine at position 105 (G105) in a member of the APOBEC protein family, including a member of the APOBEC3A subfamily (for instance, SEQ ID NO:3). In one embodiment, the substitution mutation at a position functionally equivalent to any one of Fl 02, SI 03, W104, and/or G105 can be any amino acid. In one embodiment, the deletion can be 1, 2, 3, or 4 of the amino acids at a position functionally equivalent to any one of F102, S103, W104, and/or G105. In one embodiment, deletion can be at a position functionally equivalent to W104, G105, or both W104 and G105. Suitable sequences for the region from 102-105 can be selected from, e.g., SEQ ID NO:242-410, FIMA, QVP , YLPA, EMFA, FIIA, ELYA, YLPA, NIP A, VYNA, AFRA, YYIA, FSDD, FQAG, FSAG, AVPG, FVPA, AVPA, YLIA, RYAA, LFWA, TRY A, YLIA, VLYA, RHYA, FMHA, QFPA, RRPA, RIYA, YYIA, RMLA, VLYA, RMLA, AVQA, QVWA, FVPA, FVDA, FSAA, KRAA , RRAA, FPPA, KRRA, VP A A, TDHA, QTDA, YLAA, LPAA, EMAA, MFAA, YVRA, IKLA, YIKA, QTQA, TQYA, KTNA, HLTA, LTGA, HTGA, HLGA, TGAA, HLAA, LTAA, HLTA, LTGA, QTMA, RKTA, KIYA, AYKA, MHAA, NQAA, RKAA, LMAA, LNAA , ACAA, QIAA, IMAA, FCAA, AVAA, QMAA, YDAA, LSAA, CEAA, VLAA, QQAA, RCAA, FCAA, SWAA, AGAA, RWAA, NQAA, QHAA, LAAA, MRAA, FNAA,FNAA, GCAA, SVAA, RMAA, ESAA, LMAA, MCAA, ICAA, GVAA, QHAA, among others.
[000111] Additional selectivity-enhancing alterations have been identified at amino acids other than those at positions functionally equivalent to 130-135 of SEQ ID NO:3. In one embodiment, a selectivity-enhancing alteration is one or more substitution mutations, one or more deletions, or one or more insertions, or a combination thereof, of the amino acids at positions functionally equivalent to a phenylalanine at position 102 (F102), a serine at position 103 (S103), a tryptophan at position 104 (W104), and/or a glycine at position 105 (G105) in a member of the APOBEC protein family, including a member of the APOBEC3A subfamily (for instance, SEQ ID NO:3). In one embodiment, the substitution mutation at a position functionally equivalent to any one of Fl 02, SI 03, W104, and/or G105 can be any amino acid. In one embodiment, the deletion can be 1, 2, 3, or 4 of the amino acids at a position functionally equivalent to any one of Fl 02, SI 03, W104, and/or G105. In one embodiment, deletion can be at a position functionally equivalent to W104, G105, or both W104 and G105. In one embodiment, the insertion can be the insertion of 1, 2, 3, or 4 amino acids between two amino acids at a position functionally equivalent to any one of F 102, S103, W104, or G105. In one embodiment, there can be 2 or more separate insertions, e.g., an insertion can be between Fl 02 and SI 03 and an insertion between amino acid SI 03 and W 104. The one or more substitution mutations, one or more deletions, or one or more insertions can be at any combination of positions 102-105, e.g., at one, two, three, or all four positions. Examples of substitution mutations at positions functionally equivalent to SI 03 and W 104 include, but are not limited to, S103D/W104A, S103H/W104P, S103T/W104D, S103T/W104M, S103V/W104E, S103V/W104M, and S103V/W104P.
Examples of selectivity-enhancing alterations at positions functionally equivalent to S103, W104, and G105 include, but are not limited to, substitution mutations S103V/W104P and a deletion of G105. In one embodiment, these selectivity-enhancing alterations can be present in SEQ ID NO: 14 (see, for instance, SEQ ID NO:23 and SEQ ID NO:24.
[0001121 Examples of selectivity-enhancing alterations at positions functionally equivalent to
102, 103, 104, 105, 130, 131, 132, 133, and 134 that can be present in a wild-type APOBEC3A protein, such as SEQ ID NO:3, or present in an ACD and increase the selectivity for 5mC compared to cytosine include, but are not limited to, those shown in Table 3 (see Example 1). Thus, examples of ACDs having these selectivity-enhancing alterations include, but are not limited to, SEQ ID NO: 13, 14, 15, 16, 17, 18, 19, 20, 21, and 22, and those having structural similarity to SEQ ID NO: 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22, where the amino acids at 102,
103, 104, 105, 130, 131, 132, 133, and 134 are those shown in Table 3. The structural similarity can be, but is not limited to, at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22.
[000113] Examples of selectivity-enhancing alterations at positions functionally equivalent to 102, 103, 104, 105, 130, 131, 132, 133, 134, and 135 that can be present in a wild-type APOBEC3A protein, such as SEQ ID NO:3, or present in an ACD and increase the selectivity for 5mC compared to cytosine include, but are not limited to, those shown in FIG. 8. Thus, examples of ACDs having these selectivity-enhancing alterations include, but are not limited to, SEQ ID NO: 13, 14, 15, 16, 17, 18, 19, 20, 21, and 22, and those having structural similarity to SEQ ID NO:13, 14, 15, 16, 17, 18, 19, 20, 21, or 22, where the amino acids at 102, 103, 104, 105, 130, 131, 132, 133, 134, and 135 are those shown in FIG. 8. The structural similarity can be, but is not limited to, at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22.
[000114] Examples of ACDs with additional mutations that increase selectivity for 5mC are described in International Application Publication Nos. WO 2025/072793 and WO 2025/072800. [000115] An ACD described herein having enhanced 5mC-selective deaminase activity (e.g., one or more selectivity-enhancing alterations at a position functionally equivalent to F102, 8013, W104, G105, Y130, D131, Y132, D133, P134, L135, or any combination thereof) can optionally include one or more stability-enhancing alterations. A stability-enhancing alteration can be one or more substitution mutations, one or more deletions, one or more insertions, or a combination thereof, at a position functionally equivalent to an amino acid in a member of the APOBEC protein family, including a member of the APOBEC3A subfamily (for instance, SEQ ID NO:3). A stability-enhancing alteration (e.g., substitution mutation, deletion, or insertion) is considered to be stability-enhancing if it can increase the melting temperature of an ACD by at least 1 °C, at least 2 °C, at least 3 °C, at least 4 °C, at least 5 °C, or at least 6 °C compared to the same ACD that does not have the stability-enhancing substitution mutation. A suitable method of determining the melting temperature of an ACD described herein is by fluorimetry. However, it is well within one skilled in the art to determine the melting temperature of the ACDs described herein.
[000116] Examples of stability-enhancing alterations include, but are not limited to, substitution mutation at one or more positions functionally equivalent to I17X, T19X, G25X, S45X, A59X, K60X, R74X, G108X, A126X, C171X, G188X, a deletion of amino acids 61-68 (A61-68), a deletion of amino acids 104-105 (A 104- 105), or any combination thereof, in a member of the APOBEC protein family, including a member of the APOBEC3A subfamily (for instance, SEQ ID NO:3). An example of I17X is I17T. An example of T19X is T19Y.
Examples of G25X include G25K and G25R. An example of S45X is S45W. An example of A59X is A59P. An example of K60X is K60R. An example of R74X is R74L. An example of A126X is A126C. Examples of G108X include G108C and G108A. An example of C171X is C171A. An example of G188X is G188R. Examples of ACDs having specific combinations of stability-enhancing alterations include, but are not limited to, those shown in Table 1.
[000117] Table 1 . Examples of stability mutations in ACDs.
[000118] An ACD described herein having enhanced 5mC-selective deaminase activity (e.g., one or more selectivity-enhancing alterations at a position functionally equivalent to F102, S013, W104, G105, Y130, D131, Y132, D133, P134, L135, or any combination thereof) can include one or more stability enhancing alterations selected from Hl IX, L12X, D14X, H16X, I17X, T19X, S20X, N21X, G25X, I26X, G27X, R28X, H29X, T31X, C34X, E38X, R39X, D41X, N42X, S45X, K47X, M48X, H51X, F54X, H56X, N57X, A59X, K60X, L62X, Y67X, L73X, R74X, D77X, L78X, V79X, P80X, S81X, D85X, A87X, I89X, Y90X, R91X, T93X, W94X, I96X, S97X, C101X, F102X, S103X, W104X, G105X, C106X, A107X, G108X, E109X, VI 10X, R11 IX, Al 12X, LI 14X, QI 15X, El 16X, N117X, T118X, H119X, V120X, L122X, R123X, A126X, R128X, L135X, Y136X, E138X, A139X, Q141X, M142X, D145X, A146X, A148X, D156X, E157X, K159X, C161X, D163X, T164X, D167X, Q169X, C171X, D177X, D180X, H182X, S183X, Q184X, A185X, L186X, S187X, G188X, R189X, A192X, N196X a deletion selected from AM1-L12, AE36-G53, AN61-G68, AW104- G105 , AQ195-N199 , AI26-G27, or any combination thereof, where the position number designation is functionally equivalent to the position in a wild-type APOBEC3 A (SEQ ID NO:3) and X is an amino acid substitution different from the wild-type amino acid at that position.
[000119] An ACD described herein having enhanced 5mC-selective deaminase activity (e.g., one or more selectivity-enhancing alterations at a position functionally equivalent to Fl 02, SO 13, W104, G105, Y130, D131, Y132, D133, P134, L135, or any combination thereof) can include one or more stability enhancing alterations selected from Hl IL, D14Y, H16D, I17T, T19Y, T19L, T19I, S20F, S20F, S20E, S20Y, S20I, G25R, G25V, G25K, I26R, T31I, T31W, S45W, S45R, K47P, A59P, K60R, K60G, L62P, R74L, L78H, P80R, P80T, P80S, P80G, I89R, I89T, I89H, I89R, S103R, S103Y, S103T, G108C, G108A, G108Y, E109Q, E109L, V110L, Q115K, Q115Y, E116L, T118P, R123H, A126F, A126C, E138R, E138Q, E138A, D145R, C161M, C171A, D180W, D180H, S183W, S183F, S183V, A185I, A185R, G188I, G188, G188Q, G188R, R189K, A192E or any combination thereof, where the position number designation is functionally equivalent to the position in a wild-type APOBEC3 A (SEQ ID NO:3).
[000120] In one embodiment, an ACD having the one or more selectivity-enhancing alteration described herein (e.g., a selectivity-enhancing alteration at a position functionally equivalent to F102, SO I 3, W104, G105, Y130, D131, Y132, D133, P134, L135, or any combination thereof) and the stability-enhancing alterations T19Y/R74L/C171A,
117T/T 19 Y/G25R/R74L/C 171 A, T 19 Y/G25K/S45 WR74L/G108 A/C 171 A/Gl 88R, 117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/G108C/C 171 A/Gl 88R, 117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/G108C/A126C/C 171 A/Gl 88R, or 117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/AW 104-
G105/G108C/C171A/G188R, can further include one or more stability-enhancing substitution mutations at positions functionally equivalent to those selected from one or more ofH16D, S20F, S20E, S20Y, S20I, K60G, I89R, S103T, E109L, V110L, Q115K, Q115Y, E116L, A126C, E138R, E138Q, E138A, D145R, D180H, S183V, A185R, R189K, and A192E, or any combination thereof, where the position number designation is functionally equivalent to the position in a wild-type APOBEC3A (SEQ ID NO:3)..
[000121] An ACD described herein can include a stability-enhancing alteration that is a deletion. In addition to the AN61-G68 and AW104-G105 deletions described herein, examples of other deletions are at positions functionally equivalent to AM1-L12 (the first 12 amino acids of human APOBECA3A (SEQ ID NO:3), AE36-G53, AN42, AN61-G68, AP86, AW104-G105, API 72, and AS187-N199, or any combination thereof, where the position number designation is functionally equivalent to the position in a wild-type APOBEC3 A (SEQ ID NO:3).
[000122] Some deletions can further include one or more ancillary substitution mutations. An ACD described herein can include the A61-68 deletion and the ancillary substitution mutation at (i) the position functionally equivalent to A59 where the mutation is to proline (A59P) or leucine (A59L), (ii) the position functionally equivalent to K60 where the mutation is to arginine (K60R), glutamic acid ((K60E), glutamine (K60Q), or glycine (K60G), or (iii) the position functionally equivalent to R69 (i.e., the position 69 prior to the deletion of 61-68) where the mutation is to tyrosine (R69Y), asparagine (R69N, histidine (R69H), aspartic acid (R69D), or leucine (R69L). Specific combinations of the A61-68 deletion and ancillary substitution mutations are A61-68/A59P/K60R, A61-68/A59L/R69Y, A61- 68/A59L/K60E/R69N, A61-68/A59P/K60E/R69H, A61-68/A59P/K60Q/R69H, A61- 68/A59L/R69N, A61-68/R69D, A61-68/A59L/K60R, and A61-68/A59P/K60G/R69L. An ACD described herein can include the Al 04- 105 deletion and the ancillary substitution mutation at the position functionally equivalent to Fl 02 where the mutation is to arginine (F102R), the substitution mutation at the position functionally equivalent to SI 03 where the mutation is to asparagine (S103N), or both substitution mutations F102R and S103N. In another embodiment, an ACD described herein can include both the A 104- 105 deletion and the Al-12 deletion. Additional embodiments include: an ACD having the A104-105 deletion, the Al-12 deletion, and the substitution mutation F102R; an ACD having the A104-105 deletion, the Al-12 deletion, and the substitution mutation S103N; and an ACD having the A104-105 deletion, the Al-12 deletion, and the substitution mutations F102R and S103N. [000123] Without intending to limit the particular combinations of mutations that increase selectivity and stability in an ACD having 5mC-selective deaminase activity, specific combinations encompassed by the present disclosure include, but are not limited to, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13 that further include (i) selectivity-enhancing substitution mutations Y130A, D131X, Y132X, D133X, and Y134W (e.g., the amino acids at 130, 131, 132, 133 and 134 are those shown in Table 2 (see Example 1), or (ii) selectivityenhancing substitution mutations Y130A, D131X, Y132X, D133X, and Y134W and one or more selectivity-enhancing alterations at positions functionally equivalent to Fl 02, SI 02, W104, and G105, (e.g., the amino acids at 102, 103, 104, 105, 130, 131, 132, 133 and 134 are those shown in Table 3), or (iii) selectivity-enhancing substitution mutations Y130A, D131X, Y132X, D133X, Y134W, and L135X and one or more selectivity-enhancing alterations at positions functionally equivalent to F102, S102, W104, and G105, (e.g., the amino acids at 102, 103, 104, 105, 130, 131, 132, 133, 134 and 135 are those shown in FIG. 8). In some embodiments, an ACD having enhanced 5mC-selective deaminase activity described herein (e.g., a selectivity-enhancing alteration at a position functionally equivalent to F102, SO I 3, W104, G105, Y130, D131, Y132, D133, P134, L135, or any combination thereof) can further include at least one of the following combinations of stabilizing mutations: R74L/C171A; R74L/T19Y; R74L/G25R; R74L/T19I/C171A;
R74L/T19L/C171A; R74L/T19Y/C171A/I17T; R74L/T19Y/C171A/G25A; R74L/T19Y/C171A/G25R/I17T; R74L /C171A/G25R/T19F; R74L/T19Y/C171A/G25D; R74L/T19Y/C171A/S45R; R74C/T19Y/C171A; R74L/C171A/T19F; R74L/C171A/T19W; R74L/T19Y/C171A/G108E; R74L/T19Y/C171A/G108D; R74L/T19Y/C171A/G108Q; R74L/T 19 Y/C 171 A/Gl 08 Y; R74L/T 19 Y/C 171 A/Gl 08H; R74L/T 19 Y/C 171 A/Gl 08L; R74L/T 19 Y/C 171 A/Gl 08K; R74L/T 19 Y/C 171 A/Gl 08R; R74L/T 19 Y/C 171 A/Al 26 V; R74L/T19Y/C171I; R74L/T19Y/C171A/G108M; R74L/T19Y/C171A/G108W;
R74L/T 19 Y/C 171 A/ A 126F ; R74L/T 19 Y/C 171 A/ A 1261; R74L/T 19 Y/C 171 A/ A 126L; R74L/T19Y/C171A; R74L/T19Y/C171A/S45W; R74L/T19Y/C171A/G25R;
R74L/T 19 Y/C 171 A/G25K; R74L/T 19 Y/C 171 A/Gl 88Q; R74L/T 19 Y/C 171 A/Gl 88 A; R74L/T 19 Y/C 171 A/Gl 88R; R74L/T 19 Y/C 171 A/Gl 08 A;
R74L/T19Y/C171A/G108A/G188R/G25K/S45W ; R74L/T19Y/C171A/G108C; R74L/T 19 Y/C 171 A/Gl 08 A/Gl 88R/G25K/S45 W/I 17T;
R74L/T 19 Y/C 171 A/Gl 08 A/Gl 88R/G25K/S45 W/117T/A59P/K60R/A61-68;
R74L/T 19 Y/C 171 A/Gl 08C/G188R/G25K/S45 W/Il 7T;
R74L/T 19 Y/C 171 A/Gl 08C/G188R/G25K/S45 W/117T/A59P/K60R/A61 -68/A126C;
R74L/T 19 Y/C 171 A/Gl 08C/G188R/G25K/S45 W/117T/A59P/K60R/A61-68;
T 19 Y/R74L/C 171 A; 117T/T 19 Y/G25R/R74L/C 171 A;
T 19 Y/G25K/S45 WR74L/G108 A/C 171 A/Gl 88R;
117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/G108C/C 171 A/Gl 88R;
117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/G108C/A126C/C 171 A/Gl 88R; or
117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/AW 104-
G105/G108C/C 171 A/Gl 88R.
[000124] In some embodiments, the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 130-134 a sequence selected from: AVAHW (SEQ ID NO:25), AHIYW (SEQ ID NO:26), AHEYW (SEQ ID NO:27), ARAYW ((SEQ ID NO:28), ANGFW ((SEQ ID NO:29), AKSHW (SEQ ID NO:30), AHLYW (SEQ ID NO:31), AHDFW (SEQ ID NO:32), AKTHW (SEQ ID NO:33), AHMIW (SEQ ID NO:34), AKMVW (SEQ ID NO: 35), AQAFW (SEQ ID NO: 36, ARVKW (SEQ ID NO: 37), AHRVW (SEQ ID NO:38), AKTYW (SEQ ID NO:39), AVAKW (SEQ ID NO:40), AKNFW (SEQ ID NO:41), AHARW (SEQ ID NO:42), AGPYW (SEQ ID NO:43), AKYPW (SEQ ID NO:44), AKPFW (SEQ ID NO:45), AKLIW (SEQ ID NO:46), AHVVW (SEQ ID NO:47), AGRFW (SEQ ID NO:48), AIAHW (SEQ ID NO:49), AKMYW (SEQ ID NO:50), among others in combination with one or more combinations of stability enhancing mutations selected from: at least one of the following combinations of stabilizing mutations: R74L/C 171 A/T 19 Y/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 88Q/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 88Q/G25K/S45W; R74L/C 171 A/T 19 Y/Gl 88 A/S45 W;
R74L/C 171 A/T 19 Y/Gl 88 A/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88R/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88R/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/G25K/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88 A/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G188Q/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G188 A/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188 A/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188 A/G25K/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188R/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188R/G25K/S45 W; T 19 Y/R74L/C 171 A;
117T/T 19 Y/G25R/R74L/C 171 A; T 19 Y/G25K/S45 WR74L/G108 A/C 171 A/Gl 88R;
117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/G108C/C 171 A/Gl 88R;
117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/G108C/A126C/C 171 A/Gl 88R; or 117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/AW 104-
G105/G108C/C171A/G188R In some aspects, the ACD further comprises at least one selectivity-enhancing alteration at a position functionally equivalent to Fl 02, S103, W104 and G105, for example, e g., FIMA (SEQ ID NO:404); HLTG (SEQ ID NO:242); QVPA (SEQ ID NO:405); EFNQ (SEQ ID NO:243); QTDH (SEQ ID NO:244); GLNG (SEQ ID NO:245); YLP (SEQ ID NO:406); EMFA (SEQ ID NO:407); YKQY (SEQ ID NO:246); YVRL (SEQ ID NO:247); YIKL (SEQ ID NO:248); FILA ALAHW (SEQ ID NO:408); QTQY (SEQ ID NO:249); KTNN (SEQ ID NO:250); AYDG (SEQ ID NO:251); HLTG (SEQ ID NO:242); QTMH (SEQ ID NO:252); YVQR (SEQ ID NO:253); ELYA (SEQ ID NO:409):YVEN (SEQ ID NO:254); RMLA (SEQ ID NO:410); QTVG (SEQ ID NO:255); YVQD (SEQ ID NO:256) QTQY (SEQ ID NO:249); QTYG (SEQ ID NO:257_);
YYRM AHGYW (SEQ ID NO:258): YLP A; STNN_(SEQ ID NO:259); QTRR (SEQ ID NO:260); AYEY (SEQ ID NO:261); YVND (SEQ ID NO:262); FSWG (SEQ ID NO:263); NIPA;YPFG (SEQ ID NO:264); QTLG_(SEQ ID NO:265); VYNA_; AFRA; ETKH (SEQ ID NO:266); YVEG (SEQ ID NO:267); QTMG_ (SEQ ID NO:268); AQHG_(SEQ ID NO:269); HLRG (SEQ ID NO:270); QTMH (SEQ ID NO:252); YYIA_ (SEQ ID NO:YYIA); HLYG_ (SEQ ID NO:271); YMAG (SEQ ID NO:272); YIWG_ (SEQ ID NO:273); IRQY (SEQ ID NO:274); AQMG (SEQ ID NO:275); FVPG (SEQ
ID NO: 276); and FVPA, FVPG (SEQ ID NO:276), FTDG (SEQ ID NO:277), FVDG (SEQ ID NO: 278), FVPAG (SEQ ID NO:279), FVPLG (SEQ ID NO:280), FVPPG(SEQ ID NO:280), FVPAAG(SEQ ID NO:282), FVPLAG (SEQ ID NO:283), FVPPAG (SEQ ID NO:284), FVPASG (SEQ ID NO:285), FVPLSG (SEQ ID NO:286), FVPPSG (SEQ ID NO:287), FVPAFG (SEQ ID NO:288), FVPLFG (SEQ ID NO:289), FVPPFG (SEQ ID NO:289), FVPAQG (SEQ ID NO:290), FVPLQG (SEQ ID NO:291), FVPPQG (SEQ ID NO:292), FVPADG (SEQ ID NO:293), FVPLDG (SEQ ID NO:294), FVPPDG (SEQ ID NO:295), FVPAKG (SEQ ID NO:296), FVPLKG: (SEQ ID NO:297), FVPPKG(SEQ ID NO:298), among others. Additionally, these ACD may have an additional substitution or deletion at one or more positions functionally equivalent to 102, 103, 104 and 105, for example, one or more substitution mutations, one or more deletions, or one or more insertions, or a combination thereof, of the amino acids at positions functionally equivalent to a phenylalanine at position 102 (Fl 02), a serine at position 103 (SI 03), a tryptophan at position 104 (W104), and/or a glycine at position 105 (G105) in a member of the APOB EC protein family, including a member of the APOBEC3 A subfamily (for instance, SEQ ID NO:3). In one embodiment, the substitution mutation at a position functionally equivalent to any one of F102, S103, W104, and/or G105 can be any amino acid. In one embodiment, the deletion can be 1, 2, 3, or 4 of the amino acids at a position functionally equivalent to any one of F102, S103, W104, and/or G105. In one embodiment, deletion can be at a position functionally equivalent to W104, G105, or both W104 and G105. Suitable sequences for the region from 102-105 can be selected from, e.g., SEQ ID NO:242-410, FIMA, QVPA, YLPA, EMFA, FIIA, ELYA, YLPA, NIP A, VYNA, AFRA, YYIA, FSDD, FQAG, FSAG, AVPG, FVPA, A VP A, YLIA, RYAA, LFWA, IRYA, YLIA, VLYA, RHYA, FMHA, QFPA, RRPA, RIYA, YYIA, RMLA, VLYA, RMLA, AVQA, QVWA, FVPA, FVDA, FSAA, KRAA , RRAA, FPPA, KRRA, VPAA, TDHA, QTDA, YLAA, LPAA, EMAA, MFAA, YVRA, IKLA, YIKA, QTQA, TQYA, KTNA, HLTA, LTGA, HTGA, HLGA, TGAA, HLAA, LTAA, HLTA, LTGA, QTMA, RKTA, KIYA, AYKA, MHAA, NQAA, RKAA, LMAA, LNAA , ACAA, QIAA, IMAA, FCAA, AVAA, QMAA, YDAA, LSAA, CEAA, VLAA, QQAA, RCAA, FCAA, SWAA, AGAA, RWAA, NQAA, QHAA, LAAA, MRAA, FNAA,FNAA, GCAA, SVAA, RMAA, ESAA, LMAA, MCAA, ICAA, GVAA, QHAA, among others.
[000125] In some embodiments, an ACD having enhanced 5mC-selective deaminase activity described herein(e.g., a selectivity-enhancing alteration at a position functionally equivalent to F102, S103, W104, G105, Y130, D131, Y132, D133, P134, L135, or any combination thereof) can further include at least one of the following combinations of stabilizing mutations: R74L/C171A/T19Y/G25K/S45W;
R74L/C 171 A/T 19 Y/Gl 88Q/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88Q/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 88 A/S45 W; R74L/C 171 A/T 19 Y/Gl 88 A/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 88R/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88R/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88 A/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G188Q/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188 A/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188 A/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188 A/G25K/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188R/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188R/G25K/S45 W; T 19 Y/R74L/C 171 A;
117T/T 19 Y/G25R/R74L/C 171 A; T 19 Y/G25K/S45 WR74L/G108 A/C 171 A/Gl 88R;
117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/G108C/C 171 A/Gl 88R;
117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/G108C/A126C/C 171 A/Gl 88R; or 117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/AW 104-
G105/G108C/C 171 A/Gl 88R.
[000126] Specific examples of ACDs of the present disclosure include SEQ ID NO:23, SEQ ID NO:24, and ACDs disclosed in Table 2, Table 3, and FIG 9 (SEQ ID NO:479-517, SEQ ID N0:520-605, SEQ ID NO:607-615, SEQ ID NO:635-954, SEQ ID NO:962-968). [000127] For example, specific examples of ACDs that have increased 5mC selectivity as compared with APOBEC3 A wildtype sequence include, for example, ACD comprising SEQ ID NO: 479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 489, 490, 491, 492, 493, 494, 495, 496, 497, 498, 499, 500, 501, 502, 503, 504, 505, 506, 507, 508, 509,
510, 511, 512, 513, 514, 515, 516, 517, 520, 521, 523, 524, 525, 526, 527, 528,
529, 530, 531, 532, 533, 534, 535, 536, 537, 538, 539, 540, 541, 543, 544, 545,
546, 547, 548, 549, 550, 551, 552, 553, 554, 555, 556, 557, 558, 559, 560, 561,
562, 563, 564, 565, 566, 567, 568, 569, 570, 571, 572, 573, 574, 575, 576, 577,
578, 579, 580, 581, 582, 583, 584, 585, 586, 587, 588, 589, 590, 591, 592, 593,
594, 595, 596, 597, 598, 599, 600, 601, 602, 603, 604, 605, 607, 608, 609, 610,
611, 612, 613, 614, 615, 635, 637, 638, 639, 640, 641, 642, 643, 644, 645, 646,
647, 648, 649, 650, 651, 652, 653, 654, 655, 656, 657, 658, 659, 660, 661, 662,
663, 664, 665, 666, 667, 668, 669, 670, 671, 672, 673, 674, 675, 676, 677, 678,
679, 680, 681, 682, 683, 684, 685, 686, 687, 688, 689, 690, 691, 692, 693, 694,
695, 696, 697, 698, 699, 700, 701, 702, 703, 704, 705, 706, 707, 708, 709, 710,
711, 712, 713, 714, 715, 716, 717, 718, 719, 720, 721, 722, 723, 724, 725, 726,
727, 728, 729, 730, 731, 732, 733, 734, 735, 736, 737, 738, 739, 740, 741, 742, 743, 744, 745, 746, 747, 748, 749, 750, 751, 752, 753, 754, 755, 756, 757, 758,
759, 760, 761, 762, 763, 764, 765, 766, 767, 768, 769, 770, 771, 772, 773, 774,
775, 776, 777, 778, 779, 780, 781, 782, 783, 784, 785, 786, 787, 788, 789, 790,
791, 792, 793, 794, 795, 796, 797, 798, 799, 800, 801, 802, 803, 804, 805, 806,
807, 808, 809, 810, 811, 812, 813, 814, 815, 816, 817, 818, 819, 820, 821, 822,
823, 824, 825, 826, 827, 828, 829, 830, 831, 832, 833, 834, 835, 836, 837, 838,
839, 840, 841, 842, 843, 844, 845, 846, 847, 848, 849, 850, 851, 852, 853, 854,
855, 856, 857, 858, 859, 860, 861, 862, 863, 864, 865, 866, 867, 868, 869, 870,
871, 872, 873, 874, 875, 876, 877, 878, 879, 880, 881, 882, 883, 884, 885, 886,
887, 888, 889, 890, 891, 892, 893, 894, 895, 896, 897, 898, 899, 900, 901, 902,
903, 904, 905, 906, 907, 908, 909, 910, 911, 912, 913, 914, 915, 916, 917, 918,
919, 920, 921, 922, 923, 924, 925, 926, 927, 928, 929, 930, 931, 932, 933, 934,
935, 936, 937, 938, 939, 940, 941, 942, 943, 944, 945, 946, 947, 948, 950, 951,
952, 953, 954, 955, 956, 957, 958, 959, 960, and 961. Additionally, ACDs that have increased 5mC selectivity as compared to their parental mutant strain (e.g., FIG. 9) include, for example, the ACD comprising SEQ ID NO:479, 493, 497, 498, 501, 504, 506, 507, 527, 529, 530, 533, 536, 539, 543, 545, 546, 547, 548, 549, 557, 558, 565, 566, 571, 572, 579, 580, 588, 602, 604, 605, 623, 633, 641, 643, 646, 649, 650, 652, 653, 655, 660, 661,662, 663, 664, 665 ,666, 667, 668, 669, 670, 671, 672, 673, 674, 675, 677, 680, 681, 682, 683, 684,
685, 686, 687, 689, 690, 691, 692, 693, 694, 696, 697, 698, 699, 700, 702, 703, 704, 705,
706, 707, 708, 710, 711, 712, 713, 714, 715, 716, 717, 718, 719, 720, 721, 722, 723, 724,
725, 726, 727, 728, 730, 731, 732, 733, 734, 735, 736, 737, 738, 739, 740, 741, 742, 744,
745, 746, 747, 748, 749, 750, 752, 753, 754, 755, 756, 759, 760,764, 765, 767, 768, 773, 778, 779, 780, 781, 782, 783, 784, 786, 787, 789, 790, 793, 795, 796, 797, 798, 799, 800, 802,
803, 806, 809, 810, 811, 814, 815, 817, 818, 819, 820, 821, 822, 823, 824, 827, 828, 829,
830, 831, 832, 834, 835, 836, 837, 838, 844, 851, 870, 878, 879, 881, 882, 888, 890, 896,
903, 904, 911, 918, 920, 921, 929, 933, 936, 945, 950, 955, 956, 957, 958, 959, 960, and.
961.
[000128] In some embodiments, the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 130-134, or functionally equivalent positions, a sequence selected from: AVAHW (SEQ ID NO:25) in combination with one or more combinations of stability enhancing mutations selected from Hl IX, L12X, D14X, H16X, I17X, T19X, S20X, N21X, G25X, I26X, G27X, R28X, H29X, T31X, C34X, E38X, R39X, D41X, N42X, S45X, K47X, M48X, H51X, F54X, H56X, N57X, A59X, K60X, L62X, Y67X, L73X, R74X, D77X, L78X, V79X, P80X, S81X, D85X, A87X, I89X, Y90X, R91X, T93X, W94X, I96X, S97X, C101X, F102X, S1O3X, W104X, G105X, C106X, A107X, G108X, E109X, V110X, R111X, Al 12X, LI 14X, QI 15X, El 16X, N117X, T118X, Hl 19X, V120X, L122X, R123X, A126X, R128X, L135X, Y136X, E138X, A139X, Q141X, M142X, D145X, A146X, A148X, D156X, E157X, K159X, C161X, D163X, T164X, D167X, Q169X, C171X, D177X, D180X, H182X, S183X, Q184X, A185X, L186X, S187X, G188X, R189X, A192X, N196X a deletion selected from AM1-L12, AE36-G53, AN61-G68, AW104-G105 , AQ195-N199 , AI26-G27, or any combination thereof, where the position number designation is functionally equivalent to the position in a wild-type APOBEC3A (SEQ ID NO:3) and X is an amino acid substitution different from the wild-type amino acid at that position. In some aspects, the ACD further comprises at least one selectivity-enhancing alteration at a position functionally equivalent to F102, S103, W104 and G105, for example, e.g., FVPG, FTDG, FVDG, FVPAG, FVPLG, FVPPG, FVPAAG, FVPLAG, FVPPAG, FVPASG, FVPLSG, FVPPSG, FVPAFG, FVPLFG, FVPPFG, FVPAQG, FVPLQG, FVPPQG, FVPADG, FVPLDG, FVPPDG, FVPAKG, FVPLKG, FVPPKG among others.
[000129] In some embodiments, the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 130-134, or functionally equivalent positions, a sequence selected from: AVAHW (SEQ ID NO:25 in combination with one or more combinations of stability enhancing mutations selected from: at least one of the following combinations of stabilizing mutations: R74L/C 171 A/T 19 Y/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 88Q/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88Q/G25K/S45W; R74L/C 171 A/T 19 Y/Gl 88 A/S45 W;
R74L/C 171 A/T 19 Y/Gl 88 A/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88R/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88R/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/G25K/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88 A/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G188Q/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G188 A/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188 A/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188 A/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G188R/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G188R/G25K/S45 W; T 19 Y/R74L/C 171 A;
117T/T 19 Y/G25R/R74L/C 171 A; T 19 Y/G25K/S45 WR74L/G108 A/C 171 A/Gl 88R;
117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/G108C/C 171 A/Gl 88R;
117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/G108C/A126C/C 171 A/Gl 88R; or 117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/AW 104- G105/G108C/C171A/G188R. In some aspects, the ACD further comprises at least one selectivity-enhancing alteration at a position functionally equivalent to Fl 02, S103, W104 and G105, for example, e g., FVPG, FTDG, FVDG, FVPAG, FVPLG, FVPPG, FVPAAG, FVPLAG, FVPPAG, FVPASG, FVPLSG, FVPPSG, FVPAFG, FVPLFG, FVPPFG, FVPAQG, FVPLQG, FVPPQG, FVPADG, FVPLDG, FVPPDG, FVPAKG, FVPLKG, FVPPKG among others.
[000130] In some embodiments, the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 130-134, or functionally equivalent positions, a sequence selected from: AHIYW (SEQ ID NO:26) in combination with one or more combinations of stability enhancing mutations selected from Hl IX, L12X, D14X, H16X, I17X, T19X, S20X, N21X, G25X, I26X, G27X, R28X, H29X, T31X, C34X, E38X, R39X, D41X, N42X, S45X, K47X, M48X, H51X, F54X, H56X, N57X, A59X, K60X, L62X, Y67X, L73X, R74X, D77X, L78X, V79X, P80X, S81X, D85X, A87X, I89X, Y90X, R91X, T93X, W94X, I96X, S97X, C101X, F102X, S103X, W104X, G105X, C106X, A107X, G108X, E109X, V110X, R111X, Al 12X, LI 14X, QI 15X, El 16X, N117X, T118X, Hl 19X, V120X, L122X, R123X, A126X, R128X, L135X, Y136X, E138X, A139X, Q141X, M142X, D145X, A146X, A148X, D156X, E157X, K159X, C161X, D163X, T164X, D167X, Q169X, C171X, D177X, D180X, H182X, S183X, Q184X, A185X, L186X, S187X, G188X, R189X, A192X, N196X a deletion selected from AM1-L12, AE36-G53, AN61-G68, AW104-G105 , AQ195-N199 , AI26-G27, or any combination thereof, where the position number designation is functionally equivalent to the position in a wild-type APOBEC3A (SEQ ID NO:3) and X is an amino acid substitution different from the wild-type amino acid at that position. In some aspects, the ACD further comprises at least one selectivity-enhancing alteration at a position functionally equivalent to F102, S103, W104 and G105, for example, e.g., FVPG, FTDG, FVDG, FVPAG, FVPLG, FVPPG, FVPAAG, FVPLAG, FVPPAG, FVPASG, FVPLSG, FVPPSG, FVPAFG, FVPLFG, FVPPFG, FVPAQG, FVPLQG, FVPPQG, FVPADG, FVPLDG, FVPPDG, FVPAKG, FVPLKG, FVPPKG among others.
[000131] In some embodiments, the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 130-134, or functionally equivalent positions, a sequence selected from AHIYW (SEQ ID NO:26) in combination with one or more combinations of stability enhancing mutations selected from: at least one of the following combinations of stabilizing mutations: R74L/C 171 A/T 19 Y/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 88Q/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88Q/G25K/S45W; R74L/C 171 A/T 19 Y/Gl 88 A/S45 W;
R74L/C 171 A/T 19 Y/Gl 88 A/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88R/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88R/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/G25K/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88 A/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G188Q/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G188 A/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188 A/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188 A/G25K/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188R/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188R/G25K/S45 W; T 19 Y/R74L/C 171 A;
117T/T 19 Y/G25R/R74L/C 171 A; T 19 Y/G25K/S45 WR74L/G108 A/C 171 A/Gl 88R;
117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/G108C/C 171 A/Gl 88R;
117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/G108C/A126C/C 171 A/Gl 88R; or
117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/AW 104-
G105/G108C/C171A/G188R. In some aspects, the ACD further comprises at least one selectivity-enhancing alteration at a position functionally equivalent to Fl 02, S103, W104 and G105, for example, e g., FVPG, FTDG, FVDG, FVPAG, FVPLG, FVPPG, FVPAAG, FVPLAG, FVPPAG, FVPASG, FVPLSG, FVPPSG, FVPAFG, FVPLFG, FVPPFG, FVPAQG, FVPLQG, FVPPQG, FVPADG, FVPLDG, FVPPDG, FVPAKG, FVPLKG, FVPPKG among others.
[000132] In some embodiments, the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 130-134, or functionally equivalent positions, a sequence selected from: AHEYW (SEQ ID NO:27) in combination with one or more combinations of stability enhancing mutations selected from Hl IX, L12X, D14X, H16X, I17X, T19X, S20X, N21X, G25X, I26X, G27X, R28X, H29X, T31X, C34X, E38X, R39X, D41X, N42X, S45X, K47X, M48X, H51X, F54X, H56X, N57X, A59X, K60X, L62X, Y67X, L73X, R74X, D77X, L78X, V79X, P80X, S81X, D85X, A87X, I89X, Y90X, R91X, T93X, W94X, I96X, S97X, C101X, F102X, S1O3X, W104X, G105X, C106X, A107X, G108X, E109X, V110X, R111X, Al 12X, LI 14X, QI 15X, El 16X, N117X, T118X, Hl 19X, V120X, L122X, R123X, A126X, R128X, L135X, Y136X, E138X, A139X, Q141X, M142X, D145X, A146X, A148X, D156X, E157X, K159X, C161X, D163X, T164X, D167X, Q169X, C171X, D177X, D180X, H182X, S183X, Q184X, A185X, L186X, S187X, G188X, R189X, A192X, N196X a deletion selected from AM1-L12, AE36-G53, AN61-G68, AW104-G105 , AQ195-N199 , AI26-G27, or any combination thereof, where the position number designation is functionally equivalent to the position in a wild-type APOBEC3A (SEQ ID NO:3) and X is an amino acid substitution different from the wild-type amino acid at that position. In some aspects, the ACD further comprises at least one selectivity-enhancing alteration at a position functionally equivalent to F102, S103, W104 and G105, for example, e.g., FVPG, FTDG, FVDG, FVPAG, FVPLG, FVPPG, FVPAAG, FVPLAG, FVPPAG, FVPASG, FVPLSG, FVPPSG, FVPAFG, FVPLFG, FVPPFG, FVPAQG, FVPLQG, FVPPQG, FVPADG, FVPLDG, FVPPDG, FVPAKG, FVPLKG, FVPPKG among others.
[000133] In some embodiments, the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 130-134, or functionally equivalent positions, a sequence selected from: AHEYW (SEQ ID NO:27) in combination with one or more combinations of stability enhancing mutations selected from: at least one of the following combinations of stabilizing mutations: R74L/C 171 A/T 19 Y/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 88Q/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88Q/G25K/S45W; R74L/C 171 A/T 19 Y/Gl 88 A/S45 W;
R74L/C 171 A/T 19 Y/Gl 88 A/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88R/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88R/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/G25K/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88 A/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G188Q/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G188 A/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188 A/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188 A/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G188R/G25R/S45 W; IP-281 l-PCT/531.2811 WO01
R74L/C 171 A/T 19 Y/Gl 08C/G188R/G25K/S45 W; T 19 Y/R74L/C 171 A;
117T/T 19 Y/G25R/R74L/C 171 A; T 19 Y/G25K/S45 WR74L/G108 A/C 171 A/Gl 88R;
117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/G108C/C 171 A/Gl 88R;
117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/G108C/A126C/C 171 A/Gl 88R; or 117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/AW 104- G105/G108C/C171A/G188R. In some aspects, the ACD further comprises at least one selectivity-enhancing alteration at a position functionally equivalent to Fl 02, S103, W104 and G105, for example, e g., FVPG, FTDG, FVDG, FVPAG, FVPLG, FVPPG, FVPAAG, FVPLAG, FVPPAG, FVPASG, FVPLSG, FVPPSG, FVPAFG, FVPLFG, FVPPFG, FVPAQG, FVPLQG, FVPPQG, FVPADG, FVPLDG, FVPPDG, FVPAKG, FVPLKG, FVPPKG among others.
[000134] In some embodiments, the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 130-134, or functionally equivalent positions, a sequence selected from ARAYW ((SEQ ID NO:28) in combination with one or more combinations of stability enhancing mutations selected from Hl IX, L12X, D14X, H16X, I17X, T19X, S20X, N21X, G25X, I26X, G27X, R28X, H29X, T31X, C34X, E38X, R39X, D41X, N42X, S45X, K47X, M48X, H51X, F54X, H56X, N57X, A59X, K60X, L62X, Y67X, L73X, R74X, D77X, L78X, V79X, P80X, S81X, D85X, A87X, I89X, Y90X, R91X, T93X, W94X, I96X, S97X, C101X, F102X, S103X, W104X, G105X, C106X, A107X, G108X, E109X, V110X, R111X, Al 12X, LI 14X, QI 15X, El 16X, N117X, T118X, Hl 19X, V120X, L122X, R123X, A126X, R128X, L135X, Y136X, E138X, A139X, Q141X, M142X, D145X, A146X, A148X, D156X, E157X, K159X, C161X, D163X, T164X, D167X, Q169X, C171X, D177X, D180X, H182X, S183X, Q184X, A185X, L186X, S187X, G188X, R189X, A192X, N196X a deletion selected from AM1-L12, AE36-G53, AN61-G68, AW104-G105 , AQ195-N199 , AI26-G27, or any combination thereof, where the position number designation is functionally equivalent to the position in a wild-type APOBEC3A (SEQ ID NO:3) and X is an amino acid substitution different from the wild-type amino acid at that position. In some aspects, the ACD further comprises at least one selectivity-enhancing alteration at a position functionally equivalent to F102, S103, W104 and G105, for example, e.g., FVPG, FTDG, FVDG, FVPAG, FVPLG, FVPPG, FVPAAG, FVPLAG, FVPPAG, FVPASG, FVPLSG, FVPPSG, FVPAFG, FVPLFG, FVPPFG, FVPAQG, FVPLQG, FVPPQG, FVPADG, FVPLDG, FVPPDG, FVPAKG, FVPLKG, FVPPKG among others.
[000135] In some embodiments, the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 130-134, or functionally equivalent positions, a sequence selected from: ARAYW ((SEQ ID NO:28) in combination with one or more combinations of stability enhancing mutations selected from: at least one of the following combinations of stabilizing mutations: R74L/C 171 A/T 19 Y/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 88Q/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88Q/G25K/S45W; R74L/C 171 A/T 19 Y/Gl 88 A/S45 W;
R74L/C 171 A/T 19 Y/Gl 88 A/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88R/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88R/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/G25K/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88 A/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G188Q/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188 A/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188 A/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188 A/G25K/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188R/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188R/G25K/S45 W; T 19 Y/R74L/C 171 A;
117T/T 19 Y/G25R/R74L/C 171 A; T 19 Y/G25K/S45 WR74L/G108 A/C 171 A/Gl 88R;
117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/G108C/C 171 A/Gl 88R;
117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/G108C/A126C/C 171 A/Gl 88R; or
117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/AW 104-
G105/G108C/C171A/G188R. In some aspects, the ACD further comprises at least one selectivity-enhancing alteration at a position functionally equivalent to Fl 02, S103, W104 and G105, for example, e g., FVPG, FTDG, FVDG, FVPAG, FVPLG, FVPPG, FVPAAG, FVPLAG, FVPPAG, FVPASG, FVPLSG, FVPPSG, FVPAFG, FVPLFG, FVPPFG, FVPAQG, FVPLQG, FVPPQG, FVPADG, FVPLDG, FVPPDG, FVPAKG, FVPLKG, FVPPKG among others.
[000136] In some embodiments, the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 130-134, or functionally equivalent positions, a sequence selected from ANGFW ((SEQ ID NO:29) in combination with one or more combinations of stability enhancing mutations selected from Hl IX, L12X, D14X, H16X, I17X, T19X, S20X, N21X, G25X, I26X, G27X, R28X, H29X, T31X, C34X, E38X, R39X, D41X, N42X, S45X, K47X, M48X, H51X, F54X, H56X, N57X, A59X, K60X, L62X, Y67X, L73X, R74X, D77X, L78X, V79X, P80X, S81X, D85X, A87X, I89X, Y90X, R91X, T93X, W94X, I96X, S97X, C101X, F102X, S103X, W104X, G105X, C106X, A107X, G108X, E109X, V110X, R111X, Al 12X, LI 14X, QI 15X, El 16X, N117X, T118X, Hl 19X, V120X, L122X, R123X, A126X, R128X, L135X, Y136X, E138X, A139X, Q141X, M142X, D145X, A146X, A148X, D156X, E157X, K159X, C161X, D163X, T164X, D167X, Q169X, C171X, D177X, D180X, H182X, S183X, Q184X, A185X, L186X, S187X, G188X, R189X, A192X, N196X a deletion selected from AM1-L12, AE36-G53, AN61-G68, AW104-G105 , AQ195-N199 , AI26-G27, or any combination thereof, where the position number designation is functionally equivalent to the position in a wild-type APOBEC3A (SEQ ID NO:3) and X is an amino acid substitution different from the wild-type amino acid at that position. In some aspects, the ACD further comprises at least one selectivity-enhancing alteration at a position functionally equivalent to F102, S103, W104 and G105, for example, e.g., FVPG, FTDG, FVDG, FVPAG, FVPLG, FVPPG, FVPAAG, FVPLAG, FVPPAG, FVPASG, FVPLSG, FVPPSG, FVPAFG, FVPLFG, FVPPFG, FVPAQG, FVPLQG, FVPPQG, FVPADG, FVPLDG, FVPPDG, FVPAKG, FVPLKG, FVPPKG among others.
[000137] In some embodiments, the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 130-134, or functionally equivalent positions, a sequence selected from: ANGFW ((SEQ ID NO:29) in combination with one or more combinations of stability enhancing mutations selected from: at least one of the following combinations of stabilizing mutations: R74L/C 171 A/T 19 Y/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 88Q/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88Q/G25K/S45W; R74L/C 171 A/T 19 Y/Gl 88 A/S45 W;
R74L/C 171 A/T 19 Y/Gl 88 A/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88R/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88R/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/G25K/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88 A/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G188Q/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G188 A/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188 A/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188 A/G25K/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188R/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G188R/G25K/S45 W; T 19 Y/R74L/C 171 A;
117T/T 19 Y/G25R/R74L/C 171 A; T 19 Y/G25K/S45 WR74L/G108 A/C 171 A/Gl 88R;
117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/G108C/C 171 A/Gl 88R;
117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/G108C/A126C/C 171 A/Gl 88R; or 117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/AW 104- G105/G108C/C171A/G188R. In some aspects, the ACD further comprises at least one selectivity-enhancing alteration at a position functionally equivalent to Fl 02, S103, W104 and G105, for example, e g., FVPG, FTDG, FVDG, FVPAG, FVPLG, FVPPG, FVPAAG, FVPLAG, FVPPAG, FVPASG, FVPLSG, FVPPSG, FVPAFG, FVPLFG, FVPPFG, FVPAQG, FVPLQG, FVPPQG, FVPADG, FVPLDG, FVPPDG, FVPAKG, FVPLKG, FVPPKG among others.
[000138] In some embodiments, the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 130-134, or functionally equivalent positions, a sequence selected from: AKSHW (SEQ ID NO: 30) in combination with one or more combinations of stability enhancing mutations selected from Hl IX, L12X, D14X, H16X, I17X, T19X, S20X, N21X, G25X, I26X, G27X, R28X, H29X, T31X, C34X, E38X, R39X, D41X, N42X, S45X, K47X, M48X, H51X, F54X, H56X, N57X, A59X, K60X, L62X, Y67X, L73X, R74X, D77X, L78X, V79X, P80X, S81X, D85X, A87X, I89X, Y90X, R91X, T93X, W94X, I96X, S97X, C101X, F102X, S103X, W104X, G105X, C106X, A107X, G108X, E109X, V110X, R111X, Al 12X, LI 14X, QI 15X, El 16X, N117X, T118X, Hl 19X, V120X, L122X, R123X, A126X, R128X, L135X, Y136X, E138X, A139X, Q141X, M142X, D145X, A146X, A148X, D156X, E157X, K159X, C161X, D163X, T164X, D167X, Q169X, C171X, D177X, D180X, H182X, S183X, Q184X, A185X, L186X, S187X, G188X, R189X, A192X, N196X a deletion selected from AM1-L12, AE36-G53, AN61-G68, AW104-G105 , AQ195-N199 , AI26-G27, or any combination thereof, where the position number designation is functionally equivalent to the position in a wild-type APOBEC3A (SEQ ID NO:3) and X is an amino acid substitution different from the wild-type amino acid at that position. In some aspects, the ACD further comprises at least one selectivity-enhancing alteration at a position functionally equivalent to F102, S103, W104 and G105, for example, e.g., FVPG, FTDG, FVDG, FVPAG, FVPLG, FVPPG, FVPAAG, FVPLAG, FVPPAG, FVPASG, FVPLSG, FVPPSG, FVPAFG, FVPLFG, FVPPFG, FVPAQG, FVPLQG, FVPPQG, FVPADG, FVPLDG, FVPPDG, FVPAKG, FVPLKG, FVPPKG among others.
[000139] In some embodiments, the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 130-134, or functionally equivalent positions, a sequence selected from: AKSHW (SEQ ID NO: 30) in combination with one or more combinations of stability enhancing mutations selected from: at least one of the following combinations of stabilizing mutations: R74L/C 171 A/T 19 Y/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 88Q/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88Q/G25K/S45W; R74L/C 171 A/T 19 Y/Gl 88 A/S45 W;
R74L/C 171 A/T 19 Y/Gl 88 A/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88R/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88R/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/G25K/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88 A/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G188Q/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188 A/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188 A/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188 A/G25K/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188R/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188R/G25K/S45 W; T 19 Y/R74L/C 171 A;
117T/T 19 Y/G25R/R74L/C 171 A; T 19 Y/G25K/S45 WR74L/G108 A/C 171 A/Gl 88R;
117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/G108C/C 171 A/Gl 88R;
117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/G108C/A126C/C 171 A/Gl 88R; or
117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/AW 104-
G105/G108C/C171A/G188R. In some aspects, the ACD further comprises at least one selectivity-enhancing alteration at a position functionally equivalent to Fl 02, S103, W104 and G105, for example, e g., FVPG, FTDG, FVDG, FVPAG, FVPLG, FVPPG, FVPAAG, FVPLAG, FVPPAG, FVPASG, FVPLSG, FVPPSG, FVPAFG, FVPLFG, FVPPFG, FVPAQG, FVPLQG, FVPPQG, FVPADG, FVPLDG, FVPPDG, FVPAKG, FVPLKG, FVPPKG among others.
[000140] In some embodiments, the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 130-134, or functionally equivalent positions, a sequence selected from AHLYW (SEQ ID NO: 31) in combination with one or more combinations of stability enhancing mutations selected from Hl IX, L12X, D14X, H16X, I17X, T19X, S20X, N21X, G25X, I26X, G27X, R28X, H29X, T31X, C34X, E38X, R39X, D41X, N42X, S45X, K47X, M48X, H51X, F54X, H56X, N57X, A59X, K60X, L62X, Y67X, L73X, R74X, D77X, L78X, V79X, P80X, S81X, D85X, A87X, I89X, Y90X, R91X, T93X, W94X, I96X, S97X, C101X, F102X, S1O3X, W104X, G105X, C106X, A107X, G108X, E109X, V110X, R111X, Al 12X, LI 14X, QI 15X, El 16X, N117X, T118X, Hl 19X, V120X, L122X, R123X, A126X, R128X, L135X, Y136X, E138X, A139X, Q141X, M142X, D145X, A146X, A148X, D156X, E157X, K159X, C161X, D163X, T164X, D167X, Q169X, C171X, D177X, D180X, H182X, S183X, Q184X, A185X, L186X, S187X, G188X, R189X, A192X, N196X a deletion selected from AM1-L12, AE36-G53, AN61-G68, AW104-G105 , AQ195-N199 , AI26-G27, or any combination thereof, where the position number designation is functionally equivalent to the position in a wild-type APOBEC3A (SEQ ID NO:3) and X is an amino acid substitution different from the wild-type amino acid at that position. In some aspects, the ACD further comprises at least one selectivity-enhancing alteration at a position functionally equivalent to F102, S103, W104 and G105, for example, e.g., FVPG, FTDG, FVDG, FVPAG, FVPLG, FVPPG, FVPAAG, FVPLAG, FVPPAG, FVPASG, FVPLSG, FVPPSG, FVPAFG, FVPLFG, FVPPFG, FVPAQG, FVPLQG, FVPPQG, FVPADG, FVPLDG, FVPPDG, FVPAKG, FVPLKG, FVPPKG among others.
[000141] In some embodiments, the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 130-134, or functionally equivalent positions, a sequence selected from: AHLYW (SEQ ID NO:31) in combination with one or more combinations of stability enhancing mutations selected from: at least one of the following combinations of stabilizing mutations: R74L/C 171 A/T 19 Y/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 88Q/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88Q/G25K/S45W; R74L/C 171 A/T 19 Y/Gl 88 A/S45 W;
R74L/C 171 A/T 19 Y/Gl 88 A/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88R/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88R/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/G25K/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88 A/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G188Q/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G188 A/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188 A/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188 A/G25K/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188R/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G188R/G25K/S45 W; T 19 Y/R74L/C 171 A;
117T/T 19 Y/G25R/R74L/C 171 A; T 19 Y/G25K/S45 WR74L/G108 A/C 171 A/Gl 88R;
117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/G108C/C 171 A/Gl 88R;
117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/G108C/A126C/C 171 A/Gl 88R; or 117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/AW 104- G105/G108C/C171A/G188R. In some aspects, the ACD further comprises at least one selectivity-enhancing alteration at a position functionally equivalent to Fl 02, S103, W104 and G105, for example, e g., FVPG, FTDG, FVDG, FVPAG, FVPLG, FVPPG, FVPAAG, FVPLAG, FVPPAG, FVPASG, FVPLSG, FVPPSG, FVPAFG, FVPLFG, FVPPFG, FVPAQG, FVPLQG, FVPPQG, FVPADG, FVPLDG, FVPPDG, FVPAKG, FVPLKG, FVPPKG among others.
[000142] In some embodiments, the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 130-134, or functionally equivalent positions, a sequence selected from: AHDFW (SEQ ID NO:32) in combination with one or more combinations of stability enhancing mutations selected from Hl IX, L12X, D14X, H16X, I17X, T19X, S20X, N21X, G25X, I26X, G27X, R28X, H29X, T31X, C34X, E38X, R39X, D41X, N42X, S45X, K47X, M48X, H51X, F54X, H56X, N57X, A59X, K60X, L62X, Y67X, L73X, R74X, D77X, L78X, V79X, P80X, S81X, D85X, A87X, I89X, Y90X, R91X, T93X, W94X, I96X, S97X, C101X, F102X, S103X, W104X, G105X, C106X, A107X, G108X, E109X, V110X, R111X, Al 12X, LI 14X, QI 15X, El 16X, N117X, T118X, Hl 19X, V120X, L122X, R123X, A126X, R128X, L135X, Y136X, E138X, A139X, Q141X, M142X, D145X, A146X, A148X, D156X, E157X, K159X, C161X, D163X, T164X, D167X, Q169X, C171X, D177X, D180X, H182X, S183X, Q184X, A185X, L186X, S187X, G188X, R189X, A192X, N196X a deletion selected from AM1-L12, AE36-G53, AN61-G68, AW104-G105 , AQ195-N199 , AI26-G27, or any combination thereof, where the position number designation is functionally equivalent to the position in a wild-type APOBEC3A (SEQ ID NO:3) and X is an amino acid substitution different from the wild-type amino acid at that position. In some aspects, the ACD further comprises at least one selectivity-enhancing alteration at a position functionally equivalent to F102, S103, W104 and G105, for example, e.g., FVPG, FTDG, FVDG, FVPAG, FVPLG, FVPPG, FVPAAG, FVPLAG, FVPPAG, FVPASG, FVPLSG, FVPPSG, FVPAFG, FVPLFG, FVPPFG, FVPAQG, FVPLQG, FVPPQG, FVPADG, FVPLDG, FVPPDG, FVPAKG, FVPLKG, FVPPKG among others.
[000143] In some embodiments, the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 130-134, or functionally equivalent positions, a sequence selected from: AHDFW (SEQ ID NO:32), in combination with one or more combinations of stability enhancing mutations selected from: at least one of the following combinations of stabilizing mutations: R74L/C 171 A/T 19 Y/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 88Q/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88Q/G25K/S45W; R74L/C 171 A/T 19 Y/Gl 88 A/S45 W;
R74L/C 171 A/T 19 Y/Gl 88 A/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88R/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88R/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/G25K/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88 A/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G188Q/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188 A/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188 A/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188 A/G25K/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188R/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188R/G25K/S45 W; T 19 Y/R74L/C 171 A;
117T/T 19 Y/G25R/R74L/C 171 A; T 19 Y/G25K/S45 WR74L/G108 A/C 171 A/Gl 88R;
117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/G108C/C 171 A/Gl 88R;
117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/G108C/A126C/C 171 A/Gl 88R; or
117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/AW 104-
G105/G108C/C171A/G188R. In some aspects, the ACD further comprises at least one selectivity-enhancing alteration at a position functionally equivalent to Fl 02, S103, W104 and G105, for example, e g., FVPG, FTDG, FVDG, FVPAG, FVPLG, FVPPG, FVPAAG, FVPLAG, FVPPAG, FVPASG, FVPLSG, FVPPSG, FVPAFG, FVPLFG, FVPPFG, FVPAQG, FVPLQG, FVPPQG, FVPADG, FVPLDG, FVPPDG, FVPAKG, FVPLKG, FVPPKG among others.
[000144] In some embodiments, the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 130-134, or functionally equivalent positions, a sequence selected from: AKTHW (SEQ ID NO:33), in combination with one or more combinations of stability enhancing mutations selected from Hl IX, L12X, D14X, H16X, I17X, T19X, S20X, N21X, G25X, I26X, G27X, R28X, H29X, T31X, C34X, E38X, R39X, D41X, N42X, S45X, K47X, M48X, H51X, F54X, H56X, N57X, A59X, K60X, L62X, Y67X, L73X, R74X, D77X, L78X, V79X, P80X, S81X, D85X, A87X, I89X, Y90X, R91X, T93X, W94X, I96X, S97X, C101X, F102X, S1O3X, W104X, G105X, C106X, A107X, G108X, E109X, V110X, R111X, Al 12X, LI 14X, QI 15X, El 16X, N117X, T118X, Hl 19X, V120X, L122X, R123X, A126X, R128X, L135X, Y136X, E138X, A139X, Q141X, M142X, D145X, A146X, A148X, D156X, E157X, K159X, C161X, D163X, T164X, D167X, Q169X, C171X, D177X, D180X, H182X, S183X, Q184X, A185X, L186X, S187X, G188X, R189X, A192X, N196X a deletion selected from AM1-L12, AE36-G53, AN61-G68, AW104-G105 , AQ195-N199 , AI26-G27, or any combination thereof, where the position number designation is functionally equivalent to the position in a wild-type APOBEC3A (SEQ ID NO:3) and X is an amino acid substitution different from the wild-type amino acid at that position. In some aspects, the ACD further comprises at least one selectivity-enhancing alteration at a position functionally equivalent to F102, S103, W104 and G105, for example, e.g., FVPG, FTDG, FVDG, FVPAG, FVPLG, FVPPG, FVPAAG, FVPLAG, FVPPAG, FVPASG, FVPLSG, FVPPSG, FVPAFG, FVPLFG, FVPPFG, FVPAQG, FVPLQG, FVPPQG, FVPADG, FVPLDG, FVPPDG, FVPAKG, FVPLKG, FVPPKG among others.
[000145] In some embodiments, the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 130-134, or functionally equivalent positions, a sequence selected from: AKTHW (SEQ ID NO:33), in combination with one or more combinations of stability enhancing mutations selected from: at least one of the following combinations of stabilizing mutations: R74L/C 171 A/T 19 Y/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 88Q/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88Q/G25K/S45W; R74L/C 171 A/T 19 Y/Gl 88 A/S45 W;
R74L/C 171 A/T 19 Y/Gl 88 A/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88R/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88R/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/G25K/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88 A/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G188Q/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G188 A/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188 A/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188 A/G25K/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188R/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G188R/G25K/S45 W; T 19 Y/R74L/C 171 A;
117T/T 19 Y/G25R/R74L/C 171 A; T 19 Y/G25K/S45 WR74L/G108 A/C 171 A/Gl 88R;
117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/G108C/C 171 A/Gl 88R;
117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/G108C/A126C/C 171 A/Gl 88R; or 117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/AW 104- G105/G108C/C171A/G188R In some aspects, the ACD further comprises at least one selectivity-enhancing alteration at a position functionally equivalent to Fl 02, S103, W104 and G105, for example, e g., FVPG, FTDG, FVDG, FVPAG, FVPLG, FVPPG, FVPAAG, FVPLAG, FVPPAG, FVPASG, FVPLSG, FVPPSG, FVPAFG, FVPLFG, FVPPFG, FVPAQG, FVPLQG, FVPPQG, FVPADG, FVPLDG, FVPPDG, FVPAKG, FVPLKG, FVPPKG among others.
[000146] In some embodiments, the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 130-134, or functionally equivalent positions, a sequence selected from: AHMIW (SEQ ID NO:34), in combination with one or more combinations of stability enhancing mutations selected from Hl IX, L12X, D14X, H16X, I17X, T19X, S20X, N21X, G25X, I26X, G27X, R28X, H29X, T31X, C34X, E38X, R39X, D41X, N42X, S45X, K47X, M48X, H51X, F54X, H56X, N57X, A59X, K60X, L62X, Y67X, L73X, R74X, D77X, L78X, V79X, P80X, S81X, D85X, A87X, I89X, Y90X, R91X, T93X, W94X, I96X, S97X, C101X, F102X, S103X, W104X, G105X, C106X, A107X, G108X, E109X, V110X, R111X, Al 12X, LI 14X, QI 15X, El 16X, N117X, T118X, Hl 19X, V120X, L122X, R123X, A126X, R128X, L135X, Y136X, E138X, A139X, Q141X, M142X, D145X, A146X, A148X, D156X, E157X, K159X, C161X, D163X, T164X, D167X, Q169X, C171X, D177X, D180X, H182X, S183X, Q184X, A185X, L186X, S187X, G188X, R189X, A192X, N196X a deletion selected from AM1-L12, AE36-G53, AN61-G68, AW104-G105 , AQ195-N199 , AI26-G27, or any combination thereof, where the position number designation is functionally equivalent to the position in a wild-type APOBEC3A (SEQ ID NO:3) and X is an amino acid substitution different from the wild-type amino acid at that position. In some aspects, the ACD further comprises at least one selectivity-enhancing alteration at a position functionally equivalent to F102, S103, W104 and G105, for example, e.g., FVPG, FTDG, FVDG, FVPAG, FVPLG, FVPPG, FVPAAG, FVPLAG, FVPPAG, FVPASG, FVPLSG, FVPPSG, FVPAFG, FVPLFG, FVPPFG, FVPAQG, FVPLQG, FVPPQG, FVPADG, FVPLDG, FVPPDG, FVPAKG, FVPLKG, FVPPKG among others.
[000147] In some embodiments, the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 130-134, or functionally equivalent positions, a sequence selected from: AHMIW (SEQ ID NO:34), in combination with one or more combinations of stability enhancing mutations selected from: at least one of the following combinations of stabilizing mutations: R74L/C 171 A/T 19 Y/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 88Q/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88Q/G25K/S45W; R74L/C 171 A/T 19 Y/Gl 88 A/S45 W;
R74L/C 171 A/T 19 Y/Gl 88 A/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88R/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88R/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/G25K/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88 A/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G188Q/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188 A/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188 A/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188 A/G25K/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188R/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188R/G25K/S45 W; T 19 Y/R74L/C 171 A;
117T/T 19 Y/G25R/R74L/C 171 A; T 19 Y/G25K/S45 WR74L/G108 A/C 171 A/Gl 88R;
117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/G108C/C 171 A/Gl 88R;
117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/G108C/A126C/C 171 A/Gl 88R; or
117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/AW 104-
G105/G108C/C171A/G188R. In some aspects, the ACD further comprises at least one selectivity-enhancing alteration at a position functionally equivalent to Fl 02, S103, W104 and G105, for example, e g., FVPG, FTDG, FVDG, FVPAG, FVPLG, FVPPG, FVPAAG, FVPLAG, FVPPAG, FVPASG, FVPLSG, FVPPSG, FVPAFG, FVPLFG, FVPPFG, FVPAQG, FVPLQG, FVPPQG, FVPADG, FVPLDG, FVPPDG, FVPAKG, FVPLKG, FVPPKG among others.
[000148] In some embodiments, the ACD comprises a backbone sequence relative to AKMVW (SEQ ID NO: 35), in combination with one or more combinations of stability enhancing mutations selected from Hl IX, L12X, D14X, H16X, I17X, T19X, S20X, N21X, G25X, I26X, G27X, R28X, H29X, T31X, C34X, E38X, R39X, D41X, N42X, S45X, K47X, M48X, H51X, F54X, H56X, N57X, A59X, K60X, L62X, Y67X, L73X, R74X, D77X, L78X, V79X, P80X, S81X, D85X, A87X, I89X, Y90X, R91X, T93X, W94X, I96X, S97X, C101X, F102X, S103X, W104X, G105X, C106X, A107X, G108X, E109X, V110X, R111X, A112X, L114X, Q115X, E116X, N117X, T118X, H119X, V120X, L122X, R123X, A126X, R128X, L135X, Y136X, E138X, A139X, Q141X, M142X, D145X, A146X, A148X, D156X, E157X, K159X, C161X, D163X, T164X, D167X, Q169X, C171X, D177X, D180X, H182X, S183X, Q184X, A185X, L186X, S187X, G188X, R189X, A192X, N196X a deletion selected from AM1-L12, AE36-G53, AN61-G68, AW104-G105 , AQ195-N199 , AI26-G27, or any combination thereof, where the position number designation is functionally equivalent to the position in a wild-type AP0BEC3A (SEQ ID NO:3) and X is an amino acid substitution different from the wild-type amino acid at that position. In some aspects, the ACD further comprises at least one selectivity-enhancing alteration at a position functionally equivalent to Fl 02, SI 03, W104 and G105, for example, e g., FVPG, FTDG, FVDG, FVPAG, FVPLG, FVPPG, FVPAAG, FVPLAG, FVPPAG, FVPASG, FVPLSG, FVPPSG, FVPAFG, FVPLFG, FVPPFG, FVPAQG, FVPLQG, FVPPQG, FVPADG, FVPLDG, FVPPDG, FVPAKG, FVPLKG, FVPPKG among others.
[000149] In some embodiments, the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 130-134, or functionally equivalent positions, a sequence selected from: AKMVW (SEQ ID NO: 35), in combination with one or more combinations of stability enhancing mutations selected from: at least one of the following combinations of stabilizing mutations: R74L/C 171 A/T 19 Y/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 88Q/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88Q/G25K/S45W; R74L/C 171 A/T 19 Y/Gl 88 A/S45 W;
R74L/C 171 A/T 19 Y/Gl 88 A/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88R/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88R/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/G25K/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88 A/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G188Q/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G188 A/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188 A/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188 A/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G188R/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G188R/G25K/S45 W; T 19 Y/R74L/C 171 A; 117T/T 19 Y/G25R/R74L/C 171 A; T 19 Y/G25K/S45 WR74L/G108 A/C 171 A/Gl 88R;
117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/G108C/C 171 A/Gl 88R;
117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/G108C/A126C/C 171 A/Gl 88R; or 117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/AW 104- G105/G108C/C171A/G188R. In some aspects, the ACD further comprises at least one selectivity-enhancing alteration at a position functionally equivalent to Fl 02, S103, W104 and G105, for example, e g., FVPG, FTDG, FVDG, FVPAG, FVPLG, FVPPG, FVPAAG, FVPLAG, FVPPAG, FVPASG, FVPLSG, FVPPSG, FVPAFG, FVPLFG, FVPPFG, FVPAQG, FVPLQG, FVPPQG, FVPADG, FVPLDG, FVPPDG, FVPAKG, FVPLKG, FVPPKG among others.
[000150] In some embodiments, the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 130-134, or functionally equivalent positions, a sequence selected from: AQAFW (SEQ ID NO: 36), in combination with one or more combinations of stability enhancing mutations selected from Hl IX, L12X, D14X, H16X, I17X, T19X, S20X, N21X, G25X, I26X, G27X, R28X, H29X, T31X, C34X, E38X, R39X, D41X, N42X, S45X, K47X, M48X, H51X, F54X, H56X, N57X, A59X, K60X, L62X, Y67X, L73X, R74X, D77X, L78X, V79X, P80X, S81X, D85X, A87X, I89X, Y90X, R91X, T93X, W94X, I96X, S97X, C101X, F102X, S103X, W104X, G105X, C106X, A107X, G108X, E109X, V110X, R111X, Al 12X, LI 14X, QI 15X, El 16X, N117X, T118X, Hl 19X, V120X, L122X, R123X, A126X, R128X, L135X, Y136X, E138X, A139X, Q141X, M142X, D145X, A146X, A148X, D156X, E157X, K159X, C161X, D163X, T164X, D167X, Q169X, C171X, D177X, D180X, H182X, S183X, Q184X, A185X, L186X, S187X, G188X, R189X, A192X, N196X a deletion selected from AM1-L12, AE36-G53, AN61-G68, AW104-G105 , AQ195-N199 , AI26-G27, or any combination thereof, where the position number designation is functionally equivalent to the position in a wild-type APOBEC3A (SEQ ID NO:3) and X is an amino acid substitution different from the wild-type amino acid at that position. In some aspects, the ACD further comprises at least one selectivity-enhancing alteration at a position functionally equivalent to F102, S103, W104 and G105, for example, e.g., FVPG, FTDG, FVDG, FVPAG, FVPLG, FVPPG, FVPAAG, FVPLAG, FVPPAG, FVPASG, FVPLSG, FVPPSG, FVPAFG, FVPLFG, FVPPFG, FVPAQG, FVPLQG, FVPPQG, FVPADG, FVPLDG, FVPPDG, FVPAKG, FVPLKG, FVPPKG among others.
[000151] In some embodiments, the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 130-134, or functionally equivalent positions, a sequence selected from: AQAFW (SEQ ID NO: 36), in combination with one or more combinations of stability enhancing mutations selected from: at least one of the following combinations of stabilizing mutations: R74L/C 171 A/T 19 Y/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 88Q/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88Q/G25K/S45W; R74L/C 171 A/T 19 Y/Gl 88 A/S45 W;
R74L/C 171 A/T 19 Y/Gl 88 A/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88R/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88R/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/G25K/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88 A/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G188Q/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188 A/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188 A/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188 A/G25K/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188R/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188R/G25K/S45 W; T 19 Y/R74L/C 171 A;
117T/T 19 Y/G25R/R74L/C 171 A; T 19 Y/G25K/S45 WR74L/G108 A/C 171 A/Gl 88R;
117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/G108C/C 171 A/Gl 88R;
117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/G108C/A126C/C 171 A/Gl 88R; or
117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/AW 104-
G105/G108C/C171A/G188R. In some aspects, the ACD further comprises at least one selectivity-enhancing alteration at a position functionally equivalent to Fl 02, S103, W104 and G105, for example, e g., FVPG, FTDG, FVDG, FVPAG, FVPLG, FVPPG, FVPAAG, FVPLAG, FVPPAG, FVPASG, FVPLSG, FVPPSG, FVPAFG, FVPLFG, FVPPFG, FVPAQG, FVPLQG, FVPPQG, FVPADG, FVPLDG, FVPPDG, FVPAKG, FVPLKG, FVPPKG among others.
[000152] In some embodiments, the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 130-134, or functionally equivalent positions, a sequence selected from: ARVKW (SEQ ID NO: 37), in combination with one or more combinations of stability enhancing mutations selected from Hl IX, L12X, D14X, H16X, I17X, T19X, S20X, N21X, G25X, I26X, G27X, R28X, H29X, T31X, C34X, E38X, R39X, D41X, N42X, S45X, K47X, M48X, H51X, F54X, H56X, N57X, A59X, K60X, L62X, Y67X, L73X, R74X, D77X, L78X, V79X, P80X, S81X, D85X, A87X, I89X, Y90X, R91X, T93X, W94X, I96X, S97X, C101X, F102X, S1O3X, W104X, G105X, C106X, A107X, G108X, E109X, V110X, R111X, Al 12X, LI 14X, QI 15X, El 16X, N117X, T118X, Hl 19X, V120X, L122X, R123X, A126X, R128X, L135X, Y136X, E138X, A139X, Q141X, M142X, D145X, A146X, A148X, D156X, E157X, K159X, C161X, D163X, T164X, D167X, Q169X, C171X, D177X, D180X, H182X, S183X, Q184X, A185X, L186X, S187X, G188X, R189X, A192X, N196X a deletion selected from AM1-L12, AE36-G53, AN61-G68, AW104-G105 , AQ195-N199 , AI26-G27, or any combination thereof, where the position number designation is functionally equivalent to the position in a wild-type APOBEC3A (SEQ ID NO:3) and X is an amino acid substitution different from the wild-type amino acid at that position. In some aspects, the ACD further comprises at least one selectivity-enhancing alteration at a position functionally equivalent to F102, S103, W104 and G105, for example, e.g., FVPG, FTDG, FVDG, FVPAG, FVPLG, FVPPG, FVPAAG, FVPLAG, FVPPAG, FVPASG, FVPLSG, FVPPSG, FVPAFG, FVPLFG, FVPPFG, FVPAQG, FVPLQG, FVPPQG, FVPADG, FVPLDG, FVPPDG, FVPAKG, FVPLKG, FVPPKG among others.
[000153] In some embodiments, the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 130-134, or functionally equivalent positions, a sequence selected from: ARVKW (SEQ ID NO: 37), in combination with one or more combinations of stability enhancing mutations selected from: at least one of the following combinations of stabilizing mutations: R74L/C 171 A/T 19 Y/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 88Q/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88Q/G25K/S45W; R74L/C 171 A/T 19 Y/Gl 88 A/S45 W;
R74L/C 171 A/T 19 Y/Gl 88 A/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88R/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88R/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/G25K/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88 A/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G188Q/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188 A/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188 A/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188 A/G25K/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188R/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188R/G25K/S45 W; T 19 Y/R74L/C 171 A; 117T/T 19 Y/G25R/R74L/C 171 A; T 19 Y/G25K/S45 WR74L/G108 A/C 171 A/Gl 88R;
117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/G108C/C 171 A/Gl 88R;
117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/G108C/A126C/C 171 A/Gl 88R; or 117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/AW 104- G105/G108C/C171A/G188R. In some aspects, the ACD further comprises at least one selectivity-enhancing alteration at a position functionally equivalent to Fl 02, S103, W104 and G105, for example, e g., FVPG, FTDG, FVDG, FVPAG, FVPLG, FVPPG, FVPAAG, FVPLAG, FVPPAG, FVPASG, FVPLSG, FVPPSG, FVPAFG, FVPLFG, FVPPFG, FVPAQG, FVPLQG, FVPPQG, FVPADG, FVPLDG, FVPPDG, FVPAKG, FVPLKG, FVPPKG among others.
[000154] In some embodiments, the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 130-134, or functionally equivalent positions, a sequence selected from: AHRVW (SEQ ID NO: 38), in combination with one or more combinations of stability enhancing mutations selected from Hl IX, L12X, D14X, H16X, I17X, T19X, S20X, N21X, G25X, I26X, G27X, R28X, H29X, T31X, C34X, E38X, R39X, D41X, N42X, S45X, K47X, M48X, H51X, F54X, H56X, N57X, A59X, K60X, L62X, Y67X, L73X, R74X, D77X, L78X, V79X, P80X, S81X, D85X, A87X, I89X, Y90X, R91X, T93X, W94X, I96X, S97X, C101X, F102X, S103X, W104X, G105X, C106X, A107X, G108X, E109X, V110X, R111X, Al 12X, LI 14X, QI 15X, El 16X, N117X, T118X, Hl 19X, V120X, L122X, R123X, A126X, R128X, L135X, Y136X, E138X, A139X, Q141X, M142X, D145X, A146X, A148X, D156X, E157X, K159X, C161X, D163X, T164X, D167X, Q169X, C171X, D177X, D180X, H182X, S183X, Q184X, A185X, L186X, S187X, G188X, R189X, A192X, N196X a deletion selected from AM1-L12, AE36-G53, AN61-G68, AW104-G105 , AQ195-N199 , AI26-G27, or any combination thereof, where the position number designation is functionally equivalent to the position in a wild-type APOBEC3A (SEQ ID NO:3) and X is an amino acid substitution different from the wild-type amino acid at that position. In some aspects, the ACD further comprises at least one selectivity-enhancing alteration at a position functionally equivalent to F102, S103, W104 and G105, for example, e.g., FVPG, FTDG, FVDG, FVPAG, FVPLG, FVPPG, FVPAAG, FVPLAG, FVPPAG, FVPASG, FVPLSG, FVPPSG, FVPAFG, FVPLFG, FVPPFG, FVPAQG, FVPLQG, FVPPQG, FVPADG, FVPLDG, FVPPDG, FVPAKG, FVPLKG, FVPPKG among others.
[000155] In some embodiments, the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 130-134, or functionally equivalent positions, a sequence selected from: AHRVW (SEQ ID NO: 38), in combination with one or more combinations of stability enhancing mutations selected from: at least one of the following combinations of stabilizing mutations: R74L/C 171 A/T 19 Y/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 88Q/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88Q/G25K/S45W; R74L/C 171 A/T 19 Y/Gl 88 A/S45 W;
R74L/C 171 A/T 19 Y/Gl 88 A/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88R/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88R/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/G25K/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88 A/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G188Q/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188 A/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188 A/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188 A/G25K/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188R/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188R/G25K/S45 W; T 19 Y/R74L/C 171 A;
117T/T 19 Y/G25R/R74L/C 171 A; T 19 Y/G25K/S45 WR74L/G108 A/C 171 A/Gl 88R;
117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/G108C/C 171 A/Gl 88R;
117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/G108C/A126C/C 171 A/Gl 88R; or
117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/AW 104-
G105/G108C/C171A/G188R. In some aspects, the ACD further comprises at least one selectivity-enhancing alteration at a position functionally equivalent to Fl 02, S103, W104 and G105, for example, e g., FVPG, FTDG, FVDG, FVPAG, FVPLG, FVPPG, FVPAAG, FVPLAG, FVPPAG, FVPASG, FVPLSG, FVPPSG, FVPAFG, FVPLFG, FVPPFG, FVPAQG, FVPLQG, FVPPQG, FVPADG, FVPLDG, FVPPDG, FVPAKG, FVPLKG, FVPPKG among others.
[000156] In some embodiments, the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 130-134, or functionally equivalent positions, a sequence selected from: AKTYW (SEQ ID NO: 39), in combination with one or more combinations of stability enhancing mutations selected from Hl IX, L12X, D14X, H16X, I17X, T19X, S20X, N21X, G25X, I26X, G27X, R28X, H29X, T31X, C34X, E38X, R39X, D41X, N42X, S45X, K47X, M48X, H51X, F54X, H56X, N57X, A59X, K60X, L62X, Y67X, L73X, R74X, D77X, L78X, V79X, P80X, S81X, D85X, A87X, I89X, Y90X, R91X, T93X, W94X, I96X, S97X, C101X, F102X, S1O3X, W104X, G105X, C106X, A107X, G108X, E109X, V110X, R111X, Al 12X, LI 14X, QI 15X, El 16X, N117X, T118X, Hl 19X, V120X, L122X, R123X, A126X, R128X, L135X, Y136X, E138X, A139X, Q141X, M142X, D145X, A146X, A148X, D156X, E157X, K159X, C161X, D163X, T164X, D167X, Q169X, C171X, D177X, D180X, H182X, S183X, Q184X, A185X, L186X, S187X, G188X, R189X, A192X, N196X a deletion selected from AM1-L12, AE36-G53, AN61-G68, AW104-G105 , AQ195-N199 , AI26-G27, or any combination thereof, where the position number designation is functionally equivalent to the position in a wild-type APOBEC3A (SEQ ID NO:3) and X is an amino acid substitution different from the wild-type amino acid at that position. In some aspects, the ACD further comprises at least one selectivity-enhancing alteration at a position functionally equivalent to F102, S103, W104 and G105, for example, e.g., FVPG, FTDG, FVDG, FVPAG, FVPLG, FVPPG, FVPAAG, FVPLAG, FVPPAG, FVPASG, FVPLSG, FVPPSG, FVPAFG, FVPLFG, FVPPFG, FVPAQG, FVPLQG, FVPPQG, FVPADG, FVPLDG, FVPPDG, FVPAKG, FVPLKG, FVPPKG among others.
[000157] In some embodiments, the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 130-134, or functionally equivalent positions, a sequence selected from: AKTYW (SEQ ID NO: 39), in combination with one or more combinations of stability enhancing mutations selected from: at least one of the following combinations of stabilizing mutations: R74L/C 171 A/T 19 Y/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 88Q/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88Q/G25K/S45W; R74L/C 171 A/T 19 Y/Gl 88 A/S45 W;
R74L/C 171 A/T 19 Y/Gl 88 A/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88R/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88R/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/G25K/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88 A/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G188Q/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188 A/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188 A/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188 A/G25K/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188R/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188R/G25K/S45 W; T 19 Y/R74L/C 171 A; 117T/T 19 Y/G25R/R74L/C 171 A; T 19 Y/G25K/S45 WR74L/G108 A/C 171 A/Gl 88R;
117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/G108C/C 171 A/Gl 88R;
117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/G108C/A126C/C 171 A/Gl 88R; or 117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/AW 104- G105/G108C/C171A/G188R In some aspects, the ACD further comprises at least one selectivity-enhancing alteration at a position functionally equivalent to Fl 02, S103, W104 and G105, for example, e g., FVPG, FTDG, FVDG, FVPAG, FVPLG, FVPPG, FVPAAG, FVPLAG, FVPPAG, FVPASG, FVPLSG, FVPPSG, FVPAFG, FVPLFG, FVPPFG, FVPAQG, FVPLQG, FVPPQG, FVPADG, FVPLDG, FVPPDG, FVPAKG, FVPLKG, FVPPKG among others.
[000158] In some embodiments, the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 130-134, or functionally equivalent positions, a sequence selected from: AVAKW (SEQ ID NO:40), in combination with one or more combinations of stability enhancing mutations selected from Hl IX, L12X, D14X, H16X, I17X, T19X, S20X, N21X, G25X, I26X, G27X, R28X, H29X, T31X, C34X, E38X, R39X, D41X, N42X, S45X, K47X, M48X, H51X, F54X, H56X, N57X, A59X, K60X, L62X, Y67X, L73X, R74X, D77X, L78X, V79X, P80X, S81X, D85X, A87X, I89X, Y90X, R91X, T93X, W94X, I96X, S97X, C101X, F102X, S103X, W104X, G105X, C106X, A107X, G108X, E109X, V110X, R111X, Al 12X, LI 14X, QI 15X, El 16X, N117X, T118X, Hl 19X, V120X, L122X, R123X, A126X, R128X, L135X, Y136X, E138X, A139X, Q141X, M142X, D145X, A146X, A148X, D156X, E157X, K159X, C161X, D163X, T164X, D167X, Q169X, C171X, D177X, D180X, H182X, S183X, Q184X, A185X, L186X, S187X, G188X, R189X, A192X, N196X a deletion selected from AM1-L12, AE36-G53, AN61-G68, AW104-G105 , AQ195-N199 , AI26-G27, or any combination thereof, where the position number designation is functionally equivalent to the position in a wild-type APOBEC3A (SEQ ID NO:3) and X is an amino acid substitution different from the wild-type amino acid at that position. In some aspects, the ACD further comprises at least one selectivity-enhancing alteration at a position functionally equivalent to F102, S103, W104 and G105, for example, e.g., FVPG, FTDG, FVDG, FVPAG, FVPLG, FVPPG, FVPAAG, FVPLAG, FVPPAG, FVPASG, FVPLSG, FVPPSG, FVPAFG, FVPLFG, FVPPFG, FVPAQG, FVPLQG, FVPPQG, FVPADG, FVPLDG, FVPPDG, FVPAKG, FVPLKG, FVPPKG among others.
[000159] In some embodiments, the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 130-134, or functionally equivalent positions, a sequence selected from: AVAKW (SEQ ID NO:40), in combination with one or more combinations of stability enhancing mutations selected from: at least one of the following combinations of stabilizing mutations: R74L/C 171 A/T 19 Y/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 88Q/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88Q/G25K/S45W; R74L/C 171 A/T 19 Y/Gl 88 A/S45 W;
R74L/C 171 A/T 19 Y/Gl 88 A/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88R/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88R/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/G25K/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88 A/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G188Q/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188 A/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188 A/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188 A/G25K/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188R/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188R/G25K/S45 W; T 19 Y/R74L/C 171 A;
117T/T 19 Y/G25R/R74L/C 171 A; T 19 Y/G25K/S45 WR74L/G108 A/C 171 A/Gl 88R;
117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/G108C/C 171 A/Gl 88R;
117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/G108C/A126C/C 171 A/Gl 88R; or
117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/AW 104-
G105/G108C/C171A/G188R. In some aspects, the ACD further comprises at least one selectivity-enhancing alteration at a position functionally equivalent to Fl 02, S103, W104 and G105, for example, e g., FVPG, FTDG, FVDG, FVPAG, FVPLG, FVPPG, FVPAAG, FVPLAG, FVPPAG, FVPASG, FVPLSG, FVPPSG, FVPAFG, FVPLFG, FVPPFG, FVPAQG, FVPLQG, FVPPQG, FVPADG, FVPLDG, FVPPDG, FVPAKG, FVPLKG, FVPPKG among others.
[000160] In some embodiments, the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 130-134, or functionally equivalent positions, a sequence selected from: AKNFW (SEQ ID NO:41), in combination with one or more combinations of stability enhancing mutations selected from Hl IX, L12X, D14X, H16X, I17X, T19X, S20X, N21X, G25X, I26X, G27X, R28X, H29X, T31X, C34X, E38X, R39X, D41X, N42X, S45X, K47X, M48X, H51X, F54X, H56X, N57X, A59X, K60X, L62X, Y67X, L73X, R74X, D77X, L78X, V79X, P80X, S81X, D85X, A87X, I89X, Y90X, R91X, T93X, W94X, I96X, S97X, C101X, F102X, S1O3X, W104X, G105X, C106X, A107X, G108X, E109X, V110X, R111X, Al 12X, LI 14X, QI 15X, El 16X, N117X, T118X, Hl 19X, V120X, L122X, R123X, A126X, R128X, L135X, Y136X, E138X, A139X, Q141X, M142X, D145X, A146X, A148X, D156X, E157X, K159X, C161X, D163X, T164X, D167X, Q169X, C171X, D177X, D180X, H182X, S183X, Q184X, A185X, L186X, S187X, G188X, R189X, A192X, N196X a deletion selected from AM1-L12, AE36-G53, AN61-G68, AW104-G105 , AQ195-N199 , AI26-G27, or any combination thereof, where the position number designation is functionally equivalent to the position in a wild-type APOBEC3A (SEQ ID NO:3) and X is an amino acid substitution different from the wild-type amino acid at that position. In some aspects, the ACD further comprises at least one selectivity-enhancing alteration at a position functionally equivalent to F102, S103, W104 and G105, for example, e.g., FVPG, FTDG, FVDG, FVPAG, FVPLG, FVPPG, FVPAAG, FVPLAG, FVPPAG, FVPASG, FVPLSG, FVPPSG, FVPAFG, FVPLFG, FVPPFG, FVPAQG, FVPLQG, FVPPQG, FVPADG, FVPLDG, FVPPDG, FVPAKG, FVPLKG, FVPPKG among others.
[000161] In some embodiments, the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 130-134, or functionally equivalent positions, a sequence selected from: AKNFW (SEQ ID NO:41), in combination with one or more combinations of stability enhancing mutations selected from: at least one of the following combinations of stabilizing mutations: R74L/C 171 A/T 19 Y/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 88Q/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88Q/G25K/S45W; R74L/C 171 A/T 19 Y/Gl 88 A/S45 W;
R74L/C 171 A/T 19 Y/Gl 88 A/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88R/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88R/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/G25K/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88 A/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G188Q/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188 A/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188 A/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188 A/G25K/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188R/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188R/G25K/S45 W; T 19 Y/R74L/C 171 A; 117T/T 19 Y/G25R/R74L/C 171 A; T 19 Y/G25K/S45 WR74L/G108 A/C 171 A/Gl 88R;
117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/G108C/C 171 A/Gl 88R;
117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/G108C/A126C/C 171 A/Gl 88R; or 117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/AW 104- G105/G108C/C171A/G188R. In some aspects, the ACD further comprises at least one selectivity-enhancing alteration at a position functionally equivalent to Fl 02, S103, W104 and G105, for example, e g., FVPG, FTDG, FVDG, FVPAG, FVPLG, FVPPG, FVPAAG, FVPLAG, FVPPAG, FVPASG, FVPLSG, FVPPSG, FVPAFG, FVPLFG, FVPPFG, FVPAQG, FVPLQG, FVPPQG, FVPADG, FVPLDG, FVPPDG, FVPAKG, FVPLKG, FVPPKG among others.
[000162] In some embodiments, the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 130-134, or functionally equivalent positions, a sequence selected from: AHARW (SEQ ID NO:42), in combination with one or more combinations of stability enhancing mutations selected from Hl IX, L12X, D14X, H16X, I17X, T19X, S20X, N21X, G25X, I26X, G27X, R28X, H29X, T31X, C34X, E38X, R39X, D41X, N42X, S45X, K47X, M48X, H51X, F54X, H56X, N57X, A59X, K60X, L62X, Y67X, L73X, R74X, D77X, L78X, V79X, P80X, S81X, D85X, A87X, I89X, Y90X, R91X, T93X, W94X, I96X, S97X, C101X, F102X, S103X, W104X, G105X, C106X, A107X, G108X, E109X, V110X, R111X, Al 12X, LI 14X, QI 15X, El 16X, N117X, T118X, Hl 19X, V120X, L122X, R123X, A126X, R128X, L135X, Y136X, E138X, A139X, Q141X, M142X, D145X, A146X, A148X, D156X, E157X, K159X, C161X, D163X, T164X, D167X, Q169X, C171X, D177X, D180X, H182X, S183X, Q184X, A185X, L186X, S187X, G188X, R189X, A192X, N196X a deletion selected from AM1-L12, AE36-G53, AN61-G68, AW104-G105 , AQ195-N199 , AI26-G27, or any combination thereof, where the position number designation is functionally equivalent to the position in a wild-type APOBEC3A (SEQ ID NO:3) and X is an amino acid substitution different from the wild-type amino acid at that position. In some aspects, the ACD further comprises at least one selectivity-enhancing alteration at a position functionally equivalent to F102, S103, W104 and G105, for example, e.g., FVPG, FTDG, FVDG, FVPAG, FVPLG, FVPPG, FVPAAG, FVPLAG, FVPPAG, FVPASG, FVPLSG, FVPPSG, FVPAFG, FVPLFG, FVPPFG, FVPAQG, FVPLQG, FVPPQG, FVPADG, FVPLDG, FVPPDG, FVPAKG, FVPLKG, FVPPKG among others.
[000163] In some embodiments, the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 130-134, or functionally equivalent positions, a sequence selected from: AHARW (SEQ ID NO:42), in combination with one or more combinations of stability enhancing mutations selected from: at least one of the following combinations of stabilizing mutations: R74L/C 171 A/T 19 Y/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 88Q/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88Q/G25K/S45W; R74L/C 171 A/T 19 Y/Gl 88 A/S45 W;
R74L/C 171 A/T 19 Y/Gl 88 A/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88R/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88R/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/G25K/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88 A/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G188Q/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188 A/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188 A/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188 A/G25K/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188R/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188R/G25K/S45 W; T 19 Y/R74L/C 171 A;
117T/T 19 Y/G25R/R74L/C 171 A; T 19 Y/G25K/S45 WR74L/G108 A/C 171 A/Gl 88R;
117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/G108C/C 171 A/Gl 88R;
117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/G108C/A126C/C 171 A/Gl 88R; or
117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/AW 104-
G105/G108C/C171A/G188R. In some aspects, the ACD further comprises at least one selectivity-enhancing alteration at a position functionally equivalent to Fl 02, S103, W104 and G105, for example, e g., FVPG, FTDG, FVDG, FVPAG, FVPLG, FVPPG, FVPAAG, FVPLAG, FVPPAG, FVPASG, FVPLSG, FVPPSG, FVPAFG, FVPLFG, FVPPFG, FVPAQG, FVPLQG, FVPPQG, FVPADG, FVPLDG, FVPPDG, FVPAKG, FVPLKG, FVPPKG among others.
[000164] In some embodiments, the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 130-134, or functionally equivalent positions, a sequence selected from: AGPYW (SEQ ID NO:43), in combination with one or more combinations of stability enhancing mutations selected from Hl IX, L12X, D14X, H16X, I17X, T19X, S20X, N21X, G25X, I26X, G27X, R28X, H29X, T31X, C34X, E38X, R39X, D41X, N42X, S45X, K47X, M48X, H51X, F54X, H56X, N57X, A59X, K60X, L62X, Y67X, L73X, R74X, D77X, L78X, V79X, P80X, S81X, D85X, A87X, I89X, Y90X, R91X, T93X, W94X, I96X, S97X, C101X, F102X, S1O3X, W104X, G105X, C106X, A107X, G108X, E109X, V110X, R111X, Al 12X, LI 14X, QI 15X, El 16X, N117X, T118X, Hl 19X, V120X, L122X, R123X, A126X, R128X, L135X, Y136X, E138X, A139X, Q141X, M142X, D145X, A146X, A148X, D156X, E157X, K159X, C161X, D163X, T164X, D167X, Q169X, C171X, D177X, D180X, H182X, S183X, Q184X, A185X, L186X, S187X, G188X, R189X, A192X, N196X a deletion selected from AM1-L12, AE36-G53, AN61-G68, AW104-G105 , AQ195-N199 , AI26-G27, or any combination thereof, where the position number designation is functionally equivalent to the position in a wild-type APOBEC3A (SEQ ID NO:3) and X is an amino acid substitution different from the wild-type amino acid at that position. In some aspects, the ACD further comprises at least one selectivity-enhancing alteration at a position functionally equivalent to F102, S103, W104 and G105, for example, e.g., FVPG, FTDG, FVDG, FVPAG, FVPLG, FVPPG, FVPAAG, FVPLAG, FVPPAG, FVPASG, FVPLSG, FVPPSG, FVPAFG, FVPLFG, FVPPFG, FVPAQG, FVPLQG, FVPPQG, FVPADG, FVPLDG, FVPPDG, FVPAKG, FVPLKG, FVPPKG among others.
[000165] In some embodiments, the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 130-134, or functionally equivalent positions, a sequence selected from: AGPYW (SEQ ID NO:43), in combination with one or more combinations of stability enhancing mutations selected from: at least one of the following combinations of stabilizing mutations: R74L/C 171 A/T 19 Y/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 88Q/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88Q/G25K/S45W; R74L/C 171 A/T 19 Y/Gl 88 A/S45 W;
R74L/C 171 A/T 19 Y/Gl 88 A/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88R/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88R/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/G25K/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88 A/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G188Q/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188 A/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188 A/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188 A/G25K/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188R/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188R/G25K/S45 W; T 19 Y/R74L/C 171 A; 117T/T 19 Y/G25R/R74L/C 171 A; T 19 Y/G25K/S45 WR74L/G108 A/C 171 A/Gl 88R;
117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/G108C/C 171 A/Gl 88R;
117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/G108C/A126C/C 171 A/Gl 88R; or 117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/AW 104- G105/G108C/C171A/G188R. In some aspects, the ACD further comprises at least one selectivity-enhancing alteration at a position functionally equivalent to Fl 02, S103, W104 and G105, for example, e g., FVPG, FTDG, FVDG, FVPAG, FVPLG, FVPPG, FVPAAG, FVPLAG, FVPPAG, FVPASG, FVPLSG, FVPPSG, FVPAFG, FVPLFG, FVPPFG, FVPAQG, FVPLQG, FVPPQG, FVPADG, FVPLDG, FVPPDG, FVPAKG, FVPLKG, FVPPKG among others.
[000166] In some embodiments, the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 130-134, or functionally equivalent positions, a sequence selected from: AKYPW (SEQ ID NO:44), in combination with one or more combinations of stability enhancing mutations selected from Hl IX, L12X, D14X, H16X, I17X, T19X, S20X, N21X, G25X, I26X, G27X, R28X, H29X, T31X, C34X, E38X, R39X, D41X, N42X, S45X, K47X, M48X, H51X, F54X, H56X, N57X, A59X, K60X, L62X, Y67X, L73X, R74X, D77X, L78X, V79X, P80X, S81X, D85X, A87X, I89X, Y90X, R91X, T93X, W94X, I96X, S97X, C101X, F102X, S103X, W104X, G105X, C106X, A107X, G108X, E109X, V110X, R111X, Al 12X, LI 14X, QI 15X, El 16X, N117X, T118X, Hl 19X, V120X, L122X, R123X, A126X, R128X, L135X, Y136X, E138X, A139X, Q141X, M142X, D145X, A146X, A148X, D156X, E157X, K159X, C161X, D163X, T164X, D167X, Q169X, C171X, D177X, D180X, H182X, S183X, Q184X, A185X, L186X, S187X, G188X, R189X, A192X, N196X a deletion selected from AM1-L12, AE36-G53, AN61-G68, AW104-G105 , AQ195-N199 , AI26-G27, or any combination thereof, where the position number designation is functionally equivalent to the position in a wild-type APOBEC3A (SEQ ID NO:3) and X is an amino acid substitution different from the wild-type amino acid at that position. In some aspects, the ACD further comprises at least one selectivity-enhancing alteration at a position functionally equivalent to F102, S103, W104 and G105, for example, e.g., FVPG, FTDG, FVDG, FVPAG, FVPLG, FVPPG, FVPAAG, FVPLAG, FVPPAG, FVPASG, FVPLSG, FVPPSG, FVPAFG, FVPLFG, FVPPFG, FVPAQG, FVPLQG, FVPPQG, FVPADG, FVPLDG, FVPPDG, FVPAKG, FVPLKG, FVPPKG among others. [000167] In some embodiments, the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 130-134, or functionally equivalent positions, a sequence selected from: AKYPW (SEQ ID NO:44), in combination with one or more combinations of stability enhancing mutations selected from: at least one of the following combinations of stabilizing mutations: R74L/C 171 A/T 19 Y/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 88Q/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88Q/G25K/S45W; R74L/C 171 A/T 19 Y/Gl 88 A/S45 W;
R74L/C 171 A/T 19 Y/Gl 88 A/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88R/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88R/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/G25K/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88 A/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G188Q/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188 A/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188 A/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188 A/G25K/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188R/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188R/G25K/S45 W; T 19 Y/R74L/C 171 A;
117T/T 19 Y/G25R/R74L/C 171 A; T 19 Y/G25K/S45 WR74L/G108 A/C 171 A/Gl 88R;
117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/G108C/C 171 A/Gl 88R;
117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/G108C/A126C/C 171 A/Gl 88R; or
117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/AW 104-
G105/G108C/C171A/G188R In some aspects, the ACD further comprises at least one selectivity-enhancing alteration at a position functionally equivalent to Fl 02, S103, W104 and G105, for example, e g., FVPG, FTDG, FVDG, FVPAG, FVPLG, FVPPG, FVPAAG, FVPLAG, FVPPAG, FVPASG, FVPLSG, FVPPSG, FVPAFG, FVPLFG, FVPPFG, FVPAQG, FVPLQG, FVPPQG, FVPADG, FVPLDG, FVPPDG, FVPAKG, FVPLKG, FVPPKG among others.
[000168] In some embodiments, the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 130-134, or functionally equivalent positions, a sequence selected from: AKPFW (SEQ ID NO:45), in combination with one or more combinations of stability enhancing mutations selected from Hl IX, L12X, D14X, H16X, I17X, T19X, S20X, N21X, G25X, I26X, G27X, R28X, H29X, T31X, C34X, E38X, R39X, D41X, N42X, S45X, K47X, M48X, H51X, F54X, H56X, N57X, A59X, K60X, L62X, Y67X, L73X, R74X, D77X, L78X, V79X, P80X, S81X, D85X, A87X, I89X, Y90X, R91X, T93X, W94X, I96X, S97X, C101X, F102X, S1O3X, W104X, G105X, C106X, A107X, G108X, E109X, V110X, R111X, Al 12X, LI 14X, QI 15X, El 16X, N117X, T118X, Hl 19X, V120X, L122X, R123X, A126X, R128X, L135X, Y136X, E138X, A139X, Q141X, M142X, D145X, A146X, A148X, D156X, E157X, K159X, C161X, D163X, T164X, D167X, Q169X, C171X, D177X, D180X, H182X, S183X, Q184X, A185X, L186X, S187X, G188X, R189X, A192X, N196X a deletion selected from AM1-L12, AE36-G53, AN61-G68, AW104-G105 , AQ195-N199 , AI26-G27, or any combination thereof, where the position number designation is functionally equivalent to the position in a wild-type APOBEC3A (SEQ ID NO:3) and X is an amino acid substitution different from the wild-type amino acid at that position. In some aspects, the ACD further comprises at least one selectivity-enhancing alteration at a position functionally equivalent to F102, S103, W104 and G105, for example, e.g., FVPG, FTDG, FVDG, FVPAG, FVPLG, FVPPG, FVPAAG, FVPLAG, FVPPAG, FVPASG, FVPLSG, FVPPSG, FVPAFG, FVPLFG, FVPPFG, FVPAQG, FVPLQG, FVPPQG, FVPADG, FVPLDG, FVPPDG, FVPAKG, FVPLKG, FVPPKG among others.
[000169] In some embodiments, the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 130-134, or functionally equivalent positions, a sequence selected from AKPFW (SEQ ID NO:45), in combination with one or more combinations of stability enhancing mutations selected from: at least one of the following combinations of stabilizing mutations: R74L/C 171 A/T 19 Y/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 88Q/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88Q/G25K/S45W; R74L/C 171 A/T 19 Y/Gl 88 A/S45 W;
R74L/C 171 A/T 19 Y/Gl 88 A/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88R/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88R/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/G25K/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88 A/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G188Q/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G188 A/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188 A/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G188 A/G25K/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188R/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188R/G25K/S45 W; T 19 Y/R74L/C 171 A;
117T/T 19 Y/G25R/R74L/C 171 A; T 19 Y/G25K/S45 WR74L/G108 A/C 171 A/Gl 88R;
117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/G108C/C 171 A/Gl 88R;
117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/G108C/A126C/C 171 A/Gl 88R; or 117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/AW 104- G105/G108C/C171A/G188R. In some aspects, the ACD further comprises at least one selectivity-enhancing alteration at a position functionally equivalent to Fl 02, S103, W104 and G105, for example, e g., FVPG, FTDG, FVDG, FVPAG, FVPLG, FVPPG, FVPAAG, FVPLAG, FVPPAG, FVPASG, FVPLSG, FVPPSG, FVPAFG, FVPLFG, FVPPFG, FVPAQG, FVPLQG, FVPPQG, FVPADG, FVPLDG, FVPPDG, FVPAKG, FVPLKG, FVPPKG among others.
[000170] In some embodiments, the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 130-134, or functionally equivalent positions, a sequence selected from: AKLIW (SEQ ID NO:46), in combination with one or more combinations of stability enhancing mutations selected from Hl IX, L12X, D14X, H16X, I17X, T19X, S20X, N21X, G25X, I26X, G27X, R28X, H29X, T31X, C34X, E38X, R39X, D41X, N42X, S45X, K47X, M48X, H51X, F54X, H56X, N57X, A59X, K60X, L62X, Y67X, L73X, R74X, D77X, L78X, V79X, P80X, S81X, D85X, A87X, I89X, Y90X, R91X, T93X, W94X, I96X, S97X, C101X, F102X, S103X, W104X, G105X, C106X, A107X, G108X, E109X, V110X, R111X, Al 12X, LI 14X, QI 15X, El 16X, N117X, T118X, Hl 19X, V120X, L122X, R123X, A126X, R128X, L135X, Y136X, E138X, A139X, Q141X, M142X, D145X, A146X, A148X, D156X, E157X, K159X, C161X, D163X, T164X, D167X, Q169X, C171X, D177X, D180X, H182X, S183X, Q184X, A185X, L186X, S187X, G188X, R189X, A192X, N196X a deletion selected from AM1-L12, AE36-G53, AN61-G68, AW104-G105 , AQ195-N199 , AI26-G27, or any combination thereof, where the position number designation is functionally equivalent to the position in a wild-type APOBEC3A (SEQ ID NO:3) and X is an amino acid substitution different from the wild-type amino acid at that position. In some aspects, the ACD further comprises at least one selectivity-enhancing alteration at a position functionally equivalent to F102, S103, W104 and G105, for example, e.g., FVPG, FTDG, FVDG, FVPAG, FVPLG, FVPPG, FVPAAG, FVPLAG, FVPPAG, FVPASG, FVPLSG, FVPPSG, FVPAFG, FVPLFG, FVPPFG, FVPAQG, FVPLQG, FVPPQG, FVPADG, FVPLDG, FVPPDG, FVPAKG, FVPLKG, FVPPKG among others. [000171] In some embodiments, the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 130-134, or functionally equivalent positions, a sequence selected from: AKLIW (SEQ ID NO:46), in combination with one or more combinations of stability enhancing mutations selected from: at least one of the following combinations of stabilizing mutations: R74L/C 171 A/T 19 Y/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 88Q/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88Q/G25K/S45W; R74L/C 171 A/T 19 Y/Gl 88 A/S45 W;
R74L/C 171 A/T 19 Y/Gl 88 A/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88R/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88R/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/G25K/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88 A/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G188Q/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188 A/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188 A/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188 A/G25K/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188R/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188R/G25K/S45 W; T 19 Y/R74L/C 171 A;
117T/T 19 Y/G25R/R74L/C 171 A; T 19 Y/G25K/S45 WR74L/G108 A/C 171 A/Gl 88R;
117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/G108C/C 171 A/Gl 88R;
117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/G108C/A126C/C 171 A/Gl 88R; or
117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/AW 104-
G105/G108C/C171A/G188R. In some aspects, the ACD further comprises at least one selectivity-enhancing alteration at a position functionally equivalent to Fl 02, S103, W104 and G105, for example, e g., FVPG, FTDG, FVDG, FVPAG, FVPLG, FVPPG, FVPAAG, FVPLAG, FVPPAG, FVPASG, FVPLSG, FVPPSG, FVPAFG, FVPLFG, FVPPFG, FVPAQG, FVPLQG, FVPPQG, FVPADG, FVPLDG, FVPPDG, FVPAKG, FVPLKG, FVPPKG among others.
[000172] In some embodiments, the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 130-134, or functionally equivalent positions, a sequence selected from AHVVW (SEQ ID NO:47), in combination with one or more combinations of stability enhancing mutations selected from Hl IX, L12X, D14X, H16X, I17X, T19X, S20X, N21X, G25X, I26X, G27X, R28X, H29X, T31X, C34X, E38X, R39X, D41X, N42X, S45X, K47X, M48X, H51X, F54X, H56X, N57X, A59X, K60X, L62X, Y67X, L73X, R74X, D77X, L78X, V79X, P80X, S81X, D85X, A87X, I89X, Y90X, R91X, T93X, W94X, I96X, S97X, C101X, F102X, S1O3X, W104X, G105X, C106X, A107X, G108X, E109X, V110X, R111X, Al 12X, LI 14X, QI 15X, El 16X, N117X, T118X, Hl 19X, V120X, L122X, R123X, A126X, R128X, L135X, Y136X, E138X, A139X, Q141X, M142X, D145X, A146X, A148X, D156X, E157X, K159X, C161X, D163X, T164X, D167X, Q169X, C171X, D177X, D180X, H182X, S183X, Q184X, A185X, L186X, S187X, G188X, R189X, A192X, N196X a deletion selected from AM1-L12, AE36-G53, AN61-G68, AW104-G105 , AQ195-N199 , AI26-G27, or any combination thereof, where the position number designation is functionally equivalent to the position in a wild-type APOBEC3A (SEQ ID NO:3) and X is an amino acid substitution different from the wild-type amino acid at that position. In some aspects, the ACD further comprises at least one selectivity-enhancing alteration at a position functionally equivalent to F102, S103, W104 and G105, for example, e.g., FVPG, FTDG, FVDG, FVPAG, FVPLG, FVPPG, FVPAAG, FVPLAG, FVPPAG, FVPASG, FVPLSG, FVPPSG, FVPAFG, FVPLFG, FVPPFG, FVPAQG, FVPLQG, FVPPQG, FVPADG, FVPLDG, FVPPDG, FVPAKG, FVPLKG, FVPPKG among others.
[000173] In some embodiments, the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 130-134, or functionally equivalent positions, a sequence selected from: AHVVW (SEQ ID NO:47), in combination with one or more combinations of stability enhancing mutations selected from: at least one of the following combinations of stabilizing mutations: R74L/C 171 A/T 19 Y/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 88Q/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88Q/G25K/S45W; R74L/C 171 A/T 19 Y/Gl 88 A/S45 W;
R74L/C 171 A/T 19 Y/Gl 88 A/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88R/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88R/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/G25K/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88 A/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G188Q/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G188 A/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188 A/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G188 A/G25K/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188R/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188R/G25K/S45 W; T 19 Y/R74L/C 171 A;
117T/T 19 Y/G25R/R74L/C 171 A; T 19 Y/G25K/S45 WR74L/G108 A/C 171 A/Gl 88R;
117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/G108C/C 171 A/Gl 88R;
117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/G108C/A126C/C 171 A/Gl 88R; or 117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/AW 104- G105/G108C/C171A/G188R. In some aspects, the ACD further comprises at least one selectivity-enhancing alteration at a position functionally equivalent to Fl 02, S103, W104 and G105, for example, e g., FVPG, FTDG, FVDG, FVPAG, FVPLG, FVPPG, FVPAAG, FVPLAG, FVPPAG, FVPASG, FVPLSG, FVPPSG, FVPAFG, FVPLFG, FVPPFG, FVPAQG, FVPLQG, FVPPQG, FVPADG, FVPLDG, FVPPDG, FVPAKG, FVPLKG, FVPPKG among others.
[000174] In some embodiments, the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 130-134, or functionally equivalent positions, a sequence selected from: AGRFW (SEQ ID NO:48), in combination with one or more combinations of stability enhancing mutations selected from Hl IX, L12X, D14X, H16X, I17X, T19X, S20X, N21X, G25X, I26X, G27X, R28X, H29X, T31X, C34X, E38X, R39X, D41X, N42X, S45X, K47X, M48X, H51X, F54X, H56X, N57X, A59X, K60X, L62X, Y67X, L73X, R74X, D77X, L78X, V79X, P80X, S81X, D85X, A87X, I89X, Y90X, R91X, T93X, W94X, I96X, S97X, C101X, F102X, S103X, W104X, G105X, C106X, A107X, G108X, E109X, V110X, R111X, Al 12X, LI 14X, QI 15X, El 16X, N117X, T118X, Hl 19X, V120X, L122X, R123X, A126X, R128X, L135X, Y136X, E138X, A139X, Q141X, M142X, D145X, A146X, A148X, D156X, E157X, K159X, C161X, D163X, T164X, D167X, Q169X, C171X, D177X, D180X, H182X, S183X, Q184X, A185X, L186X, S187X, G188X, R189X, A192X, N196X a deletion selected from AM1-L12, AE36-G53, AN61-G68, AW104-G105 , AQ195-N199 , AI26-G27, or any combination thereof, where the position number designation is functionally equivalent to the position in a wild-type APOBEC3A (SEQ ID NO:3) and X is an amino acid substitution different from the wild-type amino acid at that position. In some aspects, the ACD further comprises at least one selectivity-enhancing alteration at a position functionally equivalent to F102, S103, W104 and G105, for example, e.g., FVPG, FTDG, FVDG, FVPAG, FVPLG, FVPPG, FVPAAG, FVPLAG, FVPPAG, FVPASG, FVPLSG, FVPPSG, FVPAFG, FVPLFG, FVPPFG, FVPAQG, FVPLQG, FVPPQG, FVPADG, FVPLDG, FVPPDG, FVPAKG, FVPLKG, FVPPKG among others. [000175] In some embodiments, the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 130-134, or functionally equivalent positions, a sequence selected from: AGRFW (SEQ ID NO:48), in combination with one or more combinations of stability enhancing mutations selected from: at least one of the following combinations of stabilizing mutations: R74L/C 171 A/T 19 Y/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 88Q/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88Q/G25K/S45W; R74L/C 171 A/T 19 Y/Gl 88 A/S45 W;
R74L/C 171 A/T 19 Y/Gl 88 A/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88R/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88R/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/G25K/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88 A/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G188Q/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188 A/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188 A/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188 A/G25K/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188R/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188R/G25K/S45 W; T 19 Y/R74L/C 171 A;
117T/T 19 Y/G25R/R74L/C 171 A; T 19 Y/G25K/S45 WR74L/G108 A/C 171 A/Gl 88R;
117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/G108C/C 171 A/Gl 88R;
117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/G108C/A126C/C 171 A/Gl 88R; or
117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/AW 104-
G105/G108C/C171A/G188R In some aspects, the ACD further comprises at least one selectivity-enhancing alteration at a position functionally equivalent to Fl 02, S103, W104 and G105, for example, e g., FVPG, FTDG, FVDG, FVPAG, FVPLG, FVPPG, FVPAAG, FVPLAG, FVPPAG, FVPASG, FVPLSG, FVPPSG, FVPAFG, FVPLFG, FVPPFG, FVPAQG, FVPLQG, FVPPQG, FVPADG, FVPLDG, FVPPDG, FVPAKG, FVPLKG, FVPPKG among others.
[000176] In some embodiments, the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 130-134, or functionally equivalent positions, a sequence selected from: AIAHW (SEQ ID NO:49), in combination with one or more combinations of stability enhancing mutations selected from Hl IX, L12X, D14X, H16X, I17X, T19X, S20X, N21X, G25X, I26X, G27X, R28X, H29X, T31X, C34X, E38X, R39X, D41X, N42X, S45X, K47X, M48X, H51X, F54X, H56X, N57X, A59X, K60X, L62X, Y67X, L73X, R74X, D77X, L78X, V79X, P80X, S81X, D85X, A87X, I89X, Y90X, R91X, T93X, W94X, I96X, S97X, C101X, F102X, S103X, W104X, G105X, C106X, A107X, G108X, E109X, V110X, R111X, Al 12X, LI 14X, QI 15X, El 16X, N117X, T118X, Hl 19X, V120X, L122X, R123X, A126X, R128X, L135X, Y136X, E138X, A139X, Q141X, M142X, D145X, A146X, A148X, D156X, E157X, K159X, C161X, D163X, T164X, D167X, Q169X, C171X, D177X, D180X, H182X, S183X, Q184X, A185X, L186X, S187X, G188X, R189X, A192X, N196X a deletion selected from AM1-L12, AE36-G53, AN61-G68, AW104-G105 , AQ195-N199 , AI26-G27, or any combination thereof, where the position number designation is functionally equivalent to the position in a wild-type APOBEC3A (SEQ ID NO:3) and X is an amino acid substitution different from the wild-type amino acid at that position. In some aspects, the ACD further comprises at least one selectivity-enhancing alteration at a position functionally equivalent to F102, S103, W104 and G105, for example, e.g., FVPG, FTDG, FVDG, FVPAG, FVPLG, FVPPG, FVPAAG, FVPLAG, FVPPAG, FVPASG, FVPLSG, FVPPSG, FVPAFG, FVPLFG, FVPPFG, FVPAQG, FVPLQG, FVPPQG, FVPADG, FVPLDG, FVPPDG, FVPAKG, FVPLKG, FVPPKG among others.
[000177] In some embodiments, the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 130-134, or functionally equivalent positions, a sequence selected from: AIAHW (SEQ ID NO:49), in combination with one or more combinations of stability enhancing mutations selected from: at least one of the following combinations of stabilizing mutations: R74L/C 171 A/T 19 Y/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 88Q/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88Q/G25K/S45W; R74L/C 171 A/T 19 Y/Gl 88 A/S45 W;
R74L/C 171 A/T 19 Y/Gl 88 A/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88R/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88R/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/G25K/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88 A/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G188Q/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G188 A/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188 A/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G188 A/G25K/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188R/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188R/G25K/S45 W; T 19 Y/R74L/C 171 A;
117T/T 19 Y/G25R/R74L/C 171 A; T 19 Y/G25K/S45 WR74L/G108 A/C 171 A/Gl 88R;
117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/G108C/C 171 A/Gl 88R;
117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/G108C/A126C/C 171 A/Gl 88R; or 117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/AW 104- G105/G108C/C171A/G188R. In some aspects, the ACD further comprises at least one selectivity-enhancing alteration at a position functionally equivalent to Fl 02, S103, W104 and G105, for example, e g., FVPG, FTDG, FVDG, FVPAG, FVPLG, FVPPG, FVPAAG, FVPLAG, FVPPAG, FVPASG, FVPLSG, FVPPSG, FVPAFG, FVPLFG, FVPPFG, FVPAQG, FVPLQG, FVPPQG, FVPADG, FVPLDG, FVPPDG, FVPAKG, FVPLKG, FVPPKG among others.
[000178] In some embodiments, the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 130-134, or functionally equivalent positions, a sequence selected from: AKMYW (SEQ ID NO:50), in combination with one or more combinations of stability enhancing mutations selected from Hl IX, L12X, D14X, H16X, I17X, T19X, S20X, N21X, G25X, I26X, G27X, R28X, H29X, T31X, C34X, E38X, R39X, D41X, N42X, S45X, K47X, M48X, H51X, F54X, H56X, N57X, A59X, K60X, L62X, Y67X, L73X, R74X, D77X, L78X, V79X, P80X, S81X, D85X, A87X, I89X, Y90X, R91X, T93X, W94X, I96X, S97X, C101X, F102X, S103X, W104X, G105X, C106X, A107X, G108X, E109X, V110X, R111X, Al 12X, LI 14X, QI 15X, El 16X, N117X, T118X, Hl 19X, V120X, L122X, R123X, A126X, R128X, L135X, Y136X, E138X, A139X, Q141X, M142X, D145X, A146X, A148X, D156X, E157X, K159X, C161X, D163X, T164X, D167X, Q169X, C171X, D177X, D180X, H182X, S183X, Q184X, A185X, L186X, S187X, G188X, R189X, A192X, N196X a deletion selected from AM1-L12, AE36-G53, AN61-G68, AW104-G105 , AQ195-N199 , AI26-G27, or any combination thereof, where the position number designation is functionally equivalent to the position in a wild-type APOBEC3A (SEQ ID NO:3) and X is an amino acid substitution different from the wild-type amino acid at that position. In some aspects, the ACD further comprises at least one selectivity-enhancing alteration at a position functionally equivalent to F102, S103, W104 and G105, for example, e.g., FVPG, FTDG, FVDG, FVPAG, FVPLG, FVPPG, FVPAAG, FVPLAG, FVPPAG, FVPASG, FVPLSG, FVPPSG, FVPAFG, FVPLFG, FVPPFG, FVPAQG, FVPLQG, FVPPQG, FVPADG, FVPLDG, FVPPDG, FVPAKG, FVPLKG, FVPPKG among others. [000179] In some embodiments, the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 130-134, or functionally equivalent positions, a sequence selected from: AKMYW (SEQ ID NO:50), among others in combination with one or more combinations of stability enhancing mutations selected from: at least one of the following combinations of stabilizing mutations: R74L/C171A/T19Y/G25K/S45W;
R74L/C 171 A/T 19 Y/Gl 88Q/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88Q/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 88 A/S45 W; R74L/C 171 A/T 19 Y/Gl 88 A/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 88R/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88R/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/G25K/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88 A/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G188Q/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188 A/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188 A/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188 A/G25K/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188R/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188R/G25K/S45 W; T 19 Y/R74L/C 171 A;
117T/T 19 Y/G25R/R74L/C 171 A; T 19 Y/G25K/S45 WR74L/G108 A/C 171 A/Gl 88R;
117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/G108C/C 171 A/Gl 88R;
117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/G108C/A126C/C 171 A/Gl 88R; or
117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/AW 104-
G105/G108C/C171A/G188R. In some aspects, the ACD further comprises at least one selectivity-enhancing alteration at a position functionally equivalent to Fl 02, S103, W104 and G105, for example, e g., FVPG, FTDG, FVDG, FVPAG, FVPLG, FVPPG, FVPAAG, FVPLAG, FVPPAG, FVPASG, FVPLSG, FVPPSG, FVPAFG, FVPLFG, FVPPFG, FVPAQG, FVPLQG, FVPPQG, FVPADG, FVPLDG, FVPPDG, FVPAKG, FVPLKG, FVPPKG among others.
[000180] In some embodiments, the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 102-105, or functionally equivalent positions, the sequence FVPG, and from 130-134, or functionally equivalent positions, the sequence AKYPW, in combination with one or more combinations of stability enhancing mutations selected from: at least one of the following combinations of stabilizing mutations:
R74L/C 171 A/T 19 Y/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 88Q/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 88Q/G25K/S45W; R74L/C 171 A/T 19 Y/Gl 88 A/S45 W;
R74L/C 171 A/T 19 Y/Gl 88 A/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88R/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88R/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/G25K/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88 A/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G188Q/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G188 A/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188 A/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188 A/G25K/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188R/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188R/G25K/S45 W; T 19 Y/R74L/C 171 A;
117T/T 19 Y/G25R/R74L/C 171 A; T 19 Y/G25K/S45 WR74L/G108 A/C 171 A/Gl 88R;
117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/G108C/C 171 A/Gl 88R;
117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/G108C/A126C/C 171 A/Gl 88R; or 117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/AW 104-
G105/G108C/C171A/G188R.In some embodiments, the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 102-105 the sequence FVPG, and from 130-134, or functionally equivalent positions, the sequence AKYPW (SEQ ID NO:44), in combination with one or more combinations of stability enhancing mutations selected from Hl IX, L12X, D14X, H16X, I17X, T19X, S20X, N21X, G25X, I26X, G27X, R28X, H29X, T31X, C34X, E38X, R39X, D41X, N42X, S45X, K47X, M48X, H51X, F54X, H56X, N57X, A59X, K60X, L62X, Y67X, L73X, R74X, D77X, L78X, V79X, P80X, S81X, D85X, A87X, I89X, Y90X, R91X, T93X, W94X, I96X, S97X, C101X, F102X, S103X, W104X, G105X, C106X, A107X, G108X, E109X, VI 10X, R11 IX, Al 12X, LI 14X, QI 15X, El 16X, N117X, T118X, H119X, V120X, L122X, R123X, A126X, R128X, L135X, Y136X, E138X, A139X, Q141X, M142X, D145X, A146X, A148X, D156X, E157X, K159X, C161X, D163X, T164X, D167X, Q169X, C171X, D177X, D180X, H182X, S183X, Q184X, A185X, L186X, S187X, G188X, R189X, A192X, N196X a deletion selected from AM1-L12, AE36-G53, AN61-G68, AW104-G105 , AQ195-N199 , AI26-G27, or any combination thereof, where the position number designation is functionally equivalent to the position in a wild-type AP0BEC3 A (SEQ ID NO:3) and X is an amino acid substitution different from the wild-type amino acid at that position.
[000181] In some embodiments, the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 102-105 the sequence FTDG, and from 130-134, or functionally equivalent positions, the sequence AKYPW, in combination with one or more combinations of stability enhancing mutations selected from: at least one of the following combinations of stabilizing mutations: R74L/C171A/T19Y/G25K/S45W;
R74L/C 171 A/T 19 Y/Gl 88Q/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88Q/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 88 A/S45 W; R74L/C 171 A/T 19 Y/Gl 88 A/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 88R/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88R/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/G25K/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88 A/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G188Q/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188 A/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188 A/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188 A/G25K/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188R/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188R/G25K/S45 W; T 19 Y/R74L/C 171 A;
117T/T 19 Y/G25R/R74L/C 171 A; T 19 Y/G25K/S45 WR74L/G108 A/C 171 A/Gl 88R;
117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/G108C/C 171 A/Gl 88R;
117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/G108C/A126C/C 171 A/Gl 88R; or
117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/AW 104-
G105/G108C/C 171 A/Gl 88R.
[000182] In some embodiments, the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 102-105 the sequence FTDG, and from 130-134, or functionally equivalent positions, the sequence AKYPW (SEQ ID NO:44), in combination with one or more combinations of stability enhancing mutations selected from Hl IX, L12X, D14X, H16X, I17X, T19X, S20X, N21X, G25X, I26X, G27X, R28X, H29X, T31X, C34X, E38X, R39X, D41X, N42X, S45X, K47X, M48X, H51X, F54X, H56X, N57X, A59X, K60X, L62X, Y67X, L73X, R74X, D77X, L78X, V79X, P80X, S81X, D85X, A87X, I89X, Y90X, R91X, T93X, W94X, I96X, S97X, C101X, F102X, S1O3X, W104X, G105X, C106X, A107X, G108X, E109X, VI 1OX, R11 IX, Al 12X, LI 14X, QI 15X, El 16X, N117X, T118X, H119X, V120X, L122X, R123X, A126X, R128X, L135X, Y136X, E138X, A139X, Q141X, M142X, D145X, A146X, A148X, D156X, E157X, K159X, C161X, D163X, T164X, D167X, Q169X, C171X, D177X, D180X, H182X, S183X, Q184X, A185X, L186X, S187X, G188X, R189X, A192X, N196X a deletion selected from AM1-L12, AE36-G53, AN61-G68, AW104- G105 , AQ195-N199 , AI26-G27, or any combination thereof, where the position number designation is functionally equivalent to the position in a wild-type APOBEC3 A (SEQ ID NO:3) and X is an amino acid substitution different from the wild-type amino acid at that position.
[000183] In some embodiments, the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 102-105 the sequence FVDG, and from 130-134, or functionally equivalent positions, the sequence AKYPW, in combination with one or more combinations of stability enhancing mutations selected from: at least one of the following combinations of stabilizing mutations: R74L/C171A/T19Y/G25K/S45W;
R74L/C 171 A/T 19 Y/Gl 88Q/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88Q/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 88 A/S45 W; R74L/C 171 A/T 19 Y/Gl 88 A/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 88R/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88R/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/G25K/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88 A/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G188Q/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G188 A/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188 A/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188 A/G25K/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188R/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188R/G25K/S45 W; T 19 Y/R74L/C 171 A;
117T/T 19 Y/G25R/R74L/C 171 A; T 19 Y/G25K/S45 WR74L/G108 A/C 171 A/Gl 88R;
117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/G108C/C 171 A/Gl 88R; 117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/G108C/A126C/C 171 A/Gl 88R; or 117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/AW 104- G105/G108C/C 171 A/Gl 88R.
[000184] In some embodiments, the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 102-105 the sequence FVDG, and from 130-134, or functionally equivalent positions, the sequence AKYPW (SEQ ID NO:44), in combination with one or more combinations of stability enhancing mutations selected from Hl IX, L12X, D14X, H16X, I17X, T19X, S20X, N21X, G25X, I26X, G27X, R28X, H29X, T31X, C34X, E38X, R39X, D41X, N42X, S45X, K47X, M48X, H51X, F54X, H56X, N57X, A59X, K60X, L62X, Y67X, L73X, R74X, D77X, L78X, V79X, P80X, S81X, D85X, A87X, I89X, Y90X, R91X, T93X, W94X, I96X, S97X, C101X, F102X, S103X, W104X, G105X, C106X, A107X, G108X, E109X, VI 10X, R11 IX, Al 12X, LI 14X, QI 15X, El 16X, N117X, T118X, H119X, V120X, L122X, R123X, A126X, R128X, L135X, Y136X, E138X, A139X, Q141X, M142X, D145X, A146X, A148X, D156X, E157X, K159X, C161X, D163X, T164X, D167X, Q169X, C171X, D177X, D180X, H182X, S183X, Q184X, A185X, L186X, S187X, G188X, R189X, A192X, N196X a deletion selected from AM1-L12, AE36-G53, AN61-G68, AW104- G105 , AQ195-N199 , AI26-G27, or any combination thereof, where the position number designation is functionally equivalent to the position in a wild-type APOBEC3 A (SEQ ID NO:3) and X is an amino acid substitution different from the wild-type amino acid at that position.
[000185] In some embodiments, the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 102-105 the sequence FVPAG, and from 130-134, or functionally equivalent positions, the sequence AKYPW, in combination with one or more combinations of stability enhancing mutations selected from: at least one of the following combinations of stabilizing mutations: R74L/C171A/T19Y/G25K/S45W;
R74L/C 171 A/T 19 Y/Gl 88Q/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88Q/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 88 A/S45 W; R74L/C 171 A/T 19 Y/Gl 88 A/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 88R/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88R/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/G25K/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88 A/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G188Q/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188 A/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188 A/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G188 A/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G188R/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G188R/G25K/S45 W; T 19 Y/R74L/C 171 A; 117T/T 19 Y/G25R/R74L/C 171 A; T 19 Y/G25K/S45 WR74L/G108 A/C 171 A/Gl 88R; 117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/G108C/C 171 A/Gl 88R; 117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/G108C/A126C/C 171 A/Gl 88R; or 117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/AW 104- G105/G108C/C 171 A/Gl 88R.
[000186] In some embodiments, the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 102-105 the sequence FVPAG, and from 130-134, or functionally equivalent positions, the sequence AKYPW (SEQ ID NO:44), in combination with one or more combinations of stability enhancing mutations selected from Hl IX, L12X, D14X, H16X, I17X, T19X, S20X, N21X, G25X, I26X, G27X, R28X, H29X, T31X, C34X, E38X, R39X, D41X, N42X, S45X, K47X, M48X, H51X, F54X, H56X, N57X, A59X, K60X, L62X, Y67X, L73X, R74X, D77X, L78X, V79X, P80X, S81X, D85X, A87X, I89X, Y90X, R91X, T93X, W94X, I96X, S97X, C101X, F102X, S103X, W104X, G105X, C106X, A107X, G108X, E109X, VI 10X, R11 IX, Al 12X, LI 14X, QI 15X, El 16X, N117X, T118X, H119X, V120X, L122X, R123X, A126X, R128X, L135X, Y136X, E138X, A139X, Q141X, M142X, D145X, A146X, A148X, D156X, E157X, K159X, C161X, D163X, T164X, D167X, Q169X, C171X, D177X, D180X, H182X, S183X, Q184X, A185X, L186X, S187X, G188X, R189X, A192X, N196X a deletion selected from AM1-L12, AE36-G53, AN61-G68, AW104- G105 , AQ195-N199 , AI26-G27, or any combination thereof, where the position number designation is functionally equivalent to the position in a wild-type APOBEC3 A (SEQ ID NO:3) and X is an amino acid substitution different from the wild-type amino acid at that position.
[000187] In some embodiments, the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 102-105 the sequence FVPLG, and from 130-134, or functionally equivalent positions, the sequence AKYPW, in combination with one or more combinations of stability enhancing mutations selected from: at least one of the following combinations of stabilizing mutations: R74L/C171A/T19Y/G25K/S45W;
R74L/C 171 A/T 19 Y/Gl 88Q/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88Q/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 88 A/S45 W; R74L/C 171 A/T 19 Y/Gl 88 A/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 88R/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88R/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/G25K/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88 A/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G188Q/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G188 A/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188 A/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188 A/G25K/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188R/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188R/G25K/S45 W; T 19 Y/R74L/C 171 A;
117T/T 19 Y/G25R/R74L/C 171 A; T 19 Y/G25K/S45 WR74L/G108 A/C 171 A/Gl 88R;
117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/G108C/C 171 A/Gl 88R;
117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/G108C/A126C/C 171 A/Gl 88R; or
117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/AW 104-
G105/G108C/C 171 A/Gl 88R.
[000188] In some embodiments, the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 102-105 the sequence FVPLG, and from 130-134, or functionally equivalent positions, the sequence AKYPW (SEQ ID NO:44), in combination with one or more combinations of stability enhancing mutations selected from Hl IX, L12X, D14X, H16X, I17X, T19X, S20X, N21X, G25X, I26X, G27X, R28X, H29X, T31X, C34X, E38X, R39X, D41X, N42X, S45X, K47X, M48X, H51X, F54X, H56X, N57X, A59X, K60X, L62X, Y67X, L73X, R74X, D77X, L78X, V79X, P80X, S81X, D85X, A87X, I89X, Y90X, R91X, T93X, W94X, I96X, S97X, C101X, F102X, S103X, W104X, G105X, C106X, A107X, G108X, E109X, VI 10X, R11 IX, Al 12X, LI 14X, QI 15X, El 16X, N117X, T118X, H119X, V120X, L122X, R123X, A126X, R128X, L135X, Y136X, E138X, A139X, Q141X, M142X, D145X, A146X, A148X, D156X, E157X, K159X, C161X, D163X, T164X, D167X, Q169X, C171X, D177X, D180X, H182X, S183X, Q184X, A185X, L186X, S187X, G188X, R189X, A192X, N196X a deletion selected from AM1-L12, AE36-G53, AN61-G68, AW104- G105 , AQ195-N199 , AI26-G27, or any combination thereof, where the position number designation is functionally equivalent to the position in a wild-type APOBEC3 A (SEQ ID N0:3) and X is an amino acid substitution different from the wild-type amino acid at that position.
[000189] In some embodiments, the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 102-105 the sequence FVPPG, and from 130-134, or functionally equivalent positions, the sequence AKYPW, in combination with one or more combinations of stability enhancing mutations selected from: at least one of the following combinations of stabilizing mutations: R74L/C171A/T19Y/G25K/S45W;
R74L/C 171 A/T 19 Y/Gl 88Q/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88Q/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 88 A/S45 W; R74L/C 171 A/T 19 Y/Gl 88 A/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 88R/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88R/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/G25K/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88 A/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G188Q/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188 A/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188 A/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188 A/G25K/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188R/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188R/G25K/S45 W; T 19 Y/R74L/C 171 A;
117T/T 19 Y/G25R/R74L/C 171 A; T 19 Y/G25K/S45 WR74L/G108 A/C 171 A/Gl 88R;
117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/G108C/C 171 A/Gl 88R;
117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/G108C/A126C/C 171 A/Gl 88R; or
117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/AW 104-
G105/G108C/C 171 A/Gl 88R.
[000190] In some embodiments, the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 102-105 the sequence FVPPG, and from 130-134, or functionally equivalent positions, the sequence AKYPW (SEQ ID NO:44), in combination with one or more combinations of stability enhancing mutations selected from Hl IX, L12X, D14X, H16X, I17X, T19X, S20X, N21X, G25X, I26X, G27X, R28X, H29X, T31X, C34X, E38X, R39X, D41X, N42X, S45X, K47X, M48X, H51X, F54X, H56X, N57X, A59X, K60X, L62X, Y67X, L73X, R74X, D77X, L78X, V79X, P80X, S81X, D85X, A87X, I89X, Y90X, R91X, T93X, W94X, I96X, S97X, C101X, F102X, S103X, W104X, G105X, C106X, A107X, G108X, E109X, VI 10X, R11 IX, Al 12X, LI 14X, QI 15X, El 16X, N117X, T118X, H119X, V120X, L122X, R123X, A126X, R128X, L135X, Y136X, E138X, A139X, Q141X, M142X, D145X, A146X, A148X, D156X, E157X, K159X, C161X, D163X, T164X, D167X, Q169X, C171X, D177X, D180X, H182X, S183X, Q184X, A185X, L186X, S187X, G188X, R189X, A192X, N196X a deletion selected from AM1-L12, AE36-G53, AN61-G68, AW104- G105 , AQ195-N199 , AI26-G27, or any combination thereof, where the position number designation is functionally equivalent to the position in a wild-type APOBEC3 A (SEQ ID NO:3) and X is an amino acid substitution different from the wild-type amino acid at that position.
[000191] In some embodiments, the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 102-105 the sequence FVPAAG, and from 130-134, or functionally equivalent positions, the sequence AKYPW, in combination with one or more combinations of stability enhancing mutations selected from: at least one of the following combinations of stabilizing mutations: R74L/C171A/T19Y/G25K/S45W;
R74L/C 171 A/T 19 Y/Gl 88Q/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88Q/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 88 A/S45 W; R74L/C 171 A/T 19 Y/Gl 88 A/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 88R/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88R/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/G25K/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88 A/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G188Q/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G188 A/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188 A/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188 A/G25K/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188R/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188R/G25K/S45 W; T 19 Y/R74L/C 171 A;
117T/T 19 Y/G25R/R74L/C 171 A; T 19 Y/G25K/S45 WR74L/G108 A/C 171 A/Gl 88R;
117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/G108C/C 171 A/Gl 88R;
117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/G108C/A126C/C 171 A/Gl 88R; or 117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/AW 104-
G105/G108C/C 171 A/Gl 88R.
[000192] In some embodiments, the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 102-105 the sequence FVPAAG, and from 130-134, or functionally equivalent positions, the sequence AKYPW (SEQ ID NO:44), in combination with one or more combinations of stability enhancing mutations selected from Hl IX, L12X, D14X, H16X, I17X, T19X, S20X, N21X, G25X, I26X, G27X, R28X, H29X, T31X, C34X, E38X, R39X, D41X, N42X, S45X, K47X, M48X, H51X, F54X, H56X, N57X, A59X, K60X, L62X, Y67X, L73X, R74X, D77X, L78X, V79X, P80X, S81X, D85X, A87X, I89X, Y90X, R91X, T93X, W94X, I96X, S97X, C101X, F102X, S103X, W104X, G105X, C106X, A107X, G108X, E109X, VI 10X, R11 IX, Al 12X, LI 14X, QI 15X, El 16X, N117X, T118X, H119X, V120X, L122X, R123X, A126X, R128X, L135X, Y136X, E138X, A139X, Q141X, M142X, D145X, A146X, A148X, D156X, E157X, K159X, C161X, D163X, T164X, D167X, Q169X, C171X, D177X, D180X, H182X, S183X, Q184X, A185X, L186X, S187X, G188X, R189X, A192X, N196X a deletion selected from AM1-L12, AE36-G53, AN61-G68, AW104- G105 , AQ195-N199 , AI26-G27, or any combination thereof, where the position number designation is functionally equivalent to the position in a wild-type APOBEC3 A (SEQ ID NO:3) and X is an amino acid substitution different from the wild-type amino acid at that position.
[000193] In some embodiments, the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 102-105 the sequence FVPLAG, and from 130-134, or functionally equivalent positions, the sequence AKYPW, in combination with one or more combinations of stability enhancing mutations selected from: at least one of the following combinations of stabilizing mutations: R74L/C171A/T19Y/G25K/S45W;
R74L/C 171 A/T 19 Y/Gl 88Q/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88Q/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 88 A/S45 W; R74L/C 171 A/T 19 Y/Gl 88 A/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 88R/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88R/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/G25K/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88 A/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G188Q/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G188 A/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188 A/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G188 A/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G188R/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G188R/G25K/S45 W; T 19 Y/R74L/C 171 A; 117T/T 19 Y/G25R/R74L/C 171 A; T 19 Y/G25K/S45 WR74L/G108 A/C 171 A/Gl 88R; 117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/G108C/C 171 A/Gl 88R; 117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/G108C/A126C/C 171 A/Gl 88R; or 117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/AW 104- G105/G108C/C 171 A/Gl 88R.
[000194] In some embodiments, the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 102-105 the sequence FVPLAG, and from 130-134, or functionally equivalent positions, the sequence AKYPW (SEQ ID NO:44), in combination with one or more combinations of stability enhancing mutations selected from Hl IX, L12X, D14X, H16X, I17X, T19X, S20X, N21X, G25X, I26X, G27X, R28X, H29X, T31X, C34X, E38X, R39X, D41X, N42X, S45X, K47X, M48X, H51X, F54X, H56X, N57X, A59X, K60X, L62X, Y67X, L73X, R74X, D77X, L78X, V79X, P80X, S81X, D85X, A87X, I89X, Y90X, R91X, T93X, W94X, I96X, S97X, C101X, F102X, S103X, W104X, G105X, C106X, A107X, G108X, E109X, VI 10X, R11 IX, Al 12X, LI 14X, QI 15X, El 16X, N117X, T118X, H119X, V120X, L122X, R123X, A126X, R128X, L135X, Y136X, E138X, A139X, Q141X, M142X, D145X, A146X, A148X, D156X, E157X, K159X, C161X, D163X, T164X, D167X, Q169X, C171X, D177X, D180X, H182X, S183X, Q184X, A185X, L186X, S187X, G188X, R189X, A192X, N196X a deletion selected from AM1-L12, AE36-G53, AN61-G68, AW104- G105 , AQ195-N199 , AI26-G27, or any combination thereof, where the position number designation is functionally equivalent to the position in a wild-type APOBEC3 A (SEQ ID NO:3) and X is an amino acid substitution different from the wild-type amino acid at that position.
[000195] In some embodiments, the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 102-105 the sequence FVPPAG, and from 130-134, or functionally equivalent positions, the sequence AKYPW, in combination with one or more combinations of stability enhancing mutations selected from: at least one of the following combinations of stabilizing mutations: R74L/C171A/T19Y/G25K/S45W;
R74L/C 171 A/T 19 Y/Gl 88Q/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88Q/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 88 A/S45 W; R74L/C 171 A/T 19 Y/Gl 88 A/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88R/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88R/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/G25K/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88 A/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G188Q/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G188 A/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188 A/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188 A/G25K/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188R/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188R/G25K/S45 W; T 19 Y/R74L/C 171 A;
117T/T 19 Y/G25R/R74L/C 171 A; T 19 Y/G25K/S45 WR74L/G108 A/C 171 A/Gl 88R;
117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/G108C/C 171 A/Gl 88R;
117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/G108C/A126C/C 171 A/Gl 88R; or 117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/AW 104-
G105/G108C/C 171 A/Gl 88R.
[000196] In some embodiments, the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 102-105 the sequence FVPPAG, and from 130-134, or functionally equivalent positions, the sequence AKYPW (SEQ ID NO:44), in combination with one or more combinations of stability enhancing mutations selected from Hl IX, L12X, D14X, H16X, I17X, T19X, S20X, N21X, G25X, I26X, G27X, R28X, H29X, T31X, C34X, E38X, R39X, D41X, N42X, S45X, K47X, M48X, H51X, F54X, H56X, N57X, A59X, K60X, L62X, Y67X, L73X, R74X, D77X, L78X, V79X, P80X, S81X, D85X, A87X, I89X, Y90X, R91X, T93X, W94X, I96X, S97X, C101X, F102X, S103X, W104X, G105X, C106X, A107X, G108X, E109X, VI 10X, R11 IX, Al 12X, LI 14X, QI 15X, El 16X, N117X, T118X, H119X, V120X, L122X, R123X, A126X, R128X, L135X, Y136X, E138X, A139X, Q141X, M142X, D145X, A146X, A148X, D156X, E157X, K159X, C161X, D163X, T164X, D167X, Q169X, C171X, D177X, D180X, H182X, S183X, Q184X, A185X, L186X, S187X, G188X, R189X, A192X, N196X a deletion selected from AM1-L12, AE36-G53, AN61-G68, AW104- G105 , AQ195-N199 , AI26-G27, or any combination thereof, where the position number designation is functionally equivalent to the position in a wild-type APOBEC3 A (SEQ ID N0:3) and X is an amino acid substitution different from the wild-type amino acid at that position.
[000197] In some embodiments, the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 102-105 the sequence FVPASG, and from 130-134, or functionally equivalent positions, the sequence AKYPW, in combination with one or more combinations of stability enhancing mutations selected from: at least one of the following combinations of stabilizing mutations: R74L/C171A/T19Y/G25K/S45W;
R74L/C 171 A/T 19 Y/Gl 88Q/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88Q/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 88 A/S45 W; R74L/C 171 A/T 19 Y/Gl 88 A/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 88R/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88R/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/G25K/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88 A/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G188Q/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188 A/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188 A/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188 A/G25K/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188R/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188R/G25K/S45 W; T 19 Y/R74L/C 171 A;
117T/T 19 Y/G25R/R74L/C 171 A; T 19 Y/G25K/S45 WR74L/G108 A/C 171 A/Gl 88R;
117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/G108C/C 171 A/Gl 88R;
117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/G108C/A126C/C 171 A/Gl 88R; or
117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/AW 104-
G105/G108C/C 171 A/Gl 88R.
[000198] In some embodiments, the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 102-105 130-134, or functionally equivalent positions, the sequence FIMA AHIYW, in combination with one or more combinations of stability enhancing mutations selected from: at least one of the following combinations of stabilizing mutations: R74L/C 171 A/T 19 Y/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 88Q/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 88Q/G25K/S45W; R74L/C 171 A/T 19 Y/Gl 88 A/S45 W;
R74L/C 171 A/T 19 Y/Gl 88 A/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88R/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88R/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/G25K/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/G25K/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88 A/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G188Q/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G188 A/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188 A/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188 A/G25K/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188R/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188R/G25K/S45 W; T 19 Y/R74L/C 171 A;
117T/T 19 Y/G25R/R74L/C 171 A; T 19 Y/G25K/S45 WR74L/G108 A/C 171 A/Gl 88R;
117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/G108C/C 171 A/Gl 88R;
117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/G108C/A126C/C 171 A/Gl 88R; or 117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/AW 104-
G105/G108C/C 171 A/Gl 88R.
[000199] In some embodiments, the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 102-105 130-134, or functionally equivalent positions, the sequence FIMA AHIYW, in combination with one or more combinations of stability enhancing mutations selected from Hl IX, L12X, D14X, H16X, I17X, T19X, S20X, N21X, G25X, I26X, G27X, R28X, H29X, T31X, C34X, E38X, R39X, D41X, N42X, S45X, K47X, M48X, H51X, F54X, H56X, N57X, A59X, K60X, L62X, Y67X, L73X, R74X, D77X, L78X, V79X, P80X, S81X, D85X, A87X, I89X, Y90X, R91X, T93X, W94X, I96X, S97X, C101X, F102X, S103X, W104X, G105X, C106X, A107X, G108X, E109X, V110X, R111X, Al 12X, LI 14X, QI 15X, El 16X, N117X, T118X, Hl 19X, V120X, L122X, R123X, A126X, R128X, L135X, Y136X, E138X, A139X, Q141X, M142X, D145X, A146X, A148X, D156X, E157X, K159X, C161X, D163X, T164X, D167X, Q169X, C171X, D177X, D180X, H182X, S183X, Q184X, A185X, L186X, S187X, G188X, R189X, A192X, N196X a deletion selected from AM1-L12, AE36-G53, AN61-G68, AW104-G105 , AQ195-N199 , AI26-G27, or any combination thereof, where the position number designation is functionally equivalent to the position in a wild-type APOBEC3A (SEQ ID NO:3) and X is an amino acid substitution different from the wild-type amino acid at that position. In some embodiments, the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 102- 105 130-134, or functionally equivalent positions, the sequence HLTG AHEYW , in combination with one or more combinations of stability enhancing mutations selected from: at least one of the following combinations of stabilizing mutations:
R74L/C 171 A/T 19 Y/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 88Q/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 88Q/G25K/S45W; R74L/C 171 A/T 19 Y/Gl 88 A/S45 W;
R74L/C 171 A/T 19 Y/Gl 88 A/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88R/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88R/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/G25K/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88 A/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G188Q/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188 A/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188 A/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188 A/G25K/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188R/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188R/G25K/S45 W; T 19 Y/R74L/C 171 A;
117T/T 19 Y/G25R/R74L/C 171 A; T 19 Y/G25K/S45 WR74L/G108 A/C 171 A/Gl 88R;
117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/G108C/C 171 A/Gl 88R;
117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/G108C/A126C/C 171 A/Gl 88R; or
117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/AW 104-
G105/G108C/C 171 A/Gl 88R.
[000200] In some embodiments, the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 102-105 130-134, or functionally equivalent positions, the sequence HLTG AHEYW, in combination with one or more combinations of stability enhancing mutations selected from Hl IX, L12X, D14X, H16X, I17X, T19X, S20X, N21X, G25X, I26X, G27X, R28X, H29X, T31X, C34X, E38X, R39X, D41X, N42X, S45X, K47X, M48X, H51X, F54X, H56X, N57X, A59X, K60X, L62X, Y67X, L73X, R74X, D77X, L78X, V79X, P80X, S81X, D85X, A87X, I89X, Y90X, R91X, T93X, W94X, I96X, S97X, C101X, F102X, S103X, W104X, G105X, C106X, A107X, G108X, E109X, V110X, R111X, Al 12X, LI 14X, QI 15X, El 16X, N117X, T118X, Hl 19X, V120X, L122X, R123X, A126X, R128X, L135X, Y136X, E138X, A139X, Q141X, M142X, D145X, A146X, A148X, D156X, E157X, K159X, C161X, D163X, T164X, D167X, Q169X, C171X, D177X, D180X, H182X, S183X, Q184X, A185X, L186X, S187X, G188X, R189X, A192X, N196X a deletion selected from AM1-L12, AE36-G53, AN61-G68, AW104-G105 , AQ195-N199 , AI26-G27, or any combination thereof, where the position number designation is functionally equivalent to the position in a wild-type AP0BEC3A (SEQ ID NO:3) and X is an amino acid substitution different from the wild-type amino acid at that position.
[000201] In some embodiments, the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 102-105 130-134, or functionally equivalent positions, the sequence QVPA AHDFW , in combination with one or more combinations of stability enhancing mutations selected from: at least one of the following combinations of stabilizing mutations: R74L/C 171 A/T 19 Y/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 88Q/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 88Q/G25K/S45W; R74L/C 171 A/T 19 Y/Gl 88 A/S45 W;
R74L/C 171 A/T 19 Y/Gl 88 A/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88R/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88R/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/G25K/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88 A/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G188Q/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G188 A/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188 A/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188 A/G25K/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188R/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188R/G25K/S45 W; T 19 Y/R74L/C 171 A;
117T/T 19 Y/G25R/R74L/C 171 A; T 19 Y/G25K/S45 WR74L/G108 A/C 171 A/Gl 88R;
117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/G108C/C 171 A/Gl 88R;
117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/G108C/A126C/C 171 A/Gl 88R; or
117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/AW 104-
G105/G108C/C 171 A/Gl 88R.
[000202] In some embodiments, the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 102-105 130-134, or functionally equivalent positions, the sequence QVPA AHDFW, in combination with one or more combinations of stability enhancing mutations selected from Hl IX, L12X, D14X, H16X, I17X, T19X, S20X, N21X, G25X, I26X, G27X, R28X, H29X, T31X, C34X, E38X, R39X, D41X, N42X, S45X, K47X, M48X, H51X, F54X, H56X, N57X, A59X, K60X, L62X, Y67X, L73X, R74X, D77X, L78X, V79X, P80X, S81X, D85X, A87X, I89X, Y90X, R91X, T93X, W94X, I96X, S97X, C101X, F102X, S103X, W104X, G105X, C106X, A107X, G108X, E109X, V110X, R111X, Al 12X, LI 14X, QI 15X, El 16X, N117X, T118X, Hl 19X, V120X, L122X, R123X, A126X, R128X, L135X, Y136X, E138X, A139X, Q141X, M142X, D145X, A146X, A148X, D156X, E157X, K159X, C161X, D163X, T164X, D167X, Q169X, C171X, D177X, D180X, H182X, S183X, Q184X, A185X, L186X, S187X, G188X, R189X, A192X, N196X a deletion selected from AM1-L12, AE36-G53, AN61-G68, AW104-G105 , AQ195-N199 , AI26-G27, or any combination thereof, where the position number designation is functionally equivalent to the position in a wild-type APOBEC3A (SEQ ID NO:3) and X is an amino acid substitution different from the wild-type amino acid at that position.
[000203] In some embodiments, the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 102-105 130-134, or functionally equivalent positions, the sequence EFNQ ANVHW, in combination with one or more combinations of stability enhancing mutations selected from: at least one of the following combinations of stabilizing mutations: R74L/C 171 A/T 19 Y/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 88Q/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88Q/G25K/S45W; R74L/C 171 A/T 19 Y/Gl 88 A/S45 W;
R74L/C 171 A/T 19 Y/Gl 88 A/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88R/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88R/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/G25K/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88 A/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G188Q/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G188 A/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188 A/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188 A/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G188R/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G188R/G25K/S45 W; T 19 Y/R74L/C 171 A; 117T/T 19 Y/G25R/R74L/C 171 A; T 19 Y/G25K/S45 WR74L/G108 A/C 171 A/Gl 88R;
117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/G108C/C 171 A/Gl 88R;
117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/G108C/A126C/C 171 A/Gl 88R; or 117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/AW 104- G105/G108C/C 171 A/Gl 88R.
[000204] In some embodiments, the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 102-105 130-134, or functionally equivalent positions, the sequence EFNQ ANVHW, in combination with one or more combinations of stability enhancing mutations selected from Hl IX, L12X, D14X, H16X, I17X, T19X, S20X, N21X, G25X, I26X, G27X, R28X, H29X, T31X, C34X, E38X, R39X, D41X, N42X, S45X, K47X, M48X, H51X, F54X, H56X, N57X, A59X, K60X, L62X, Y67X, L73X, R74X, D77X, L78X, V79X, P80X, S81X, D85X, A87X, I89X, Y90X, R91X, T93X, W94X, I96X, S97X, C101X, F102X, S103X, W104X, G105X, C106X, A107X, G108X, E109X, V110X, R111X, Al 12X, LI 14X, QI 15X, El 16X, N117X, T118X, Hl 19X, V120X, L122X, R123X, A126X, R128X, L135X, Y136X, E138X, A139X, Q141X, M142X, D145X, A146X, A148X, D156X, E157X, K159X, C161X, D163X, T164X, D167X, Q169X, C171X, D177X, D180X, H182X, S183X, Q184X, A185X, L186X, S187X, G188X, R189X, A192X, N196X a deletion selected from AM1-L12, AE36-G53, AN61-G68, AW104-G105 , AQ195-N199 , AI26-G27, or any combination thereof, where the position number designation is functionally equivalent to the position in a wild-type APOBEC3A (SEQ ID NO:3) and X is an amino acid substitution different from the wild-type amino acid at that position.
[000205] In some embodiments, the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 102-105 130-134, or functionally equivalent positions, the sequence QTDH AAEHW, in combination with one or more combinations of stability enhancing mutations selected from: at least one of the following combinations of stabilizing mutations: R74L/C 171 A/T 19 Y/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 88Q/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88Q/G25K/S45W; R74L/C 171 A/T 19 Y/Gl 88 A/S45 W;
R74L/C 171 A/T 19 Y/Gl 88 A/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88R/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88R/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/G25K/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88 A/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G188Q/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G188 A/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188 A/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188 A/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G188R/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G188R/G25K/S45 W; T 19 Y/R74L/C 171 A; 117T/T 19 Y/G25R/R74L/C 171 A; T 19 Y/G25K/S45 WR74L/G108 A/C 171 A/Gl 88R; 117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/G108C/C 171 A/Gl 88R;
117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/G108C/A126C/C 171 A/Gl 88R; or 117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/AW 104- G105/G108C/C 171 A/Gl 88R.
[000206] In some embodiments, the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 102-105 130-134, or functionally equivalent positions, the sequence QTDH AAEHW, in combination with one or more combinations of stability enhancing mutations selected from Hl IX, L12X, D14X, H16X, I17X, T19X, S20X, N21X, G25X, I26X, G27X, R28X, H29X, T31X, C34X, E38X, R39X, D41X, N42X, S45X, K47X, M48X, H51X, F54X, H56X, N57X, A59X, K60X, L62X, Y67X, L73X, R74X, D77X, L78X, V79X, P80X, S81X, D85X, A87X, I89X, Y90X, R91X, T93X, W94X, I96X, S97X, C101X, F102X, S103X, W104X, G105X, C106X, A107X, G108X, E109X, V110X, R111X, Al 12X, LI 14X, QI 15X, El 16X, N117X, T118X, Hl 19X, V120X, L122X, R123X, A126X, R128X, L135X, Y136X, E138X, A139X, Q141X, M142X, D145X, A146X, A148X, D156X, E157X, K159X, C161X, D163X, T164X, D167X, Q169X, C171X, D177X, D180X, H182X, S183X, Q184X, A185X, L186X, S187X, G188X, R189X, A192X, N196X a deletion selected from AM1-L12, AE36-G53, AN61-G68, AW104-G105 , AQ195-N199 , AI26-G27, or any combination thereof, where the position number designation is functionally equivalent to the position in a wild-type APOBEC3A (SEQ ID NO:3) and X is an amino acid substitution different from the wild-type amino acid at that position.
[000207] In some embodiments, the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 102-105 130-134, or functionally equivalent positions, the sequence GLNG AHEYW, in combination with one or more combinations of stability enhancing mutations selected from: at least one of the following combinations of stabilizing mutations: R74L/C 171 A/T 19 Y/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 88Q/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88Q/G25K/S45W; R74L/C 171 A/T 19 Y/Gl 88 A/S45 W; R74L/C 171 A/T 19 Y/Gl 88 A/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88R/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88R/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/G25K/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88 A/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G188Q/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G188 A/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188 A/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188 A/G25K/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188R/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188R/G25K/S45 W; T 19 Y/R74L/C 171 A;
117T/T 19 Y/G25R/R74L/C 171 A; T 19 Y/G25K/S45 WR74L/G108 A/C 171 A/Gl 88R;
117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/G108C/C 171 A/Gl 88R;
117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/G108C/A126C/C 171 A/Gl 88R; or 117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/AW 104-
G105/G108C/C 171 A/Gl 88R.
[000208] In some embodiments, the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 102-105 130-134, or functionally equivalent positions, the sequence GLNG AHEYW, in combination with one or more combinations of stability enhancing mutations selected from Hl IX, L12X, D14X, H16X, I17X, T19X, S20X, N21X, G25X, I26X, G27X, R28X, H29X, T31X, C34X, E38X, R39X, D41X, N42X, S45X, K47X, M48X, H51X, F54X, H56X, N57X, A59X, K60X, L62X, Y67X, L73X, R74X, D77X, L78X, V79X, P80X, S81X, D85X, A87X, I89X, Y90X, R91X, T93X, W94X, I96X, S97X, C101X, F102X, S103X, W104X, G105X, C106X, A107X, G108X, E109X, V110X, R111X, Al 12X, LI 14X, QI 15X, El 16X, N117X, T118X, Hl 19X, V120X, L122X, R123X, A126X, R128X, L135X, Y136X, E138X, A139X, Q141X, M142X, D145X, A146X, A148X, D156X, E157X, K159X, C161X, D163X, T164X, D167X, Q169X, C171X, D177X, D180X, H182X, S183X, Q184X, A185X, L186X, S187X, G188X, R189X, A192X, N196X a deletion selected from AM1-L12, AE36-G53, AN61-G68, AW104-G105 , AQ195-N199 , AI26-G27, or any combination thereof, where the position number designation is functionally equivalent to the position in a wild-type APOBEC3A (SEQ ID NO:3) and X is an amino acid substitution different from the wild-type amino acid at that position.
[000209] In some embodiments, the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 102-105 130-134, or functionally equivalent positions, the sequence YLPA ANGFW, in combination with one or more combinations of stability enhancing mutations selected from: at least one of the following combinations of stabilizing mutations: R74L/C 171 A/T 19 Y/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 88Q/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88Q/G25K/S45W; R74L/C 171 A/T 19 Y/Gl 88 A/S45 W;
R74L/C 171 A/T 19 Y/Gl 88 A/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88R/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88R/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/G25K/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88 A/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G188Q/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188 A/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188 A/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188 A/G25K/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188R/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188R/G25K/S45 W; T 19 Y/R74L/C 171 A;
117T/T 19 Y/G25R/R74L/C 171 A; T 19 Y/G25K/S45 WR74L/G108 A/C 171 A/Gl 88R;
117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/G108C/C 171 A/Gl 88R;
117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/G108C/A126C/C 171 A/Gl 88R; or
117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/AW 104-
G105/G108C/C 171 A/Gl 88R.
[000210] In some embodiments, the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 102-105 130-134, or functionally equivalent positions, the sequence YLPA ANGFW, in combination with one or more combinations of stability enhancing mutations selected from Hl IX, L12X, D14X, H16X, I17X, T19X, S20X, N21X, G25X, I26X, G27X, R28X, H29X, T31X, C34X, E38X, R39X, D41X, N42X, S45X, K47X, M48X, H51X, F54X, H56X, N57X, A59X, K60X, L62X, Y67X, L73X, R74X, D77X, L78X, V79X, P80X, S81X, D85X, A87X, I89X, Y90X, R91X, T93X, W94X, I96X, S97X, C101X, F102X, S103X, W104X, G105X, C106X, A107X, G108X, E109X, V110X, R111X, Al 12X, LI 14X, QI 15X, El 16X, N117X, T118X, Hl 19X, V120X, L122X, R123X, A126X, R128X, L135X, Y136X, E138X, A139X, Q141X, M142X, D145X, A146X, A148X, D156X, E157X, K159X, C161X, D163X, T164X, D167X, Q169X, C171X, D177X, D180X, H182X, S183X, Q184X, A185X, L186X, S187X, G188X, R189X, A192X, N196X a deletion selected from AM1-L12, AE36-G53, AN61-G68, AW104-G105 , AQ195-N199 , AI26-G27, or any combination thereof, where the position number designation is functionally equivalent to the position in a wild-type APOBEC3A (SEQ ID NO:3) and X is an amino acid substitution different from the wild-type amino acid at that position.
[000211] In some embodiments, the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 102-105 130-134, or functionally equivalent positions, the sequence EMFA ANDFW , in combination with one or more combinations of stability enhancing mutations selected from: at least one of the following combinations of stabilizing mutations: R74L/C 171 A/T 19 Y/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 88Q/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88Q/G25K/S45W; R74L/C 171 A/T 19 Y/Gl 88 A/S45 W;
R74L/C 171 A/T 19 Y/Gl 88 A/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88R/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88R/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/G25K/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88 A/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G188Q/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188 A/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188 A/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188 A/G25K/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188R/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188R/G25K/S45 W; T 19 Y/R74L/C 171 A;
117T/T 19 Y/G25R/R74L/C 171 A; T 19 Y/G25K/S45 WR74L/G108 A/C 171 A/Gl 88R;
117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/G108C/C 171 A/Gl 88R;
117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/G108C/A126C/C 171 A/Gl 88R; or 117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/AW 104-
G105/G108C/C 171 A/Gl 88R. [000212] In some embodiments, the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 102-105 130-134, or functionally equivalent positions, the sequence EMFA ANDFW, in combination with one or more combinations of stability enhancing mutations selected from Hl IX, L12X, D14X, H16X, I17X, T19X, S20X, N21X, G25X, I26X, G27X, R28X, H29X, T31X, C34X, E38X, R39X, D41X, N42X, S45X, K47X, M48X, H51X, F54X, H56X, N57X, A59X, K60X, L62X, Y67X, L73X, R74X, D77X, L78X, V79X, P80X, S81X, D85X, A87X, I89X, Y90X, R91X, T93X, W94X, I96X, S97X, C101X, F102X, S103X, W104X, G105X, C106X, A107X, G108X, E109X, V110X, R111X, Al 12X, LI 14X, QI 15X, El 16X, N117X, T118X, Hl 19X, V120X, L122X, R123X, A126X, R128X, L135X, Y136X, E138X, A139X, Q141X, M142X, D145X, A146X, A148X, D156X, E157X, K159X, C161X, D163X, T164X, D167X, Q169X, C171X, D177X, D180X, H182X, S183X, Q184X, A185X, L186X, S187X, G188X, R189X, A192X, N196X a deletion selected from AM1-L12, AE36-G53, AN61-G68, AW104-G105 , AQ195-N199 , AI26-G27, or any combination thereof, where the position number designation is functionally equivalent to the position in a wild-type APOBEC3A (SEQ ID NO:3) and X is an amino acid substitution different from the wild-type amino acid at that position.
[000213] In some embodiments, the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 102-105 130-134, or functionally equivalent positions, the sequence YKQY AGEYW, in combination with one or more combinations of stability enhancing mutations selected from: at least one of the following combinations of stabilizing mutations: R74L/C 171 A/T 19 Y/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 88Q/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88Q/G25K/S45W; R74L/C 171 A/T 19 Y/Gl 88 A/S45 W;
R74L/C 171 A/T 19 Y/Gl 88 A/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88R/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88R/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/G25K/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88 A/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G188Q/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G188 A/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188 A/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188 A/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G188R/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188R/G25K/S45 W; T 19 Y/R74L/C 171 A;
117T/T 19 Y/G25R/R74L/C 171 A; T 19 Y/G25K/S45 WR74L/G108 A/C 171 A/Gl 88R;
117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/G108C/C 171 A/Gl 88R;
117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/G108C/A126C/C 171 A/Gl 88R; or 117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/AW 104- G105/G108C/C 171 A/Gl 88R.
[000214] In some embodiments, the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 102-105 130-134, or functionally equivalent positions, the sequence YKQY AGEYW, in combination with one or more combinations of stability enhancing mutations selected from Hl IX, L12X, D14X, H16X, I17X, T19X, S20X, N21X, G25X, I26X, G27X, R28X, H29X, T31X, C34X, E38X, R39X, D41X, N42X, S45X, K47X, M48X, H51X, F54X, H56X, N57X, A59X, K60X, L62X, Y67X, L73X, R74X, D77X, L78X, V79X, P80X, S81X, D85X, A87X, I89X, Y90X, R91X, T93X, W94X, I96X, S97X, C101X, F102X, S103X, W104X, G105X, C106X, A107X, G108X, E109X, V110X, R111X, Al 12X, LI 14X, QI 15X, El 16X, N117X, T118X, Hl 19X, V120X, L122X, R123X, A126X, R128X, L135X, Y136X, E138X, A139X, Q141X, M142X, D145X, A146X, A148X, D156X, E157X, K159X, C161X, D163X, T164X, D167X, Q169X, C171X, D177X, D180X, H182X, S183X, Q184X, A185X, L186X, S187X, G188X, R189X, A192X, N196X a deletion selected from AM1-L12, AE36-G53, AN61-G68, AW104-G105 , AQ195-N199 , AI26-G27, or any combination thereof, where the position number designation is functionally equivalent to the position in a wild-type APOBEC3A (SEQ ID NO:3) and X is an amino acid substitution different from the wild-type amino acid at that position.
[000215] In some embodiments, the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 102-105 130-134, or functionally equivalent positions, the sequence YVRL AVPFW, in combination with one or more combinations of stability enhancing mutations selected from: at least one of the following combinations of stabilizing mutations: R74L/C 171 A/T 19 Y/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 88Q/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88Q/G25K/S45W; R74L/C 171 A/T 19 Y/Gl 88 A/S45 W;
R74L/C 171 A/T 19 Y/Gl 88 A/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88R/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88R/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88 A/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G188Q/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G188 A/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188 A/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188 A/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G188R/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G188R/G25K/S45 W; T 19 Y/R74L/C 171 A; 117T/T 19 Y/G25R/R74L/C 171 A; T 19 Y/G25K/S45 WR74L/G108 A/C 171 A/Gl 88R; 117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/G108C/C 171 A/Gl 88R;
117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/G108C/A126C/C 171 A/Gl 88R; or 117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/AW 104- G105/G108C/C 171 A/Gl 88R.
[000216] In some embodiments, the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 102-105 130-134, or functionally equivalent positions, the sequence YVRL AVPFW, in combination with one or more combinations of stability enhancing mutations selected from Hl IX, L12X, D14X, H16X, I17X, T19X, S20X, N21X, G25X, I26X, G27X, R28X, H29X, T31X, C34X, E38X, R39X, D41X, N42X, S45X, K47X, M48X, H51X, F54X, H56X, N57X, A59X, K60X, L62X, Y67X, L73X, R74X, D77X, L78X, V79X, P80X, S81X, D85X, A87X, I89X, Y90X, R91X, T93X, W94X, I96X, S97X, C101X, F102X, S103X, W104X, G105X, C106X, A107X, G108X, E109X, V110X, R111X, Al 12X, LI 14X, QI 15X, El 16X, N117X, T118X, Hl 19X, V120X, L122X, R123X, A126X, R128X, L135X, Y136X, E138X, A139X, Q141X, M142X, D145X, A146X, A148X, D156X, E157X, K159X, C161X, D163X, T164X, D167X, Q169X, C171X, D177X, D180X, H182X, S183X, Q184X, A185X, L186X, S187X, G188X, R189X, A192X, N196X a deletion selected from AM1-L12, AE36-G53, AN61-G68, AW104-G105 , AQ195-N199 , AI26-G27, or any combination thereof, where the position number designation is functionally equivalent to the position in a wild-type APOBEC3A (SEQ ID NO:3) and X is an amino acid substitution different from the wild-type amino acid at that position.
[000217] In some embodiments, the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 102-105 130-134, or functionally equivalent positions, the sequence YIKL AVAHW, in combination with one or more combinations of stability enhancing mutations selected from: at least one of the following combinations of stabilizing mutations: R74L/C 171 A/T 19 Y/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 88Q/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88Q/G25K/S45W; R74L/C 171 A/T 19 Y/Gl 88 A/S45 W;
R74L/C 171 A/T 19 Y/Gl 88 A/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88R/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88R/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/G25K/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88 A/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G188Q/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G188 A/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188 A/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188 A/G25K/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188R/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188R/G25K/S45 W; T 19 Y/R74L/C 171 A;
117T/T 19 Y/G25R/R74L/C 171 A; T 19 Y/G25K/S45 WR74L/G108 A/C 171 A/Gl 88R;
117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/G108C/C 171 A/Gl 88R;
117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/G108C/A126C/C 171 A/Gl 88R; or
117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/AW 104-
G105/G108C/C 171 A/Gl 88R.
[000218] In some embodiments, the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 102-105 130-134, or functionally equivalent positions, the sequence YIKL AVAHW, in combination with one or more combinations of stability enhancing mutations selected from Hl IX, L12X, D14X, H16X, I17X, T19X, S20X, N21X, G25X, I26X, G27X, R28X, H29X, T31X, C34X, E38X, R39X, D41X, N42X, S45X, K47X, M48X, H51X, F54X, H56X, N57X, A59X, K60X, L62X, Y67X, L73X, R74X, D77X, L78X, V79X, P80X, S81X, D85X, A87X, I89X, Y90X, R91X, T93X, W94X, I96X, S97X, C101X, F102X, S103X, W104X, G105X, C106X, A107X, G108X, E109X, V110X, R111X, Al 12X, LI 14X, QI 15X, El 16X, N117X, T118X, Hl 19X, V120X, L122X, R123X, A126X, R128X, L135X, Y136X, E138X, A139X, Q141X, M142X, D145X, A146X, A148X, D156X, E157X, K159X, C161X, D163X, T164X, D167X, Q169X, C171X, D177X, D180X, H182X, S183X, Q184X, A185X, L186X, S187X, G188X, R189X, A192X, N196X a deletion selected from AM1-L12, AE36-G53, AN61-G68, AW104-G105 , AQ195-N199 , AI26-G27, or any combination thereof, where the position number designation is functionally equivalent to the position in a wild-type APOBEC3A (SEQ ID NO:3) and X is an amino acid substitution different from the wild-type amino acid at that position.
[000219] In some embodiments, the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 102-105 130-134, or functionally equivalent positions, the sequence FILA ALAHW, in combination with one or more combinations of stability enhancing mutations selected from: at least one of the following combinations of stabilizing mutations: R74L/C 171 A/T 19 Y/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 88Q/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88Q/G25K/S45W; R74L/C 171 A/T 19 Y/Gl 88 A/S45 W;
R74L/C 171 A/T 19 Y/Gl 88 A/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88R/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88R/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/G25K/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88 A/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G188Q/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188 A/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188 A/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188 A/G25K/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188R/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188R/G25K/S45 W; T 19 Y/R74L/C 171 A;
117T/T 19 Y/G25R/R74L/C 171 A; T 19 Y/G25K/S45 WR74L/G108 A/C 171 A/Gl 88R;
117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/G108C/C 171 A/Gl 88R;
117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/G108C/A126C/C 171 A/Gl 88R; or
117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/AW 104-
G105/G108C/C 171 A/Gl 88R.
[000220] In some embodiments, the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 102-105 130-134, or functionally equivalent positions, the sequence FILA ALAHW, in combination with one or more combinations of stability enhancing mutations selected from Hl IX, L12X, D14X, H16X, I17X, T19X, S20X, N21X, G25X, I26X, G27X, R28X, H29X, T31X, C34X, E38X, R39X, D41X, N42X, S45X, K47X, M48X, H51X, F54X, H56X, N57X, A59X, K60X, L62X, Y67X, L73X, R74X, D77X, L78X, V79X, P80X, S81X, D85X, A87X, I89X, Y90X, R91X, T93X, W94X, I96X, S97X, C101X, F102X, S103X, W104X, G105X, C106X, A107X, G108X, E109X, V110X, R111X, Al 12X, LI 14X, QI 15X, El 16X, N117X, T118X, Hl 19X, V120X, L122X, R123X, A126X, R128X, L135X, Y136X, E138X, A139X, Q141X, M142X, D145X, A146X, A148X, D156X, E157X, K159X, C161X, D163X, T164X, D167X, Q169X, C171X, D177X, D180X, H182X, S183X, Q184X, A185X, L186X, S187X, G188X, R189X, A192X, N196X a deletion selected from AM1-L12, AE36-G53, AN61-G68, AW104-G105 , AQ195-N199 , AI26-G27, or any combination thereof, where the position number designation is functionally equivalent to the position in a wild-type APOBEC3A (SEQ ID NO:3) and X is an amino acid substitution different from the wild-type amino acid at that position.
[000221] In some embodiments, the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 102-105 130-134, or functionally equivalent positions, the sequence QTQY ARVKW, in combination with one or more combinations of stability enhancing mutations selected from: at least one of the following combinations of stabilizing mutations: R74L/C 171 A/T 19 Y/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 88Q/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88Q/G25K/S45W; R74L/C 171 A/T 19 Y/Gl 88 A/S45 W;
R74L/C 171 A/T 19 Y/Gl 88 A/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88R/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88R/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/G25K/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88 A/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G188Q/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G188 A/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188 A/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188 A/G25K/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188R/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188R/G25K/S45 W; T 19 Y/R74L/C 171 A;
117T/T 19 Y/G25R/R74L/C 171 A; T 19 Y/G25K/S45 WR74L/G108 A/C 171 A/Gl 88R;
117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/G108C/C 171 A/Gl 88R;
117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/G108C/A126C/C 171 A/Gl 88R; or 117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/AW 104-
G105/G108C/C 171 A/Gl 88R.
[000222] In some embodiments, the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 102-105 130-134, or functionally equivalent positions, the sequence QTQY ARVKW, in combination with one or more combinations of stability enhancing mutations selected from Hl IX, L12X, D14X, H16X, I17X, T19X, S20X, N21X, G25X, I26X, G27X, R28X, H29X, T31X, C34X, E38X, R39X, D41X, N42X, S45X, K47X, M48X, H51X, F54X, H56X, N57X, A59X, K60X, L62X, Y67X, L73X, R74X, D77X, L78X, V79X, P80X, S81X, D85X, A87X, I89X, Y90X, R91X, T93X, W94X, I96X, S97X, C101X, F102X, S103X, W104X, G105X, C106X, A107X, G108X, E109X, V110X, R111X, Al 12X, LI 14X, QI 15X, El 16X, N117X, T118X, Hl 19X, V120X, L122X, R123X, A126X, R128X, L135X, Y136X, E138X, A139X, Q141X, M142X, D145X, A146X, A148X, D156X, E157X, K159X, C161X, D163X, T164X, D167X, Q169X, C171X, D177X, D180X, H182X, S183X, Q184X, A185X, L186X, S187X, G188X, R189X, A192X, N196X a deletion selected from AM1-L12, AE36-G53, AN61-G68, AW104-G105 , AQ195-N199 , AI26-G27, or any combination thereof, where the position number designation is functionally equivalent to the position in a wild-type APOBEC3A (SEQ ID NO:3) and X is an amino acid substitution different from the wild-type amino acid at that position.
[000223] In some embodiments, the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 102-105 130-134, or functionally equivalent positions, the sequence KTNN ASEHW, in combination with one or more combinations of stability enhancing mutations selected from: at least one of the following combinations of stabilizing mutations: R74L/C 171 A/T 19 Y/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 88Q/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88Q/G25K/S45W; R74L/C 171 A/T 19 Y/Gl 88 A/S45 W;
R74L/C 171 A/T 19 Y/Gl 88 A/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88R/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88R/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/G25K/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88 A/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G188Q/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G188 A/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G188 A/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188 A/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G188R/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G188R/G25K/S45 W; T 19 Y/R74L/C 171 A; 117T/T 19 Y/G25R/R74L/C 171 A; T 19 Y/G25K/S45 WR74L/G108 A/C 171 A/Gl 88R; 117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/G108C/C 171 A/Gl 88R;
117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/G108C/A126C/C 171 A/Gl 88R; or 117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/AW 104- G105/G108C/C 171 A/Gl 88R.
[000224] In some embodiments, the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 102-105 130-134, or functionally equivalent positions, the sequence KTNN ASEHW, in combination with one or more combinations of stability enhancing mutations selected from Hl IX, L12X, D14X, H16X, I17X, T19X, S20X, N21X, G25X, I26X, G27X, R28X, H29X, T31X, C34X, E38X, R39X, D41X, N42X, S45X, K47X, M48X, H51X, F54X, H56X, N57X, A59X, K60X, L62X, Y67X, L73X, R74X, D77X, L78X, V79X, P80X, S81X, D85X, A87X, I89X, Y90X, R91X, T93X, W94X, I96X, S97X, C101X, F102X, S103X, W104X, G105X, C106X, A107X, G108X, E109X, V110X, R111X, Al 12X, LI 14X, QI 15X, El 16X, N117X, T118X, Hl 19X, V120X, L122X, R123X, A126X, R128X, L135X, Y136X, E138X, A139X, Q141X, M142X, D145X, A146X, A148X, D156X, E157X, K159X, C161X, D163X, T164X, D167X, Q169X, C171X, D177X, D180X, H182X, S183X, Q184X, A185X, L186X, S187X, G188X, R189X, A192X, N196X a deletion selected from AM1-L12, AE36-G53, AN61-G68, AW104-G105 , AQ195-N199 , AI26-G27, or any combination thereof, where the position number designation is functionally equivalent to the position in a wild-type APOBEC3A (SEQ ID NO:3) and X is an amino acid substitution different from the wild-type amino acid at that position.
[000225] In some embodiments, the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 102-105 130-134, or functionally equivalent positions, the sequence AYDG AHEYW, in combination with one or more combinations of stability enhancing mutations selected from: at least one of the following combinations of stabilizing mutations: R74L/C 171 A/T 19 Y/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 88Q/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88Q/G25K/S45W; R74L/C 171 A/T 19 Y/Gl 88 A/S45 W;
R74L/C 171 A/T 19 Y/Gl 88 A/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88R/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88R/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/S45 W;
I l l R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/G25K/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88 A/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G188Q/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G188 A/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188 A/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188 A/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G188R/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G188R/G25K/S45 W; T 19 Y/R74L/C 171 A; 117T/T 19 Y/G25R/R74L/C 171 A; T 19 Y/G25K/S45 WR74L/G108 A/C 171 A/Gl 88R; 117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/G108C/C 171 A/Gl 88R;
117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/G108C/A126C/C 171 A/Gl 88R; or 117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/AW 104- G105/G108C/C 171 A/Gl 88R.
[000226] In some embodiments, the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 102-105 130-134, or functionally equivalent positions, the sequence AYDG AHEYW, in combination with one or more combinations of stability enhancing mutations selected from Hl IX, L12X, D14X, H16X, I17X, T19X, S20X, N21X, G25X, I26X, G27X, R28X, H29X, T31X, C34X, E38X, R39X, D41X, N42X, S45X, K47X, M48X, H51X, F54X, H56X, N57X, A59X, K60X, L62X, Y67X, L73X, R74X, D77X, L78X, V79X, P80X, S81X, D85X, A87X, I89X, Y90X, R91X, T93X, W94X, I96X, S97X, C101X, F102X, S103X, W104X, G105X, C106X, A107X, G108X, E109X, V110X, R111X, Al 12X, LI 14X, QI 15X, El 16X, N117X, T118X, Hl 19X, V120X, L122X, R123X, A126X, R128X, L135X, Y136X, E138X, A139X, Q141X, M142X, D145X, A146X, A148X, D156X, E157X, K159X, C161X, D163X, T164X, D167X, Q169X, C171X, D177X, D180X, H182X, S183X, Q184X, A185X, L186X, S187X, G188X, R189X, A192X, N196X a deletion selected from AM1-L12, AE36-G53, AN61-G68, AW104-G105 , AQ195-N199 , AI26-G27, or any combination thereof, where the position number designation is functionally equivalent to the position in a wild-type APOBEC3A (SEQ ID NO:3) and X is an amino acid substitution different from the wild-type amino acid at that position.
[000227] In some embodiments, the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 102-105 130-134, or functionally equivalent positions, the sequence HLTG AVAYW, in combination with one or more combinations of stability enhancing mutations selected from: at least one of the following combinations of stabilizing mutations: R74L/C 171 A/T 19 Y/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 88Q/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88Q/G25K/S45W; R74L/C 171 A/T 19 Y/Gl 88 A/S45 W;
R74L/C 171 A/T 19 Y/Gl 88 A/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88R/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88R/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/G25K/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88 A/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G188Q/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188 A/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188 A/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188 A/G25K/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188R/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188R/G25K/S45 W; T 19 Y/R74L/C 171 A;
117T/T 19 Y/G25R/R74L/C 171 A; T 19 Y/G25K/S45 WR74L/G108 A/C 171 A/Gl 88R;
117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/G108C/C 171 A/Gl 88R;
117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/G108C/A126C/C 171 A/Gl 88R; or
117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/AW 104-
G105/G108C/C 171 A/Gl 88R.
[000228] In some embodiments, the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 102-105 130-134, or functionally equivalent positions, the sequence HLTG AVAYW, in combination with one or more combinations of stability enhancing mutations selected from Hl IX, L12X, D14X, H16X, I17X, T19X, S20X, N21X, G25X, I26X, G27X, R28X, H29X, T31X, C34X, E38X, R39X, D41X, N42X, S45X, K47X, M48X, H51X, F54X, H56X, N57X, A59X, K60X, L62X, Y67X, L73X, R74X, D77X, L78X, V79X, P80X, S81X, D85X, A87X, I89X, Y90X, R91X, T93X, W94X, I96X, S97X, C101X, F102X, S103X, W104X, G105X, C106X, A107X, G108X, E109X, V110X, R111X, Al 12X, LI 14X, QI 15X, El 16X, N117X, T118X, Hl 19X, V120X, L122X, R123X, A126X, R128X, L135X, Y136X, E138X, A139X, Q141X, M142X, D145X, A146X, A148X, D156X, E157X, K159X, C161X, D163X, T164X, D167X, Q169X, C171X, D177X, D180X, H182X, S183X, Q184X, A185X, L186X, S187X, G188X, R189X, A192X, N196X a deletion selected from AM1-L12, AE36-G53, AN61-G68, AW104-G105 , AQ195-N199 , AI26-G27, or any combination thereof, where the position number designation is functionally equivalent to the position in a wild-type AP0BEC3A (SEQ ID NO:3) and X is an amino acid substitution different from the wild-type amino acid at that position.
[000229] In some embodiments, the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 102-105 130-134, or functionally equivalent positions, the sequence QTMH AHQYW, in combination with one or more combinations of stability enhancing mutations selected from: at least one of the following combinations of stabilizing mutations: R74L/C 171 A/T 19 Y/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 88Q/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 88Q/G25K/S45W; R74L/C 171 A/T 19 Y/Gl 88 A/S45 W;
R74L/C 171 A/T 19 Y/Gl 88 A/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88R/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88R/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/G25K/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88 A/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G188Q/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188 A/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188 A/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188 A/G25K/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188R/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188R/G25K/S45 W; T 19 Y/R74L/C 171 A;
117T/T 19 Y/G25R/R74L/C 171 A; T 19 Y/G25K/S45 WR74L/G108 A/C 171 A/Gl 88R;
117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/G108C/C 171 A/Gl 88R;
117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/G108C/A126C/C 171 A/Gl 88R; or
117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/AW 104-
G105/G108C/C 171 A/Gl 88R.
[000230] In some embodiments, the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 102-105 130-134, or functionally equivalent positions, the sequence QTMH AHQYW, in combination with one or more combinations of stability enhancing mutations selected from Hl IX, L12X, D14X, H16X, I17X, T19X, S20X, N21X, G25X, I26X, G27X, R28X, H29X, T31X, C34X, E38X, R39X, D41X, N42X, S45X, K47X, M48X, H51X, F54X, H56X, N57X, A59X, K60X, L62X, Y67X, L73X, R74X, D77X, L78X, V79X, P80X, S81X, D85X, A87X, I89X, Y90X, R91X, T93X, W94X, I96X, S97X, C101X, F102X, S1O3X, W104X, G105X, C106X, A107X, G108X, E109X, V110X, R111X, Al 12X, LI 14X, QI 15X, El 16X, N117X, T118X, Hl 19X, V120X, L122X, R123X, A126X, R128X, L135X, Y136X, E138X, A139X, Q141X, M142X, D145X, A146X, A148X, D156X, E157X, K159X, C161X, D163X, T164X, D167X, Q169X, C171X, D177X, D180X, H182X, S183X, Q184X, A185X, L186X, S187X, G188X, R189X, A192X, N196X a deletion selected from AM1-L12, AE36-G53, AN61-G68, AW104-G105 , AQ195-N199 , AI26-G27, or any combination thereof, where the position number designation is functionally equivalent to the position in a wild-type APOBEC3A (SEQ ID NO:3) and X is an amino acid substitution different from the wild-type amino acid at that position.
[000231] In some embodiments, the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 102-105 130-134, or functionally equivalent positions, the sequence YVQR ANVYW, in combination with one or more combinations of stability enhancing mutations selected from: at least one of the following combinations of stabilizing mutations: R74L/C 171 A/T 19 Y/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 88Q/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88Q/G25K/S45W; R74L/C 171 A/T 19 Y/Gl 88 A/S45 W;
R74L/C 171 A/T 19 Y/Gl 88 A/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88R/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88R/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/G25K/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88 A/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G188Q/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G188 A/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188 A/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188 A/G25K/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188R/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188R/G25K/S45 W; T 19 Y/R74L/C 171 A;
117T/T 19 Y/G25R/R74L/C 171 A; T 19 Y/G25K/S45 WR74L/G108 A/C 171 A/Gl 88R;
117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/G108C/C 171 A/Gl 88R; 117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/G108C/A126C/C 171 A/Gl 88R; or 117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/AW 104- G105/G108C/C 171 A/Gl 88R.
[000232] In some embodiments, the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 102-105 130-134, or functionally equivalent positions, the sequence YVQR ANVYW, in combination with one or more combinations of stability enhancing mutations selected from Hl IX, L12X, D14X, H16X, I17X, T19X, S20X, N21X, G25X, I26X, G27X, R28X, H29X, T31X, C34X, E38X, R39X, D41X, N42X, S45X, K47X, M48X, H51X, F54X, H56X, N57X, A59X, K60X, L62X, Y67X, L73X, R74X, D77X, L78X, V79X, P80X, S81X, D85X, A87X, I89X, Y90X, R91X, T93X, W94X, I96X, S97X, C101X, F102X, S103X, W104X, G105X, C106X, A107X, G108X, E109X, V110X, R111X, Al 12X, LI 14X, QI 15X, El 16X, N117X, T118X, Hl 19X, V120X, L122X, R123X, A126X, R128X, L135X, Y136X, E138X, A139X, Q141X, M142X, D145X, A146X, A148X, D156X, E157X, K159X, C161X, D163X, T164X, D167X, Q169X, C171X, D177X, D180X, H182X, S183X, Q184X, A185X, L186X, S187X, G188X, R189X, A192X, N196X a deletion selected from AM1-L12, AE36-G53, AN61-G68, AW104-G105 , AQ195-N199 , AI26-G27, or any combination thereof, where the position number designation is functionally equivalent to the position in a wild-type APOBEC3A (SEQ ID NO:3) and X is an amino acid substitution different from the wild-type amino acid at that position.
[000233] In some embodiments, the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 102-105 130-134, or functionally equivalent positions, the sequence ELYA AHIYW, in combination with one or more combinations of stability enhancing mutations selected from: at least one of the following combinations of stabilizing mutations: R74L/C 171 A/T 19 Y/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 88Q/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88Q/G25K/S45W; R74L/C 171 A/T 19 Y/Gl 88 A/S45 W;
R74L/C 171 A/T 19 Y/Gl 88 A/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88R/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88R/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/G25K/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88 A/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G188Q/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G188 A/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188 A/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188 A/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G188R/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G188R/G25K/S45 W; T 19 Y/R74L/C 171 A; 117T/T 19 Y/G25R/R74L/C 171 A; T 19 Y/G25K/S45 WR74L/G108 A/C 171 A/Gl 88R; 117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/G108C/C 171 A/Gl 88R;
117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/G108C/A126C/C 171 A/Gl 88R; or 117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/AW 104- G105/G108C/C 171 A/Gl 88R.
[000234] In some embodiments, the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 102-105 130-134, or functionally equivalent positions, the sequence ELYA AHIYW, in combination with one or more combinations of stability enhancing mutations selected from Hl IX, L12X, D14X, H16X, I17X, T19X, S20X, N21X, G25X, I26X, G27X, R28X, H29X, T31X, C34X, E38X, R39X, D41X, N42X, S45X, K47X, M48X, H51X, F54X, H56X, N57X, A59X, K60X, L62X, Y67X, L73X, R74X, D77X, L78X, V79X, P80X, S81X, D85X, A87X, I89X, Y90X, R91X, T93X, W94X, I96X, S97X, C101X, F102X, S103X, W104X, G105X, C106X, A107X, G108X, E109X, V110X, R111X, Al 12X, LI 14X, QI 15X, El 16X, N117X, T118X, Hl 19X, V120X, L122X, R123X, A126X, R128X, L135X, Y136X, E138X, A139X, Q141X, M142X, D145X, A146X, A148X, D156X, E157X, K159X, C161X, D163X, T164X, D167X, Q169X, C171X, D177X, D180X, H182X, S183X, Q184X, A185X, L186X, S187X, G188X, R189X, A192X, N196X a deletion selected from AM1-L12, AE36-G53, AN61-G68, AW104-G105 , AQ195-N199 , AI26-G27, or any combination thereof, where the position number designation is functionally equivalent to the position in a wild-type APOBEC3A (SEQ ID NO:3) and X is an amino acid substitution different from the wild-type amino acid at that position.
[000235] In some embodiments, the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 102-105 130-134, or functionally equivalent positions, the sequence YVEN ANGFW , in combination with one or more combinations of stability enhancing mutations selected from: at least one of the following combinations of stabilizing mutations: R74L/C 171 A/T 19 Y/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 88Q/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88Q/G25K/S45W; R74L/C 171 A/T 19 Y/Gl 88 A/S45 W;
R74L/C 171 A/T 19 Y/Gl 88 A/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88R/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88R/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/G25K/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88 A/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G188Q/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G188 A/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188 A/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188 A/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G188R/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G188R/G25K/S45 W; T 19 Y/R74L/C 171 A; 117T/T 19 Y/G25R/R74L/C 171 A; T 19 Y/G25K/S45 WR74L/G108 A/C 171 A/Gl 88R; 117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/G108C/C 171 A/Gl 88R;
117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/G108C/A126C/C 171 A/Gl 88R; or 117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/AW 104- G105/G108C/C 171 A/Gl 88R.
[000236] In some embodiments, the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 102-105 130-134, or functionally equivalent positions, the sequence YVEN ANGFW, in combination with one or more combinations of stability enhancing mutations selected from Hl IX, L12X, D14X, H16X, I17X, T19X, S20X, N21X, G25X, I26X, G27X, R28X, H29X, T31X, C34X, E38X, R39X, D41X, N42X, S45X, K47X, M48X, H51X, F54X, H56X, N57X, A59X, K60X, L62X, Y67X, L73X, R74X, D77X, L78X, V79X, P80X, S81X, D85X, A87X, I89X, Y90X, R91X, T93X, W94X, I96X, S97X, C101X, F102X, S103X, W104X, G105X, C106X, A107X, G108X, E109X, V110X, R111X, Al 12X, LI 14X, QI 15X, El 16X, N117X, T118X, Hl 19X, V120X, L122X, R123X, A126X, R128X, L135X, Y136X, E138X, A139X, Q141X, M142X, D145X, A146X, A148X, D156X, E157X, K159X, C161X, D163X, T164X, D167X, Q169X, C171X, D177X, D180X, H182X, S183X, Q184X, A185X, L186X, S187X, G188X, R189X, A192X, N196X a deletion selected from AM1-L12, AE36-G53, AN61-G68, AW104-G105 , AQ195-N199 , AI26-G27, or any combination thereof, where the position number designation is functionally equivalent to the position in a wild-type APOBEC3A (SEQ ID NO:3) and X is an amino acid substitution different from the wild-type amino acid at that position. [000237] In some embodiments, the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 102-105 130-134, or functionally equivalent positions, the sequence RMLA AAEHW, in combination with one or more combinations of stability enhancing mutations selected from: at least one of the following combinations of stabilizing mutations: R74L/C 171 A/T 19 Y/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 88Q/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88Q/G25K/S45W; R74L/C 171 A/T 19 Y/Gl 88 A/S45 W;
R74L/C 171 A/T 19 Y/Gl 88 A/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88R/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88R/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/G25K/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88 A/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G188Q/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188 A/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188 A/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188 A/G25K/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188R/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188R/G25K/S45 W; T 19 Y/R74L/C 171 A;
117T/T 19 Y/G25R/R74L/C 171 A; T 19 Y/G25K/S45 WR74L/G108 A/C 171 A/Gl 88R;
117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/G108C/C 171 A/Gl 88R;
117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/G108C/A126C/C 171 A/Gl 88R; or
117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/AW 104-
G105/G108C/C 171 A/Gl 88R.
[000238] In some embodiments, the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 102-105 130-134, or functionally equivalent positions, the sequence RMLA AAEHW, in combination with one or more combinations of stability enhancing mutations selected from Hl IX, L12X, D14X, H16X, I17X, T19X, S20X, N21X, G25X, I26X, G27X, R28X, H29X, T31X, C34X, E38X, R39X, D41X, N42X, S45X, K47X, M48X, H51X, F54X, H56X, N57X, A59X, K60X, L62X, Y67X, L73X, R74X, D77X, L78X, V79X, P80X, S81X, D85X, A87X, I89X, Y90X, R91X, T93X, W94X, I96X, S97X, C101X, F102X, S103X, W104X, G105X, C106X, A107X, G108X, E109X, V110X, R111X, Al 12X, LI 14X, QI 15X, El 16X, N117X, T118X, Hl 19X, V120X, L122X, R123X, A126X, R128X, L135X, Y136X, E138X, A139X, Q141X, M142X, D145X, A146X, A148X, D156X, E157X, K159X, C161X, D163X, T164X, D167X, Q169X, C171X, D177X, D180X, H182X, S183X, Q184X, A185X, L186X, S187X, G188X, R189X, A192X, N196X a deletion selected from AM1-L12, AE36-G53, AN61-G68, AW104-G105 , AQ195-N199 , AI26-G27, or any combination thereof, where the position number designation is functionally equivalent to the position in a wild-type APOBEC3A (SEQ ID NO:3) and X is an amino acid substitution different from the wild-type amino acid at that position.
[000239] In some embodiments, the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 102-105 130-134, or functionally equivalent positions, the sequence QTVG AKSHW , in combination with one or more combinations of stability enhancing mutations selected from: at least one of the following combinations of stabilizing mutations: R74L/C 171 A/T 19 Y/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 88Q/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 88Q/G25K/S45W; R74L/C 171 A/T 19 Y/Gl 88 A/S45 W;
R74L/C 171 A/T 19 Y/Gl 88 A/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88R/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88R/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/G25K/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88 A/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G188Q/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G188 A/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188 A/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188 A/G25K/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188R/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188R/G25K/S45 W; T 19 Y/R74L/C 171 A;
117T/T 19 Y/G25R/R74L/C 171 A; T 19 Y/G25K/S45 WR74L/G108 A/C 171 A/Gl 88R;
117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/G108C/C 171 A/Gl 88R;
117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/G108C/A126C/C 171 A/Gl 88R; or
117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/AW 104-
G105/G108C/C 171 A/Gl 88R.
[000240] In some embodiments, the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 102-105 130-134, or functionally equivalent positions, the sequence QTVG AKSHW, in combination with one or more combinations of stability enhancing mutations selected from Hl IX, L12X, D14X, H16X, I17X, T19X, S20X, N21X, G25X, I26X, G27X, R28X, H29X, T31X, C34X, E38X, R39X, D41X, N42X, S45X, K47X, M48X, H51X, F54X, H56X, N57X, A59X, K60X, L62X, Y67X, L73X, R74X, D77X, L78X, V79X, P80X, S81X, D85X, A87X, I89X, Y90X, R91X, T93X, W94X, I96X, S97X, C101X, F102X, S103X, W104X, G105X, C106X, A107X, G108X, E109X, V110X, R111X, Al 12X, LI 14X, QI 15X, El 16X, N117X, T118X, Hl 19X, V120X, L122X, R123X, A126X, R128X, L135X, Y136X, E138X, A139X, Q141X, M142X, D145X, A146X, A148X, D156X, E157X, K159X, C161X, D163X, T164X, D167X, Q169X, C171X, D177X, D180X, H182X, S183X, Q184X, A185X, L186X, S187X, G188X, R189X, A192X, N196X a deletion selected from AM1-L12, AE36-G53, AN61-G68, AW104-G105 , AQ195-N199 , AI26-G27, or any combination thereof, where the position number designation is functionally equivalent to the position in a wild-type APOBEC3A (SEQ ID NO:3) and X is an amino acid substitution different from the wild-type amino acid at that position.
[000241] In some embodiments, the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 102-105 130-134, or functionally equivalent positions, the sequence YVQD AHEYW, in combination with one or more combinations of stability enhancing mutations selected from: at least one of the following combinations of stabilizing mutations: R74L/C 171 A/T 19 Y/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 88Q/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88Q/G25K/S45W; R74L/C 171 A/T 19 Y/Gl 88 A/S45 W;
R74L/C 171 A/T 19 Y/Gl 88 A/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88R/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88R/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/G25K/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88 A/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G188Q/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G188 A/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188 A/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188 A/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G188R/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G188R/G25K/S45 W; T 19 Y/R74L/C 171 A; 117T/T 19 Y/G25R/R74L/C 171 A; T 19 Y/G25K/S45 WR74L/G108 A/C 171 A/Gl 88R;
117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/G108C/C 171 A/Gl 88R;
117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/G108C/A126C/C 171 A/Gl 88R; or 117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/AW 104- G105/G108C/C 171 A/Gl 88R.
[000242] In some embodiments, the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 102-105 130-134, or functionally equivalent positions, the sequence YVQD AHEYW, in combination with one or more combinations of stability enhancing mutations selected from Hl IX, L12X, D14X, H16X, I17X, T19X, S20X, N21X, G25X, I26X, G27X, R28X, H29X, T31X, C34X, E38X, R39X, D41X, N42X, S45X, K47X, M48X, H51X, F54X, H56X, N57X, A59X, K60X, L62X, Y67X, L73X, R74X, D77X, L78X, V79X, P80X, S81X, D85X, A87X, I89X, Y90X, R91X, T93X, W94X, I96X, S97X, C101X, F102X, S103X, W104X, G105X, C106X, A107X, G108X, E109X, V110X, R111X, Al 12X, LI 14X, QI 15X, El 16X, N117X, T118X, Hl 19X, V120X, L122X, R123X, A126X, R128X, L135X, Y136X, E138X, A139X, Q141X, M142X, D145X, A146X, A148X, D156X, E157X, K159X, C161X, D163X, T164X, D167X, Q169X, C171X, D177X, D180X, H182X, S183X, Q184X, A185X, L186X, S187X, G188X, R189X, A192X, N196X a deletion selected from AM1-L12, AE36-G53, AN61-G68, AW104-G105 , AQ195-N199 , AI26-G27, or any combination thereof, where the position number designation is functionally equivalent to the position in a wild-type APOBEC3A (SEQ ID NO:3) and X is an amino acid substitution different from the wild-type amino acid at that position.
[000243] In some embodiments, the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 102-105 130-134, or functionally equivalent positions, the sequence QTQY AHEYW, in combination with one or more combinations of stability enhancing mutations selected from: at least one of the following combinations of stabilizing mutations: R74L/C 171 A/T 19 Y/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 88Q/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88Q/G25K/S45W; R74L/C 171 A/T 19 Y/Gl 88 A/S45 W;
R74L/C 171 A/T 19 Y/Gl 88 A/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88R/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88R/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/G25K/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88 A/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G188Q/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G188 A/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188 A/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188 A/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G188R/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G188R/G25K/S45 W; T 19 Y/R74L/C 171 A; 117T/T 19 Y/G25R/R74L/C 171 A; T 19 Y/G25K/S45 WR74L/G108 A/C 171 A/Gl 88R; 117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/G108C/C 171 A/Gl 88R;
117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/G108C/A126C/C 171 A/Gl 88R; or 117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/AW 104- G105/G108C/C 171 A/Gl 88R.
[000244] In some embodiments, the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 102-105 130-134, or functionally equivalent positions, the sequence QTQY AHEYW, in combination with one or more combinations of stability enhancing mutations selected from Hl IX, L12X, D14X, H16X, I17X, T19X, S20X, N21X, G25X, I26X, G27X, R28X, H29X, T31X, C34X, E38X, R39X, D41X, N42X, S45X, K47X, M48X, H51X, F54X, H56X, N57X, A59X, K60X, L62X, Y67X, L73X, R74X, D77X, L78X, V79X, P80X, S81X, D85X, A87X, I89X, Y90X, R91X, T93X, W94X, I96X, S97X, C101X, F102X, S103X, W104X, G105X, C106X, A107X, G108X, E109X, V110X, R111X, Al 12X, LI 14X, QI 15X, El 16X, N117X, T118X, Hl 19X, V120X, L122X, R123X, A126X, R128X, L135X, Y136X, E138X, A139X, Q141X, M142X, D145X, A146X, A148X, D156X, E157X, K159X, C161X, D163X, T164X, D167X, Q169X, C171X, D177X, D180X, H182X, S183X, Q184X, A185X, L186X, S187X, G188X, R189X, A192X, N196X a deletion selected from AM1-L12, AE36-G53, AN61-G68, AW104-G105 , AQ195-N199 , AI26-G27, or any combination thereof, where the position number designation is functionally equivalent to the position in a wild-type APOBEC3A (SEQ ID NO:3) and X is an amino acid substitution different from the wild-type amino acid at that position.
[000245] In some embodiments, the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 102-105 130-134, or functionally equivalent positions, the sequence QTYG AHLYW, in combination with one or more combinations of stability enhancing mutations selected from: at least one of the following combinations of stabilizing mutations: R74L/C 171 A/T 19 Y/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 88Q/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88Q/G25K/S45W; R74L/C 171 A/T 19 Y/Gl 88 A/S45 W; R74L/C 171 A/T 19 Y/Gl 88 A/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88R/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88R/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/G25K/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88 A/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G188Q/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G188 A/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188 A/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188 A/G25K/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188R/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188R/G25K/S45 W; T 19 Y/R74L/C 171 A;
117T/T 19 Y/G25R/R74L/C 171 A; T 19 Y/G25K/S45 WR74L/G108 A/C 171 A/Gl 88R;
117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/G108C/C 171 A/Gl 88R;
117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/G108C/A126C/C 171 A/Gl 88R; or 117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/AW 104-
G105/G108C/C 171 A/Gl 88R.
[000246] In some embodiments, the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 102-105 130-134, or functionally equivalent positions, the sequence QTYG AHLYW, in combination with one or more combinations of stability enhancing mutations selected from Hl IX, L12X, D14X, H16X, I17X, T19X, S20X, N21X, G25X, I26X, G27X, R28X, H29X, T31X, C34X, E38X, R39X, D41X, N42X, S45X, K47X, M48X, H51X, F54X, H56X, N57X, A59X, K60X, L62X, Y67X, L73X, R74X, D77X, L78X, V79X, P80X, S81X, D85X, A87X, I89X, Y90X, R91X, T93X, W94X, I96X, S97X, C101X, F102X, S103X, W104X, G105X, C106X, A107X, G108X, E109X, V110X, R111X, Al 12X, LI 14X, QI 15X, El 16X, N117X, T118X, Hl 19X, V120X, L122X, R123X, A126X, R128X, L135X, Y136X, E138X, A139X, Q141X, M142X, D145X, A146X, A148X, D156X, E157X, K159X, C161X, D163X, T164X, D167X, Q169X, C171X, D177X, D180X, H182X, S183X, Q184X, A185X, L186X, S187X, G188X, R189X, A192X, N196X a deletion selected from AM1-L12, AE36-G53, AN61-G68, AW104-G105 , AQ195-N199 , AI26-G27, or any combination thereof, where the position number designation is functionally equivalent to the position in a wild-type AP0BEC3A (SEQ ID NO:3) and X is an amino acid substitution different from the wild-type amino acid at that position.
[000247] In some embodiments, the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 102-105 130-134, or functionally equivalent positions, the sequence YYRM AHGYW, in combination with one or more combinations of stability enhancing mutations selected from: at least one of the following combinations of stabilizing mutations: R74L/C 171 A/T 19 Y/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 88Q/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88Q/G25K/S45W; R74L/C 171 A/T 19 Y/Gl 88 A/S45 W;
R74L/C 171 A/T 19 Y/Gl 88 A/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88R/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88R/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/G25K/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88 A/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G188Q/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188 A/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188 A/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188 A/G25K/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188R/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188R/G25K/S45 W; T 19 Y/R74L/C 171 A;
117T/T 19 Y/G25R/R74L/C 171 A; T 19 Y/G25K/S45 WR74L/G108 A/C 171 A/Gl 88R;
117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/G108C/C 171 A/Gl 88R;
117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/G108C/A126C/C 171 A/Gl 88R; or
117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/AW 104-
G105/G108C/C 171 A/Gl 88R.
[000248] In some embodiments, the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 102-105 130-134, or functionally equivalent positions, the sequence YYRM AHGYW, in combination with one or more combinations of stability enhancing mutations selected from Hl IX, L12X, D14X, H16X, I17X, T19X, S20X, N21X, G25X, I26X, G27X, R28X, H29X, T31X, C34X, E38X, R39X, D41X, N42X, S45X, K47X, M48X, H51X, F54X, H56X, N57X, A59X, K60X, L62X, Y67X, L73X, R74X, D77X, L78X, V79X, P80X, S81X, D85X, A87X, I89X, Y90X, R91X, T93X, W94X, I96X, S97X, C101X, F102X, S1O3X, W104X, G105X, C106X, A107X, G108X, E109X, V110X, R111X, Al 12X, LI 14X, QI 15X, El 16X, N117X, T118X, Hl 19X, V120X, L122X, R123X, A126X, R128X, L135X, Y136X, E138X, A139X, Q141X, M142X, D145X, A146X, A148X, D156X, E157X, K159X, C161X, D163X, T164X, D167X, Q169X, C171X, D177X, D180X, H182X, S183X, Q184X, A185X, L186X, S187X, G188X, R189X, A192X, N196X a deletion selected from AM1-L12, AE36-G53, AN61-G68, AW104-G105 , AQ195-N199 , AI26-G27, or any combination thereof, where the position number designation is functionally equivalent to the position in a wild-type APOBEC3A (SEQ ID NO:3) and X is an amino acid substitution different from the wild-type amino acid at that position.
[000249] In some embodiments, the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 102-105 130-134, or functionally equivalent positions, the sequence YLPD AKGFW, in combination with one or more combinations of stability enhancing mutations selected from: at least one of the following combinations of stabilizing mutations: R74L/C 171 A/T 19 Y/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 88Q/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88Q/G25K/S45W; R74L/C 171 A/T 19 Y/Gl 88 A/S45 W;
R74L/C 171 A/T 19 Y/Gl 88 A/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88R/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88R/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/G25K/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88 A/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G188Q/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188 A/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188 A/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188 A/G25K/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188R/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188R/G25K/S45 W; T 19 Y/R74L/C 171 A;
117T/T 19 Y/G25R/R74L/C 171 A; T 19 Y/G25K/S45 WR74L/G108 A/C 171 A/Gl 88R;
117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/G108C/C 171 A/Gl 88R;
117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/G108C/A126C/C 171 A/Gl 88R; or 117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/AW 104-
G105/G108C/C 171 A/Gl 88R. [000250] In some embodiments, the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 102-105 130-134, or functionally equivalent positions, the sequence YLPD AKGFW, in combination with one or more combinations of stability enhancing mutations selected from Hl IX, L12X, D14X, H16X, I17X, T19X, S20X, N21X, G25X, I26X, G27X, R28X, H29X, T31X, C34X, E38X, R39X, D41X, N42X, S45X, K47X, M48X, H51X, F54X, H56X, N57X, A59X, K60X, L62X, Y67X, L73X, R74X, D77X, L78X, V79X, P80X, S81X, D85X, A87X, I89X, Y90X, R91X, T93X, W94X, I96X, S97X, C101X, F102X, S103X, W104X, G105X, C106X, A107X, G108X, E109X, V110X, R111X, Al 12X, LI 14X, QI 15X, El 16X, N117X, T118X, Hl 19X, V120X, L122X, R123X, A126X, R128X, L135X, Y136X, E138X, A139X, Q141X, M142X, D145X, A146X, A148X, D156X, E157X, K159X, C161X, D163X, T164X, D167X, Q169X, C171X, D177X, D180X, H182X, S183X, Q184X, A185X, L186X, S187X, G188X, R189X, A192X, N196X a deletion selected from AM1-L12, AE36-G53, AN61-G68, AW104-G105 , AQ195-N199 , AI26-G27, or any combination thereof, where the position number designation is functionally equivalent to the position in a wild-type APOBEC3A (SEQ ID NO:3) and X is an amino acid substitution different from the wild-type amino acid at that position.
[000251] In some embodiments, the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 102-105 130-134, or functionally equivalent positions, the sequence STNN AHIYW, in combination with one or more combinations of stability enhancing mutations selected from: at least one of the following combinations of stabilizing mutations: R74L/C 171 A/T 19 Y/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 88Q/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88Q/G25K/S45W; R74L/C 171 A/T 19 Y/Gl 88 A/S45 W;
R74L/C 171 A/T 19 Y/Gl 88 A/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88R/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88R/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/G25K/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88 A/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G188Q/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G188 A/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188 A/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188 A/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G188R/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188R/G25K/S45 W; T 19 Y/R74L/C 171 A;
117T/T 19 Y/G25R/R74L/C 171 A; T 19 Y/G25K/S45 WR74L/G108 A/C 171 A/Gl 88R;
117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/G108C/C 171 A/Gl 88R;
117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/G108C/A126C/C 171 A/Gl 88R; or 117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/AW 104- G105/G108C/C 171 A/Gl 88R.
[000252] In some embodiments, the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 102-105 130-134, or functionally equivalent positions, the sequence STNN AHIYW, in combination with one or more combinations of stability enhancing mutations selected from Hl IX, L12X, D14X, H16X, I17X, T19X, S20X, N21X, G25X, I26X, G27X, R28X, H29X, T31X, C34X, E38X, R39X, D41X, N42X, S45X, K47X, M48X, H51X, F54X, H56X, N57X, A59X, K60X, L62X, Y67X, L73X, R74X, D77X, L78X, V79X, P80X, S81X, D85X, A87X, I89X, Y90X, R91X, T93X, W94X, I96X, S97X, C101X, F102X, S103X, W104X, G105X, C106X, A107X, G108X, E109X, V110X, R111X, Al 12X, LI 14X, QI 15X, El 16X, N117X, T118X, Hl 19X, V120X, L122X, R123X, A126X, R128X, L135X, Y136X, E138X, A139X, Q141X, M142X, D145X, A146X, A148X, D156X, E157X, K159X, C161X, D163X, T164X, D167X, Q169X, C171X, D177X, D180X, H182X, S183X, Q184X, A185X, L186X, S187X, G188X, R189X, A192X, N196X a deletion selected from AM1-L12, AE36-G53, AN61-G68, AW104-G105 , AQ195-N199 , AI26-G27, or any combination thereof, where the position number designation is functionally equivalent to the position in a wild-type APOBEC3A (SEQ ID NO:3) and X is an amino acid substitution different from the wild-type amino acid at that position.
[000253] In some embodiments, the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 102-105 130-134, or functionally equivalent positions, the sequence QTRR AHQYW, in combination with one or more combinations of stability enhancing mutations selected from: at least one of the following combinations of stabilizing mutations: R74L/C 171 A/T 19 Y/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 88Q/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88Q/G25K/S45W; R74L/C 171 A/T 19 Y/Gl 88 A/S45 W;
R74L/C 171 A/T 19 Y/Gl 88 A/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88R/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88R/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88 A/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G188Q/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G188 A/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188 A/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188 A/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G188R/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G188R/G25K/S45 W; T 19 Y/R74L/C 171 A; 117T/T 19 Y/G25R/R74L/C 171 A; T 19 Y/G25K/S45 WR74L/G108 A/C 171 A/Gl 88R; 117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/G108C/C 171 A/Gl 88R;
117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/G108C/A126C/C 171 A/Gl 88R; or 117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/AW 104-
G105/G108C/C 171 A/Gl 88R.
[000254] In some embodiments, the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 102-105 130-134, or functionally equivalent positions, the sequence QTRR AHQYW, in combination with one or more combinations of stability enhancing mutations selected from Hl IX, L12X, D14X, H16X, I17X, T19X, S20X, N21X, G25X, I26X, G27X, R28X, H29X, T31X, C34X, E38X, R39X, D41X, N42X, S45X, K47X, M48X, H51X, F54X, H56X, N57X, A59X, K60X, L62X, Y67X, L73X, R74X, D77X, L78X, V79X, P80X, S81X, D85X, A87X, I89X, Y90X, R91X, T93X, W94X, I96X, S97X, C101X, F102X, S103X, W104X, G105X, C106X, A107X, G108X, E109X, V110X, R111X, Al 12X, LI 14X, QI 15X, El 16X, N117X, T118X, Hl 19X, V120X, L122X, R123X, A126X, R128X, L135X, Y136X, E138X, A139X, Q141X, M142X, D145X, A146X, A148X, D156X, E157X, K159X, C161X, D163X, T164X, D167X, Q169X, C171X, D177X, D180X, H182X, S183X, Q184X, A185X, L186X, S187X, G188X, R189X, A192X, N196X a deletion selected from AM1-L12, AE36-G53, AN61-G68, AW104-G105 , AQ195-N199 , AI26-G27, or any combination thereof, where the position number designation is functionally equivalent to the position in a wild-type APOBEC3A (SEQ ID NO:3) and X is an amino acid substitution different from the wild-type amino acid at that position. In some embodiments, the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 102- 105 130-134, or functionally equivalent positions, the sequence , in combination with one or more combinations of stability enhancing mutations selected from: at least one of the following combinations of stabilizing mutations: R74L/C171A/T19Y/G25K/S45W; R74L/C 171 A/T 19 Y/Gl 88Q/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88Q/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 88 A/S45 W; R74L/C 171 A/T 19 Y/Gl 88 A/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 88R/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88R/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/G25K/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88 A/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G188Q/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G188 A/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188 A/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188 A/G25K/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188R/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188R/G25K/S45 W; T 19 Y/R74L/C 171 A;
117T/T 19 Y/G25R/R74L/C 171 A; T 19 Y/G25K/S45 WR74L/G108 A/C 171 A/Gl 88R;
117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/G108C/C 171 A/Gl 88R;
117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/G108C/A126C/C 171 A/Gl 88R; or
117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/AW 104-
G105/G108C/C 171 A/Gl 88R.
[000255] In some embodiments, the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 102-105 130-134, or functionally equivalent positions, the sequence AYEY AHIYW, in combination with one or more combinations of stability enhancing mutations selected from Hl IX, L12X, D14X, H16X, I17X, T19X, S20X, N21X, G25X, I26X, G27X, R28X, H29X, T31X, C34X, E38X, R39X, D41X, N42X, S45X, K47X, M48X, H51X, F54X, H56X, N57X, A59X, K60X, L62X, Y67X, L73X, R74X, D77X, L78X, V79X, P80X, S81X, D85X, A87X, I89X, Y90X, R91X, T93X, W94X, I96X, S97X, C101X, F102X, S103X, W104X, G105X, C106X, A107X, G108X, E109X, V110X, R111X, Al 12X, LI 14X, QI 15X, El 16X, N117X, T118X, Hl 19X, V120X, L122X, R123X, A126X, R128X, L135X, Y136X, E138X, A139X, Q141X, M142X, D145X, A146X, A148X, D156X, E157X, K159X, C161X, D163X, T164X, D167X, Q169X, C171X, D177X, D180X, H182X, S183X, Q184X, A185X, L186X, S187X, G188X, R189X, A192X, N196X a deletion selected from AM1-L12, AE36-G53, AN61-G68, AW104-G105 , AQ195-N199 , AI26-G27, or any combination thereof, where the position number designation is functionally equivalent to the position in a wild-type AP0BEC3A (SEQ ID NO:3) and X is an amino acid substitution different from the wild-type amino acid at that position.
[000256] In some embodiments, the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 102-105 130-134, or functionally equivalent positions, the sequence AYEY AHIYW, in combination with one or more combinations of stability enhancing mutations selected from: at least one of the following combinations of stabilizing mutations: R74L/C 171 A/T 19 Y/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 88Q/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88Q/G25K/S45W; R74L/C 171 A/T 19 Y/Gl 88 A/S45 W;
R74L/C 171 A/T 19 Y/Gl 88 A/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88R/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88R/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/G25K/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88 A/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G188Q/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188 A/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188 A/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188 A/G25K/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188R/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188R/G25K/S45 W; T 19 Y/R74L/C 171 A;
117T/T 19 Y/G25R/R74L/C 171 A; T 19 Y/G25K/S45 WR74L/G108 A/C 171 A/Gl 88R;
117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/G108C/C 171 A/Gl 88R;
117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/G108C/A126C/C 171 A/Gl 88R; or
117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/AW 104-
G105/G108C/C 171 A/Gl 88R.
[000257] In some embodiments, the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 102-105 130-134, or functionally equivalent positions, the sequence YVND AHLYW, in combination with one or more combinations of stability enhancing mutations selected from Hl IX, L12X, D14X, H16X, I17X, T19X, S20X, N21X, G25X, I26X, G27X, R28X, H29X, T31X, C34X, E38X, R39X, D41X, N42X, S45X, K47X, M48X, H51X, F54X, H56X, N57X, A59X, K60X, L62X, Y67X, L73X, R74X, D77X, L78X, V79X, P80X, S81X, D85X, A87X, I89X, Y90X, R91X, T93X, W94X, I96X, S97X, C101X, F102X, S1O3X, W104X, G105X, C106X, A107X, G108X, E109X, V110X, R111X, Al 12X, LI 14X, QI 15X, El 16X, N117X, T118X, Hl 19X, V120X, L122X, R123X, A126X, R128X, L135X, Y136X, E138X, A139X, Q141X, M142X, D145X, A146X, A148X, D156X, E157X, K159X, C161X, D163X, T164X, D167X, Q169X, C171X, D177X, D180X, H182X, S183X, Q184X, A185X, L186X, S187X, G188X, R189X, A192X, N196X a deletion selected from AM1-L12, AE36-G53, AN61-G68, AW104-G105 , AQ195-N199 , AI26-G27, or any combination thereof, where the position number designation is functionally equivalent to the position in a wild-type APOBEC3A (SEQ ID NO:3) and X is an amino acid substitution different from the wild-type amino acid at that position.
[000258] In some embodiments, the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 102-105 130-134, or functionally equivalent positions, the sequence YVND AHLYW, in combination with one or more combinations of stability enhancing mutations selected from: at least one of the following combinations of stabilizing mutations: R74L/C 171 A/T 19 Y/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 88Q/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88Q/G25K/S45W; R74L/C 171 A/T 19 Y/Gl 88 A/S45 W;
R74L/C 171 A/T 19 Y/Gl 88 A/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88R/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88R/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/G25K/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88 A/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G188Q/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188 A/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188 A/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188 A/G25K/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188R/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188R/G25K/S45 W; T 19 Y/R74L/C 171 A;
117T/T 19 Y/G25R/R74L/C 171 A; T 19 Y/G25K/S45 WR74L/G108 A/C 171 A/Gl 88R;
117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/G108C/C 171 A/Gl 88R;
117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/G108C/A126C/C 171 A/Gl 88R; or 117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/AW 104-
G105/G108C/C 171 A/Gl 88R. [000259] In some embodiments, the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 102-105 130-134, or functionally equivalent positions, the sequence FSWG ANVHW, in combination with one or more combinations of stability enhancing mutations selected from Hl IX, L12X, D14X, H16X, I17X, T19X, S20X, N21X, G25X, I26X, G27X, R28X, H29X, T31X, C34X, E38X, R39X, D41X, N42X, S45X, K47X, M48X, H51X, F54X, H56X, N57X, A59X, K60X, L62X, Y67X, L73X, R74X, D77X, L78X, V79X, P80X, S81X, D85X, A87X, I89X, Y90X, R91X, T93X, W94X, I96X, S97X, C101X, F102X, S103X, W104X, G105X, C106X, A107X, G108X, E109X, V110X, R111X, Al 12X, LI 14X, QI 15X, El 16X, N117X, T118X, Hl 19X, V120X, L122X, R123X, A126X, R128X, L135X, Y136X, E138X, A139X, Q141X, M142X, D145X, A146X, A148X, D156X, E157X, K159X, C161X, D163X, T164X, D167X, Q169X, C171X, D177X, D180X, H182X, S183X, Q184X, A185X, L186X, S187X, G188X, R189X, A192X, N196X a deletion selected from AM1-L12, AE36-G53, AN61-G68, AW104-G105 , AQ195-N199 , AI26-G27, or any combination thereof, where the position number designation is functionally equivalent to the position in a wild-type APOBEC3A (SEQ ID NO:3) and X is an amino acid substitution different from the wild-type amino acid at that position.
[000260] In some embodiments, the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 102-105 130-134, or functionally equivalent positions, the sequence FSWG ANVHW , in combination with one or more combinations of stability enhancing mutations selected from: at least one of the following combinations of stabilizing mutations: R74L/C 171 A/T 19 Y/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 88Q/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88Q/G25K/S45W; R74L/C 171 A/T 19 Y/Gl 88 A/S45 W;
R74L/C 171 A/T 19 Y/Gl 88 A/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88R/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88R/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/G25K/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88 A/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G188Q/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G188 A/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188 A/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188 A/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G188R/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188R/G25K/S45 W; T 19 Y/R74L/C 171 A;
117T/T 19 Y/G25R/R74L/C 171 A; T 19 Y/G25K/S45 WR74L/G108 A/C 171 A/Gl 88R;
117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/G108C/C 171 A/Gl 88R;
117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/G108C/A126C/C 171 A/Gl 88R; or 117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/AW 104- G105/G108C/C 171 A/Gl 88R.
[000261] In some embodiments, the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 102-105 130-134, or functionally equivalent positions, the sequence NIPD ARAYW, in combination with one or more combinations of stability enhancing mutations selected from Hl IX, L12X, D14X, H16X, I17X, T19X, S20X, N21X, G25X, I26X, G27X, R28X, H29X, T31X, C34X, E38X, R39X, D41X, N42X, S45X, K47X, M48X, H51X, F54X, H56X, N57X, A59X, K60X, L62X, Y67X, L73X, R74X, D77X, L78X, V79X, P80X, S81X, D85X, A87X, I89X, Y90X, R91X, T93X, W94X, I96X, S97X, C101X, F102X, S103X, W104X, G105X, C106X, A107X, G108X, E109X, V110X, R111X, Al 12X, LI 14X, QI 15X, El 16X, N117X, T118X, Hl 19X, V120X, L122X, R123X, A126X, R128X, L135X, Y136X, E138X, A139X, Q141X, M142X, D145X, A146X, A148X, D156X, E157X, K159X, C161X, D163X, T164X, D167X, Q169X, C171X, D177X, D180X, H182X, S183X, Q184X, A185X, L186X, S187X, G188X, R189X, A192X, N196X a deletion selected from AM1-L12, AE36-G53, AN61-G68, AW104-G105 , AQ195-N199 , AI26-G27, or any combination thereof, where the position number designation is functionally equivalent to the position in a wild-type APOBEC3A (SEQ ID NO:3) and X is an amino acid substitution different from the wild-type amino acid at that position. In some embodiments, the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 102- 105 130-134, or functionally equivalent positions, the sequence NIPD ARAYW, in combination with one or more combinations of stability enhancing mutations selected from: at least one of the following combinations of stabilizing mutations:
R74L/C 171 A/T 19 Y/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 88Q/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88Q/G25K/S45W; R74L/C 171 A/T 19 Y/Gl 88 A/S45 W; R74L/C 171 A/T 19 Y/Gl 88 A/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88R/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88R/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88 A/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G188Q/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G188 A/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188 A/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188 A/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G188R/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G188R/G25K/S45 W; T 19 Y/R74L/C 171 A; 117T/T 19 Y/G25R/R74L/C 171 A; T 19 Y/G25K/S45 WR74L/G108 A/C 171 A/Gl 88R; 117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/G108C/C 171 A/Gl 88R;
117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/G108C/A126C/C 171 A/Gl 88R; or 117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/AW 104- G105/G108C/C 171 A/Gl 88R.
[000262] In some embodiments, the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 102-105 130-134, or functionally equivalent positions, the sequence YPFG ANGFW, in combination with one or more combinations of stability enhancing mutations selected from Hl IX, L12X, D14X, H16X, I17X, T19X, S20X, N21X, G25X, I26X, G27X, R28X, H29X, T31X, C34X, E38X, R39X, D41X, N42X, S45X, K47X, M48X, H51X, F54X, H56X, N57X, A59X, K60X, L62X, Y67X, L73X, R74X, D77X, L78X, V79X, P80X, S81X, D85X, A87X, I89X, Y90X, R91X, T93X, W94X, I96X, S97X, C101X, F102X, S103X, W104X, G105X, C106X, A107X, G108X, E109X, V110X, R111X, Al 12X, LI 14X, QI 15X, El 16X, N117X, T118X, Hl 19X, V120X, L122X, R123X, A126X, R128X, L135X, Y136X, E138X, A139X, Q141X, M142X, D145X, A146X, A148X, D156X, E157X, K159X, C161X, D163X, T164X, D167X, Q169X, C171X, D177X, D180X, H182X, S183X, Q184X, A185X, L186X, S187X, G188X, R189X, A192X, N196X a deletion selected from AM1-L12, AE36-G53, AN61-G68, AW104-G105 , AQ195-N199 , AI26-G27, or any combination thereof, where the position number designation is functionally equivalent to the position in a wild-type APOBEC3A (SEQ ID NO:3) and X is an amino acid substitution different from the wild-type amino acid at that position.
[000263] In some embodiments, the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 102-105 130-134, or functionally equivalent positions, the sequence YPFG ANGFW, in combination with one or more combinations of stability enhancing mutations selected from: at least one of the following combinations of stabilizing mutations: R74L/C 171 A/T 19 Y/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 88Q/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88Q/G25K/S45W; R74L/C 171 A/T 19 Y/Gl 88 A/S45 W;
R74L/C 171 A/T 19 Y/Gl 88 A/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88R/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88R/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/G25K/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88 A/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G188Q/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G188 A/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188 A/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188 A/G25K/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188R/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188R/G25K/S45 W; T 19 Y/R74L/C 171 A;
117T/T 19 Y/G25R/R74L/C 171 A; T 19 Y/G25K/S45 WR74L/G108 A/C 171 A/Gl 88R;
117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/G108C/C 171 A/Gl 88R;
117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/G108C/A126C/C 171 A/Gl 88R; or
117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/AW 104-
G105/G108C/C 171 A/Gl 88R.
[000264] In some embodiments, the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 102-105 130-134, or functionally equivalent positions, the sequence QTLG ANAFW, in combination with one or more combinations of stability enhancing mutations selected from Hl IX, L12X, D14X, H16X, I17X, T19X, S20X, N21X, G25X, I26X, G27X, R28X, H29X, T31X, C34X, E38X, R39X, D41X, N42X, S45X, K47X, M48X, H51X, F54X, H56X, N57X, A59X, K60X, L62X, Y67X, L73X, R74X, D77X, L78X, V79X, P80X, S81X, D85X, A87X, I89X, Y90X, R91X, T93X, W94X, I96X, S97X, C101X, F102X, S103X, W104X, G105X, C106X, A107X, G108X, E109X, V110X, R111X, Al 12X, LI 14X, QI 15X, El 16X, N117X, T118X, Hl 19X, V120X, L122X, R123X, A126X, R128X, L135X, Y136X, E138X, A139X, Q141X, M142X, D145X, A146X, A148X, D156X, E157X, K159X, C161X, D163X, T164X, D167X, Q169X, C171X, D177X, D180X, H182X, S183X, Q184X, A185X, L186X, S187X, G188X, R189X, A192X, N196X a deletion selected from AM1-L12, AE36-G53, AN61-G68, AW104-G105 , AQ195-N199 , AI26-G27, or any combination thereof, where the position number designation is functionally equivalent to the position in a wild-type AP0BEC3A (SEQ ID NO:3) and X is an amino acid substitution different from the wild-type amino acid at that position.
[000265] In some embodiments, the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 102-105 130-134, or functionally equivalent positions, the sequence QTLG ANAFW, in combination with one or more combinations of stability enhancing mutations selected from: at least one of the following combinations of stabilizing mutations: R74L/C 171 A/T 19 Y/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 88Q/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88Q/G25K/S45W; R74L/C 171 A/T 19 Y/Gl 88 A/S45 W;
R74L/C 171 A/T 19 Y/Gl 88 A/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88R/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88R/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/G25K/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88 A/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G188Q/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188 A/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188 A/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188 A/G25K/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188R/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188R/G25K/S45 W; T 19 Y/R74L/C 171 A;
117T/T 19 Y/G25R/R74L/C 171 A; T 19 Y/G25K/S45 WR74L/G108 A/C 171 A/Gl 88R;
117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/G108C/C 171 A/Gl 88R;
117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/G108C/A126C/C 171 A/Gl 88R; or
117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/AW 104-
G105/G108C/C 171 A/Gl 88R.
[000266] In some embodiments, the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 102-105 130-134, or functionally equivalent positions, the sequence VYND ARAYW, in combination with one or more combinations of stability enhancing mutations selected from Hl IX, L12X, D14X, H16X, I17X, T19X, S20X, N21X, G25X, I26X, G27X, R28X, H29X, T31X, C34X, E38X, R39X, D41X, N42X, S45X, K47X, M48X, H51X, F54X, H56X, N57X, A59X, K60X, L62X, Y67X, L73X, R74X, D77X, L78X, V79X, P80X, S81X, D85X, A87X, I89X, Y90X, R91X, T93X, W94X, I96X, S97X, C101X, F102X, S1O3X, W104X, G105X, C106X, A107X, G108X, E109X, V110X, R111X, Al 12X, LI 14X, QI 15X, El 16X, N117X, T118X, Hl 19X, V120X, L122X, R123X, A126X, R128X, L135X, Y136X, E138X, A139X, Q141X, M142X, D145X, A146X, A148X, D156X, E157X, K159X, C161X, D163X, T164X, D167X, Q169X, C171X, D177X, D180X, H182X, S183X, Q184X, A185X, L186X, S187X, G188X, R189X, A192X, N196X a deletion selected from AM1-L12, AE36-G53, AN61-G68, AW104-G105 , AQ195-N199 , AI26-G27, or any combination thereof, where the position number designation is functionally equivalent to the position in a wild-type APOBEC3A (SEQ ID NO:3) and X is an amino acid substitution different from the wild-type amino acid at that position.
[000267] In some embodiments, the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 102-105 130-134, or functionally equivalent positions, the sequence VYND ARAYW, in combination with one or more combinations of stability enhancing mutations selected from: at least one of the following combinations of stabilizing mutations: R74L/C 171 A/T 19 Y/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 88Q/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88Q/G25K/S45W; R74L/C 171 A/T 19 Y/Gl 88 A/S45 W;
R74L/C 171 A/T 19 Y/Gl 88 A/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88R/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88R/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/G25K/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88 A/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G188Q/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G188 A/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188 A/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188 A/G25K/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188R/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188R/G25K/S45 W; T 19 Y/R74L/C 171 A;
117T/T 19 Y/G25R/R74L/C 171 A; T 19 Y/G25K/S45 WR74L/G108 A/C 171 A/Gl 88R;
117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/G108C/C 171 A/Gl 88R;
117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/G108C/A126C/C 171 A/Gl 88R; or 117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/AW 104-
G105/G108C/C 171 A/Gl 88R.
[000268] In some embodiments, the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 102-105 130-134, or functionally equivalent positions, the sequence AFRD ASKHW, in combination with one or more combinations of stability enhancing mutations selected from Hl IX, L12X, D14X, H16X, I17X, T19X, S20X, N21X, G25X, I26X, G27X, R28X, H29X, T31X, C34X, E38X, R39X, D41X, N42X, S45X, K47X, M48X, H51X, F54X, H56X, N57X, A59X, K60X, L62X, Y67X, L73X, R74X, D77X, L78X, V79X, P80X, S81X, D85X, A87X, I89X, Y90X, R91X, T93X, W94X, I96X, S97X, C101X, F102X, S103X, W104X, G105X, C106X, A107X, G108X, E109X, V110X, R111X, Al 12X, LI 14X, QI 15X, El 16X, N117X, T118X, Hl 19X, V120X, L122X, R123X, A126X, R128X, L135X, Y136X, E138X, A139X, Q141X, M142X, D145X, A146X, A148X, D156X, E157X, K159X, C161X, D163X, T164X, D167X, Q169X, C171X, D177X, D180X, H182X, S183X, Q184X, A185X, L186X, S187X, G188X, R189X, A192X, N196X a deletion selected from AM1-L12, AE36-G53, AN61-G68, AW104-G105 , AQ195-N199 , AI26-G27, or any combination thereof, where the position number designation is functionally equivalent to the position in a wild-type APOBEC3A (SEQ ID NO:3) and X is an amino acid substitution different from the wild-type amino acid at that position. In some embodiments, the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 102- 105 130-134, or functionally equivalent positions, the sequence AFRD ASKHW , in combination with one or more combinations of stability enhancing mutations selected from: at least one of the following combinations of stabilizing mutations:
R74L/C 171 A/T 19 Y/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 88Q/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88Q/G25K/S45W; R74L/C 171 A/T 19 Y/Gl 88 A/S45 W; R74L/C 171 A/T 19 Y/Gl 88 A/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88R/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88R/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/G25K/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88 A/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G188Q/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G188 A/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G188 A/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188 A/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G188R/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G188R/G25K/S45 W; T 19 Y/R74L/C 171 A; 117T/T 19 Y/G25R/R74L/C 171 A; T 19 Y/G25K/S45 WR74L/G108 A/C 171 A/Gl 88R; 117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/G108C/C 171 A/Gl 88R;
117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/G108C/A126C/C 171 A/Gl 88R; or 117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/AW 104- G105/G108C/C 171 A/Gl 88R.
[000269] In some embodiments, the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 102-105 130-134, or functionally equivalent positions, the sequence ETKH ALAHW, in combination with one or more combinations of stability enhancing mutations selected from Hl IX, L12X, D14X, H16X, I17X, T19X, S20X, N21X, G25X, I26X, G27X, R28X, H29X, T31X, C34X, E38X, R39X, D41X, N42X, S45X, K47X, M48X, H51X, F54X, H56X, N57X, A59X, K60X, L62X, Y67X, L73X, R74X, D77X, L78X, V79X, P80X, S81X, D85X, A87X, I89X, Y90X, R91X, T93X, W94X, I96X, S97X, C101X, F102X, S103X, W104X, G105X, C106X, A107X, G108X, E109X, V110X, R111X, Al 12X, LI 14X, QI 15X, El 16X, N117X, T118X, Hl 19X, V120X, L122X, R123X, A126X, R128X, L135X, Y136X, E138X, A139X, Q141X, M142X, D145X, A146X, A148X, D156X, E157X, K159X, C161X, D163X, T164X, D167X, Q169X, C171X, D177X, D180X, H182X, S183X, Q184X, A185X, L186X, S187X, G188X, R189X, A192X, N196X a deletion selected from AM1-L12, AE36-G53, AN61-G68, AW104-G105 , AQ195-N199 , AI26-G27, or any combination thereof, where the position number designation is functionally equivalent to the position in a wild-type APOBEC3A (SEQ ID NO:3) and X is an amino acid substitution different from the wild-type amino acid at that position.
[000270] In some embodiments, the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 102-105 130-134, or functionally equivalent positions, the sequence ETKH ALAHW, in combination with one or more combinations of stability enhancing mutations selected from: at least one of the following combinations of stabilizing mutations: R74L/C 171 A/T 19 Y/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 88Q/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88Q/G25K/S45W; R74L/C 171 A/T 19 Y/Gl 88 A/S45 W;
R74L/C 171 A/T 19 Y/Gl 88 A/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88R/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88R/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/G25K/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88 A/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G188Q/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G188 A/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188 A/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188 A/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G188R/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G188R/G25K/S45 W; T 19 Y/R74L/C 171 A; 117T/T 19 Y/G25R/R74L/C 171 A; T 19 Y/G25K/S45 WR74L/G108 A/C 171 A/Gl 88R; 117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/G108C/C 171 A/Gl 88R;
117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/G108C/A126C/C 171 A/Gl 88R; or 117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/AW 104- G105/G108C/C 171 A/Gl 88R.
[000271] In some embodiments, the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 102-105 130-134, or functionally equivalent positions, the sequence YVEG AHLYW, in combination with one or more combinations of stability enhancing mutations selected from Hl IX, L12X, D14X, H16X, I17X, T19X, S20X, N21X, G25X, I26X, G27X, R28X, H29X, T31X, C34X, E38X, R39X, D41X, N42X, S45X, K47X, M48X, H51X, F54X, H56X, N57X, A59X, K60X, L62X, Y67X, L73X, R74X, D77X, L78X, V79X, P80X, S81X, D85X, A87X, I89X, Y90X, R91X, T93X, W94X, I96X, S97X, C101X, F102X, S103X, W104X, G105X, C106X, A107X, G108X, E109X, V110X, R111X, Al 12X, LI 14X, QI 15X, El 16X, N117X, T118X, Hl 19X, V120X, L122X, R123X, A126X, R128X, L135X, Y136X, E138X, A139X, Q141X, M142X, D145X, A146X, A148X, D156X, E157X, K159X, C161X, D163X, T164X, D167X, Q169X, C171X, D177X, D180X, H182X, S183X, Q184X, A185X, L186X, S187X, G188X, R189X, A192X, N196X a deletion selected from AM1-L12, AE36-G53, AN61-G68, AW104-G105 , AQ195-N199 , AI26-G27, or any combination thereof, where the position number designation is functionally equivalent to the position in a wild-type APOBEC3A (SEQ ID NO:3) and X is an amino acid substitution different from the wild-type amino acid at that position.
[000272] In some embodiments, the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 102-105 130-134, or functionally equivalent positions, the sequence YVEG AHLYW, in combination with one or more combinations of stability enhancing mutations selected from: at least one of the following combinations of stabilizing mutations: R74L/C 171 A/T 19 Y/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 88Q/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88Q/G25K/S45W; R74L/C 171 A/T 19 Y/Gl 88 A/S45 W;
R74L/C 171 A/T 19 Y/Gl 88 A/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88R/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88R/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/G25K/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88 A/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G188Q/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188 A/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188 A/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188 A/G25K/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188R/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188R/G25K/S45 W; T 19 Y/R74L/C 171 A;
117T/T 19 Y/G25R/R74L/C 171 A; T 19 Y/G25K/S45 WR74L/G108 A/C 171 A/Gl 88R;
117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/G108C/C 171 A/Gl 88R;
117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/G108C/A126C/C 171 A/Gl 88R; or
117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/AW 104-
G105/G108C/C 171 A/Gl 88R.
[000273] In some embodiments, the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 102-105 130-134, or functionally equivalent positions, the sequence QTMG AHQYW, in combination with one or more combinations of stability enhancing mutations selected from Hl IX, L12X, D14X, H16X, I17X, T19X, S20X, N21X, G25X, I26X, G27X, R28X, H29X, T31X, C34X, E38X, R39X, D41X, N42X, S45X, K47X, M48X, H51X, F54X, H56X, N57X, A59X, K60X, L62X, Y67X, L73X, R74X, D77X, L78X, V79X, P80X, S81X, D85X, A87X, I89X, Y90X, R91X, T93X, W94X, I96X, S97X, C101X, F102X, S103X, W104X, G105X, C106X, A107X, G108X, E109X, V110X, R111X, Al 12X, LI 14X, QI 15X, El 16X, N117X, T118X, Hl 19X, V120X, L122X, R123X, A126X, R128X, L135X, Y136X, E138X, A139X, Q141X, M142X, D145X, A146X, A148X, D156X, E157X, K159X, C161X, D163X, T164X, D167X, Q169X, C171X, D177X, D180X, H182X, S183X, Q184X, A185X, L186X, S187X, G188X, R189X, A192X, N196X a deletion selected from AM1-L12, AE36-G53, AN61-G68, AW104-G105 , AQ195-N199 , AI26-G27, or any combination thereof, where the position number designation is functionally equivalent to the position in a wild-type AP0BEC3A (SEQ ID NO:3) and X is an amino acid substitution different from the wild-type amino acid at that position.
[000274] In some embodiments, the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 102-105 130-134, or functionally equivalent positions, the sequence QTMG AHQYW, in combination with one or more combinations of stability enhancing mutations selected from: at least one of the following combinations of stabilizing mutations: R74L/C 171 A/T 19 Y/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 88Q/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 88Q/G25K/S45W; R74L/C 171 A/T 19 Y/Gl 88 A/S45 W;
R74L/C 171 A/T 19 Y/Gl 88 A/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88R/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88R/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/G25K/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88 A/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G188Q/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188 A/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188 A/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188 A/G25K/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188R/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188R/G25K/S45 W; T 19 Y/R74L/C 171 A;
117T/T 19 Y/G25R/R74L/C 171 A; T 19 Y/G25K/S45 WR74L/G108 A/C 171 A/Gl 88R;
117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/G108C/C 171 A/Gl 88R;
117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/G108C/A126C/C 171 A/Gl 88R; or
117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/AW 104-
G105/G108C/C 171 A/Gl 88R.
[000275] In some embodiments, the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 102-105 130-134, or functionally equivalent positions, the sequence AQHG AHEYW, in combination with one or more combinations of stability enhancing mutations selected from Hl IX, L12X, D14X, H16X, I17X, T19X, S20X, N21X, G25X, I26X, G27X, R28X, H29X, T31X, C34X, E38X, R39X, D41X, N42X, S45X, K47X, M48X, H51X, F54X, H56X, N57X, A59X, K60X, L62X, Y67X, L73X, R74X, D77X, L78X, V79X, P80X, S81X, D85X, A87X, I89X, Y90X, R91X, T93X, W94X, I96X, S97X, C101X, F102X, S1O3X, W104X, G105X, C106X, A107X, G108X, E109X, V110X, R111X, Al 12X, LI 14X, QI 15X, El 16X, N117X, T118X, Hl 19X, V120X, L122X, R123X, A126X, R128X, L135X, Y136X, E138X, A139X, Q141X, M142X, D145X, A146X, A148X, D156X, E157X, K159X, C161X, D163X, T164X, D167X, Q169X, C171X, D177X, D180X, H182X, S183X, Q184X, A185X, L186X, S187X, G188X, R189X, A192X, N196X a deletion selected from AM1-L12, AE36-G53, AN61-G68, AW104-G105 , AQ195-N199 , AI26-G27, or any combination thereof, where the position number designation is functionally equivalent to the position in a wild-type APOBEC3A (SEQ ID NO:3) and X is an amino acid substitution different from the wild-type amino acid at that position.
[000276] In some embodiments, the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 102-105 130-134, or functionally equivalent positions, the sequence AQHG AHEYW, in combination with one or more combinations of stability enhancing mutations selected from: at least one of the following combinations of stabilizing mutations: R74L/C 171 A/T 19 Y/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 88Q/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88Q/G25K/S45W; R74L/C 171 A/T 19 Y/Gl 88 A/S45 W;
R74L/C 171 A/T 19 Y/Gl 88 A/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88R/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88R/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/G25K/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88 A/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G188Q/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G188 A/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188 A/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188 A/G25K/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188R/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188R/G25K/S45 W; T 19 Y/R74L/C 171 A;
117T/T 19 Y/G25R/R74L/C 171 A; T 19 Y/G25K/S45 WR74L/G108 A/C 171 A/Gl 88R;
117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/G108C/C 171 A/Gl 88R; 117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/G108C/A126C/C 171 A/Gl 88R; or 117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/AW 104- G105/G108C/C 171 A/Gl 88R.
[000277] In some embodiments, the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 102-105 130-134, or functionally equivalent positions, the sequence HLRG AHEYW, in combination with one or more combinations of stability enhancing mutations selected from Hl IX, L12X, D14X, H16X, I17X, T19X, S20X, N21X, G25X, I26X, G27X, R28X, H29X, T31X, C34X, E38X, R39X, D41X, N42X, S45X, K47X, M48X, H51X, F54X, H56X, N57X, A59X, K60X, L62X, Y67X, L73X, R74X, D77X, L78X, V79X, P80X, S81X, D85X, A87X, I89X, Y90X, R91X, T93X, W94X, I96X, S97X, C101X, F102X, S103X, W104X, G105X, C106X, A107X, G108X, E109X, V110X, R111X, Al 12X, LI 14X, QI 15X, El 16X, N117X, T118X, Hl 19X, V120X, L122X, R123X, A126X, R128X, L135X, Y136X, E138X, A139X, Q141X, M142X, D145X, A146X, A148X, D156X, E157X, K159X, C161X, D163X, T164X, D167X, Q169X, C171X, D177X, D180X, H182X, S183X, Q184X, A185X, L186X, S187X, G188X, R189X, A192X, N196X a deletion selected from AM1-L12, AE36-G53, AN61-G68, AW104-G105 , AQ195-N199 , AI26-G27, or any combination thereof, where the position number designation is functionally equivalent to the position in a wild-type APOBEC3A (SEQ ID NO:3) and X is an amino acid substitution different from the wild-type amino acid at that position.
[000278] In some embodiments, the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 102-105 130-134, or functionally equivalent positions, the sequence HLRG AHEYW, in combination with one or more combinations of stability enhancing mutations selected from: at least one of the following combinations of stabilizing mutations: R74L/C 171 A/T 19 Y/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 88Q/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88Q/G25K/S45W; R74L/C 171 A/T 19 Y/Gl 88 A/S45 W;
R74L/C 171 A/T 19 Y/Gl 88 A/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88R/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88R/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/G25K/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88 A/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G188Q/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G188 A/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188 A/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G188 A/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G188R/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G188R/G25K/S45 W; T 19 Y/R74L/C 171 A; 117T/T 19 Y/G25R/R74L/C 171 A; T 19 Y/G25K/S45 WR74L/G108 A/C 171 A/Gl 88R; 117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/G108C/C 171 A/Gl 88R; 117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/G108C/A126C/C 171 A/Gl 88R; or 117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/AW 104- G105/G108C/C 171 A/Gl 88R.
[000279] In some embodiments, the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 102-105 130-134, or functionally equivalent positions, the sequence QTMH ANAFW, in combination with one or more combinations of stability enhancing mutations selected from Hl IX, L12X, D14X, H16X, I17X, T19X, S20X, N21X, G25X, I26X, G27X, R28X, H29X, T31X, C34X, E38X, R39X, D41X, N42X, S45X, K47X, M48X, H51X, F54X, H56X, N57X, A59X, K60X, L62X, Y67X, L73X, R74X, D77X, L78X, V79X, P80X, S81X, D85X, A87X, I89X, Y90X, R91X, T93X, W94X, I96X, S97X, C101X, F102X, S103X, W104X, G105X, C106X, A107X, G108X, E109X, V110X, R111X, Al 12X, LI 14X, QI 15X, El 16X, N117X, T118X, Hl 19X, V120X, L122X, R123X, A126X, R128X, L135X, Y136X, E138X, A139X, Q141X, M142X, D145X, A146X, A148X, D156X, E157X, K159X, C161X, D163X, T164X, D167X, Q169X, C171X, D177X, D180X, H182X, S183X, Q184X, A185X, L186X, S187X, G188X, R189X, A192X, N196X a deletion selected from AM1-L12, AE36-G53, AN61-G68, AW104-G105 , AQ195-N199 , AI26-G27, or any combination thereof, where the position number designation is functionally equivalent to the position in a wild-type APOBEC3A (SEQ ID NO:3) and X is an amino acid substitution different from the wild-type amino acid at that position.
[000280] In some embodiments, the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 102-105 130-134, or functionally equivalent positions, the sequence QTMH ANAFW, in combination with one or more combinations of stability enhancing mutations selected from: at least one of the following combinations of stabilizing mutations: R74L/C 171 A/T 19 Y/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 88Q/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88Q/G25K/S45W; R74L/C 171 A/T 19 Y/Gl 88 A/S45 W;
R74L/C 171 A/T 19 Y/Gl 88 A/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88R/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88R/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/G25K/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88 A/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G188Q/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G188 A/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188 A/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188 A/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G188R/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G188R/G25K/S45 W; T 19 Y/R74L/C 171 A; 117T/T 19 Y/G25R/R74L/C 171 A; T 19 Y/G25K/S45 WR74L/G108 A/C 171 A/Gl 88R; 117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/G108C/C 171 A/Gl 88R;
117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/G108C/A126C/C 171 A/Gl 88R; or 117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/AW 104- G105/G108C/C 171 A/Gl 88R.
[000281] In some embodiments, the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 102-105 130-134, or functionally equivalent positions, the sequence YYID AGRHW, in combination with one or more combinations of stability enhancing mutations selected from Hl IX, L12X, D14X, H16X, I17X, T19X, S20X, N21X, G25X, I26X, G27X, R28X, H29X, T31X, C34X, E38X, R39X, D41X, N42X, S45X, K47X, M48X, H51X, F54X, H56X, N57X, A59X, K60X, L62X, Y67X, L73X, R74X, D77X, L78X, V79X, P80X, S81X, D85X, A87X, I89X, Y90X, R91X, T93X, W94X, I96X, S97X, C101X, F102X, S103X, W104X, G105X, C106X, A107X, G108X, E109X, V110X, R111X, Al 12X, LI 14X, QI 15X, El 16X, N117X, T118X, Hl 19X, V120X, L122X, R123X, A126X, R128X, L135X, Y136X, E138X, A139X, Q141X, M142X, D145X, A146X, A148X, D156X, E157X, K159X, C161X, D163X, T164X, D167X, Q169X, C171X, D177X, D180X, H182X, S183X, Q184X, A185X, L186X, S187X, G188X, R189X, A192X, N196X a deletion selected from AM1-L12, AE36-G53, AN61-G68, AW104-G105 , AQ195-N199 , AI26-G27, or any combination thereof, where the position number designation is functionally equivalent to the position in a wild-type APOBEC3A (SEQ ID NO:3) and X is an amino acid substitution different from the wild-type amino acid at that position. [000282] In some embodiments, the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 102-105 130-134, or functionally equivalent positions, the sequence YYID AGRHW, in combination with one or more combinations of stability enhancing mutations selected from: at least one of the following combinations of stabilizing mutations: R74L/C 171 A/T 19 Y/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 88Q/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88Q/G25K/S45W; R74L/C 171 A/T 19 Y/Gl 88 A/S45 W;
R74L/C 171 A/T 19 Y/Gl 88 A/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88R/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88R/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/G25K/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88 A/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G188Q/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188 A/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188 A/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188 A/G25K/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188R/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188R/G25K/S45 W; T 19 Y/R74L/C 171 A;
117T/T 19 Y/G25R/R74L/C 171 A; T 19 Y/G25K/S45 WR74L/G108 A/C 171 A/Gl 88R;
117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/G108C/C 171 A/Gl 88R;
117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/G108C/A126C/C 171 A/Gl 88R; or
117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/AW 104-
G105/G108C/C 171 A/Gl 88R.
[000283] In some embodiments, the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 102-105 130-134, or functionally equivalent positions, the sequence HLYG ARAYW, in combination with one or more combinations of stability enhancing mutations selected from Hl IX, L12X, D14X, H16X, I17X, T19X, S20X, N21X, G25X, I26X, G27X, R28X, H29X, T31X, C34X, E38X, R39X, D41X, N42X, S45X, K47X, M48X, H51X, F54X, H56X, N57X, A59X, K60X, L62X, Y67X, L73X, R74X, D77X, L78X, V79X, P80X, S81X, D85X, A87X, I89X, Y90X, R91X, T93X, W94X, I96X, S97X, C101X, F102X, S103X, W104X, G105X, C106X, A107X, G108X, E109X, V110X, R111X, Al 12X, LI 14X, QI 15X, El 16X, N117X, T118X, Hl 19X, V120X, L122X, R123X, A126X, R128X, L135X, Y136X, E138X, A139X, Q141X, M142X, D145X, A146X, A148X, D156X, E157X, K159X, C161X, D163X, T164X, D167X, Q169X, C171X, D177X, D180X, H182X, S183X, Q184X, A185X, L186X, S187X, G188X, R189X, A192X, N196X a deletion selected from AM1-L12, AE36-G53, AN61-G68, AW104-G105 , AQ195-N199 , AI26-G27, or any combination thereof, where the position number designation is functionally equivalent to the position in a wild-type AP0BEC3A (SEQ ID NO:3) and X is an amino acid substitution different from the wild-type amino acid at that position. In some embodiments, the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 102- 105 130-134, or functionally equivalent positions, the sequence HLYG ARAYW, in combination with one or more combinations of stability enhancing mutations selected from: at least one of the following combinations of stabilizing mutations:
R74L/C 171 A/T 19 Y/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 88Q/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88Q/G25K/S45W; R74L/C 171 A/T 19 Y/Gl 88 A/S45 W; R74L/C 171 A/T 19 Y/Gl 88 A/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88R/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88R/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/G25K/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88 A/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G188Q/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G188 A/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188 A/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188 A/G25K/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188R/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188R/G25K/S45 W; T 19 Y/R74L/C 171 A;
117T/T 19 Y/G25R/R74L/C 171 A; T 19 Y/G25K/S45 WR74L/G108 A/C 171 A/Gl 88R;
117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/G108C/C 171 A/Gl 88R;
117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/G108C/A126C/C 171 A/Gl 88R; or 117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/AW 104-
G105/G108C/C 171 A/Gl 88R.
[000284] In some embodiments, the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 102-105 130-134, or functionally equivalent positions, the sequence YMAG AREYW, in combination with one or more combinations of stability enhancing mutations selected from Hl IX, L12X, D14X, H16X, I17X, T19X, S20X, N21X, G25X, I26X, G27X, R28X, H29X, T31X, C34X, E38X, R39X, D41X, N42X, S45X, K47X, M48X, H51X, F54X, H56X, N57X, A59X, K60X, L62X, Y67X, L73X, R74X, D77X, L78X, V79X, P80X, S81X, D85X, A87X, I89X, Y90X, R91X, T93X, W94X, I96X, S97X, C101X, F102X, S103X, W104X, G105X, C106X, A107X, G108X, E109X, V110X, R111X, Al 12X, LI 14X, QI 15X, El 16X, N117X, T118X, Hl 19X, V120X, L122X, R123X, A126X, R128X, L135X, Y136X, E138X, A139X, Q141X, M142X, D145X, A146X, A148X, D156X, E157X, K159X, C161X, D163X, T164X, D167X, Q169X, C171X, D177X, D180X, H182X, S183X, Q184X, A185X, L186X, S187X, G188X, R189X, A192X, N196X a deletion selected from AM1-L12, AE36-G53, AN61-G68, AW104-G105 , AQ195-N199 , AI26-G27, or any combination thereof, where the position number designation is functionally equivalent to the position in a wild-type APOBEC3A (SEQ ID NO:3) and X is an amino acid substitution different from the wild-type amino acid at that position.
[000285] In some embodiments, the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 102-105 130-134, or functionally equivalent positions, the sequence YMAG AREYW, in combination with one or more combinations of stability enhancing mutations selected from: at least one of the following combinations of stabilizing mutations: R74L/C 171 A/T 19 Y/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 88Q/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88Q/G25K/S45W; R74L/C 171 A/T 19 Y/Gl 88 A/S45 W;
R74L/C 171 A/T 19 Y/Gl 88 A/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88R/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88R/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/G25K/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88 A/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G188Q/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G188 A/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188 A/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188 A/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G188R/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G188R/G25K/S45 W; T 19 Y/R74L/C 171 A; 117T/T 19 Y/G25R/R74L/C 171 A; T 19 Y/G25K/S45 WR74L/G108 A/C 171 A/Gl 88R;
117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/G108C/C 171 A/Gl 88R;
117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/G108C/A126C/C 171 A/Gl 88R; or 117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/AW 104- G105/G108C/C 171 A/Gl 88R.
[000286] In some embodiments, the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 102-105 130-134, or functionally equivalent positions, the sequence YIWG ARAYW, in combination with one or more combinations of stability enhancing mutations selected from Hl IX, L12X, D14X, H16X, I17X, T19X, S20X, N21X, G25X, I26X, G27X, R28X, H29X, T31X, C34X, E38X, R39X, D41X, N42X, S45X, K47X, M48X, H51X, F54X, H56X, N57X, A59X, K60X, L62X, Y67X, L73X, R74X, D77X, L78X, V79X, P80X, S81X, D85X, A87X, I89X, Y90X, R91X, T93X, W94X, I96X, S97X, C101X, F102X, S103X, W104X, G105X, C106X, A107X, G108X, E109X, V110X, R111X, Al 12X, LI 14X, QI 15X, El 16X, N117X, T118X, Hl 19X, V120X, L122X, R123X, A126X, R128X, L135X, Y136X, E138X, A139X, Q141X, M142X, D145X, A146X, A148X, D156X, E157X, K159X, C161X, D163X, T164X, D167X, Q169X, C171X, D177X, D180X, H182X, S183X, Q184X, A185X, L186X, S187X, G188X, R189X, A192X, N196X a deletion selected from AM1-L12, AE36-G53, AN61-G68, AW104-G105 , AQ195-N199 , AI26-G27, or any combination thereof, where the position number designation is functionally equivalent to the position in a wild-type APOBEC3A (SEQ ID NO:3) and X is an amino acid substitution different from the wild-type amino acid at that position.
[000287] In some embodiments, the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 102-105 130-134, or functionally equivalent positions, the sequence YIWG ARAYW, in combination with one or more combinations of stability enhancing mutations selected from: at least one of the following combinations of stabilizing mutations: R74L/C 171 A/T 19 Y/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 88Q/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88Q/G25K/S45W; R74L/C 171 A/T 19 Y/Gl 88 A/S45 W;
R74L/C 171 A/T 19 Y/Gl 88 A/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88R/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88R/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/G25K/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88 A/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G188Q/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G188 A/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188 A/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188 A/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G188R/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G188R/G25K/S45 W; T 19 Y/R74L/C 171 A; 117T/T 19 Y/G25R/R74L/C 171 A; T 19 Y/G25K/S45 WR74L/G108 A/C 171 A/Gl 88R; 117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/G108C/C 171 A/Gl 88R;
117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/G108C/A126C/C 171 A/Gl 88R; or 117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/AW 104- G105/G108C/C 171 A/Gl 88R.
[000288] In some embodiments, the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 102-105 130-134, or functionally equivalent positions, the sequence IRQY ANAFW, in combination with one or more combinations of stability enhancing mutations selected from Hl IX, L12X, D14X, H16X, I17X, T19X, S20X, N21X, G25X, I26X, G27X, R28X, H29X, T31X, C34X, E38X, R39X, D41X, N42X, S45X, K47X, M48X, H51X, F54X, H56X, N57X, A59X, K60X, L62X, Y67X, L73X, R74X, D77X, L78X, V79X, P80X, S81X, D85X, A87X, I89X, Y90X, R91X, T93X, W94X, I96X, S97X, C101X, F102X, S103X, W104X, G105X, C106X, A107X, G108X, E109X, V110X, R111X, Al 12X, LI 14X, QI 15X, El 16X, N117X, T118X, Hl 19X, V120X, L122X, R123X, A126X, R128X, L135X, Y136X, E138X, A139X, Q141X, M142X, D145X, A146X, A148X, D156X, E157X, K159X, C161X, D163X, T164X, D167X, Q169X, C171X, D177X, D180X, H182X, S183X, Q184X, A185X, L186X, S187X, G188X, R189X, A192X, N196X a deletion selected from AM1-L12, AE36-G53, AN61-G68, AW104-G105 , AQ195-N199 , AI26-G27, or any combination thereof, where the position number designation is functionally equivalent to the position in a wild-type APOBEC3A (SEQ ID NO:3) and X is an amino acid substitution different from the wild-type amino acid at that position.
[000289] In some embodiments, the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 102-105 130-134, or functionally equivalent positions, the sequence IRQY ANAFW, in combination with one or more combinations of stability enhancing mutations selected from: at least one of the following combinations of stabilizing mutations: R74L/C 171 A/T 19 Y/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 88Q/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88Q/G25K/S45W; R74L/C 171 A/T 19 Y/Gl 88 A/S45 W; R74L/C 171 A/T 19 Y/Gl 88 A/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88R/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88R/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/G25K/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88 A/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G188Q/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G188 A/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188 A/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188 A/G25K/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188R/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188R/G25K/S45 W; T 19 Y/R74L/C 171 A;
117T/T 19 Y/G25R/R74L/C 171 A; T 19 Y/G25K/S45 WR74L/G108 A/C 171 A/Gl 88R;
117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/G108C/C 171 A/Gl 88R;
117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/G108C/A126C/C 171 A/Gl 88R; or 117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/AW 104-
G105/G108C/C 171 A/Gl 88R.
[000290] In some embodiments, the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 102-105 130-134, or functionally equivalent positions, the sequence AQMG ARAYW, in combination with one or more combinations of stability enhancing mutations selected from Hl IX, L12X, D14X, H16X, I17X, T19X, S20X, N21X, G25X, I26X, G27X, R28X, H29X, T31X, C34X, E38X, R39X, D41X, N42X, S45X, K47X, M48X, H51X, F54X, H56X, N57X, A59X, K60X, L62X, Y67X, L73X, R74X, D77X, L78X, V79X, P80X, S81X, D85X, A87X, I89X, Y90X, R91X, T93X, W94X, I96X, S97X, C101X, F102X, S103X, W104X, G105X, C106X, A107X, G108X, E109X, V110X, R111X, Al 12X, LI 14X, QI 15X, El 16X, N117X, T118X, Hl 19X, V120X, L122X, R123X, A126X, R128X, L135X, Y136X, E138X, A139X, Q141X, M142X, D145X, A146X, A148X, D156X, E157X, K159X, C161X, D163X, T164X, D167X, Q169X, C171X, D177X, D180X, H182X, S183X, Q184X, A185X, L186X, S187X, G188X, R189X, A192X, N196X a deletion selected from AM1-L12, AE36-G53, AN61-G68, AW104-G105 , AQ195-N199 , AI26-G27, or any combination thereof, where the position number designation is functionally equivalent to the position in a wild-type AP0BEC3A (SEQ ID NO:3) and X is an amino acid substitution different from the wild-type amino acid at that position.
[000291] In some embodiments, the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 102-105 130-134, or functionally equivalent positions, the sequence AQMG ARAYW, in combination with one or more combinations of stability enhancing mutations selected from: at least one of the following combinations of stabilizing mutations: R74L/C 171 A/T 19 Y/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 88Q/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88Q/G25K/S45W; R74L/C 171 A/T 19 Y/Gl 88 A/S45 W;
R74L/C 171 A/T 19 Y/Gl 88 A/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88R/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88R/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/G25K/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88 A/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G188Q/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188 A/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188 A/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188 A/G25K/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188R/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188R/G25K/S45 W; T 19 Y/R74L/C 171 A;
117T/T 19 Y/G25R/R74L/C 171 A; T 19 Y/G25K/S45 WR74L/G108 A/C 171 A/Gl 88R;
117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/G108C/C 171 A/Gl 88R;
117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/G108C/A126C/C 171 A/Gl 88R; or
117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/AW 104-
G105/G108C/C 171 A/Gl 88R.
[000292]
[000293] In some embodiments, the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 102-105 the sequence FVPASG, and from 130-134, or functionally equivalent positions, the sequence AKYPW (SEQ ID NO:44), in combination with one or more combinations of stability enhancing mutations selected from Hl IX, L12X, D14X, H16X, I17X, T19X, S20X, N21X, G25X, I26X, G27X, R28X, H29X, T31X, C34X, E38X, R39X, D41X, N42X, S45X, K47X, M48X, H51X, F54X, H56X, N57X, A59X, K60X, L62X, Y67X, L73X, R74X, D77X, L78X, V79X, P80X, S81X, D85X, A87X, I89X, Y90X, R91X, T93X, W94X, I96X, S97X, C101X, F102X, S1O3X, W104X, G105X, C106X, A107X, G108X, E109X, VI 1OX, R11 IX, Al 12X, LI 14X, QI 15X, El 16X, N117X, T118X, H119X, V120X, L122X, R123X, A126X, R128X, L135X, Y136X, E138X, A139X, Q141X, M142X, D145X, A146X, A148X, D156X, E157X, K159X, C161X, D163X, T164X, D167X, Q169X, C171X, D177X, D180X, H182X, S183X, Q184X, A185X, L186X, S187X, G188X, R189X, A192X, N196X a deletion selected from AM1-L12, AE36-G53, AN61-G68, AW104- G105 , AQ195-N199 , AI26-G27, or any combination thereof, where the position number designation is functionally equivalent to the position in a wild-type APOBEC3 A (SEQ ID NO:3) and X is an amino acid substitution different from the wild-type amino acid at that position.
[000294] In some embodiments, the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 102-105 the sequence FVPLSG, and from 130-134, or functionally equivalent positions, the sequence AKYPW, in combination with one or more combinations of stability enhancing mutations selected from: at least one of the following combinations of stabilizing mutations: R74L/C171A/T19Y/G25K/S45W;
R74L/C 171 A/T 19 Y/Gl 88Q/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88Q/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 88 A/S45 W; R74L/C 171 A/T 19 Y/Gl 88 A/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 88R/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88R/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/G25K/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88 A/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G188Q/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G188 A/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188 A/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188 A/G25K/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188R/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188R/G25K/S45 W; T 19 Y/R74L/C 171 A;
117T/T 19 Y/G25R/R74L/C 171 A; T 19 Y/G25K/S45 WR74L/G108 A/C 171 A/Gl 88R;
117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/G108C/C 171 A/Gl 88R;
117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/G108C/A126C/C 171 A/Gl 88R; or 117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/AW 104-
G105/G108C/C 171 A/Gl 88R.
[000295] In some embodiments, the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 102-105 the sequence FVPLSG, and from 130-134, or functionally equivalent positions, the sequence AKYPW (SEQ ID NO:44), in combination with one or more combinations of stability enhancing mutations selected from Hl IX, L12X, D14X, H16X, I17X, T19X, S20X, N21X, G25X, I26X, G27X, R28X, H29X, T31X, C34X, E38X, R39X, D41X, N42X, S45X, K47X, M48X, H51X, F54X, H56X, N57X, A59X, K60X, L62X, Y67X, L73X, R74X, D77X, L78X, V79X, P80X, S81X, D85X, A87X, I89X, Y90X, R91X, T93X, W94X, I96X, S97X, C101X, F102X, S103X, W104X, G105X, C106X, A107X, G108X, E109X, VI 10X, R11 IX, Al 12X, LI 14X, QI 15X, El 16X, N117X, T118X, H119X, V120X, L122X, R123X, A126X, R128X, L135X, Y136X, E138X, A139X, Q141X, M142X, D145X, A146X, A148X, D156X, E157X, K159X, C161X, D163X, T164X, D167X, Q169X, C171X, D177X, D180X, H182X, S183X, Q184X, A185X, L186X, S187X, G188X, R189X, A192X, N196X a deletion selected from AM1-L12, AE36-G53, AN61-G68, AW104- G105 , AQ195-N199 , AI26-G27, or any combination thereof, where the position number designation is functionally equivalent to the position in a wild-type APOBEC3 A (SEQ ID NO:3) and X is an amino acid substitution different from the wild-type amino acid at that position.
[000296] In some embodiments, the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 102-105 the sequence FVPPSG, and from 130-134, or functionally equivalent positions, the sequence AKYPW, in combination with one or more combinations of stability enhancing mutations selected from: at least one of the following combinations of stabilizing mutations: R74L/C171A/T19Y/G25K/S45W;
R74L/C 171 A/T 19 Y/Gl 88Q/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88Q/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 88 A/S45 W; R74L/C 171 A/T 19 Y/Gl 88 A/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 88R/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88R/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/G25K/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88 A/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G188Q/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G188 A/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188 A/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G188 A/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G188R/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G188R/G25K/S45 W; T 19 Y/R74L/C 171 A; 117T/T 19 Y/G25R/R74L/C 171 A; T 19 Y/G25K/S45 WR74L/G108 A/C 171 A/Gl 88R; 117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/G108C/C 171 A/Gl 88R; 117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/G108C/A126C/C 171 A/Gl 88R; or 117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/AW 104- G105/G108C/C 171 A/Gl 88R.
[000297] In some embodiments, the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 102-105 the sequence FVPPSG, and from 130-134, or functionally equivalent positions, the sequence AKYPW (SEQ ID NO:44), in combination with one or more combinations of stability enhancing mutations selected from Hl IX, L12X, D14X, H16X, I17X, T19X, S20X, N21X, G25X, I26X, G27X, R28X, H29X, T31X, C34X, E38X, R39X, D41X, N42X, S45X, K47X, M48X, H51X, F54X, H56X, N57X, A59X, K60X, L62X, Y67X, L73X, R74X, D77X, L78X, V79X, P80X, S81X, D85X, A87X, I89X, Y90X, R91X, T93X, W94X, I96X, S97X, C101X, F102X, S103X, W104X, G105X, C106X, A107X, G108X, E109X, VI 10X, R11 IX, Al 12X, LI 14X, QI 15X, El 16X, N117X, T118X, H119X, V120X, L122X, R123X, A126X, R128X, L135X, Y136X, E138X, A139X, Q141X, M142X, D145X, A146X, A148X, D156X, E157X, K159X, C161X, D163X, T164X, D167X, Q169X, C171X, D177X, D180X, H182X, S183X, Q184X, A185X, L186X, S187X, G188X, R189X, A192X, N196X a deletion selected from AM1-L12, AE36-G53, AN61-G68, AW104- G105 , AQ195-N199 , AI26-G27, or any combination thereof, where the position number designation is functionally equivalent to the position in a wild-type APOBEC3 A (SEQ ID NO:3) and X is an amino acid substitution different from the wild-type amino acid at that position.
[000298] In some embodiments, the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 102-105 the sequence FVPAFG, and from 130-134, or functionally equivalent positions, the sequence AKYPW, in combination with one or more combinations of stability enhancing mutations selected from: at least one of the following combinations of stabilizing mutations: R74L/C171A/T19Y/G25K/S45W;
R74L/C 171 A/T 19 Y/Gl 88Q/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88Q/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 88 A/S45 W; R74L/C 171 A/T 19 Y/Gl 88 A/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88R/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88R/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/G25K/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88 A/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G188Q/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G188 A/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188 A/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188 A/G25K/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188R/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188R/G25K/S45 W; T 19 Y/R74L/C 171 A;
117T/T 19 Y/G25R/R74L/C 171 A; T 19 Y/G25K/S45 WR74L/G108 A/C 171 A/Gl 88R;
117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/G108C/C 171 A/Gl 88R;
117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/G108C/A126C/C 171 A/Gl 88R; or 117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/AW 104-
G105/G108C/C 171 A/Gl 88R.
[000299] In some embodiments, the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 102-105 the sequence FVPAFG, and from 130-134, or functionally equivalent positions, the sequence AKYPW (SEQ ID NO:44), in combination with one or more combinations of stability enhancing mutations selected from Hl IX, L12X, D14X, H16X, I17X, T19X, S20X, N21X, G25X, I26X, G27X, R28X, H29X, T31X, C34X, E38X, R39X, D41X, N42X, S45X, K47X, M48X, H51X, F54X, H56X, N57X, A59X, K60X, L62X, Y67X, L73X, R74X, D77X, L78X, V79X, P80X, S81X, D85X, A87X, I89X, Y90X, R91X, T93X, W94X, I96X, S97X, C101X, F102X, S103X, W104X, G105X, C106X, A107X, G108X, E109X, VI 10X, R11 IX, Al 12X, LI 14X, QI 15X, El 16X, N117X, T118X, H119X, V120X, L122X, R123X, A126X, R128X, L135X, Y136X, E138X, A139X, Q141X, M142X, D145X, A146X, A148X, D156X, E157X, K159X, C161X, D163X, T164X, D167X, Q169X, C171X, D177X, D180X, H182X, S183X, Q184X, A185X, L186X, S187X, G188X, R189X, A192X, N196X a deletion selected from AM1-L12, AE36-G53, AN61-G68, AW104- G105 , AQ195-N199 , AI26-G27, or any combination thereof, where the position number designation is functionally equivalent to the position in a wild-type APOBEC3 A (SEQ ID N0:3) and X is an amino acid substitution different from the wild-type amino acid at that position.
[000300] In some embodiments, the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 102-105 the sequence FVPLFG, and from 130-134, or functionally equivalent positions, the sequence AKYPW, in combination with one or more combinations of stability enhancing mutations selected from: at least one of the following combinations of stabilizing mutations: R74L/C171A/T19Y/G25K/S45W;
R74L/C 171 A/T 19 Y/Gl 88Q/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88Q/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 88 A/S45 W; R74L/C 171 A/T 19 Y/Gl 88 A/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 88R/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88R/G25K/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/G25K/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88 A/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G188Q/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188 A/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188 A/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188 A/G25K/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188R/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188R/G25K/S45 W; T 19 Y/R74L/C 171 A;
117T/T 19 Y/G25R/R74L/C 171 A; T 19 Y/G25K/S45 WR74L/G108 A/C 171 A/Gl 88R;
117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/G108C/C 171 A/Gl 88R;
117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/G108C/A126C/C 171 A/Gl 88R; or
117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/AW 104-
G105/G108C/C 171 A/Gl 88R.
[000301] In some embodiments, the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 102-105 the sequence FVPLFG, and from 130-134, or functionally equivalent positions, the sequence AKYPW (SEQ ID NO:44), in combination with one or more combinations of stability enhancing mutations selected from Hl IX, L12X, D14X, H16X, I17X, T19X, S20X, N21X, G25X, I26X, G27X, R28X, H29X, T31X, C34X, E38X, R39X, D41X, N42X, S45X, K47X, M48X, H51X, F54X, H56X, N57X, A59X, K60X, L62X, Y67X, L73X, R74X, D77X, L78X, V79X, P80X, S81X, D85X, A87X, I89X, Y90X, R91X, T93X, W94X, I96X, S97X, C101X, F102X, S103X, W104X, G105X, C106X, A107X, G108X, E109X, VI 10X, R11 IX, Al 12X, LI 14X, QI 15X, El 16X, N117X, T118X, H119X, V120X, L122X, R123X, A126X, R128X, L135X, Y136X, E138X, A139X, Q141X, M142X, D145X, A146X, A148X, D156X, E157X, K159X, C161X, D163X, T164X, D167X, Q169X, C171X, D177X, D180X, H182X, S183X, Q184X, A185X, L186X, S187X, G188X, R189X, A192X, N196X a deletion selected from AM1-L12, AE36-G53, AN61-G68, AW104- G105 , AQ195-N199 , AI26-G27, or any combination thereof, where the position number designation is functionally equivalent to the position in a wild-type APOBEC3 A (SEQ ID NO:3) and X is an amino acid substitution different from the wild-type amino acid at that position.
[000302] In some embodiments, the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 102-105 the sequence FVPPFG, and from 130-134, or functionally equivalent positions, the sequence AKYPW, in combination with one or more combinations of stability enhancing mutations selected from: at least one of the following combinations of stabilizing mutations: R74L/C171A/T19Y/G25K/S45W;
R74L/C 171 A/T 19 Y/Gl 88Q/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88Q/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 88 A/S45 W; R74L/C 171 A/T 19 Y/Gl 88 A/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 88R/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88R/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/G25K/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88 A/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G188Q/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G188 A/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188 A/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188 A/G25K/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188R/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188R/G25K/S45 W; T 19 Y/R74L/C 171 A;
117T/T 19 Y/G25R/R74L/C 171 A; T 19 Y/G25K/S45 WR74L/G108 A/C 171 A/Gl 88R;
117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/G108C/C 171 A/Gl 88R;
117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/G108C/A126C/C 171 A/Gl 88R; or 117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/AW 104-
G105/G108C/C 171 A/Gl 88R.
[000303] In some embodiments, the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 102-105 the sequence FVPPFG, and from 130-134, or functionally equivalent positions, the sequence AKYPW (SEQ ID NO:44), in combination with one or more combinations of stability enhancing mutations selected from Hl IX, L12X, D14X, H16X, I17X, T19X, S20X, N21X, G25X, I26X, G27X, R28X, H29X, T31X, C34X, E38X, R39X, D41X, N42X, S45X, K47X, M48X, H51X, F54X, H56X, N57X, A59X, K60X, L62X, Y67X, L73X, R74X, D77X, L78X, V79X, P80X, S81X, D85X, A87X, I89X, Y90X, R91X, T93X, W94X, I96X, S97X, C101X, F102X, S103X, W104X, G105X, C106X, A107X, G108X, E109X, VI 10X, R11 IX, Al 12X, LI 14X, QI 15X, El 16X, N117X, T118X, H119X, V120X, L122X, R123X, A126X, R128X, L135X, Y136X, E138X, A139X, Q141X, M142X, D145X, A146X, A148X, D156X, E157X, K159X, C161X, D163X, T164X, D167X, Q169X, C171X, D177X, D180X, H182X, S183X, Q184X, A185X, L186X, S187X, G188X, R189X, A192X, N196X a deletion selected from AM1-L12, AE36-G53, AN61-G68, AW104- G105 , AQ195-N199 , AI26-G27, or any combination thereof, where the position number designation is functionally equivalent to the position in a wild-type APOBEC3 A (SEQ ID NO:3) and X is an amino acid substitution different from the wild-type amino acid at that position.
[000304] In some embodiments, the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 102-105 the sequence FVPAQG, and from 130-134, or functionally equivalent positions, the sequence AKYPW, in combination with one or more combinations of stability enhancing mutations selected from: at least one of the following combinations of stabilizing mutations: R74L/C171A/T19Y/G25K/S45W;
R74L/C 171 A/T 19 Y/Gl 88Q/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88Q/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 88 A/S45 W; R74L/C 171 A/T 19 Y/Gl 88 A/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 88R/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88R/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/G25K/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88 A/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G188Q/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G188 A/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188 A/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G188 A/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G188R/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G188R/G25K/S45 W; T 19 Y/R74L/C 171 A; 117T/T 19 Y/G25R/R74L/C 171 A; T 19 Y/G25K/S45 WR74L/G108 A/C 171 A/Gl 88R; 117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/G108C/C 171 A/Gl 88R; 117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/G108C/A126C/C 171 A/Gl 88R; or 117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/AW 104- G105/G108C/C 171 A/Gl 88R.
[000305] In some embodiments, the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 102-105 the sequence FVPAQG, and from 130-134, or functionally equivalent positions, the sequence AKYPW (SEQ ID NO:44), in combination with one or more combinations of stability enhancing mutations selected from Hl IX, L12X, D14X, H16X, I17X, T19X, S20X, N21X, G25X, I26X, G27X, R28X, H29X, T31X, C34X, E38X, R39X, D41X, N42X, S45X, K47X, M48X, H51X, F54X, H56X, N57X, A59X, K60X, L62X, Y67X, L73X, R74X, D77X, L78X, V79X, P80X, S81X, D85X, A87X, I89X, Y90X, R91X, T93X, W94X, I96X, S97X, C101X, F102X, S103X, W104X, G105X, C106X, A107X, G108X, E109X, VI 10X, R11 IX, Al 12X, LI 14X, QI 15X, El 16X, N117X, T118X, H119X, V120X, L122X, R123X, A126X, R128X, L135X, Y136X, E138X, A139X, Q141X, M142X, D145X, A146X, A148X, D156X, E157X, K159X, C161X, D163X, T164X, D167X, Q169X, C171X, D177X, D180X, H182X, S183X, Q184X, A185X, L186X, S187X, G188X, R189X, A192X, N196X a deletion selected from AM1-L12, AE36-G53, AN61-G68, AW104- G105 , AQ195-N199 , AI26-G27, or any combination thereof, where the position number designation is functionally equivalent to the position in a wild-type APOBEC3 A (SEQ ID NO:3) and X is an amino acid substitution different from the wild-type amino acid at that position.
[000306] In some embodiments, the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 102-105 the sequence FVPLQG, and from 130-134, or functionally equivalent positions, the sequence AKYPW, in combination with one or more combinations of stability enhancing mutations selected from: at least one of the following combinations of stabilizing mutations: R74L/C171A/T19Y/G25K/S45W;
R74L/C 171 A/T 19 Y/Gl 88Q/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88Q/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 88 A/S45 W; R74L/C 171 A/T 19 Y/Gl 88 A/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88R/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88R/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/G25K/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88 A/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G188Q/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G188 A/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188 A/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188 A/G25K/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188R/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188R/G25K/S45 W; T 19 Y/R74L/C 171 A;
117T/T 19 Y/G25R/R74L/C 171 A; T 19 Y/G25K/S45 WR74L/G108 A/C 171 A/Gl 88R;
117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/G108C/C 171 A/Gl 88R;
117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/G108C/A126C/C 171 A/Gl 88R; or 117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/AW 104-
G105/G108C/C 171 A/Gl 88R.
[000307] In some embodiments, the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 102-105 the sequence FVPLQG, and from 130-134, or functionally equivalent positions, the sequence AKYPW (SEQ ID NO:44), in combination with one or more combinations of stability enhancing mutations selected from Hl IX, L12X, D14X, H16X, I17X, T19X, S20X, N21X, G25X, I26X, G27X, R28X, H29X, T31X, C34X, E38X, R39X, D41X, N42X, S45X, K47X, M48X, H51X, F54X, H56X, N57X, A59X, K60X, L62X, Y67X, L73X, R74X, D77X, L78X, V79X, P80X, S81X, D85X, A87X, I89X, Y90X, R91X, T93X, W94X, I96X, S97X, C101X, F102X, S103X, W104X, G105X, C106X, A107X, G108X, E109X, VI 10X, R11 IX, Al 12X, LI 14X, QI 15X, El 16X, N117X, T118X, H119X, V120X, L122X, R123X, A126X, R128X, L135X, Y136X, E138X, A139X, Q141X, M142X, D145X, A146X, A148X, D156X, E157X, K159X, C161X, D163X, T164X, D167X, Q169X, C171X, D177X, D180X, H182X, S183X, Q184X, A185X, L186X, S187X, G188X, R189X, A192X, N196X a deletion selected from AM1-L12, AE36-G53, AN61-G68, AW104- G105 , AQ195-N199 , AI26-G27, or any combination thereof, where the position number designation is functionally equivalent to the position in a wild-type APOBEC3 A (SEQ ID N0:3) and X is an amino acid substitution different from the wild-type amino acid at that position.
[000308] In some embodiments, the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 102-105 the sequence FVPPQG, and from 130-134, or functionally equivalent positions, the sequence AKYPW, in combination with one or more combinations of stability enhancing mutations selected from: at least one of the following combinations of stabilizing mutations: R74L/C171A/T19Y/G25K/S45W;
R74L/C 171 A/T 19 Y/Gl 88Q/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88Q/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 88 A/S45 W; R74L/C 171 A/T 19 Y/Gl 88 A/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 88R/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88R/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/G25K/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88 A/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G188Q/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188 A/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188 A/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188 A/G25K/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188R/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188R/G25K/S45 W; T 19 Y/R74L/C 171 A;
117T/T 19 Y/G25R/R74L/C 171 A; T 19 Y/G25K/S45 WR74L/G108 A/C 171 A/Gl 88R;
117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/G108C/C 171 A/Gl 88R;
117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/G108C/A126C/C 171 A/Gl 88R; or
117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/AW 104-
G105/G108C/C 171 A/Gl 88R.
[000309] In some embodiments, the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 102-105 the sequence FVPPQG, and from 130-134, or functionally equivalent positions, the sequence AKYPW (SEQ ID NO:44), in combination with one or more combinations of stability enhancing mutations selected from Hl IX, L12X, D14X, H16X, I17X, T19X, S20X, N21X, G25X, I26X, G27X, R28X, H29X, T31X, C34X, E38X, R39X, D41X, N42X, S45X, K47X, M48X, H51X, F54X, H56X, N57X, A59X, K60X, L62X, Y67X, L73X, R74X, D77X, L78X, V79X, P80X, S81X, D85X, A87X, I89X, Y90X, R91X, T93X, W94X, I96X, S97X, C101X, F102X, S1O3X, W104X, G105X, C106X, A107X, G108X, E109X, VI 1OX, R11 IX, Al 12X, LI 14X, QI 15X, El 16X, N117X, T118X, H119X, V120X, L122X, R123X, A126X, R128X, L135X, Y136X, E138X, A139X, Q141X, M142X, D145X, A146X, A148X, D156X, E157X, K159X, C161X, D163X, T164X, D167X, Q169X, C171X, D177X, D180X, H182X, S183X, Q184X, A185X, L186X, S187X, G188X, R189X, A192X, N196X a deletion selected from AM1-L12, AE36-G53, AN61-G68, AW104- G105 , AQ195-N199 , AI26-G27, or any combination thereof, where the position number designation is functionally equivalent to the position in a wild-type APOBEC3 A (SEQ ID NO:3) and X is an amino acid substitution different from the wild-type amino acid at that position.
[000310] In some embodiments, the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 102-105 the sequence FVPADG, and from 130-134, or functionally equivalent positions, the sequence AKYPW, in combination with one or more combinations of stability enhancing mutations selected from: at least one of the following combinations of stabilizing mutations: R74L/C171A/T19Y/G25K/S45W;
R74L/C 171 A/T 19 Y/Gl 88Q/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88Q/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 88 A/S45 W; R74L/C 171 A/T 19 Y/Gl 88 A/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 88R/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88R/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/G25K/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88 A/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G188Q/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G188 A/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188 A/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188 A/G25K/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188R/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188R/G25K/S45 W; T 19 Y/R74L/C 171 A;
117T/T 19 Y/G25R/R74L/C 171 A; T 19 Y/G25K/S45 WR74L/G108 A/C 171 A/Gl 88R;
117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/G108C/C 171 A/Gl 88R;
117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/G108C/A126C/C 171 A/Gl 88R; or 117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/AW 104-
G105/G108C/C 171 A/Gl 88R.
[000311] In some embodiments, the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 102-105 the sequence FVPADG, and from 130-134, or functionally equivalent positions, the sequence AKYPW (SEQ ID NO:44), in combination with one or more combinations of stability enhancing mutations selected from Hl IX, L12X, D14X, H16X, I17X, T19X, S20X, N21X, G25X, I26X, G27X, R28X, H29X, T31X, C34X, E38X, R39X, D41X, N42X, S45X, K47X, M48X, H51X, F54X, H56X, N57X, A59X, K60X, L62X, Y67X, L73X, R74X, D77X, L78X, V79X, P80X, S81X, D85X, A87X, I89X, Y90X, R91X, T93X, W94X, I96X, S97X, C101X, F102X, S103X, W104X, G105X, C106X, A107X, G108X, E109X, VI 10X, R11 IX, Al 12X, LI 14X, QI 15X, El 16X, N117X, T118X, H119X, V120X, L122X, R123X, A126X, R128X, L135X, Y136X, E138X, A139X, Q141X, M142X, D145X, A146X, A148X, D156X, E157X, K159X, C161X, D163X, T164X, D167X, Q169X, C171X, D177X, D180X, H182X, S183X, Q184X, A185X, L186X, S187X, G188X, R189X, A192X, N196X a deletion selected from AM1-L12, AE36-G53, AN61-G68, AW104- G105 , AQ195-N199 , AI26-G27, or any combination thereof, where the position number designation is functionally equivalent to the position in a wild-type APOBEC3 A (SEQ ID NO:3) and X is an amino acid substitution different from the wild-type amino acid at that position.
[000312] In some embodiments, the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 102-105 the sequence FVPLDG, and from 130-134, or functionally equivalent positions, the sequence AKYPW, in combination with one or more combinations of stability enhancing mutations selected from: at least one of the following combinations of stabilizing mutations: R74L/C171A/T19Y/G25K/S45W;
R74L/C 171 A/T 19 Y/Gl 88Q/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88Q/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 88 A/S45 W; R74L/C 171 A/T 19 Y/Gl 88 A/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 88R/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88R/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/G25K/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88 A/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G188Q/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G188 A/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188 A/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G188 A/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G188R/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G188R/G25K/S45 W; T 19 Y/R74L/C 171 A; 117T/T 19 Y/G25R/R74L/C 171 A; T 19 Y/G25K/S45 WR74L/G108 A/C 171 A/Gl 88R; 117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/G108C/C 171 A/Gl 88R; 117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/G108C/A126C/C 171 A/Gl 88R; or 117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/AW 104- G105/G108C/C 171 A/Gl 88R.
[000313] In some embodiments, the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 102-105 the sequence FVPLDG, and from 130-134, or functionally equivalent positions, the sequence AKYPW (SEQ ID NO:44), in combination with one or more combinations of stability enhancing mutations selected from Hl IX, L12X, D14X, H16X, I17X, T19X, S20X, N21X, G25X, I26X, G27X, R28X, H29X, T31X, C34X, E38X, R39X, D41X, N42X, S45X, K47X, M48X, H51X, F54X, H56X, N57X, A59X, K60X, L62X, Y67X, L73X, R74X, D77X, L78X, V79X, P80X, S81X, D85X, A87X, I89X, Y90X, R91X, T93X, W94X, I96X, S97X, C101X, F102X, S103X, W104X, G105X, C106X, A107X, G108X, E109X, VI 10X, R11 IX, Al 12X, LI 14X, QI 15X, El 16X, N117X, T118X, H119X, V120X, L122X, R123X, A126X, R128X, L135X, Y136X, E138X, A139X, Q141X, M142X, D145X, A146X, A148X, D156X, E157X, K159X, C161X, D163X, T164X, D167X, Q169X, C171X, D177X, D180X, H182X, S183X, Q184X, A185X, L186X, S187X, G188X, R189X, A192X, N196X a deletion selected from AM1-L12, AE36-G53, AN61-G68, AW104- G105 , AQ195-N199 , AI26-G27, or any combination thereof, where the position number designation is functionally equivalent to the position in a wild-type APOBEC3 A (SEQ ID NO:3) and X is an amino acid substitution different from the wild-type amino acid at that position.
[000314] In some embodiments, the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 102-105 the sequence FVPPDG, and from 130-134, or functionally equivalent positions, the sequence AKYPW, in combination with one or more combinations of stability enhancing mutations selected from: at least one of the following combinations of stabilizing mutations: R74L/C171A/T19Y/G25K/S45W;
R74L/C 171 A/T 19 Y/Gl 88Q/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88Q/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 88 A/S45 W; R74L/C 171 A/T 19 Y/Gl 88 A/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88R/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88R/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/G25K/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88 A/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G188Q/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G188 A/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188 A/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188 A/G25K/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188R/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188R/G25K/S45 W; T 19 Y/R74L/C 171 A;
117T/T 19 Y/G25R/R74L/C 171 A; T 19 Y/G25K/S45 WR74L/G108 A/C 171 A/Gl 88R;
117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/G108C/C 171 A/Gl 88R;
117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/G108C/A126C/C 171 A/Gl 88R; or 117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/AW 104-
G105/G108C/C 171 A/Gl 88R.
[000315] In some embodiments, the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 102-105 the sequence FVPPDG, and from 130-134, or functionally equivalent positions, the sequence AKYPW (SEQ ID NO:44), in combination with one or more combinations of stability enhancing mutations selected from Hl IX, L12X, D14X, H16X, I17X, T19X, S20X, N21X, G25X, I26X, G27X, R28X, H29X, T31X, C34X, E38X, R39X, D41X, N42X, S45X, K47X, M48X, H51X, F54X, H56X, N57X, A59X, K60X, L62X, Y67X, L73X, R74X, D77X, L78X, V79X, P80X, S81X, D85X, A87X, I89X, Y90X, R91X, T93X, W94X, I96X, S97X, C101X, F102X, S103X, W104X, G105X, C106X, A107X, G108X, E109X, VI 10X, R11 IX, Al 12X, LI 14X, QI 15X, El 16X, N117X, T118X, H119X, V120X, L122X, R123X, A126X, R128X, L135X, Y136X, E138X, A139X, Q141X, M142X, D145X, A146X, A148X, D156X, E157X, K159X, C161X, D163X, T164X, D167X, Q169X, C171X, D177X, D180X, H182X, S183X, Q184X, A185X, L186X, S187X, G188X, R189X, A192X, N196X a deletion selected from AM1-L12, AE36-G53, AN61-G68, AW104- G105 , AQ195-N199 , AI26-G27, or any combination thereof, where the position number designation is functionally equivalent to the position in a wild-type APOBEC3 A (SEQ ID N0:3) and X is an amino acid substitution different from the wild-type amino acid at that position.
[000316] In some embodiments, the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 102-105 the sequence FVPAKG, and from 130-134, or functionally equivalent positions, the sequence AKYPW, in combination with one or more combinations of stability enhancing mutations selected from: at least one of the following combinations of stabilizing mutations: R74L/C171A/T19Y/G25K/S45W;
R74L/C 171 A/T 19 Y/Gl 88Q/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88Q/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 88 A/S45 W; R74L/C 171 A/T 19 Y/Gl 88 A/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 88R/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88R/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/G25K/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88 A/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G188Q/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188 A/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188 A/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188 A/G25K/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188R/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188R/G25K/S45 W; T 19 Y/R74L/C 171 A;
117T/T 19 Y/G25R/R74L/C 171 A; T 19 Y/G25K/S45 WR74L/G108 A/C 171 A/Gl 88R;
117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/G108C/C 171 A/Gl 88R;
117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/G108C/A126C/C 171 A/Gl 88R; or
117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/AW 104-
G105/G108C/C 171 A/Gl 88R.
[000317] In some embodiments, the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 102-105 the sequence FVPAKG, and from 130-134, or functionally equivalent positions, the sequence AKYPW (SEQ ID NO:44), in combination with one or more combinations of stability enhancing mutations selected from Hl IX, L12X, D14X, H16X, I17X, T19X, S20X, N21X, G25X, I26X, G27X, R28X, H29X, T31X, C34X, E38X, R39X, D41X, N42X, S45X, K47X, M48X, H51X, F54X, H56X, N57X, A59X, K60X, L62X, Y67X, L73X, R74X, D77X, L78X, V79X, P80X, S81X, D85X, A87X, I89X, Y90X, R91X, T93X, W94X, I96X, S97X, C101X, F102X, S103X, W104X, G105X, C106X, A107X, G108X, E109X, VI 10X, R11 IX, Al 12X, LI 14X, QI 15X, El 16X, N117X, T118X, H119X, V120X, L122X, R123X, A126X, R128X, L135X, Y136X, E138X, A139X, Q141X, M142X, D145X, A146X, A148X, D156X, E157X, K159X, C161X, D163X, T164X, D167X, Q169X, C171X, D177X, D180X, H182X, S183X, Q184X, A185X, L186X, S187X, G188X, R189X, A192X, N196X a deletion selected from AM1-L12, AE36-G53, AN61-G68, AW104- G105 , AQ195-N199 , AI26-G27, or any combination thereof, where the position number designation is functionally equivalent to the position in a wild-type APOBEC3 A (SEQ ID NO:3) and X is an amino acid substitution different from the wild-type amino acid at that position.
[000318] In some embodiments, the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 102-105 the sequence FVPLKG, and from 130-134, or functionally equivalent positions, the sequence AKYPW, in combination with one or more combinations of stability enhancing mutations selected from: at least one of the following combinations of stabilizing mutations: R74L/C171A/T19Y/G25K/S45W;
R74L/C 171 A/T 19 Y/Gl 88Q/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88Q/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 88 A/S45 W; R74L/C 171 A/T 19 Y/Gl 88 A/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 88R/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88R/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/G25K/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88 A/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G188Q/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G188 A/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188 A/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188 A/G25K/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188R/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188R/G25K/S45 W; T 19 Y/R74L/C 171 A;
117T/T 19 Y/G25R/R74L/C 171 A; T 19 Y/G25K/S45 WR74L/G108 A/C 171 A/Gl 88R;
117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/G108C/C 171 A/Gl 88R;
117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/G108C/A126C/C 171 A/Gl 88R; or 117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/AW 104-
G105/G108C/C 171 A/Gl 88R.
[000319] In some embodiments, the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 102-105 the sequence FVPLKG, and from 130-134, or functionally equivalent positions, the sequence AKYPW (SEQ ID NO:44), in combination with one or more combinations of stability enhancing mutations selected from Hl IX, L12X, D14X, H16X, I17X, T19X, S20X, N21X, G25X, I26X, G27X, R28X, H29X, T31X, C34X, E38X, R39X, D41X, N42X, S45X, K47X, M48X, H51X, F54X, H56X, N57X, A59X, K60X, L62X, Y67X, L73X, R74X, D77X, L78X, V79X, P80X, S81X, D85X, A87X, I89X, Y90X, R91X, T93X, W94X, I96X, S97X, C101X, F102X, S103X, W104X, G105X, C106X, A107X, G108X, E109X, VI 10X, R11 IX, Al 12X, LI 14X, QI 15X, El 16X, N117X, T118X, H119X, V120X, L122X, R123X, A126X, R128X, L135X, Y136X, E138X, A139X, Q141X, M142X, D145X, A146X, A148X, D156X, E157X, K159X, C161X, D163X, T164X, D167X, Q169X, C171X, D177X, D180X, H182X, S183X, Q184X, A185X, L186X, S187X, G188X, R189X, A192X, N196X a deletion selected from AM1-L12, AE36-G53, AN61-G68, AW104- G105 , AQ195-N199 , AI26-G27, or any combination thereof, where the position number designation is functionally equivalent to the position in a wild-type APOBEC3 A (SEQ ID NO:3) and X is an amino acid substitution different from the wild-type amino acid at that position.
[000320] In some embodiments, the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 102-105 the sequence FVPPKG, and from 130-134, or functionally equivalent positions, the sequence AKYPW, in combination with one or more combinations of stability enhancing mutations selected from: at least one of the following combinations of stabilizing mutations: R74L/C171A/T19Y/G25K/S45W;
R74L/C 171 A/T 19 Y/Gl 88Q/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88Q/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 88 A/S45 W; R74L/C 171 A/T 19 Y/Gl 88 A/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 88R/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88R/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/G25K/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88 A/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G188Q/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G188 A/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188 A/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G188 A/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G188R/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G188R/G25K/S45 W; T 19 Y/R74L/C 171 A; 117T/T 19 Y/G25R/R74L/C 171 A; T 19 Y/G25K/S45 WR74L/G108 A/C 171 A/Gl 88R; 117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/G108C/C 171 A/Gl 88R; 117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/G108C/A126C/C 171 A/Gl 88R; or 117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/AW 104- G105/G108C/C 171 A/Gl 88R.
[000321] In some embodiments, the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 102-105 the sequence FVPPKG, and from 130-134, or functionally equivalent positions, the sequence AKYPW (SEQ ID NO:44), in combination with one or more combinations of stability enhancing mutations selected from Hl IX, L12X, D14X, H16X, I17X, T19X, S20X, N21X, G25X, I26X, G27X, R28X, H29X, T31X, C34X, E38X, R39X, D41X, N42X, S45X, K47X, M48X, H51X, F54X, H56X, N57X, A59X, K60X, L62X, Y67X, L73X, R74X, D77X, L78X, V79X, P80X, S81X, D85X, A87X, I89X, Y90X, R91X, T93X, W94X, I96X, S97X, C101X, F102X, S103X, W104X, G105X, C106X, A107X, G108X, E109X, VI 10X, R11 IX, Al 12X, LI 14X, QI 15X, El 16X, N117X, T118X, H119X, V120X, L122X, R123X, A126X, R128X, L135X, Y136X, E138X, A139X, Q141X, M142X, D145X, A146X, A148X, D156X, E157X, K159X, C161X, D163X, T164X, D167X, Q169X, C171X, D177X, D180X, H182X, S183X, Q184X, A185X, L186X, S187X, G188X, R189X, A192X, N196X a deletion selected from AM1-L12, AE36-G53, AN61-G68, AW104- G105 , AQ195-N199 , AI26-G27, or any combination thereof, where the position number designation is functionally equivalent to the position in a wild-type APOBEC3 A (SEQ ID NO:3) and X is an amino acid substitution different from the wild-type amino acid at that position.
[000322] In some embodiments, the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 102-105 the sequence FVPA, and from 130-134, or functionally equivalent positions, the sequence AKYPW (SEQ ID NO:44), in combination with one or more combinations of stability enhancing mutations selected from Hl IX, L12X, D14X, H16X, I17X, T19X, S20X, N21X, G25X, I26X, G27X, R28X, H29X, T31X, C34X, E38X, R39X, D41X, N42X, S45X, K47X, M48X, H51X, F54X, H56X, N57X, A59X, K60X, L62X, Y67X, L73X, R74X, D77X, L78X, V79X, P80X, S81X, D85X, A87X, I89X, Y90X, R91X, T93X, W94X, I96X, S97X, C101X, F102X, S1O3X, W104X, G105X, C106X, A107X, G108X, E109X, VI 1OX, R11 IX, Al 12X, LI 14X, QI 15X, El 16X, N117X, T118X, H119X, V120X, L122X, R123X, A126X, R128X, L135X, Y136X, E138X, A139X, Q141X, M142X, D145X, A146X, A148X, D156X, E157X, K159X, C161X, D163X, T164X, D167X, Q169X, C171X, D177X, D180X, H182X, S183X, Q184X, A185X, L186X, S187X, G188X, R189X, A192X, N196X a deletion selected from AM1-L12, AE36-G53, AN61-G68, AW104- G105 , AQ195-N199 , AI26-G27, or any combination thereof, where the position number designation is functionally equivalent to the position in a wild-type APOBEC3 A (SEQ ID NO:3) and X is an amino acid substitution different from the wild-type amino acid at that position.
[000323] In some embodiments, the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 102-105 the sequence FVPA, and from 130-134, or functionally equivalent positions, the sequence AKYPW, in combination with one or more combinations of stability enhancing mutations selected from: at least one of the following combinations of stabilizing mutations: R74L/C171A/T19Y/G25K/S45W;
R74L/C 171 A/T 19 Y/Gl 88Q/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88Q/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 88 A/S45 W; R74L/C 171 A/T 19 Y/Gl 88 A/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 88R/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 88R/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88Q/G25K/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88 A/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/S45 W; R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08 A/Gl 88R/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G25R/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G25K/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G188Q/S45 W; R74L/C 171 A/T 19 Y/Gl 08C/G188 A/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188 A/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188 A/G25K/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188R/G25R/S45 W;
R74L/C 171 A/T 19 Y/Gl 08C/G188R/G25K/S45 W; T 19 Y/R74L/C 171 A;
117T/T 19 Y/G25R/R74L/C 171 A; T 19 Y/G25K/S45 WR74L/G108 A/C 171 A/Gl 88R;
117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/G108C/C 171 A/Gl 88R;
117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/G108C/A126C/C 171 A/Gl 88R; or 117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/AW 104-
G1 05/G108C/C 171 A/Gl 88R.
[000324] In some embodiments, the ACD comprises a backbone sequence relative to SEQ ID NO:3 comprising from 102-105 the sequence FVPA, and from 130-134, or functionally equivalent positions, the sequence AKYPW (SEQ ID NO:44), in combination with one or more combinations of stability enhancing mutations selected from Hl IX, L12X, D14X, H16X, I17X, T19X, S20X, N21X, G25X, I26X, G27X, R28X, H29X, T31X, C34X, E38X, R39X, D41X, N42X, S45X, K47X, M48X, H51X, F54X, H56X, N57X, A59X, K60X, L62X, Y67X, L73X, R74X, D77X, L78X, V79X, P80X, S81X, D85X, A87X, I89X, Y90X, R91X, T93X, W94X, I96X, S97X, C101X, F102X, S103X, W104X, G105X, C106X, A107X, G108X, E109X, VI 10X, R11 IX, Al 12X, LI 14X, QI 15X, El 16X, N117X, T118X, H119X, V120X, L122X, R123X, A126X, R128X, L135X, Y136X, E138X, A139X, Q141X, M142X, D145X, A146X, A148X, D156X, E157X, K159X, C161X, D163X, T164X, D167X, Q169X, C171X, D177X, D180X, H182X, S183X, Q184X, A185X, L186X, S187X, G188X, R189X, A192X, N196X a deletion selected from AM1-L12, AE36-G53, AN61-G68, AW104- G105 , AQ195-N199 , AI26-G27, or any combination thereof, where the position number designation is functionally equivalent to the position in a wild-type APOBEC3 A (SEQ ID NO:3) and X is an amino acid substitution different from the wild-type amino acid at that position.
[000325]
[000326] An ACD described herein can include additional mutations. Typically, additional mutations do not unduly alter the activity of the altered cytidine deaminase. One or more additional mutations can be a conservative mutation. The specification discloses at FIG. 2 an alignment between a human APOBEC3A (SEQ ID NO:3, referred to as sp|P3194111-199 in the figure), and other APOBEC3 A proteins from other primates. The identical amino acids are marked with an (asterisk), a (colon) indicates conservation between amino acids of strongly similar properties, and a (period) indicates conservation between groups of weakly similar properties. The skilled person would expect that many conservative substitutions in the areas that are identical or similar between the protein in the alignment would be likely to result in an active protein. Likewise, the skilled person would expect that many non-conservative substitutions in the areas that are identical or similar between the proteins in the alignment would be more likely to result in an inactive protein. Moreover, the location of the highly conserved spatial arrangement of the catalytic center residues of the ZDD motif is disclosed. The ZDD motif includes the H and two C residues which are believed to coordinate a Zn atom and the E residue which polarizes a water molecule near the Zn-atom for catalysis. As discussed herein the active site includes conserved residues such as arginine at position 28, histidine, asparagine, or arginine at position 29, serine or threonine, preferably threonine, at position 31, asparagine or aspartic acid at position 57, tryptophan at position 98, serine or threonine at position 99, and arginine or lysine at position 189 of SEQ ID N0:3 (Kouno et al., 2017, Nat. Comm, 8: 15024, DOI: 10.1038/ncomms 15024). The active site also includes tyrosine or phenylalanine at position 130, asparagine or tyrosine at position 131, and asparagine, tyrosine, or phenylalanine, tyrosine, at position 132; however, as described herein these residues can be the locations of selectivity-enhancing alterations. [000327] The present disclosure also provides dimerized ACDs (dACDs) including any of the ACDs described herein (International Application Pub. No. WO 2025-072800). The dACD may be a heterodimer or a homodimer. The two ACDs of a dACD are typically covalently attached.
[000328] ACD-helicase complexes including fusion proteins
[000329] Described herein further are ACD-helicase complexes, for example, a fusion protein comprising the disclosed 5mC specific ACDs conjugated to helicase for a one-step, enzymatic method for mapping modified cytosines, at single base resolution on double stranded DNA (dsDNA). These ACD-helicase complexes (e.g. fusion proteins) provide benefits for certain embodiments of methods described herein where a gentler denaturation step, no denaturing step, a reduction of washes, or a combination thereof, is helpful for library preparation to provide enough DNA for analysis or to not alter the substrate (e.g. flow cell conditions), including, for example, cell free DNA preparations, PCR-free library preparations, on-flow cell preparations, arrays, etc. The working examples provided herein describe an altered cytidine deaminase complex, e.g., an ACD conjugated to helicase in a fusion protein, that uniquely provides deaminase activity on dsDNA to be able to detect 5mC at single base resolution. The engineered ACDs described herein (e.g. based on APOBEC3 A) that have increased 5mC specificity have greater activity on single-stranded DNA than on double-stranded DNA. Complexing an ACD of the present disclosure with helicase allows for the use in applications requiring dsDNA, for instance, where providing single-stranded DNA is not possible or deleterious to the DNA output or methodology workflow. Library preparation methods that require ssDNA can complicate some workflows as it requires denaturation steps and washes (to remove the denaturant) which may not be compatible with the deamination reaction or other methodology conditions for some assays. For example, a flow cell surface may be detrimentally effected by denaturants, thus use of an ACD-helicase complex provides an advantage. Likewise, PCR free library preparations do not include a PCR step to increase the DNA input before sequencing and thus require maintaining as much DNA from the original sample as possible, and the use of an ACD-helicase complex reduces the loss of DNA.
[000330] In one aspect, we demonstrate that we can either use a less stringent denaturation step or eliminate denaturation step in the workflow by using 5mC selective mutants (e.g. ACD described herein) fused to helicase that allows for deamination of 5mC in dsDNA. As demonstrated in the examples, the inventors have developed a fusion protein of a 5mC selective altered cytidine deaminase described herein to helicases (using certain linkers between the protein, e.g., in the examples, GS or HIS). The ACD-helicase complex allowed for the development of a soluble protein (FIG. 10) that had 5-mC specific activity on single stranded DNA (FIG. 11). Furthermore, ACD-helicase fusion was active and able to unwind dsDNA while maintaining selective 5mC deaminase activity (FIG.14, FIG 15). This fusion enzyme thus is a further improvement that is useful in applications where removing the denaturation would hinder the assay or require an extra washing step which would further reduce the viable DNA for running on the assay, for example, for the direct on-flow cell deamination for methylation detection as well as PCR-free one-step enzymatic detection.
[000331] As the ACDs described herein preferentially function on single-stranded DNA, methods using these ACDs are aided by inclusion of a denaturation step for ACD to be able to act on the DNA, which adds extra complexity to an assay. Moreover, the current workflow for the direct on-flow cell deamination includes two additional steps: extension and denaturation before deamination. Not only is an on-flow cell workflow more complicated, but adding denaturants, e.g., sodium hydroxide, DMSO, and/or betaine, or using high temperature for DNA denaturation can be detrimental to the flow cell. Therefore, an ACD- helicase fusion was developed using one of our engineered ACDs described herein that has activity on double stranded DNA (dsDNA). The working examples demonstrate that it is possible to deaminate dsDNA with 5mC selectivity by using an ACD fusedi with a helicase. Further, the working examples demonstrates different protein fusions of ACD and helicase that are efficiently expressed and purified using a bacterial expression system (FIG. 10), for example, SEQ ID NO:962 and NO:963 (ACD fused to CaTe helicase). However, the present disclosure contemplates that any of the ACDs described herein can be fused in a similar manner to helicases and have deaminase activity on dsDNA. FIG. 1 lb and FIG. 12a demonstrate that the ACD fusion with helicase is still active and able to deaminate 5mC present on an oligonucleotide. FIG. 13 demonstrates that the helicase was still functionally active after fusion to the ACD. After confirmation of both helicase and deaminase activity of fusion proteins, the activity of the new constructs was tested on model dsDNA by NGS oligo assay. Remarkably, ACD-helicase fusion proteins were able to deaminate dsDNA while retaining the specificity towards 5mC (FIG. 14), and also demonstrated deamination of a more complex substrate (FIG. 15).
[000332] Helicases
[000333] Provided herein are fusion proteins of a helicase and an altered cytidine deaminase described herein , compositions that include a fusion protein, and methods of using a fusion protein. Suitable helicases are known in the art, as described more herein and below. Any ACD described herein can be fused to a helicase.
[000334] Wild-type helicases directionally separate double-stranded nucleic acids, commonly during replication of double-stranded DNA. During DNA replication, each separated DNA strand is amplified using a polymerase to prepare two daughter strands from a single double-stranded parent template. . While some passive helicases exist, most typically require energy to break the hydrogen bonds between base pairs. This energy is usually provided by ATP hydrolysis (Johnson DS, Bai L, Smith BY, Patel SS, Wang MD. Singlemolecule studies reveal dynamics of DNA unwinding by the ring-shaped T7 helicase. Cell. 2007 Jun 29; 129(7): 1299-309. doi:10.1016/j.cell.2007.04.038. PMID: 17604719; PMCID: PMC2699903.). Helicases can move along a segment of double-stranded DNA, leaving behind a window of opened single-stranded DNA. Helicases can move in a 3 ' to 5 ' direction, or in a 5 ' to 3 ' direction. Helicase movement can lead with the N-terminus or the C-terminus of the protein. Some helicases are localized to the negative strand of a nucleic acid, while others are localized to the positive strand. Each of these properties may be considered when selecting a helicase to use for a given function or in a given context.
[000335] Helicases are regarded as vital to all life forms, and have been identified in eukaryotes, prokaryotes, archaea, and viruses. There are six superfamilies of helicases (SF1- SF6), defined by rough structure and shared sequence motifs (Singleton, Martin R et al. “Structure and mechanism of helicases and nucleic acid translocases.” Annual review of biochemistry vol. 76 (2007): 23-50. doi: 10.1146/annurev.biochem.76.052305.115300). Helicases belonging to SF3-SF6 form a ring around a nucleic acid as they process along the strands. Helicases frequently form multimers, such as homo-hexamers. In other instances, helicases are active as monomers or dimers. Other numbers of subunits are possible and are considered, particularly for the modified proteins of this disclosure. While it is typically thought that, in nature, multimeric helicases exist as homo-tetramers or homodimers, formation of hetero-tetramers and heterodimers is possible.
[000336] The helicases of this disclosure may be mutated to change their function. For example, mutations may be made to a protein sequence to result in a helicase with faster or slower processive movement, depending on the desired activity of the resultant enzyme. [000337] In some aspects where a helicase having more than one monomer is used, any suitable number of monomers may be altered, such as two, three, four, five, six, seven, or eight monomers. In embodiments, the alteration may be present on each monomer.
Alternately, a modification may be present on only a subset of the monomers, resulting in a hetero-multimeric helicase. In particular, when a helicase is modified to include a large structural change, such as in attachment of a cytidine deaminase, the multimeric helicase assembly may have improved structural stability when only a portion of the monomers include the structural change. For example, it may be desirable to form a mutant DnaB helicase assembly wherein two or three of the six monomers are fused to a cytidine deaminase. In embodiments wherein a dimeric helicase is used, one or both of the monomers may be altered.
[000338] Expression cassette components such as promoters, polyadenylation signals, and post-transcriptional elements may be selected to achieve a desired level of expression of the fusion protein.
[000339] In certain embodiments, a helicase is attached to a cytidine deaminase by an amino acid linker. Amino acid linkers are described herein.
[000340] In one or more embodiments, the helicase includes an ATP-dependent helicase. In one or more embodiments, the helicase does not include an ATP-dependent helicase. [000341] In the following amino acid motifs for helicases, the character “X” is used to represent any amino acid. The character “h” is used to represent any hydrophobic amino acid, typically either valine, leucine, isoleucine, phenylalanine, methionine, or tryptophan. The character “y” is used to represent any hydrophilic amino acid, typically either serine, threonine, histidine, aspartate, glutamine, glutamate, lysine, arginine, or asparagine.
[000342] In one or more embodiments, the amino acid sequence of a helicase includes a subset of the amino acids of a member of the helicase Superfamily 1, including Motif I: hhXGXAGyGKS (SEQ ID NO:439), Motif la: XXhXXyy (SEQ ID NO:440), Motif II: hhhDEXy (SEQ ID NO:441), Motif III: hhhhGDXyQ (SEQ ID NO:442), Motif IV: xxhXyyXR (SEQ ID NO:443), Motif V: XXThXXXQGhyhyyV (SEQ ID NO:444), Motif VI: VAhTRXyy (SEQ ID NO:445), or a combination thereof. (Singleton, Martin R et al. “Structure and mechanism of helicases and nucleic acid translocases.” Annual review of biochemistry vol. 76 (2007): 23-50., Hall, M C, and S W Matson. “Helicase motifs: the engine that powers DNA unwinding.” Molecular microbiology vol. 34,5 (1999): 867-77.) Particularly, the inventors tested UvrD homologs, which are part of the SF1 family, as described more below.
In some aspects, the helicases are selected from UvrD homologues from different organisms, for example; CaTe: Caldanaerobacter tengcongensis (SEQ ID NO:427), GeSt: Geobacillus Stereothermophilus (SEQ ID NO:429), & ThAm: Thermovibrio ammonificans (SEQ ID NO:430), among others. As described in the working examples, CaTe proved to have the highest level of expression when fused to the ACD, and therefore was selected for further study. However, not to be bound by any theory, other UvrD homologues can be used in the practice of this disclosure, including, but not limited to, for example, Bacillus stearolhermophilus, Bst pcrA, (SEQ ID NO:431), .Staphylococcus aureus (SEQ ID NO:432); Helicobacter pylori (annotated as HP1478 or JHP1371, SEQ ID NO:433), Mycobacterium tuberculosis (SEQ ID NO:434), Saccharomyces cerevisiae, (Srs2 helicase, SEQ ID NO: 435); Thermus aqualicus, (SEQ ID NO/436); Haemophilus influenzae (HiUvrD, SEQ ID NO:437) and Helicobacter pylori (HpUvrD, SEQ ID NO:438), among others. One skilled in the art can alter the helicase-ACD fusion with other known homologs using molecular biology skills practiced by a skilled artisan.
[000343] In one or more embodiments, the amino acid sequence of a helicase includes a subset of the amino acid of a member of the helicase Superfamily 2, including Motif EhhXXXyGXGKT (SEQ ID NO:446), Motif la: XhhhXPyy (SEQ ID NO:447), Motif II: hhhDEXH (SEQ ID NO:448), Motif III: hXhSAThhh (SEQ ID NO:449), Motif IV: hhFXXyXy (SEQ ID NO:450), Motif V: hXXTXXXXXGhyhXyh (SEQ ID NO:451), Motif VI: QXXGRXXR (SEQ ID NO:452), or a combination thereof. (Singleton, Martin R et al. “Structure and mechanism of helicases and nucleic acid translocases.” Annual review of biochemistry vol. 76 (2007): 23-50., Hall, M C, and S W Matson. “Helicase motifs: the engine that powers DNA unwinding.” Molecular microbiology vol. 34,5 (1999): 867-77.) [000344] In one or more embodiments, the amino acid sequence of a helicase includes a subset of the amino acids of a member of the helicase Superfamily 3, including Motif A: hhhXGPXGTGKS (SEQ ID NO:453), Motif B: hhXhDD (SEQ ID NO:454), Motif C: hhhTTN (SEQ ID NO:455), or a combination thereof. (Singleton, Martin R et al. “Structure and mechanism of helicases and nucleic acid translocases.” Annual review of biochemistry vol. 76 (2007): 23-50., Hall, M C, and S W Matson. “Helicase motifs: the engine that powers DNA unwinding.” Molecular microbiology vol. 34,5 (1999): 867-77.)
[000345] In one or more embodiments, the amino acid sequence of a helicase includes a subset of the amino acid of the helicase Family 4, including Motif 1 : XhXhhXARXXhGKT (SEQ ID NO:456 Motif la: VLXhSLEM (SEQ ID NO:457), Motif 2, hlhhDYL (SEQ ID NO:458), Motif 3, IXXIXXyLKAhAXyLXPhXXhXQ (SEQ ID NO:459), Motif 4, PXXXDLRXSGXIXQXADXIh (SEQ ID NO:460), or a combination thereof. (Singleton, Martin R et al. “Structure and mechanism of helicases and nucleic acid translocases.” Annual review of biochemistry vol. 76 (2007): 23-50., Hall, M C, and S W Matson. “Helicase motifs: the engine that powers DNA unwinding.” Molecular microbiology vol. 34,5 (1999): 867-77. [000346] In one or more embodiments, the amino acid sequence of a helicase includes a Walker motif, including a Walker A motif: GXXXXGKTS (SEQ ID NO:461) and/or a Walker B motif: RKXXXGXXXLhhhDE (SEQ ID NO:462).
[000347] In one or more embodiments, the helicase includes a RecQ family helicase. RecQ family helicases are a subfamily of helicase Superfamily 2 and are typically identified by a conserved RecQ motif. Examples of suitable RecQ family helicases include RecQ from E. coli (UniProt:P15043, SEQ ID NO:463), BLM helicase from H. sapiens (UniProt:B7ZKN7, SEQ ID NO:464), RecQ from B. subtilis (UniProt:O34748, SEQ ID NO:465), WRN helicase from H. sapiens (UniProt:W14191, SEQ ID NO:466), Sgsl helicase from S. cerevisiae (UniProt:P35187, SEQ ID NO:467), and DDM1 helicase from A. thaliana (UniProt:Q9XFH4, SEQ ID NO:468).
[000348] In one or more embodiments, the helicase may be of bacterial or archaeal origin. In one or more embodiments, the helicase is RadA from S. pneumoniae (UniProt:Q8DRP0, SEQ ID NO:469), MCM from AL. thermoautotrophicum (Uniprot:O27798, SEQ ID NO:470), or the MCM4,6,7 complex from 5. pombe (UniProt:P29458, P49731, 075001, SEQ ID NOs:471-473).
[000349] In one or more embodiments, the helicase is a mammalian helicase. Examples of suitable mammalian helicase include BLM DNA helicase from H. sapiens (UniProt:B7ZKN7, SEQ ID NO:474), DnaB helicase from H. sapiens (UniProt:Q8NG08, SEQ ID NO:475), DnaB helicase from P. paniscus (UniProt:A0A2R8ZJKl, SEQ ID NO:476), DnaB helicase from P. abelii (UniProt:A0A2R8ZJKl, SEQ ID NO:477), DnaB helicase from P. troglodytes (UniProt:A0A2J8KTTl, SEQ ID NO:478), DNA helicase B from G. gorilla (UniProt:G3RJF6, SEQ ID NO: 964), DNA helicase B from fascicularis (UniProt: Al A7N9CCZ5, SEQ ID NO:965), and DNA helicase B from P. tephrosceles (UniProt:A0A8C9LS01, SEQ ID NO:966).
[000350] In some embodiments, the helicase is or has structural similarity to a reference helicase. Examples of a reference helicase include, but are not limited to, the helicase proteins of SEQ NO:474-478 and 964-966. As used herein, a helicase may be "structurally similar" to a reference helicase if the amino acid sequence of the helicase possesses a specified amount of sequence similarity and/or sequence identity compared to the reference helicase. Structural similarity of two amino acid sequences can be determined as described herein. A candidate helicase that has structural similarity with a reference helicase and the helicase activity described herein is a helicase that is expected to be useful in the disclosed methods. In one embodiment, the amino acid sequence of a helicase protein having sequence similarity to a reference sequence may include conservative substitutions of amino acids present in that reference sequence. Conservative substitutions are described herein.
[000351] Thus, as used herein, reference to a helicase as described herein, such as reference to the amino acid sequence of one or more SEQ ID NOs of a cytidine deaminase described herein, such as SEQ ID NOs: 474-478 and 964-966, can include a protein with at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence similarity to the reference UDG.
Alternatively, as used herein, reference to a helicase as described herein, such as reference to the amino acid sequence of one or more SEQ ID NOs described herein, such as SEQ ID NOs:57-62, can include a protein with at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity to the reference UDG.-In some aspects, suitable helicases include, for example, CaTe (SEQ ID NO:427), UniProt Seq: Q8RC43 (SEQ ID NO:428), SEQ ID NO:429 , or ThAM (SEQ ID NO:430). In one aspect, the helicase is SEQ ID NO:427. In another aspect, the helicase is SEQ ID NO:428. In another aspect, the helicase is SEQ ID NO:429. In another aspect, the helicase is SEQ ID NO:430.
[000352] Methods of helicase-cytidine deaminase attachment
[000353] In certain embodiments, it may be desirable for a helicase and a cytidine deaminase to be in close physical proximity during treatment of a nucleic acid. As described herein, the altered cytidine deaminases of this disclosure can preferentially deaminate 5mC. The catalytic activity of these proteins may be significantly higher on single-stranded nucleic acids than double-stranded nucleic acids. In some instances, it may be desirable for the cytidine deaminases of this disclosure to demonstrate improved catalytic activity on doublestranded nucleic acids. To achieve this, a protein complex comprising a cytidine deaminase, such as an altered cytidine deaminase, and an enzyme that can separate the two strands of a double-stranded nucleic acid, such as a helicase, may be used. In some embodiments, the protein complex comprises an ACD attached to an enzyme that can separate the two strands of a double-stranded nucleic acid, such as a helicase. When the ACD is spatially near an enzyme such as a helicase, the ACD may act on the strands separated by the helicase. In some embodiments, a region of separated single strands may re-form a double-stranded structure after a protein complex such as a helicase-cytidine deaminase fusion has moved away.
[000354] In some embodiments, an ACD may be attached to a helicase to form a helicase- altered cytidine deaminase complex (ACD-helicase). The ACD-helicase complex may preferably be a fusion protein comprising the ACD linked to the helicase by a linker. In some embodiments, treatment of a dsDNA substrate with a helicase-altered cytidine deaminase complex (including a fusion protein) may exhibit higher levels of 5mC deamination than a comparable treatment of the ACD alone or treatment wherein the helicase and altered cytidine deaminase are not attached. In some aspects, the level of 5mC deamination of dsDNA of the ACD-helicase complex (e.g. fusion protein) may be an increase in 5mC deamination of at least 5%, at least 10%, at least 15%, least 20%, at least 25%, least 30%, least 40%, least 50%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% as compared to the ACD alone.
[000355] In some further aspects, the level of 5mC deamination of dsDNA of the ACD- helicase complex (e.g., fusion protein) may be an increase in 5mC deamination of at least 5%, at least 10%, at least 15%, least 20%, at least 25%, least 30%, least 40%, least 50%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% as compared to a reaction wherein the ACD and helicase are not attached.
[000356] In some embodiments, an ACD-helicase complex may be recombinantly expressed. In some embodiments, the helicase and the altered cytidine deaminase are each expressed as separate recombinant proteins and prepared as a helicase-cytidine deaminase protein complex. In some other embodiments, the helicase and the cytidine deaminase may be expressed as a fusion protein.
[000357] In some aspects, it may be desirable to provide to a cell a polynucleotide encoding a fusion protein of an altered cytidine deaminase described herein and a helicase in combination with a nucleic acid construct encoding an unmodified helicase. The cell may then be incubated in conditions suitable for recombinant expression of both constructs simultaneously, and the resultant protein may be purified from cell lysate or culture media. In this manner, a hetero-multimeric helicase including a combination of modified and unmodified monomers may be obtained.
[000358] In some preferred aspects, the helicase-cytidine deaminase complex may be a fusion protein. In some embodiments, the cytidine deaminase and the helicase may be attached N-terminus to N-terminus, N-terminus to C-terminus, or C-terminus to C-terminus. The helicase and the cytidine deaminase may be attached in any suitable order. For example, the helicase may be attached to the C-terminus of the cytidine deaminase.
[000359] In some embodiments, the helicase may be attached to a non-terminal region of the altered cytidine deaminase. As used herein, attachment of a protein to a “non-terminal region” of another protein describes attachment of a protein at any residue other than the N- terminal residue and the C-terminal residue. Alternately, the cytidine deaminase may be attached to a non-terminal region of the helicase. Suitable loops may be identified by their high b-factor.
[000360] In embodiments wherein the ACD-helicase complex is a fusion protein, the fusion protein may include any additional components. For example, the fusion protein may include a tag, for example, a purification tag. Suitable tags are known in the art and include, but are not limited to, a His-tag, a FLAG-tag, or a Myc-tag. The fusion protein may be further modified to include a reactive handle such as a free cystine. In some embodiments, the fusion protein may include a fluorescent protein or another detectable marker.
[000361] In embodiments wherein the helicase-ACD complex is a fusion protein, they may be connected by any suitable linker. The physical proximity of the helicase to the ACD may play a role in the efficiency of 5mC deamination. For example, when the ACD and the helicase are far apart, the ACD may not access the ssDNA window opened by the helicase with a high frequency, leading to a lower rate of 5mC deamination. It should be understood by one of ordinary skill in the art that selection of a linker for a bifunctional fusion protein involves considerable inventive effort.
[000362] In some embodiments, it may be preferable to have a structured linker, such as an alpha helical linker. In some other embodiments, it may be preferable to have an unstructured linker, such as a glycine-serine linker. The linker may have any suitable length, for example, the linker may include at least two amino acids, at least four amino acids, at least six amino acids, at least ten amino acids, at least 12 amino acids, at least 16 amino acids, at least 20 amino acids, at least 30 amino acids, or at least 40 amino acids. The linker may be at most 100 amino acids, at most 80 amino acids, at most 60 amino acids, at most 40 amino acids, at most 20 amino acids, or at most ten amino acids.
[000363] For example, suitable amino acid linkers include, but are not limited to, flexible linkers or ridged linkers (e.g., SEQ ID NO:967-995). Suitable flexible linkers include, but are not limited to, a Glycine linker (referred to as a G-linker) or a linker composed predominately of the amino acids Glycine and Serine (referred to as a GS-linker). Such linkers of the present disclosure can be of various lengths, for example, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 amino acids in length, and can in some aspects range from 2-50 amino acids, preferably from 5-25 amino acids. In some embodiments, the linker comprises an amino acid sequence selected from the group consisting of GGSGGS (SEQ ID NO:975), i.e., (GGS)2 (SEQ ID NO: 976); GGSGGSGGS, i.e., (GGS)3 (SEQ ID NO: 977); GGSGGSGGSGGS, i.e., (GGS)4 (SEQ ID NO: 978); and GGSGGS GGSGGSGGS, i.e., (GGS)5 (SEQ ID NO: 979), Gly-Gly (GG), GGG, GGGG (SEQ ID NO: 980), GGGGG (SEQ ID NO: 981), and GGGGGG (SEQ ID NO: 982). In some embodiments, the linker is (GGGGS)n, wherein n is 1 to 5 (SEQ ID NO:983); (GGGGGS)n, wherein n is 1 to 5 (SEQ ID NO:984); GGGGS (SEQ ID NO:985); GGGGGS (SEQ ID NO:986);
GGGGGSGGGGGSGGGGGS (SEQ ID NO: 987); GGGGS GGGGS GGGGS (SEQ ID NO:988); GGSGGGGSGGGGSGGGGS (SEQ ID NO:989); or PGGGG (SEQ ID NO:990). In some preferred aspects, the linker is SSGGSSGGS (SEQ ID NO:967), GSGSGSGSGSS (SEQ ID NO:968), (GS)n, where n is an integer from 1-20, (SG)n where n is an integer from 1-20, (GGGGS)n (SEQ ID NO:969), where n is 1-6, (GGSG)n (SEQ ID NO:970)where n is 1-10, (GGGS)n, (SEQ ID NO:971) wherein n is 1-10; GGGGGGGGG (SEQ ID NO:972), (GGGGS)n,(SEQ ID NO:973) n is 1-10, Gn, wherein n is 1-20, GSAGSAAGSGEF (SEQ ID NO:974), among others.
[000364] In another aspect, the linker is a more rigid linker, for example, Helix linker (ALGGAAAAAAS (SEQ ID NO: 995). Other suitable linkers are known in the art and easily used in the practice of this disclosure, including, for example, KESGSVSSEQLAQFRSLD (SEQ ID NO: 991), EGKSSGSGSESKS (SEQ ID NO: 992), SSGNSNANSRGPSFSSGLVPLSLRGSH (SEQ ID NO:993), (EAAAK)n, n is 1-5, (SEQ ID NO:994), among others.
[000365] In some embodiments, the enzymes of a helicase-cytidine deaminase complex are covalently attached. In some embodiments, each protein may be provided with a modification that enables attachment. For example, one protein may be provided attached to an avidin moiety, such as streptavidin or neutravidin, and the other protein may be provided attached to a biotin moiety.
[000366] Attachment moieties may be attached to each protein by any suitable method. The helicase and/or the ACD may be recombinantly expressed as a fusion to a peptide attachment moiety. In some embodiments, an ACD and a helicase may be obtained as independent proteins and further modified to include a peptide attachment moiety. For instance, each protein may be separately recombinantly expressed, or they may be coexpressed and co-purified. A peptide attachment moiety may include any additional protein or peptide that is usually capable of forming a covalent bond with a partner. Other attachment systems may be suitable, including systems that do not form a covalent bond, but form a strong bond with a dissociation constant of less than, for example, 10 nM. Examples of suitable attachment pairs include Spy Tag/Spy Catcher, SnoopTag/SnoopCatcher, biotin/avidin, and leucine zippers.
[000367] Alternately or additionally, the helicase and/or the ACD may be recombinantly expressed or provided as a fusion to a reactive handle. A reactive handle may be further reacted to enable attachment of the helicase to the ACD.
In embodiments where a reactive handle is used, the reactive handle may be attached to any suitable portion of the cytidine deaminase and/or the helicase. The reactive handle may be attached to the N-terminus, the C-terminus, or a non-terminal region of the cytidine deaminase. The reactive handle may be attached to N-terminus, the C-terminus, or a nonterminal region of the helicase.
[000368]
[000369] In nature, helicases often work in concert with additional enzymes when acting on dsDNA (Seo, Yeon-Soo, and Young-Hoon Kang. “The Human Replicative Helicase, the CMG Complex, as a Target for Anti-cancer Therapy.” Frontiers in molecular biosciences vol. 5 26. 29 Mar. 2018, doi: 10.3389/fmolb.2018.00026). In some embodiments, it may be advantageous to include additional enzymes in the compositions and methods described herein to improve helicase activity. Examples of enzymes that may be used include, but are not limited to, topoisomerases. Topoisomerases that may be used include DNA topoisomerase 1 from H. sapiens (UniProt:Pl 1387, SEQ ID NO:) and DNA topoisomerase 2- a from H. sapiens (UniProt:Pl 1388, SEQ ID NO:). In embodiments wherein an ATP- dependent helicase is used, it may be advantageous to include a high concentration of ATP. In some embodiments, the helicase may require additional cofactors for use. Any suitable cofactors may be used to improve activity of the helicase. [000370] Polynucleotides encoding ACDs or fusion proteins
[000371] ACDs and fusion proteins described herein also may be identified in terms of the polynucleotide that encodes the protein. Thus, this disclosure provides polynucleotides that encode an ACD, a helicase, or an ACD-helicase fusion protein described herein or hybridize, under standard hybridization conditions, to a polynucleotide that encodes an ACD described herein, and the complements of such polynucleotide sequences.
[000372] A polynucleotide as described herein can include any polynucleotide that encodes an ACD of the present disclosure, a helicase of the present disclosure, or an ACD-helicase fusion protein of the present disclosure. Thus, the nucleotide sequence of the polynucleotide may be deduced from the amino acid sequence that is to be encoded by the polynucleotide. An ACD, helicase, or fusion protein can be encoded by multiple codons, and certain translation systems (e.g., prokaryotic or eukaryotic cells) often exhibit codon bias, e.g., different organisms often prefer one of the several synonymous codons that encode the same amino acid. As such, polynucleotides presented herein are optionally "codon optimized," meaning that the polynucleotides are synthesized to include codons that are preferred by the particular translation system being employed to express the protein. For example, when it is desirable to express the protein in a bacterial cell (or even a particular strain of bacteria), the polynucleotide can be synthesized to include codons most frequently found in the genome of that bacterial cell, for efficient expression of the ACD, helicase, or fusion protein. A similar strategy can be employed when it is desirable to express the ACD, helicase, or fusion protein in a eukaryotic cell, e.g., the nucleic acid can include codons preferred by that eukaryotic cell. [000373] A polynucleotide described herein may also, advantageously, be included in a suitable expression vector to express the ACD, helicase, or fusion protein encoded therefrom in a suitable host. Incorporation of cloned DNA into a suitable expression vector for subsequent transformation of a host cell and subsequent selection of the transformed cells is well known to those skilled in the art as provided in Sambrook et al. (1989), Molecular cloning: A Laboratory Manual, Cold Spring Harbor Laboratory. Suitable host cells include, but are not limited to, E. coli and S. cerevisiae.
[000374] Such an expression vector includes a vector having a polynucleotide described herein operably linked to heterologous regulatory sequences, such as promoter regions, that are capable of effecting expression of said DNA fragments. The term "operably linked" refers to a juxtaposition wherein the components described are in a relationship permitting them to function in their intended manner. Such vectors may be transformed into a suitable host cell to provide for the expression of an altered cytidine deaminase. [000375] The nucleic acid molecule may encode a mature protein or a protein having a pro-sequence, including encoding a leader sequence on the preprotein which is then cleaved by the host cell to form a mature protein. The vectors may be, for example, plasmid, virus or phage vectors provided with an origin of replication, and optionally a promoter for the expression of said nucleotide and optionally a regulator of the promoter. The vectors may contain one or more selectable markers, such as, for example, an antibiotic resistance gene. [000376] Regulatory elements required for expression include promoter sequences to bind RNA polymerase and to direct an appropriate level of transcription initiation and also translation initiation sequences for ribosome binding. For example, a bacterial expression vector may include a promoter such as the lac promoter and for translation initiation the Shine-Dalgarno sequence and the start codon AUG. Similarly, a eukaryotic expression vector may include a heterologous or homologous promoter for RNA polymerase II, a downstream polyadenylation signal, the start codon AUG, and a termination codon for detachment of the ribosome. Such vectors may be obtained commercially or be assembled from the sequences described by methods well known in the art.
[000377] Transcription of DNA encoding an ACD may be optimized by including an enhancer sequence in the vector. Enhancers are cis-acting elements of DNA that act on a promoter to increase the level of transcription. Vectors will also generally include origins of replication in addition to the selectable markers.
[000378] Polynucleotides encoding the fusions proteins described herein can be prepared using any available molecular cloning techniques. Preferably, a polynucleotide encoding a fusion of a helicase and an ACD may be prepared using a scarless or semi-scarless gene assembly technique. Examples of suitable techniques include, but are not limited to, Golden Gate assembly, Gibson assembly, and restriction enzyme ligation. Template polynucleotides encoding a helicase and/or an ACD may be obtained from a source as described herein. [000379] Polynucleotides encoding additional protein components desired for preparation of an ACD or ACD-helicase fusion may be obtained from a source and subjected to mutagenesis or cloning to prepare the altered proteins described herein. Examples of additional protein components include linkers, affinity tags, protein conjugation tags, and additional enzymes.
[000380] Making and isolating ACDs, helicases, or ACD-helicase fusion proteins [000381] Generally, polynucleotides encoding an ACD or fusion protein as presented herein can be made by cloning, recombination, in vitro synthesis, in vitro amplification and/or other available methods. A variety of recombinant methods can be used for expressing an expression vector that encodes an ACD, helicase, or fusion protein presented herein. Methods for making recombinant polynucleotides, expression, and isolation of expressed products are well known and described in the art.
[000382] Polynucleotides encoding wild type cytidine deaminases can be obtained from a source and subjected to mutagenesis to introduce one or more substitution mutations, deletions, and/or insertions described herein. Polynucleotides encoding a helicase can be obtained from a source and subjected to mutagenesis to introduce alterations, for instance. Addition of a tag such as a His-tag, a FLAG-tag, or a Myc-tag. Alternatively, the polynucleotides can be synthetically generated. In general, any available mutagenesis procedure can be used for making an ACD. helicase, or fusion protein described herein. Procedures that can be used include, but are not limited to: site-directed point mutagenesis, in vitro or in vivo homologous recombination, oligonucleotide-directed mutagenesis, mutagenesis by total gene synthesis, and many others known to persons skilled in the art.
[000383] Additional useful references for mutation, recombinant, and in vitro nucleic acid manipulation methods (including cloning, expression, PCR, and the like) include Berger and Kimmel, Guide to Molecular Cloning Techniques, Methods in Enzymology volume 152 Academic Press, Inc., San Diego, Calif. (Berger); Kaufman et al. (2003) Handbook of Molecular and Cellular Methods in Biology and Medicine Second Edition Ceske (ed) CRC Press (Kaufman); The Nucleic Acid Protocols Handbook Ralph Rapley (ed) (2000) Cold Spring Harbor, Humana Press Inc (Rapley); Chen et al. (ed) PCR Cloning Protocols, Second Edition (Methods in Molecular Biology, volume 192) Humana Press; and in Viljoen et al. (2005) Molecular Diagnostic PCR Handbook, Springer, ISBN 1402034032.
[000384] In addition, many kits are commercially available for the purification of plasmids or other relevant nucleic acids from cells. An isolated polynucleotide can be further manipulated to produce other polynucleotides, used to transfect or transform cells, incorporated into related vectors and introduced into cells for expression, and/or the like. Typical cloning vectors contain transcription and translation terminators, transcription and translation initiation sequences, and promoters useful for regulation of the expression of the particular target nucleic acid. The vectors optionally include generic expression cassettes containing at least one independent terminator sequence, sequences permitting replication of the cassette in eukaryotes, or prokaryotes, or both, (e.g., shuttle vectors) and selection markers for both prokaryotic and eukaryotic systems. Vectors are suitable for replication and integration in prokaryotes, eukaryotes, or both. [000385] Other useful references, e.g., for cell isolation and culture (e.g., for subsequent nucleic acid isolation) include Freshney (1994) Culture of Animal Cells, a Manual of Basic Technique, third edition, Wiley-Liss, New York and the references cited therein; Payne et al. (1992) Plant Cell and Tissue Culture in Liquid Systems John Wiley & Sons, Inc. New York, N.Y.; Gamborg and Phillips (eds) (1995) Plant Cell, Tissue and Organ Culture; Fundamental Methods Springer Lab Manual, Springer-Verlag (Berlin Heidelberg New York); and Atlas and Parks (eds) The Handbook of Microbiological Media (1993) CRC Press, Boca Raton, Fla. Construction of vectors containing a nucleic acid encoding an ACD described herein employs standard ligation techniques known in the art. See, e.g., Sambrook et al, Molecular Cloning: A Laboratory Manual. , Cold Spring Harbor Laboratory Press (1989) or Ausubel, R.M., ed. Current Protocols in Molecular Biology (1994).
[000386] A variety of protein isolation and detection methods are known and can be used to isolate an ACD, a helicase, or a fusion protein, e.g., from recombinant cultures of cells expressing the recombinant cytidine deaminase presented herein. A variety of protein isolation and detection methods are well known in the art, including, e.g., those set forth in R. Scopes, Protein Purification, Springer-Verlag, N.Y. (1982); Deutscher, Methods in Enzymology Vol. 182: Guide to Protein Purification, Academic Press, Inc. N.Y. (1990);
Sandana (1997) Bioseparation of Proteins, Academic Press, Inc.; Bollag et al. (1996) Protein Methods, 2nd Edition Wiley-Liss, NY; Walker (1996) The Protein Protocols Handbook Humana Press, NJ, Harris and Angal (1990) Protein Purification Applications: A Practical Approach IRL Press at Oxford, Oxford, England; Harris and Angal Protein Purification Methods: A Practical Approach IRL Press at Oxford, Oxford, England; Scopes (1993) Protein Purification: Principles and Practice 3rd Edition Springer Verlag, NY; Janson and Ryden (1998) Protein Purification: Principles, High Resolution Methods and Applications, Second Edition Wiley-VCH, NY; and Walker (1998) Protein Protocols on CD-ROM Humana Press, NJ; and the references cited therein. Additional details regarding protein purification and detection methods can be found in Satinder Ahuja ed., Handbook of Bioseparations, Academic Press (2000).
[000387] An ACD, helicase, or fusion-protein or polynucleotide can be isolated.
[000388] Methods of use
[000389] The ACDs provided by the present disclosure, including ACD-helicase complexes, can be easily integrated into essentially any application for identifying modified cytosines. For instance, ACDs can be integrated into applications that include sequencing library preparation. Examples of sequencing library preparation include, but are not limited to, whole genome, accessible (e.g., ATAC), conformational state (e.g., HiC), and reduced representation bisulfite sequencing (RRBS). It can be particularly useful in essentially any application using low input DNA or RNA such as, but not limited to, single cell combinatorial indexing (sci) methods like sci-WGS-seq, sci-MET-seq, sci-ATAC-seq, and sci-RNA-seq, and cell free DNA-based methods. Specific applications include, but are not limited to, identifying one or more patterns of cytosine modification such as determining methylation on CpG islands and reduced representation bisulfite sequencing (RRBS); variant calling, including somatic variant calling, SNV/indel, copy number variation (CNV), short tandem repeats (STR), and structural variants (SV); detecting differentially methylated regions (DMRs); measuring methylation at promoters; and detecting tumor DNA (International Application Publication No. WO 2023/196572).
[000390] The ACDs and ACD-helicase complexes (e.g., fusion protein) provided by the present disclosure can be easily integrated into essentially any application that includes locus-specific methylation profiling. Typical locus-specific detection of epigenetic methylated cytosines, such as 5mC, require the use of 5mC-specific antibodies, or multi-step chemical or chemoenzymatic transformations that lead to deamination of C or 5mC to U/T to enable differentiation of the two C-isoforms. When combined with various in vitro detection methodologies, these approaches can be strong approaches to detect 5mC at defined loci. However, these methods can be confounded by antibody cross-reactivity and stability, or the toxicity and complex workflows required by chemical and chemoenzymatic approaches. Use of an ACD or ACD-helicase complexes (e.g., fusion proteins) described herein in a single enzymatic deamination protocol permits selective conversion of 5mC to T that is compatible with a number of in vitro diagnostic modalities, resulting in locus-specific detection of 5mC. [000391] Instead of using destructive methods for identifying methylated cytosines, integrating the altered cytidine deaminases provided by the present disclosure into methods for identifying modified cytosines, such as sequencing library production and locus-specific methylation profiling, results in the more efficient enzyme-catalyzed conversion of modified cytosines, thereby permitting better sequencing data and better retention of genetic information, which is demonstrated by high variant calling performance (International Application Publication No. WO 2023/196572). Furthermore, as an enzymatic method for conversion, the use of the ACDs or ACD fusion proteins (e.g., fusion protein) enables high coverage uniformity and low sample damage, which results in lower nucleic acid input requirements. A multitude of sequencing library methods are known to a skilled person that can be used in the construction of whole-genome or targeted libraries. [000392] In general, methods for using an ACD of the present disclosure include contacting target nucleic acids, e.g., DNA or RNA, with the enzyme, under conditions suitable for conversion of modified cytidines. Because amplification of DNA does not preserve the modification status of cytidine (e.g., the methylation status of 5mC is not retained), use of an ACD typically occurs before amplification of target DNA. Target nucleic acids can be contacted with an ACD at essentially any time in a method before an amplification, provided the DNA is single-stranded. For instance, target nucleic acids can be contacted with an ACD while the nucleic acids are inside a fixed or unfixed cell or nucleus, after isolation of genomic or cell free DNA or mRNA, before or after fragmentation, or before or after tagmentation. In some embodiments, target nucleic acids can be present on a solid support, such as a bead, a well, or a flow cell, when contacted with an ACD. The skilled person will recognize that target nucleic acids can be contacted with ACD after addition of a universal sequence and/or an adapter, provided the universal sequence and/or an adapter is not added by amplification.
[000393] A method for using an ACD can include the optional step of comparison of the treated target nucleic acid with an untreated nucleic acid or comparison of the treated target nucleic acid with a nucleic acid treated with a wild-type cytidine deaminase. For instance, in embodiments where the treated nucleic acid is sequenced, the sequence can be compared to a reference sequence thereby permitting easy identification of point mutations and inference of modified cytosines. Thus, C to T point mutations can be easily identified, and these point mutations are inferred as 5mC positions. In embodiments where the treated nucleic acid is not sequenced, the nucleic acid can be treated with an ACD and compared to the nucleic acid that is untreated, i.e., not contacted with an ACD. Here the read-out typically depends on the assay method, for instance when an amplification is used the relative amounts of amplification can be easily identified and the presence or absence of a 5mC or a pattern of cytosine modification at a predetermined sequence inferred (International Application Publication No. WO 2023/196572).
[000394] Reaction conditions suitable for conversion of modified cytidines by a cytidine deaminase described herein include, but are not limited to, a substrate of target nucleic acid that is double-stranded (ds) DNA, single-stranded (ss) DNA, or RNA suspected of including at least one modified cytidine, buffer, pH, temperature of the reaction, time of the reaction, and concentration of the ACD and/or ss DNA or RNA substrate. In one embodiment, double-stranded (ds) DNA can be denatured and exposed to an ACD. Methods for denaturing dsDNA are known and routine, and include but are not limited to heat treatment, chemical treatment, such as NaOH, formamide, DMSO, or N, N-dimethylformamide (DMF), or a combination thereof. In one embodiment, denaturation of the dsDNA is optional, such as when the dsDNA is exposed to an ACD-helicase complex.
[000395] Target nucleic acids useful in the methods of the present disclosure are described herein. A modified cytidine present on a substrate single-stranded (ss) DNA or RNA includes, but is not limited to, 5-methyl cytosine (5mC), 5 -hydroxymethyl cytosine (5hmC), 5-formyl cytosine (5fC), and 5-carboxy cytosine (5CaC). In one embodiment, the modified cytidine is 5-methyl cytosine. Methods that use double stranded target DNA for generating a sequencing library can be modified to include denaturation to convert the double stranded target DNA to ssDNA. In some embodiments, dsDNA that is used in a tagmentation reaction or for adapter attachment can be denatured and then treated with an ACD. Conditions for denaturation are known and routine. In those embodiments where ssDNA is contacted with an ACD and subsequently used in a process that requires dsDNA, e.g., addition of universal adapters by tagmentation or ligation, the ssDNA can be converted to dsDNA using routine methods.
[000396] In some embodiments, an ACD or ACD-helicase complex (e.g., fusion protein) as presented herein can be used to differentiate between 5-methyl cytosine (5mC) and 5- hydroxymethyl cytosine (5hmC). In such an embodiment, a sample of DNA suspected of including single-stranded DNA comprising at least one 5-methyl cytosine (5mC) or 5- hydroxymethyl cytosine (5hmC) is modified to prevent an ACD from converting 5hmC to thymidine. Methods for blocking deaminase activity are known in the art, and any one of a number of methods can be used to protect 5hmC from deaminase activity. As one example, target DNA can be treated to modify 5hmC but not 5mC such that 5hmC is an unsuitable substrate for cytidine deaminase activity. In a specific example, a glucosyltransferase enzyme can be used to glucosylate 5hmC but not 5mC. Glucosyltransferase enzymes are known to those of skill in the art, and include, for example, p-glucosyltransferase (PGT). By way of example, the enzyme T4 p-glucosyltransferase is commercially available (PGT, NEB) and can be used for modification of 5hmC. Methods for using a PGT to glucosylate 5hmC are known in the art, and can be used in conjunction with the use of altered cytidine deaminase enzymes as presented here. For example, a sample of DNA can be treated with a PGT to glucosylate 5hmC in the sample DNA prior to treating the DNA with the altered cytidine deaminase enzyme. By treating the sample DNA with a PGT, 5hmC is protected from the deaminase activity of the altered cytidine deaminase enzyme. Thus, 5mC will be detected in downstream readout, such as sequencing, PCR, array, and the like, as a thymidine. In contrast, any protected 5hmC sites will be detected as cytosine in the same readout. Enzymes, buffers, and conditions for performing glucosylation of 5hmC are known in the art, as exemplified by the methods disclosed in Schutsky et al., Nature biotechnology, 10.1038/nbt.4204. 8 Oct. 2018, doi: 10.1038/nbt.4204.
[000397] In some embodiments, an ACD or ACD-helicase complex (e.g., fusion protein) as presented herein can be used in in vitro diagnostic (IVD) approaches for profiling methylation in a locus-specific manner. Current methods for methylation biomarker detection typically include digestion of genomic DNA with methylation-sensitive enzymes and then quantitative PCR (qPCR) at a locus of interest to quantify the extent of restriction enzyme digestion, and therefore the percent methylation at that site. This is followed by mismatchsensitive qPCR of bisulfite-treated DNA, where 5mC is read out as a lack of 5mC>T conversion. These methods, however, have drawbacks. The recognition site of the methylsensitive restriction enzyme must be present in the methylated region of the target locus. Bisulfite treatment requires a large quantity of starting DNA and results in conversion to a low complexity genome (unmethylated cytosines - which represent the majority of cytosines in the genome - are converted to U and read as T). This reduced complexity of the genomic template constrains the design of qPCR primers that hybridize specifically to the locus of interest. During bisulfite conversion, DNA is intrinsically damaged or lost, which can hinder downstream analysis. DNA damage decreases coverage uniformity of the genome, which can lead to bias coverage. Furthermore, incomplete bisulfite conversion has the potential to adversely affect results, since it can exaggerate DNA methylation levels (Sam et al., PLoS One. 2018; 13(6); Ehrich et al., Nucleic Acids Res. Oxford University Press; 2007;35: e29). [000398] 5mC to T conversion by the altered cytidine deaminases described herein obviates the need for restriction enzymes or bisulfite treatment, and preserves DNA complexity. The resulting modifications of one or more cytosines can be detected using established in vitro diagnostic (IVD) approaches for profiling methylation in a locus-specific manner. Examples of approaches include detection of 5mC loci via amplification, e.g., quantitative PCT (qPCR), detection of 5mC loci using a CRISPR-based system, e.g., CRISPR-Casl2, spatial detection of 5mC using molecular cytogenic methods, e.g., fluorescence in situ hybridization (FISH), and array-based detection of 5mC (International Application Publication No. WO 2023/196572). In one embodiment, in vitro diagnostic (IVD) approaches for profiling methylation in a locus-specific manner use one or more primers to anneal to a predetermined sequence that may include one or more modified cytosines. After treatment of target nucleic acids with an ACD or ACD-helicase complex (e.g., fusion protein), the modified cytosines present in the target nucleic acids are converted as described herein (e.g., 5mC is converted to T), and primers can be easily designed to anneal with higher affinity to a predetermined sequence when it includes nucleotides resulting from the deaminase treatment (e.g., a T nucleotide where a 5mC was present prior to treatment). For example, primers used for an amplification bind with greater affinity to a nucleic acid that includes T nucleotides where 5mC nucleotides were present prior to treatment (International Application Publication No. WO 2023/196572, incorporated by reference regarding primers). The annealing of a primer to a predetermined sequence that includes the expected 5mC to T conversion(s) allows one to infer the location of a modified cytosine in the untreated target nucleic acid. A primer that binds with greater affinity to a nucleic acid that includes T nucleotides where 5mC nucleotides were present prior to treatment can include at least 1, at least 2, at least 3, at least 4 or at least 5 nucleotides that will base-pair with a nucleotide that results from conversion of 5mC to T, i.e., an adenine (A), and when amplification is used, then a second primer for the reverse strand that has a T instead of guanine (G).
[000399] In some embodiments, target nucleic acids obtained from a subject can be treated with an ACD or ACD-helicase complex (e.g., fusion protein) to result in converted nucleic acids, and a pattern of cytosine modification can be identified in the converted nucleic acids. The pattern of cytosine modification can optionally be compared with the pattern of cytosine modification in a reference nucleic acid. In embodiments where a pattern of cytosine modification correlates with a disease or condition, the method can be used in diagnostic or prognostic applications. For instance, the subject can have or be at risk of having a disease or condition, and the reference nucleic acid can be from a normal subject, e.g., a subject that does not have and is not at risk for the disease or condition. The pattern of cytosine modification can be associated with a disease or condition (e.g., the target nucleic acid can be a predetermined sequence), and identification in the subject of a pattern of cytosine modification associated with a disease or condition can indicate the subject has or is at risk of having the disease or condition. For instance, a pattern of cytosine modification can be linked in-cv.s to a coding region that is correlated with a disease or condition and identification of that pattern, or absence of that pattern, in the subject can be used for diagnosis or prognosis. In one embodiment, the coding region can be one that is transcriptionally active or transcriptionally inactive in a reference nucleic acid. The comparison of the converted nucleic acid to the reference nucleic acid can include determining if the pattern of cytosine modification of the converted nucleic acid indicates the coding region is transcriptionally active or transcriptionally inactive in the subject. When that coding region is associated with a disease or condition, the status of transcriptional activity can be used for diagnosis or prognosis.
[000400] Comparison of a pattern of cytosine modification in a subject can also be used in identifying changes in a pattern of cytosine modification in a subject over time. For instance, a subject can have a disease or conditions and is undergoing treatment, or a subject had a disease or condition and is cured (e.g., the subject was treated and no signs of the disease or condition are present) or in remission (e.g., the subject was treated and signs of the disease or condition are reduced). Target nucleic acids from the subject at different times, e.g., before treatment started, during treatment, after treatment is stopped, can be compared and a pattern of cytosine modification of a sequence, e.g., a predetermined sequence compared and used to determine the progress of a treatment or the status of the disease or condition in the subject. [000401] In some embodiments where detection of 5mC nucleotides uses amplification, the use of a polymerase that disfavors uracil can aid in reducing the amplification of treated target nucleic acids that include spurious C to U conversion that may result from use of an ACD. B-family polymerases are known to exhibit “uracil read-ahead” function which causes stalling of the polymerase at uracil residues (Greagg et al., 1999, PNAS USA 96(16):9045- 50). Examples of B-family polymerases that disfavor uracil include archaeal B-family polymerases from Pyrococcus furiosus (Pfu), Thermococcus kodakarensis (KOD), Thermococcus litoralis (Tli/Vent), Pyrococcus woe sei (Pwo), and Thermococcus fumicolans (Tfu). Other examples of uracil-disfavoring polymerases include Phusion™, Q5®, and Kapa HiFi™. In other embodiments where amplification of nucleic acids containing uracil nucleotides is desired, the use of a uracil tolerant polymerase can be used. Examples of uracil-tolerant polymerases include PhusionUTM, Q5U®, KapaUTM, Taq, and Dpo4.
[000402] Wild-type cytidine deaminases typically function at near-neutral pH, e.g., pH 7. ACDs described herein can have increased activity at below neutral pH. In some embodiments, the pH of a reaction that includes an ACD described herein can be no greater than pH 8, no greater than 7.8, no greater than 7.7, no greater than 7.6, no greater than 7.5, no greater than 7.4, no greater than 7.3, no greater than 7.2, no greater than 7.1, no greater than 7.0, no greater than pH 6.7, no greater than pH 6.5, no greater than pH 6.3, no greater than pH 6.1, no greater than pH 6.0. In some embodiments, the pH of a reaction that includes an ACD described herein can be at least pH 5.1, at least pH 5.3, at least pH 5.5, at least pH 5.7, at least pH 5.9, at least pH 6.1, at least pH 6.3, at least pH 6.5, at least pH 6.7, at least pH 6.9, at least pH 7.0, at least pH 7.1, at least pH 7.2, at least pH 7.3, at least pH 7.4, at least pH 7.5. In some embodiments, the pH of a reaction that includes an ACD described herein can be no greater than pH 7.5, no greater than pH 7.3, or no greater than pH 7.1. Examples of ranges of pH in a reaction include at least 6 to no greater than 8, at least 6.5 to no greater than 8, at least 7 to no greater than 8, or at least 6.9 to no greater than 7.6, e.g., pH of about 7.0 to about 7.5. The activity of an ACD to deaminate a 5mC oligonucleotide substrate can be an increased catalytic activity that is at least 10-fold greater, at least 50-fold greater, or at least 100-fold greater when comparing activity.
[000403] It is expected that an ACD can function in essentially any buffer. Examples of useful buffers include, but are not limited to: a citrate buffer, such as the citrate buffer available from Thermo Fisher Scientific (Cat. No. #005000); sodium acetate buffer, Bis TrisPropane HC1; and Tris-HCl Tris. Examples of other buffers include, but are not limited to, Bicine, DIPSO, glycylglycine, HEPES, imidazole, malonate, MES, MOPS, PB, phosphate, PIPES, SPG, succinate, TAPS, TAPSO, trincine. In some embodiments a reducing agent such as dithiothreitol (DTT), TCEP, can be present. In some embodiments a divalent cation may be included, for example, Zinc. In some embodiments, a divalent cation is not included. [000404] A deamination reaction can occur at a temperature of 25°C to 75°C, such as 37°C or 50°C. Suitable temperature ranges include from about 37°C to 75 °C, alternatively about 42 °C to 75°C, alternatively about 48 °C- 75 °C, about 50°C to 75 °C, alternatively about 50°C to 65 °C, and any temperature or range between. Some ACDs described herein preferentially deaminate a modified cytosine to thymidine at a faster rate than deamination of cytosine to uracil. Thus, in some embodiments the time of reaction can be used to maximize the difference of deamination of modified cytosine versus deamination of cytosine. In one embodiment, the reaction can proceed for at least 15 minutes, at least 30 minutes, at least 45 minutes, at least 60 minutes, at least 90 minutes, at least 120 minutes, or at least 150 minutes, and for no greater than 15 minutes, no greater than 30 minutes, no greater than 45 minutes, no greater than 60 minutes, no greater than 90 minutes, no greater than 120 minutes, no greater than 150 minutes, or no greater than 180 minutes.
[000405] In one embodiment, a deamination reaction can include an ACD at a concentration from at least 0.05 micromolar (pM) to no greater than 5 pM. For instance, the concentration of the enzyme can be at least 0.05, at least 0.1 pM, at least 0.2 pM, at least 0.3 pM, at least 0.4 pM, or at least 0.5 pM, at least 0.6 pM, at least 0.7 pM, at least 0.8 pM, at least 0.9 pM, at least 1.0 pM and/or no greater than 5 pM, no greater than 4 pM, no greater than 3 M, no greater than 2 M, no greater than 1 pM, or 0.5 pM. In one embodiment, a deamination reaction can include nucleic acids at a concentration of at least 1 picomolar (pM) to no greater than 2 pM. For instance, the concentration of nucleic acids can be at least 1 pM, at least 3 pM, at least 6 pM, at least 10 pM, at least 100 pM, at least 1 nanomolar (nM), at least 40 nM, at least 400 nM, at least 500 nM, at least, 600 nM, at least 700 nM, at least 800 nM, at least 900 nM, or 1 pM, and/or no greater than 1 pM, no greater than 900 nM, no greater than 800 nM, no greater than 700 nM, no greater than 600 nM, no greater than 500 nM, no greater than 400 nM, no greater than 40 nM, no greater than 1 nM, no greater than 100 nM, no greater than 6 nM, or no greater than 3 nM. In one embodiment, a deamination reaction can include nucleic acids at an amount of at least 3 picograms (pg) to no greater than 3 micrograms (pg). For instance, the concentration of nucleic acids can be at least 3 pg, at least 6 pg, at least 10 pg, at least 50 pg, at least 100 pg, at least 250 pg, at least 500 pg, at least 750 pg, at least 1 nanogram (ng), at least 2.5 ng, at least 5 ng, at least 50 ng, at least 100 ng, at least 250 ng, at least 500 ng, at least 750 ng, or at least 1 pg. For instance, the concentration of nucleic acids can be no greater than 3 ug, no greater than 1 pg, no greater than 750 ng, no greater than 500 ng, no greater than 250 ng, no greater than 100 ng, no greater than 50 ng, no greater than 10 ng, no greater than 5 ng, no greater than 2.5 ng, no greater than 1 ng, no greater than 750 pg, no greater than 500 pg, no greater than 250 pg, no greater than 100 pg, no greater than 50 pg, no greater than 10 pg, or no greater than 6 pg. [000406] The substrate of an ACD described herein can be a single stranded nucleic acid, such as single stranded DNA (ssDNA). ssDNA is susceptible to the formation of secondary structure such as hairpins, which can reduce accessibility of reaction sites to an ACD and increase the production of false positives. Accordingly, some embodiments a method of using an ACD can include the use of one or more denaturant to reduce the formation of secondary structure. Examples of denaturants include NaOH, DMSO, betaine, and combinations thereof.
[000407] Stability-enhancing substitution mutations increase the thermal melting point of an ACD, and increased temperature optimums are highly desirable because it decreases DNA secondary structures by opening reaction sites that would be otherwise inaccessible due to secondary structure resulting in decreased false positive rate; stabilizes the enzyme in reaction conditions, which permits longer incubations and increased conversion; increases reaction kinetics, which allows for more tightly controlled conditions; and improves characteristics for commercialization, including increased shelf life, robustness in the assay, manufacturability, etc.
[000408] In one embodiment, a deamination reaction can include an RNAse. RNase A has been implicated in increasing activity of cytidine deaminases (Bransteitter et al., Proceedings of the National Academy of Sciences of the United States of America 100, no. 7 (2003): 4102-7. doi.org/10.1073/pnas.0730835100). When activity of an ACD of the present disclosure was determined in the presence of RNAse A the opposite was observed. When RNAse A was included in the reaction, an ACD having 5mC-selective deaminase activity had reduced activity, and the reduced activity was more pronounced for off-target cytosine deamination. Thus, RNAse A resulted in greater selectivity for deamination of 5mC compared to C. An RNAse A can be included in a deamination reaction at a concentration from at least 1 microgram/milliliter (ug/ml) to no greater than 20 pM. For instance, the concentration of RNAse A can be at least 1 ug/ml , at least 2 ug/ml, at least 3 ug/ml, at least 4 ug/ml, 5 ug/ml, 6 ug/ml, 7 ug/ml, 8 ug/ml, or 9 ug/ml, and/or no greater than 50 ug/ml, no greater than 40 ug/ml, no greater than 30 ug/ml, no greater than 20 ug/ml, no greater than 19 ug/ml, no greater than 18 ug/ml, no greater than 17 ug/ml, no greater than 16 ug/ml, no greater than 15 ug/ml, no greater than 14 ug/ml, no greater than 13 ug/ml, no greater than 12 ug/ml, or no greater than 11 ug/ml. In one embodiment, the concentration of RNAse A is from 2 ug/ml to 13 ug/ml, or from 5 ug/ml to 10 ug/ml.
[000409] In some embodiments, an ACD may deaminate cytosine at a low level compared to the deamination of 5mC, resulting in false positives. Methods for reducing the deamination of cytosine are available. Methods that include protective approaches (e.g., an additional treatment before use of an ACD) are described in International Application Pub. Nos. WO 2024/073043 and WO 2025/137222. Methods that include corrective approaches (e.g., an additional treatment after use of an ACD) are described in International Application Pub. Nos. WO 2024/118903, WO 2024/073047, WO2024/147904, and WO 2024/249466. Methods that include an additional treatment during use of an ACD are described in International Application Pub. No. WO 2024/069581.
[000410] In some aspects, the present ACDs or ACD-helicase complexes (e.g., fusion proteins) can be used in different library preparation kits, including library kits that are designed for use with Illumina sequencing platforms or are compatible with Illumina’s sequencing workflows. Examples of Illumina sequencing platforms include, without limitation, the MiSeq™, HiSeq™, NextSeq™, MiniSeq™, NovaSeq™ and iSeq™ platforms (Illumina, Inc., San Diego, Calif.).
[000411] For example, in one aspect, the ACDs or ACD-helicase complexes (e.g., fusion proteins) may be used in a PCR-free library preparation kit. Such a kit, includes, for example, Lotus DNA Library preparation kit (IDT,) which is suitable for use in whole genome sequencing, PCR-free sequencing, SNV and indel detection, low frequency somatic variation RNA-Seq starting with full-length, cDNA input, metagenomic sequencing, among others, or Illumina’s DNA PCR-Free Prep workflow that uses tagmentation (Illumina, San Diego, CA).
[000412] Suitably, the ACDs or ACD-helicase complexes may be used in library preparation workflows in which the input DNA is physically or enzymatically fragmented. Physical fragmentation includes sheering using acoustics, nebulization, centrifugal force, needles, or hydrodynamics, e.g. mechanically sheering (e.g., Covaris). Enzymatic fragmentation includes DNA digestion into fragments using enzymes, including, for example, fragmentation via fragmentases, restriction digestion, or other means to obtain DNA fragments of suitable size for sequencing. Enzymatic methods may be followed by a step of end-repair and A-tailing to allow for the next steps in library preparation, e.g., ligation of adaptors.
[000413] Further, the ACDs or ACD-helicase complexes (e.g., fusion proteins) may be used in library preparation workflows that use tagmentation (e.g., Illumina DNA library preparation kits) which allows for the simultaneous fragmenting and tagging of the target DNA with adapter sequences using a transposase enzyme, which inserts adapter sequences into the DNA at random locations, effectively fragmenting the DNA while adding sequences that can be used in library preparation.
[000414] Further, the ACDs or the ACD-helicase complexes (e.g. fusion proteins) described herein can be used in arrays for detection of methylation status.
[000415] In some aspects, the ACDs or ACD-helicase complexes described herein may be used in methods and kits in which DNA is fragmented and prepared for sequencing wherein the kits and/or methods use physical, enzymatic or tagmentation for sheering the DNA. Further adaptors for sequencing can be added as known in the art, including ligation and tagmentation, among others.
[000416] In another aspect, the ACD-helicase complexes, (e.g. fusion proteins) can be used in assays and methodologies in which dsDNA is to be assayed for methylation status, e.g., on-flow cell sequencing, or cell-free DNA preparations, etc., in which minimizing harsh chemical steps (e.g., denaturing) and/or the minimizing the number of wash steps is helpful in preserving the integrity of the substrate (e.g., flow cell) or the amount of DNA within the library for subsequent sequencing.
[000417] The ACD-helicase complexes (e.g. fusion proteins) provide benefits for certain embodiments of the method where a gentler denaturation step, no denaturing step, or a reduction of washes is needed to allow for the library preparation to provide enough DNA for analysis or to not alter the substrate (e.g., flow cell surface or conditions).
[000418] The ACD conjugated to helicase, e.g., in a fusion protein, uniquely provides deaminase activity on double stranded DNA to be able to detect 5mC on a single base resolution.
[000419] In one aspect, the ACD-helicase complexes (e.g. fusion proteins) can be used for an on-flow cell library preparation. Since library preparation is done on the flow cell, harsh denaturants like DMSO can alter the surface of the flow cell and thus lead to loss of DNA libraries on the flow cell and reduced signal for subsequent sequencing. Further, on-flow cell library preparation relies on dsDNA seeding on the flow cell, and as such, requires a deaminase that can work with the on-flow cell system. Thus, the ACD-helicase complexes (e.g. fusion proteins) described herein can be used in methods for on-flow cell library preparation and subsequent sequencing as described in PCT/US2025/021759, the contents of which are incorporated by reference in its entirety with regard to the method of preparing a library on-flow cell and subsequent sequencing of said library.
[000420] Further, the ACD-helicase complexes (e.g. fusion proteins) can be used in aspects for PCR-free library preparation, or for cell-free DNA library preparation, where the input DNA is low and the number of steps and/or washes need to be minimized as to not reduce the amount of DNA that will be sequenced. Requiring denaturation of the dsDNA before deamination typically includes at least one additional wash step to remove the denaturant, which can lead to a reduction in the amount of DNA left to prepare the library preparation (as every wash step within a workflow results in some loss of DNA). Thus, every additional step and/or wash increases the chance for loss of DNA and increases the risk of not meeting the minimal amount of DNA for accurate sequencing. Thus, if there is no PCR step to increase the DNA input before sequencing, it is recommended to maintain as much DNA from the original sample as possible. As such, the ACD-helicase complexes (e.g. fusion proteins) described herein can be used either with a gentler denaturant, or no denaturant step in the library preparation workflow, allowing for maintenance of the minimal amounts of input DNA for accurate sequencing. [000421] In some aspects, the present invention provides a sequencing kit comprising a flow cell including a substrate having depressions separated by interstitial regions; first and second primers immobilized within each of the depressions; and first and second transposome complexes immobilized within each of the depressions, over the interstitial regions, or both within each of the depressions and over the interstitial regions; an extension mix; and an enzymatic methylation conversion mix comprising the ACD or ACD-helicase complex and a buffer solution.
[000422] The kit may be used in suitable methods. For example, one method comprises initiating tagmentation of a DNA sample using first and second transposome complexes asymmetrically attached in a flow cell, thereby forming partially adapted hybridized fragments including a first partially adapted DNA fragment that is immobilized, at its 5’ end, to a substrate of the flow cell, and a second partially adapted DNA fragment that is removably attached to the substrate; removing a transposase enzyme from each of the first and second transposome complexes; initiating an extension reaction of the partially adapted hybridized fragments to form fully adapted hybridized fragments; initiating enzymatic methylation conversion of the first fully adapted DNA fragment using the ACD-helicase complex described herein.
[000423] The extension mix may includes nucleotides, a polymerase, and a buffer agent. The buffer agent may include any of the neutral buffers set forth herein (e.g., Tris), ammonium sulfate, betaine, a metal co-factor (e.g., Mg2+), a surfactant (e.g., TWEEN polysorbates, TRITON™ X-100 (a non-ionic surfactant from Dow)), and/or a co-solvent (e.g., dimethylsulfoxide). An example extension mix includes from about 0.1 mM to about 0.5 mM of the nucleotides, from about 40 U/mL to about 80 U/mL of the polymerase, from about 15 mM to about 25 mM of the neutral buffer, from about 5 mM to about 15 mM of ammonium sulfate (e.g., about 10 mM ammonium sulfate), from about 1.8 M to about 2.2 M of the betaine (e.g., about 2 M betaine), from about 2 mM to about 5.5 mM of the metal cofactor, from about 0.1% to about 0.4% of the surfactant, and from about 1.0% to about 2.0% of the co-solvent. The extension mix does not include a recombinase, as it is not desirable for the fully adapted DNA fragments to be amplified immediately upon being formed via extension.
[000424] The enzymatic methylation conversion mix includes a liquid carrier and an altered cytidine deaminase (ACD) as described herein or a Helicase- ACD complex described herein. The liquid carrier in the enzymatic methylation conversion mix may be a buffer having a pH lower than 7 (e.g., ranging from 5.1 to 6.5). Examples of suitable buffers include, but are not limited to: a citrate buffer, a sodium acetate buffer, Bis Tris-Propane HC1, and Tris-HCl Tris. Examples of other buffers include, but are not limited to, Bicine, DIPSO (3 -[N,N-Bis(2-hydroxyethylamino)-2-hydroxy-l -propanesulfonic acid), glycylglycine, HEPES (2-[4-(2-hydroxyethyl)piperazin-l-yl]ethanesulfonic acid), imidazole, malonate, MES (2-(N-morpholino)ethanesulfonic acid), MOPS (3-(N- morpholino)propanesulfonic acid), phosphate, PIPES (1,4-Piperazinedi ethanesulfonic acid), SPG (succinic acid, sodium dihydrogen phosphate, and glycine in the molar ratio 2:7:7), succinate, TAPS (N-[Tris(hydroxymethyl)methyl]-3-aminopropanesulfonic acid), TAPSO (2- Hydroxy-3-[tris(hydroxymethyl)methylamino]-l-propanesulfonic acid), trincine. In some examples, a reducing agent such as dithiothreitol (DTT) can be present. In some examples, a divalent cation is not included.
[000425] A method of making an array is also provided. The method can include the steps of: providing a sample comprising a template nucleic acid; generating a complementary copy of the template nucleic acid; subjecting the template nucleic acid and the complementary copy to ACD or ACD-helicase treatment to convert methylated cytosine residues (5mC) in the template nucleic acid into T, and coupling the template and the complementary copy of the template to the solid support. The method of making an array can include the steps of: providing a solid support with a plurality of sites; providing a sample comprising a template nucleic acid; generating a complementary copy of the template nucleic acid, the generating being directed by an oligonucleotide primer using a nucleic acid polymerase, wherein the generating produces a complementary copy of the template nucleic acid; subjecting the template nucleic acid to ACD treatment to convert methylated cytosine residues in the template nucleic acid into T residues, resulting in a ACD-converted template nucleic acid; and coupling the template and the complementary copy of the template to the solid support. In certain aspects of the method, at least one of the sites comprises a capture probe. In certain aspects, the capture probe comprises a nucleotide sequence complementary to the template or a nucleotide sequence complementary to a complementary copy of the template. In other aspects, an oligonucleotide complementary to the capture probe is attached to the template or complementary copy of the template.
[000426] Target nucleic acids
[000427] The target nucleic acids contacted with an ACD and used in the methods, compositions, and kits provided herein may be essentially any nucleic acid of known or unknown sequence. Sequencing may result in determination of the sequence of the whole or a part of the target molecule. In one embodiment, target nucleic acids can be processed into templates suitable for amplification by the placement of universal amplification sequences, e.g., sequences present in a universal adaptor, at the ends of each target fragment.
[000428] Target nucleic acids are typically derived from primary nucleic acids present in a sample, such as a biological sample. The primary nucleic acids may originate as DNA or RNA. DNA primary nucleic acids may originate in double-stranded DNA (dsDNA) form (e.g., genomic DNA, genomic DNA fragments, cell-free DNA, and the like) from a sample or may originate in single-stranded form from a sample. RNA primary nucleic acids may be mRNA or non-coding RNA, e.g., microRNA or small interfering RNA. The precise sequence of the polynucleotide molecules from a primary nucleic acid sample is generally not material to the disclosure and may be known or unknown.
[000429] The primary nucleic acid molecules may represent the entire genetic complement of an organism, e.g., genomic DNA molecules which include both intron and exon sequences, as well as non-coding regulatory sequences such as promoter and enhancer sequences. The primary nucleic acid molecules may represent the entire genetic complement of specific cells of an organism, e.g., from tumor cells, where the genomic DNA molecules which include both intron and exon sequences, as well as non-coding regulatory sequences such as promoter and enhancer sequences. In one embodiment, particular subsets of genomic DNA can be used, such as, for example, particular chromosomes, DNA associated with open chromatin, DNA associated with closed chromatin, or one or more specific sequences such as a region of a specific gene (e.g., targeted sequencing). In one embodiment, the primary nucleic acid molecules may represent a particular subset of DNA, e.g., DNA having a specific sequence that anneals with a primer such as one used for targeted sequencing or target enrichment. In one embodiment, a particular subset of DNA can be used, such as cell-free DNA, which can include DNA of the subject including DNA from normal cells, DNA from diseased cells such as tumor cells, and/or DNA from fetal cells.
[000430] The primary nucleic acid molecules may represent the entire transcriptome of cells of an organism, e.g., mRNA molecules. The primary nucleic acid molecules may represent the entire transcriptome of specific cells of an organism, e.g., from tumor cells or for instance the cells of a tissue. In one embodiment, the primary nucleic acid molecules may represent a particular subset of mRNA, e.g., mRNA having a specific sequence that anneals with a primer such as one used for targeted sequencing or target enrichment.
[000431] A sample, such as a biological sample, can include nucleic acid molecules obtained from biopsies, tumors, scrapings, swabs, blood, mucus, urine, stool, plasma, semen, hair, laser capture micro-dissections, surgical resections, and other clinical or laboratory obtained samples. In some embodiments, the sample can be an epidemiological, agricultural, forensic or pathogenic sample. In some embodiments, the sample can include cultured cells. In some embodiments, the sample can include nucleic acid molecules obtained from an animal such as a human or mammalian source. In another embodiment, the sample can include nucleic acid molecules obtained from a non-mammalian source such as a plant, bacteria, virus, or fungus. In some embodiments, the source of the nucleic acid molecules may be an archived or extinct sample or species.
[000432] Additional non-limiting examples of sources of biological samples can include whole organisms as well as a sample obtained from a patient. The biological sample can be obtained from any biological fluid or tissue and can be in a variety of forms, including fluid, e.g., liquid or gas, tissue, solid tissue, and preserved forms of such a fluid or tissue, such as dried, frozen, and fixed forms. The sample may be of any biological tissue, cells or fluid. Such samples include, but are not limited to, sputum, blood, serum, plasma, blood cells (e.g., white cells), ascitic fluid, urine, saliva, tears, sputum, vaginal fluid (discharge), washings obtained during a medical procedure (e.g., pelvic or other washings obtained during biopsy, endoscopy or surgery), tissue, nipple aspirate, core or fine needle biopsy samples, cellcontaining body fluids, peritoneal fluid, and pleural fluid, or cells therefrom, and free floating nucleic acids such as cell-free circulating DNA (cfDNA). In one embodiment, a biological sample is a liquid biopsy that includes cfDNA. Biological samples may also include sections of tissues such as frozen or fixed sections taken for histological purposes or micro-dissected cells or extracellular parts thereof. In some embodiments, the sample can be a blood sample, such as, for example, a whole blood sample. In another example, the sample is an unprocessed dried blood spot (DBS) sample. In yet another example, the sample is a formalin-fixed paraffin-embedded (FFPE) sample. In yet another example, the sample is a saliva sample. In yet another example, the sample is a dried saliva spot (DSS) sample.
[000433] Exemplary biological samples from which target nucleic acids can be derived include, for example, those from a eukaryote, for instance a mammal, such as a rodent, mouse, rat, rabbit, guinea pig, ungulate, horse, sheep, pig, goat, cow, cat, dog, primate, human or non-human primate; a plant, such as Arabidopsis thaliana. corn, sorghum, oat, wheat, rice, canola, or soybean; an algae, such as Chlamydomonas reinhardlii: a nematode such as Caenorhabditis elegans an insect, such as Drosophila melanogaster, mosquito, fruit fly, honey bee or spider; a fish, such as zebrafish; a reptile; an amphibian, such as a frog or Xenopus laevis: a Dictyostelium discoideum: a fungi, such as Pneumocystis carinii. Takifugu rubripes. yeast, Saccharamoyces cerevisiae. or Schizosaccharomyces pombe: or a protozoan such as Plasmodium falciparum. Target nucleic acids can also be derived from a prokaryote such as a bacterium, Escherichia coli. Staphylococcus o Mycoplasma pneumoniae, ' an archaeon; a virus such as Hepatitis C virus or human immunodeficiency virus; or a viroid. Target nucleic acids can be derived from a homogeneous culture or population of organisms described herein or alternatively from a collection of several different organisms, for example, in a community or ecosystem.
[000434] In some embodiments, a biological sample includes tissue that is processed to obtain the desired primary nucleic acids. In some embodiments, cells are used obtain the desired primary nucleic acids. In some embodiments, nuclei are used to obtain the desired primary nucleic acids. The method can further include dissociating cells, and/or isolating nuclei from cells. Methods for isolating cells and nuclei from tissue are available (WO 2019/236599).
[000435] In some embodiments, nucleic acids present in tissue, in cells, or in isolated nuclei can be processed depending on the desired read-out. For instance, nucleic acids can be fixed during processing, and useful fixation methods are available (WO 2019/236599). Fixation can be useful to preserve a sample or maintain contiguity of analytes from a sample, a cell, or a nucleus. Fixation methods preserve and stabilize tissue, cell, and nucleus morphology and architecture, inactivates proteolytic enzymes, strengthens samples, cells, and nuclei so they can withstand further processing and staining, and protects against contamination. Examples of methods where fixation can be useful include, but are not limited to, whole genome sequencing of isolated nuclei and chromosome conformation capture methods such as Hi-C. Common methods of fixation include perfusion, immersion, freezing, and drying (Srinivasan et al., Am J Pathol. 2002 Dec; 161(6): 1961-1971. doi:
10.1016/S0002-9440(10)64472-0). In some embodiments such as whole genome sequencing, isolated nuclei can be processed to dissociate nucleosomes from DNA while leaving the nuclei intact, and methods for generating nucleosome-free nuclei are available (WO 2018/018008). In some embodiments, processing of nucleic acids can include purification. Purification, also referred to as clean up, of nucleic acids aids in reducing or removing compounds such as, but not limited to, proteins, buffers, and undesired nucleic acids (for instance, removal of RNA when DNA is the target nucleic acid). Methods for purification of nucleic acids are known and include, for instance, solid-phase reversible immobilization (SPRI) or magnetic beads.
[000436] In some embodiments, primary nucleic acids in bulk, e.g., from a plurality of cells, can be used to produce a sequencing library as described herein. In other embodiments, individual cells or nuclei can be used as sources of primary nucleic acids to obtain sequence information from single cells and nuclei. Many different single cell library preparation methods are known in the art, including, but not limited to, Drop-seq, Seq-well, and single cell combinatorial indexing ("sci-") methods. Companies providing single cell products and related technologies include, but are not limited to, Illumina, 10X Genomics, Takara Biosciences, BD Biosciences, Bio-Rad Laboratories, Icellbio, Isoplexis, CellSee, NanoSelect, and Dolomite Bio. Sci-seq is a methodological framework that employs splitpool barcoding to uniquely label the nucleic acid contents of large numbers of single cells or nuclei. Typically, the number of nuclei or cells can be at least two. The upper limit is dependent on the practical limitations of equipment (e.g., multi-well plates, number of indexes) used in other steps of the methods as described herein. The number of nuclei or cells that can be used is not intended to be limiting and can number in the billions.
[000437] The target nucleic acids used in the methods and compositions of the present disclosure can be derived by fragmentation. Random fragmentation refers to the fragmentation of a polynucleotide molecule from a primary nucleic acid sample in a nonordered fashion by enzymatic, chemical, or mechanical methods. Such fragmentation methods are known in the art and use standard methods (Sambrook and Russell, Molecular Cloning, A Laboratory Manual, third edition). Moreover, random fragmentation is designed to produce fragments irrespective of the sequence identity or position of nucleotides comprising and/or surrounding the break. In one embodiment, the random fragmentation is by mechanical means such as nebulization or sonication to produce fragments of about 50 base pairs in length to about 1500 base pairs in length, still more particularly 50-700 base pairs in length, yet more particularly 50-400 base pairs in length. Most particularly, the method is used to generate smaller fragments of from 50-150 base pairs in length.
[000438] Fragmentation of polynucleotide molecules by mechanical means (nebulization, sonication, and Hydroshear, for example) results in fragments with a heterogeneous mix of blunt and 3'- and 5'-overhanging ends. It is therefore desirable to repair the fragment ends using methods or kits (such as the Lucigen DNA terminator End Repair Kit) known in the art to generate ends that are optimal for insertion, for example, into blunt sites of cloning vectors. In a particular embodiment, the fragment ends of the population of nucleic acids are blunt ended. More particularly, the fragment ends are blunt ended and phosphorylated. The phosphate moiety can be introduced via enzymatic treatment, for example, using polynucleotide kinase. [000439] In a particular embodiment, the target fragment sequences are prepared with single overhanging nucleotides by, for example, activity of certain types of DNA polymerase such as Taq polymerase or KI enow exo minus polymerase which has a non-templatedependent terminal transferase activity that adds a single deoxynucleotide, for example, deoxyadenosine (A) to the 3' ends of a DNA molecule, for example, a PCR product. Such enzymes can be used to add a single nucleotide 'A' to the blunt ended 3' terminus of each strand of the double-stranded target fragments. Thus, an 'A' could be added to the 3' terminus of each end repaired strand of the double-stranded target fragments by reaction with Taq or Klenow exo minus polymerase, while the universal adapter polynucleotide construct could be a T-construct with a compatible 'T' overhang present on the 3' terminus of each region of double-stranded nucleic acid of the universal adapter. This end modification also prevents self-ligation of both vector and target such that there is a bias towards formation of target nucleic acids having a universal adapter at each end.
[000440] In one embodiment, fragmentation can be accomplished using a process often referred to as tagmentation. Tagmentation uses a transposome complex and combines into a single step fragmentation and ligation to add universal adapters (WO 2016/130704). A transposome complex is a transposase bound to a transposase recognition site and can insert the transposase recognition site into a target nucleic acid in a process sometimes termed "tagmentation." In some such insertion events, one strand of the transposase recognition site may be transferred into the target nucleic acid. Such a strand is referred to as a "transferred strand." In one embodiment, a transposome complex includes a dimeric transposase having two subunits, and two non-contiguous transposon sequences. In another embodiment, a transposase includes a dimeric transposase having two subunits, and a contiguous transposon sequence.
[000441] Examples of transposon sequences useful with the methods, compositions, and kits described herein are provided in U.S. Patent Application Pub. No. 2012/0208705, U.S. Patent Application Pub. No. 2012/0208724 and Int. Patent Application Pub. No. WO 2012/061832. In some embodiments, a transposon sequence includes a first transposase recognition site and a second transposase recognition site.
[000442] A population of target nucleic acids can have an average strand length that is desired or appropriate for a particular application of the methods, compositions, or kits set forth herein. For example, the average strand length can be less than about 100,000 nucleotides, 50,000 nucleotides, 10,000 nucleotides, 5,000 nucleotides, 1,000 nucleotides, 500 nucleotides, 100 nucleotides, or 50 nucleotides. Alternatively or additionally, the average strand length can be greater than about 10 nucleotides, 50 nucleotides, 100 nucleotides, 500 nucleotides, 1,000 nucleotides, 5,000 nucleotides, 10,000 nucleotides, 50,000 nucleotides, or 100,000 nucleotides. The average strand length for a population of target nucleic acids can be in a range between a maximum and minimum value set forth herein. It will be understood that amplicons generated at an amplification site (or otherwise made or used herein) can have an average strand length that is in a range between an upper and lower limit selected from those exemplified above.
[000443] In some cases, a population of target nucleic acids can be produced under conditions or otherwise configured to have a maximum length for its members. For example, the maximum length for the members that are used in one or more steps of a method set forth herein or that are present in a particular composition can be less than 100,000 nucleotides, less than 50,000 nucleotides, less than 10,000 nucleotides, less than 5,000 nucleotides, less than 1,000 nucleotides, less than 500 nucleotides, less than 100 nucleotides, or less than 50 nucleotides. Alternatively or additionally, a population of target nucleic acids can be produced under conditions or otherwise configured to have a minimum length for its members. For example, the minimum length for the members that are used in one or more steps of a method set forth herein or that are present in a particular composition can be more than 10 nucleotides, more than 50 nucleotides, more than 100 nucleotides, more than 500 nucleotides, more than 1,000 nucleotides, more than 5,000 nucleotides, more than 10,000 nucleotides, more than 50,000 nucleotides, or more than 100,000 nucleotides. The maximum and minimum strand length for target nucleic acids in a population can be in a range between a maximum and minimum value set forth above. It will be understood that amplicons generated at an amplification site (or otherwise made or used herein) can have maximum and/or minimum strand lengths in a range between the upper and lower limits exemplified above.
[000444] In some embodiments, a sample can be enriched for sequences of interest, e.g., a predetermined sequence. For example, a subset of genes or regions of the genome are isolated and sequenced, or a subset of genes or regions of the genome are interrogated by other methods, such as a locus-specific in vitro diagnostic method. A predetermined sequence can be, for instance, one that can have a pattern of cytosine modification.
[000445] In some embodiments, target enrichment works by capturing genomic regions of interest by hybridization to target-specific probes that can be used to physically separate target DNA that has hybridized to bait probes from all other DNA in solution, which are then washed away. For example, some methods of enrichment use biotinylated probes, which are then isolated by magnetic pulldown with streptavidin-coated magnetic particles. In another example, some methods of enrichment use analyte arrays, also known as microarrays, that allow for the hybridization of predetermined sequences.
[000446] Enrichment can occur, for example, prior to treatment with ACD. In such embodiments, enriching a nucleic acid of interest, or a fragment thereof, such as enriching DNA in a sample, may include any suitable enrichment techniques. In some embodiments, enrichment of DNA may include enrichment through molecular inversion probes, in solution capture, pulldown probes, bait sets, standard PCR, multiplex PCR, hybrid capture, endonuclease digestion, DNase I hypersensitivity, and selective circularization. Enrichment can be achieved through negative selection of nucleic acids by eliminating undesired material. This sort of enrichment includes 'footprinting' techniques or 'subtractive' hybrid capture. During the former, the target sample is safe from nuclease activity through the protection of protein or by single and double stranded arrangements. During the latter, nucleic acids that bind ‘bait’ probes are eliminated.
[000447] In some embodiments, enriching can comprise amplification using target-specific primers. In some embodiments, amplification is performed subsequent to another form of enrichment. Typically, however, in embodiments where amplification is used for enrichment, the amplification step occurs after treatment with deaminase, to preserve methylation status of the target DNA. In some such embodiments, amplification can include PCR amplification or genome-wide amplification.
[000448] In some embodiments, enrichment can occur after treatment with an ACD. Typically, methods used to identity methylated cytosines result in the loss of DNA complexity due to conversion of unmethylated DNA bases to uracil, resulting in a 3 -base genome and limits the use of sequences that specifically hybridize to a predetermined sequence. Accordingly, typical methods for identifying methylated cytosines are more difficult to use in methods that include enrichment, such as hybrid-enrichment sequencing and amplicon-based targeted sequencing, after conversion of methylated cytosines. In contrast, because of the 5mC to T conversion by ACDs, only a small percentage of cytosines are methylated and expected to be converted by an ACD. Examples of enrichment-based methods that can be used after treatment of a target nucleic acid with an ACD include but are not limited to analyte arrays, use of primers for selective amplification, CRISPR-Cas systems, and molecular cytogenic techniques such as FISH. Examples of arrays include, for instance, methylation arrays for interrogation of selected methylation sites across a genome (e.g., the Infmium Methylation EPIC BeadChip, Illumina). [000449] Attachment of Universal Adapters
[000450] In some embodiments, a target nucleic acid used in a method, composition, or kit described herein can include a universal adapter attached to each end. A target nucleic acid having a universal adapter at each end can be referred to as a "modified target nucleic acid." Methods for attaching a universal adapter to each end of a target nucleic acid used in a method described herein are known to the person skilled in the art. The attachment can be through tagmentation using transposase complexes (WO 2016/130704), or through standard library preparation techniques using ligation (U.S. Pat. Pub. No. 2018/0305753). Attachment of a universal adapter to the ends of a target nucleic acid can occur before or after treatment of the target nucleic acid with an ACD.
[000451] In one embodiment, double-stranded target nucleic acids from a sample, e.g., a fragmented sample that has been contacted with an ACD and converted from single-stranded to double-stranded nucleic acids, are treated by first ligating identical universal adaptor molecules to the 5' and 3' ends of the double-stranded target nucleic acids. In one embodiment, the universal adapters are "matched" adapters or Y-adapters because the two strands of the adaptors are formed by annealing complementary polynucleotide strands. In one embodiment, the universal adapters used in the method of the disclosure are referred to as "mismatched" adaptors because the adaptors include a region of sequence mismatch, i.e., they are not formed by annealing fully complementary polynucleotide strands. The general features of mismatched adaptors are further described in Gormley et al., U.S. Pat. No.
7,741,463, and Bignell et al., U.S. Pat. No. 8,053,192,). The universal adaptor typically includes universal capture binding sequences that aid in immobilizing the target nucleic acids on an array for subsequent sequencing, and universal primer binding sites useful for the sequencing. In another embodiment, double-stranded target nucleic acids from a sample, a sample that has been contacted with an ACD and converted from single-stranded to doublestranded nucleic acids, are subjected to tagmentation with a transposome complex that inserts a universal adapter, or sequences that can be used to add a universal adapter, into a target nucleic acid.
[000452] A universal adapter can optionally include at least one index. An index can be used as a marker characteristic of the source of particular target nucleic acids on a flow cell (U.S. Pat. No. 8,053,192). Generally, the index is a synthetic sequence of nucleotides that is part of the universal adapter which is added to the target nucleic acids as part of the library preparation step. Accordingly, an index is a nucleic acid sequence which is attached to each of the target molecules of a particular sample, the presence of which is indicative of, or is used to identify, the sample or source from which the target molecules were isolated.
[000453] In one embodiment, an index may be up to 20 nucleotides in length, such as 1-10 nucleotides in length or 4-6 nucleotides in length. A four nucleotide index gives a possibility of multiplexing 256 samples on the same array, a six base index enables 4096 samples to be processed on the same array.
[000454] The precise nucleotide sequence of the universal adapters is generally not material to the disclosure and may be selected by the user such that the desired sequence elements are ultimately included in the common sequences of the plurality of different modified target nucleic acids, for example, to provide for the universal capture binding sequences for immobilizing the target nucleic acids on an array for subsequent sequencing, and binding sites for particular sets of universal amplification primers and/or sequencing primers. Additional sequence elements may be included, for example, to provide binding sites for sequencing primers which will ultimately be used in sequencing of target nucleic acids in the library, sequencing of an index, or products derived from amplification of the target nucleic acids in the library, for example on a solid support.
[000455] In order to prepare a library of deaminase-treated DNA for analysis using a sequencing platform, it may be useful to make additional modifications to the target DNA, either prior to or after treatment with ACD. In some embodiments, single-stranded deaminase-treated DNA is prepared for sequencing using a single-stranded library preparation method, as is known in the art. Such methods include, but are not limited to, template switching based second strand synthesis, adapters containing a single-stranded splint overhang, and the like. Reagents for performing single-stranded library preparation methods are commercially available. Examples include xGen ssDNA & Low-Input DNA Library Prep Kit (Integrated DNA Technologies catalog number 10009859), previously sold as Accel-NGS (Swift Biosciences), NGS Single Stranded DNA Library Prep Kit (BioDynami catalog number 30082). Another example includes single-reaction single-stranded library (SRSLY) as set forth in Troll et al., BMC Genomics 20, 1023 (2019).
[000456] In some embodiments, library preparation modifications are made to doublestranded target DNA prior to treatment with an ACD. Methods for library preparation of double-stranded DNA template are known in the art, and include Y-adaptor ligation, transposome-based tagmentation, and the like. It will be appreciated by those of skill in the art that methods of double-strand library preparation often include one or more amplification steps using for example, PCR. In such methods, the amplification step may be deferred until after ACD treatment, to preserve the methylation status of the template strand. For example, in Y-adapter ligation methods, the Y-adapters can be ligated to the double-stranded template, after which the adapter-ligated template DNA is denatured and treated with an ACD as described elsewhere herein. Following treatment with an ACD, the resulting treated singlestrand DNA molecules can be amplified using PCR, bridge amplification, and other methods as are commonly known in the art.
[000457] Preparation of Immobilized Samples for Sequencing
[000458] The library of modified target nucleic acids, e.g., target nucleic acids having universal adapters at each end, can be prepared for sequencing. Methods for attaching modified target nucleic acids to a substrate are known in the art. In one embodiment, modified fragments are enriched using a plurality of capture oligonucleotides having specificity for the modified fragments, and the capture oligonucleotides can be immobilized on a surface of a solid substrate such as a flow cell or a bead. For instance, capture oligonucleotides can include a first member of a universal binding pair, and where a second member of the binding pair is immobilized on a surface of a solid substrate. Likewise, methods for amplifying immobilized target nucleic acids include, but are not limited to, bridge amplification and exclusion amplification (also referred to as kinetic exclusion amplification). Methods for immobilizing and amplifying prior to sequencing are described in, for instance, Bignell et al. (US 8,053,192), Gunderson et al. (W02016/130704), Shen et al. (US 8,895,249), and Pipenburg et al. (US 9,309,502).
[000459] A pooled sample can be immobilized in preparation for sequencing. Sequencing can be performed as an array of single molecules or can be amplified prior to sequencing. The amplification can be carried out using one or more immobilized primers. The immobilized primer(s) can be, for instance, a lawn on a planar surface, or on a pool of beads. The pool of beads can be isolated into an emulsion with a single bead in each "compartment" of the emulsion. At a concentration of only one template per "compartment," only a single template is amplified on each bead.
[000460] The term "solid-phase amplification" as used herein refers to any nucleic acid amplification reaction carried out on or in association with a solid support such that all or a portion of the amplified products are immobilized on the solid support as they are formed. In particular, the term encompasses solid-phase polymerase chain reaction (solid-phase PCR) and solid phase isothermal amplification which are reactions analogous to standard solution phase amplification, except that one or both of the forward and reverse amplification primers is/are immobilized on the solid support. Solid phase PCR covers systems such as emulsions, where one primer is anchored to a bead and the other is in free solution, and colony formation in solid phase gel matrices wherein one primer is anchored to the surface, and one is in free solution.
[000461] In some embodiments, the solid support includes a patterned surface. A "patterned surface" refers to an arrangement of different regions in or on an exposed layer of a solid support. For example, one or more of the regions can be features where one or more amplification primers are present. The features can be separated by interstitial regions where amplification primers are not present. In some embodiments, the pattern can be an x-y format of features that are in rows and columns. In some embodiments, the pattern can be a repeating arrangement of features and/or interstitial regions. In some embodiments, the pattern can be a random arrangement of features and/or interstitial regions. Exemplary patterned surfaces that can be used in the methods and compositions set forth herein are described in U.S. Pat. Nos. 8,778,848, 8,778,849 and 9,079,148, and U.S. Pat. Appl. Pub. No. 2014/0243224.
[000462] In some embodiments, the solid support includes an array of wells or depressions in a surface. This may be fabricated as is generally known in the art using a variety of techniques, including, but not limited to, photolithography, stamping techniques, molding techniques and micro-etching techniques. As will be appreciated by those of skill in the art, the technique used will depend on the composition and shape of the array substrate.
[000463] The features in a patterned surface can be wells in an array of wells (e.g., microwells or nanowells) on glass, silicon, plastic or other suitable solid supports with patterned, covalently-linked gel such as poly(N-(5-azidoacetamidylpentyl)acrylamide-co- acrylamide) (PAZAM, see, for example, US Pub. No. 2013/184796, WO 2016/066586, and WO 2015/002813). The process creates gel pads used for sequencing that can be stable over sequencing runs with a large number of cycles. The covalent linking of the polymer to the wells is helpful for maintaining the gel in the structured features throughout the lifetime of the structured substrate during a variety of uses. However, in many embodiments the gel need not be covalently linked to the wells. For example, in some conditions silane free acrylamide (SFA, see, for example, US Pat. No. 8,563,477) which is not covalently attached to any part of the structured substrate, can be used as the gel material.
[000464] In particular embodiments, a structured substrate can be made by patterning a solid support material with wells (e.g., microwells or nanowells), coating the patterned support with a gel material (e.g., PAZAM, SFA, or chemically modified variants thereof, such as the azidolyzed version of SFA (azido-SFA)) and polishing the gel coated support, for example via chemical or mechanical polishing, thereby retaining gel in the wells but removing or inactivating substantially all of the gel from the interstitial regions on the surface of the structured substrate between the wells. Primer nucleic acids can be attached to gel material. A solution of modified target nucleic acids can then be contacted with the polished substrate such that individual modified target nucleic acids will seed individual wells via interactions with primers attached to the gel material; however, the target nucleic acids will not occupy the interstitial regions due to absence or inactivity of the gel material.
Amplification of the modified target nucleic acids will be confined to the wells since absence or inactivity of gel in the interstitial regions prevents outward migration of the growing nucleic acid colony. The process can be conveniently manufactured, being scalable and utilizing conventional micro- or nanofabrication methods.
[000465] Although the disclosure encompasses "solid-phase" amplification methods in which only one amplification primer is immobilized (the other primer usually being present in free solution), in one embodiment the solid support is provided with both the forward and the reverse primers immobilized. In practice, there will be a plurality of identical forward primers and/or a plurality of identical reverse primers immobilized on the solid support, since the amplification process requires an excess of primers to sustain amplification. References herein to forward and reverse primers are to be interpreted accordingly as encompassing a plurality of such primers unless the context indicates otherwise.
[000466] As will be appreciated by the skilled reader, any given amplification reaction requires at least one type of forward primer and at least one type of reverse primer specific for the template to be amplified. However, in certain embodiments the forward and reverse primers may include template-specific portions of identical sequence, and may have entirely identical nucleotide sequence and structure (including any non-nucleotide modifications). In other words, it is possible to carry out solid-phase amplification using only one type of primer, and such single-primer methods are encompassed within the scope of the disclosure. Other embodiments may use forward and reverse primers which contain identical templatespecific sequences but which differ in some other structural features. For example, one type of primer may contain a non-nucleotide modification which is not present in the other.
[000467] Primers for solid-phase amplification are preferably immobilized by single point covalent attachment to the solid support at or near the 5' end of the primer, leaving the template-specific portion of the primer free to anneal to its cognate template and the 3' hydroxyl group free for primer extension. Any suitable covalent attachment means known in the art may be used for this purpose. The chosen attachment chemistry will depend on the nature of the solid support, and any derivatization or functionalization applied to it. The primer itself may include a moiety, which may be a non-nucleotide chemical modification, to facilitate attachment. In a particular embodiment, the primer may include a sulphur- containing nucleophile, such as phosphorothioate or thiophosphate, at the 5' end. In the case of solid-supported polyacrylamide hydrogels, this nucleophile will bind to a bromoacetamide group present in the hydrogel. A more particular means of attaching primers and templates to a solid support is via 5' phosphorothioate attachment to a hydrogel comprised of polymerized acrylamide and N-(5-bromoacetamidylpentyl) acrylamide (BRAPA), as described in Int. Pub. No. WO 05/065814.
[000468] Certain embodiments of the disclosure may make use of solid supports that include an inert substrate or matrix (e.g., glass slides, polymer beads, etc.) which has been "functionalized," for example by application of a layer or coating of an intermediate material including reactive groups which permit covalent attachment to biomolecules, such as polynucleotides. Examples of such supports include, but are not limited to, polyacrylamide hydrogels supported on an inert substrate such as glass. In such embodiments, the biomolecules (e.g., polynucleotides) may be directly covalently attached to the intermediate material (e.g., the hydrogel), but the intermediate material may itself be non-covalently attached to the substrate or matrix (e.g., the glass substrate). The term "covalent attachment to a solid support" is to be interpreted accordingly as encompassing this type of arrangement. [000469] The pooled samples may be amplified on beads wherein each bead contains a forward and reverse amplification primer. In one embodiment, a library of modified target nucleic acids is used to prepare clustered arrays of nucleic acid colonies, analogous to those described in U.S. Pub. No. 2005/0100900, U.S. Pat. No. 7,115,400, WO 00/18957 and WO 98/44151 by solid-phase amplification and more particularly solid phase isothermal amplification. The terms "cluster" and "colony" are used interchangeably herein to refer to a discrete site on a solid support including a plurality of identical immobilized nucleic acid strands and a plurality of identical immobilized complementary nucleic acid strands. The term "clustered array" refers to an array formed from such clusters or colonies. In this context, the term "array" is not to be understood as requiring an ordered arrangement of clusters.
[000470] The term "solid phase," "solid support," or "surface" is used to mean either a planar surface or array, such as a flow cell, slide, chip, microchip, array, microarray, wafer, panel, charge pad, and/or web. The planar structure can be a single surface structure having a single surface of sample/reaction sites. The planar structure can be a dual surface structure. One example of a dual surface structure includes a top substrate having a top surface of sample/reactions sites, a bottom substrate having a bottom surface of sample/reactions sites, and a spacer layer separating the top substrate and the bottom substrate. The solid support or solid surface can be open to direct application of a fluid. One example of an open solid support or open solid surface is an open flow cell having a single surface structure without an inlet port. In some examples, the solid support is not necessarily planar, such as, for example, the surface of a well, tube, or other vessel. Nonlimiting examples include the surface of a microcentrifuge tube, a well of a multiwell plate, and the like. In some instances, the planar array has primers are attached to a flat surface, for example, glass, silica or plastic microscope slides or similar flow cell devices; beads, wherein either one or two primers are attached to the beads and the beads are amplified; or an array of beads on a surface after the beads have been amplified.
[000471] Further, the terms “solid support,” “solid surface,” and other grammatical equivalents herein refer to any substrate that is appropriate for or can be modified to be appropriate for the attachment of enzymes, nucleic acids, and complexes thereof. As will be appreciated by those in the art, the number of possible substrates is very large. Possible substrates include, but are not limited to, glass and modified or functionalized glass, polymers (including acrylics, polystyrene and copolymers of styrene and other materials, polypropylene, polyethylene, polybutylene, polyurethanes, polytetrafluoroethylene (e.g., TEFLON™ from Chemours), polyamides (i.e., nylon)), polysaccharides, nitrocellulose, ceramics, resins, silica or silica-based materials including silicon and modified silicon, carbon, metals, optical fiber bundles, quartz, metal oxides, inorganic oxides, other suitable transparent materials, other suitable non-transparent materials, other suitable translucent materials, and combinations thereof. The composition and geometry of the solid support can vary with its use.
[000472] In some examples, the solid support comprises one or more surfaces of a flowcell or flow cell. In accordance with definition set forth herein, the term “flowcell” or “flow cell” refers to a solid surface across which one or more fluid reagents can be flowed. Examples of flow cells and related fluidic systems and detection platforms that can be readily used in the methods of the present disclosure are described, for example, in Bentley et al., Nature 456:53-59 (2008), WO 2004/018497 A2; U.S. Pat. No. 7,057,026 B2; WO 1991/06678 Al; WO 2007/123744 A2; U.S. Pat. No. 7,329,492 B2; U.S. Pat. No. 7,211,414 B2; U.S. Pat. No. 7,315,019 B2; U.S. Pat. No. 7,405,281 B2, and U.S. Pat. Pub. 2008/0108082 Al, each of which is incorporated herein by reference in its entirety. In some examples, the flow cells can be one or more flow lanes. For flow cells having a plurality of flow lanes, each of the flow lanes can be independently accessed or two or more flow lanes can be accessed as a group.
[000473] In some examples, the solid support or solid surface is a non-planar structure, such as beads, microspheres, and/or inner and/or outer surface of a tube or vessel. The terms “beads”, “microspheres,” or “particles” or grammatical equivalents herein, refer to small discrete particles. Suitable bead compositions include, but are not limited to, plastics, ceramics, glass, polystyrene, methylstyrene, acrylic polymers, paramagnetic materials, thoria sol, carbon graphite, titanium dioxide, latex, polysaccharide (e.g., DEXTRAN™ , SEPHAROSE™, cellulose), polyamides, cross-linked micelles, TEFLON™, as well as any other materials outlined herein for solid supports may all be used. “Microsphere Detection Guide” from Bangs Laboratories, Fishers Ind. is a helpful guide. In certain examples, the microspheres are magnetic microspheres or beads. The beads need not be spherical; as irregular particles may be used. Alternatively or additionally, the beads may be porous. The bead sizes range from nanometers, i.e. 100 nm, to millimeters, i.e. 1 mm, with beads from about 0.2 micron to about 200 microns being preferred, and from about 0.5 to about 5 micron being particularly preferred, although in some examples smaller or larger beads may be used. [000474] The term tagmentation refers to a process in which the DNA sample strands are cleaved/fragmented and tagged (e.g., with the adapters) for analysis. Tagmentation is an in vitro transposition reaction.
[000475] Transposase or Transposase Enzyme refers to an enzyme that is capable of forming a functional complex with a transposon end-containing composition (e.g., transposons, transposon ends, transposon end compositions) and catalyzing insertion or transposition of the transposon end-containing composition into the double-stranded DNA sample with which it is incubated, for example, in the in vitro transposition reaction (i.e., tagmentation). A transposase, as presented herein, can also include integrases from retrotransposons and retroviruses. Although many examples described herein refer to Tn5 transposase and/or hyperactive Tn5 transposase, it will be appreciated that any transposase that is capable of inserting a transposon end with sufficient efficiency to 5 ’-tag and fragment the DNA sample for its intended purpose can be used.
[000476] Transposome / Transposome Complex refers to an entity formed between a transposase enzyme and a nucleic acid. Typically, the nucleic acid is a double stranded nucleic acid including a transposase integration recognition site. For example, the transposome complex can be the product of incubating a transposase enzyme with double- stranded transposon DNA under conditions that support non-covalent complex formation. Double-stranded transposon DNA can include, for example, Tn5 DNA, a portion of Tn5 DNA, a transposon end composition, a mixture of transposon end compositions or other double-stranded DNAs capable of interacting with a transposase, such as the hyperactive Tn5 transposase
[000477] Clustered arrays can be prepared using either a process of thermocycling, as described in WO 98/44151, or a process whereby the temperature is maintained as a constant, and the cycles of extension and denaturing are performed using changes of reagents. Such isothermal amplification methods are described in patent application numbers WO 02/46456 and U.S. Pub. No. 2008/0009420.
[000478] It will be appreciated that any of the amplification methodologies described herein or generally known in the art may be used with universal or target-specific primers to amplify immobilized DNA fragments. Suitable methods for amplification include, but are not limited to, the polymerase chain reaction (PCR), strand displacement amplification (SDA), transcription mediated amplification (TMA) and nucleic acid sequence-based amplification (NASBA), as described in U.S. Pat. No. 8,003,354. The above amplification methods may be employed to amplify one or more nucleic acids of interest. For example, PCR, including multiplex PCR, SDA, TMA, NASBA and the like may be utilized to amplify immobilized DNA fragments. In some embodiments, primers directed specifically to the polynucleotide of interest are included in the amplification reaction.
[000479] Other suitable methods for amplification of polynucleotides may include oligonucleotide extension and ligation, rolling circle amplification (RCA) (Lizardi et al., Nat. Genet. 19:225-232 (1998)) and oligonucleotide ligation assay (OLA) (See generally U.S. Pat. Nos. 7,582,420, 5,185,243, 5,679,524 and 5,573,907; EP 0 320 308 Bl; EP 0 336 731 Bl; EP 0 439 182 Bl; WO 90/01069; WO 89/12696; and WO 89/09835) technologies. It will be appreciated that these amplification methodologies may be designed to amplify immobilized DNA fragments. For example, in some embodiments, the amplification method may include ligation probe amplification or oligonucleotide ligation assay (OLA) reactions that contain primers directed specifically to the nucleic acid of interest. In some embodiments, the amplification method may include a primer extension-ligation reaction that contains primers directed specifically to the nucleic acid of interest. As a non-limiting example of primer extension and ligation primers that may be specifically designed to amplify a nucleic acid of interest, the amplification may include primers used for the GoldenGate assay (Illumina, Inc., San Diego, CA) as exemplified by U.S. Pat. No. 7,582,420 and 7,611,869. [000480] DNA nanoballs can also be used in combination with methods, systems, compositions and kits as described herein. Methods for creating and using DNA nanoballs for genomic sequencing can be found at, for example, US patents and publications U.S. Pat. No. 7,910,354, 2009/0264299, 2009/0011943, 2009/0005252, 2009/0155781, 2009/0118488 and as described in, for example, Drmanac et al. (2010, Science 327(5961): 78-81). Briefly, following production of modified target nucleic acids, the modified target nucleic acids are circularized and amplified by rolling circle amplification (Lizardi et al., 1998. Nat. Genet. 19:225-232; US 2007/0099208 Al). The extended concatemeric structure of the amplicons promotes coiling creates compact DNA nanoballs. The DNA nanoballs can be captured on substrates, preferably to create an ordered or patterned array such that distance between each nanoball is maintained thereby allowing sequencing of the separate DNA nanoballs. In some embodiments such as those used by Complete Genomics (Mountain View, Calif.), consecutive rounds of adapter addition, amplification, and digestion are carried out prior to circularization to produce head to tail constructs having several target nucleic acids separated by adapter sequences.
[000481] Exemplary isothermal amplification methods that may be used in a method of the present disclosure include, but are not limited to, Multiple Displacement Amplification (MDA) as exemplified by, for example Dean et al., Proc. Natl. Acad. Sci. USA 99:5261-66 (2002) or isothermal strand displacement nucleic acid amplification exemplified by, for example U.S. Pat. No. 6,214,587. Other non-PCR-based methods that may be used in the present disclosure include, for example, strand displacement amplification (SDA) which is described in, for example Walker et al., Molecular Methods for Virus Detection, Academic Press, Inc., 1995; U.S. Pat. Nos. 5,455,166, and 5,130,238, and Walker et al., Nucl. Acids Res. 20: 1691-96 (1992) or hyper-branched strand displacement amplification which is described in, for example Lage et al., Genome Res. 13:294-307 (2003). Isothermal amplification methods may be used with, for instance, the strand-displacing Phi 29 polymerase or Bst DNA polymerase large fragment, 5'->3 ' exo- for random primer amplification of genomic DNA. The use of these polymerases takes advantage of their high processivity and strand displacing activity. High processivity allows the polymerases to produce fragments that are 10-20 kb in length. As set forth above, smaller fragments may be produced under isothermal conditions using polymerases having low processivity and stranddisplacing activity such as Klenow polymerase. Additional description of amplification reactions, conditions and components are set forth in detail in the disclosure of U.S. Patent No. 7,670,810. [000482] In some embodiments, isothermal amplification can be performed using exclusion amplification, , also referred to as kinetic exclusion amplification. A nucleic acid library of the present disclosure can be made using a method that includes a step of reacting an amplification reagent to produce a plurality of amplification sites that each includes a substantially clonal population of amplicons from an individual target nucleic acid that has seeded the site. In some embodiments, the amplification reaction proceeds until a sufficient number of amplicons are generated to fill the capacity of the respective amplification site. Filling an already seeded site to capacity in this way inhibits target nucleic acids from landing and amplifying at the site thereby producing a clonal population of amplicons at the site. In some embodiments, apparent clonality can be achieved even if an amplification site is not filled to capacity prior to a second target nucleic acid arriving at the site. Under some conditions, amplification of a first target nucleic acid can proceed to a point that a sufficient number of copies are made to effectively outcompete or overwhelm production of copies from a second target nucleic acid that is transported to the site. For example, in an embodiment that uses a bridge amplification process on a circular feature that is smaller than 500 nm in diameter, it has been determined that after 14 cycles of exponential amplification for a first target nucleic acid, contamination from a second target nucleic acid at the same site will produce an insufficient number of contaminating amplicons to adversely impact sequencing-by-synthesis analysis on an Illumina sequencing platform.
[000483] In some embodiments, amplification sites in an array can be, but need not be, entirely clonal. Rather, for some applications, an individual amplification site can be predominantly populated with amplicons from a first modified target nucleic acid and can also have a low level of contaminating amplicons from a second modified target nucleic acid. An array can have one or more amplification sites that have a low level of contaminating amplicons so long as the level of contamination does not have an unacceptable impact on a subsequent use of the array. For example, when the array is to be used in a detection application, an acceptable level of contamination would be a level that does not impact signal to noise or resolution of the detection technique in an unacceptable way. Accordingly, apparent clonality will generally be relevant to a particular use or application of an array made by the methods set forth herein. Exemplary levels of contamination that can be acceptable at an individual amplification site for particular applications include, but are not limited to, at most 0.1%, 0.5%, 1%, 5%, 10% or 25% contaminating amplicons. An array can include one or more amplification sites having these exemplary levels of contaminating amplicons. For example, up to 5%, 10%, 25%, 50%, 75%, or even 100% of the amplification sites in an array can have some contaminating amplicons. It will be understood that in an array or other collection of sites, at least 50%, 75%, 80%, 85%, 90%, 95% or 99% or more of the sites can be clonal or apparently clonal.
[000484] In some embodiments, kinetic exclusion can occur when a process occurs at a sufficiently rapid rate to effectively exclude another event or process from occurring. Take for example the making of a nucleic acid array where sites of the array are randomly seeded with modified target nucleic acids from a solution and copies of the modified target nucleic acids are generated in an amplification process to fill each of the seeded sites to capacity. In accordance with the kinetic exclusion methods of the present disclosure, the seeding and amplification processes can proceed simultaneously under conditions where the amplification rate exceeds the seeding rate. As such, the relatively rapid rate at which copies are made at a site that has been seeded by a first target nucleic acid will effectively exclude a second nucleic acid from seeding the site for amplification. Kinetic exclusion amplification methods can be performed as described in detail in the disclosure of U.S. Pat. Appl. Pub. No. 2013/0338042.
[000485] Kinetic exclusion can exploit a relatively slow rate for initiating amplification (e.g., a slow rate of making a first copy of a modified target nucleic acids) vs. a relatively rapid rate for making subsequent copies of the modified target nucleic acids (or of the first copy of the modified target nucleic acids). In the example of the previous paragraph, kinetic exclusion occurs due to the relatively slow rate of modified target nucleic acids seeding (e.g., relatively slow diffusion or transport) vs. the relatively rapid rate at which amplification occurs to fill the site with copies of the modified target nucleic acid seed. In another exemplary embodiment, kinetic exclusion can occur due to a delay in the formation of a first copy of a modified target nucleic acid that has seeded a site (e.g., delayed or slow activation) vs. the relatively rapid rate at which subsequent copies are made to fill the site. In this example, an individual site may have been seeded with several different modified target nucleic acids (e.g., several modified target nucleic acids can be present at each site prior to amplification). However, first copy formation for any given modified target nucleic acid can be activated randomly such that the average rate of first copy formation is relatively slow compared to the rate at which subsequent copies are generated. In this case, although an individual site may have been seeded with several different modified target nucleic acids, kinetic exclusion will allow only one of those to be amplified. More specifically, once a first modified target nucleic acid has been activated for amplification, the site will rapidly fill to capacity with its copies, thereby preventing copies of a second modified target nucleic acid from being made at the site.
[000486] In one embodiment, the method is carried out to simultaneously (i) transport modified target nucleic acids to amplification sites at an average transport rate, and (ii) amplify the modified target nucleic acids that are at the amplification sites at an average amplification rate, wherein the average amplification rate exceeds the average transport rate (U.S. Pat. No. 9,169,513). Accordingly, kinetic exclusion can be achieved in such embodiments by using a relatively slow rate of transport. For example, a sufficiently low concentration of modified target nucleic acids can be selected to achieve a desired average transport rate, lower concentrations resulting in slower average rates of transport. Alternatively or additionally, a high viscosity solution and/or presence of molecular crowding reagents in the solution can be used to reduce transport rates. Examples of useful molecular crowding reagents include, but are not limited to, polyethylene glycol (PEG), ficoll, dextran, or polyvinyl alcohol. Exemplary molecular crowding reagents and formulations are set forth in U.S. Pat. No. 7,399,590. Another factor that can be adjusted to achieve a desired transport rate is the average size of the target nucleic acids.
[000487] An amplification reagent can include further components that facilitate amplicon formation, and in some cases increase the rate of amplicon formation. An example is a recombinase. Recombinase can facilitate amplicon formation by allowing repeated invasion/extension. More specifically, recombinase can facilitate invasion of a modified target nucleic acid by the polymerase and extension of a primer by the polymerase using the modified target nucleic acid as a template for amplicon formation. This process can be repeated as a chain reaction where amplicons produced from each round of invasion/extension serve as templates in a subsequent round. The process can occur more rapidly than standard PCR since a denaturation cycle (e.g., via heating or chemical denaturation) is not required. As such, recombinase-facilitated amplification can be carried out isothermally. It is generally desirable to include ATP, or other nucleotides (or in some cases non-hydrolyzable analogs thereof) in a recombinase-facilitated amplification reagent to facilitate amplification. A mixture of recombinase and single-stranded binding (SSB) protein is particularly useful as SSB can further facilitate amplification. Exemplary formulations for recombinase-facilitated amplification include those sold commercially as TwistAmp kits by TwistDx (Cambridge, UK). Useful components of recombinase-facilitated amplification reagent and reaction conditions are set forth in US Pat. No. 5,223,414 and US Pat. No. 7,399,590. [000488] Another example of a component that can be included in an amplification reagent to facilitate amplicon formation and in some cases to increase the rate of amplicon formation is a helicase. Helicase can facilitate amplicon formation by allowing a chain reaction of amplicon formation. The process can occur more rapidly than standard PCR since a denaturation cycle (e.g., via heating or chemical denaturation) is not required. As such, helicase-facilitated amplification can be carried out isothermally. A mixture of helicase and single-stranded binding (SSB) protein is particularly useful as SSB can further facilitate amplification. Exemplary formulations for helicase-facilitated amplification include those sold commercially as IsoAmp kits from Biohelix (Beverly, MA). Further, examples of useful formulations that include a helicase protein are described in US Pat. No. 7,399,590 and US Pat. No. 7,829,284.
[000489] Yet another example of a component that can be included in an amplification reagent to facilitate amplicon formation and in some cases increase the rate of amplicon formation is an origin binding protein.
[000490] Methods of Sequencing
[000491] Following attachment of modified target nucleic acids to a surface, the sequence of the immobilized and amplified modified target nucleic acids is determined. Sequencing can be carried out using any suitable sequencing technique, and methods for determining the sequence of immobilized and amplified modified target nucleic acids, including strand resynthesis, are known in the art and are described in, for instance, Bignell et al. (US 8,053,192), Gunderson et al. (W02016/130704), Shen et al. (US 8,895,249), and Pipenburg et al. (US 9,309,502).
[000492] The methods described herein can be used in conjunction with a variety of nucleic acid sequencing techniques. Particularly applicable techniques are those wherein nucleic acids are attached at fixed locations in an array such that their relative positions do not change and wherein the array is repeatedly imaged. Embodiments in which images are obtained in different color channels, for example, coinciding with different labels used to distinguish one nucleotide base type from another are particularly applicable. In some embodiments, the process to determine the nucleotide sequence of a modified target nucleic acid can be an automated process. Preferred embodiments include sequencing-by-synthesis ("SBS") techniques.
[000493] SBS techniques generally involve the enzymatic extension of a nascent nucleic acid strand through the iterative addition of nucleotides against a template strand. In traditional methods of SBS, a single nucleotide monomer may be provided to a target nucleotide in the presence of a polymerase in each delivery. However, in the methods described herein, more than one type of nucleotide monomer can be provided to a target nucleic acid in the presence of a polymerase in a delivery.
[000494] In one embodiment, a nucleotide monomer includes locked nucleic acids (LNAs) or bridged nucleic acids (BNAs). The use of LNAs or BNAs in a nucleotide monomer increases hybridization strength between a nucleotide monomer and a sequencing primer sequence present on an immobilized modified modified target nucleic acid.
[000495] SBS can use nucleotide monomers that have a terminator moiety or those that lack any terminator moieties. Methods using nucleotide monomers lacking terminators include, for example, pyrosequencing and sequencing using y-phosphate-labeled nucleotides, as set forth in further detail herein. In methods using nucleotide monomers lacking terminators, the number of nucleotides added in each cycle is generally variable and dependent upon the template sequence and the mode of nucleotide delivery. For SBS techniques that use nucleotide monomers having a terminator moiety, the terminator can be effectively irreversible under the sequencing conditions used as is the case for traditional Sanger sequencing which utilizes dideoxynucleotides, or the terminator can be reversible as is the case for sequencing methods developed by Solexa (now Illumina, Inc.).
[000496] SBS techniques can use nucleotide monomers that have a label moiety or those that lack a label moiety. Accordingly, incorporation events can be detected based on a characteristic of the label, such as fluorescence of the label; a characteristic of the nucleotide monomer such as molecular weight or charge; a byproduct of incorporation of the nucleotide, such as release of pyrophosphate; or the like. In embodiments where two or more different nucleotides are present in a sequencing reagent, the different nucleotides can be distinguishable from each other, or alternatively the two or more different labels can be the indistinguishable under the detection techniques being used. For example, the different nucleotides present in a sequencing reagent can have different labels and they can be distinguished using appropriate optics as exemplified by the sequencing methods developed by Solexa (now Illumina, Inc.).
[000497] Examples described herein can be used with any suitable sequencing chemistry, such as sequencing by synthesis (SBS), sequencing by binding, sequencing by ligation, or nanopore sequencing.
[000498] SBS can be performed with or without the use of reversible terminators. For example, SBS can be initiated by contacting the target nucleic acids with one or more nucleotides (e.g., labeled, synthetic, modified, or a combination thereof), DNA polymerase, etc. Those features where a primer is extended using the target nucleic acid as the template will incorporate a labeled nucleotide that can be detected. The incorporation time used in a sequencing run can be significantly reduced using altered polymerases. Optionally, the labeled nucleotides can further include a reversible termination property that terminates further primer extension once a nucleotide has been added to a primer. For example, a nucleotide analog having a reversible terminator moiety can be added to a primer such that subsequent extension cannot occur until a deblocking agent is delivered to remove the moiety. Thus, for examples that use reversible termination, a deblocking reagent can be delivered to the flow cell (before or after detection occurs). Washes can be carried out between the various delivery steps. The cycle can then be repeated n times to extend the primer by n nucleotides, thereby detecting a sequence of length n. Exemplary SBS procedures, fluidic systems, and detection platforms that can be readily adapted for use with an array produced by the methods of the present disclosure are described, for example, in Bentley et al., Nature 456:53-59 (2008); WO 2004/018497 A2; WO 1991/006678 Al; WO 2007/123744 Al; U.S. Pat. Nos. 7,057,026 B2, 7,329,492 B2, 7,211,414 B2, 7,315,019 B2, 7,405,281 B2, and 8,343,746 B2. Sequence reads can be generated using instruments such as MINISEQ™, MISEQ™, NEXTSEQ™, HISEQX™, and NOVASEQ™ sequencing instruments from Illumina, Inc. (San Diego, CA).
[000499] One example of SBS is termed sequencing by binding. One implementation of sequencing by binding includes cycles of initiating sequencing of a template with a reversible blocker on the 3’ end to prevent additional bases from incorporating, interrogating the template by flooding the flow cell with fluorescently tagged bases that do not include a blocker and measuring an emitted signal of bound bases, activating the 3’ end via removal of the reversible blocker, and incorporating the complementary base from unlabeled, blocked nucleotides. Reads using sequencing by binding can be generated from using instruments such as ONSO™ sequencing instruments from Pacific Biosciences of California, Inc. (Menlo Park, CA). Another implementation of sequencing by binding could be sequencing by avidity. In sequencing by avidity, fluorescent dye labeled cores, termed avidites, are used. One potential cycle of sequencing by avidity includes providing a reagent of polymerase and reversibly terminated nucleotides to templates immobilized on a solid surface, de-blocking the incorporated nucleotides, flowing a set of four types of avidites, washing away unbound avidites, detecting the incorporated bases/nucleotides, and removing the bound avidites. The steps in the cycle of sequencing by avidity may be performed in other orders. Sequencing by avidity is described in Arslan, S., Garcia, F.J., Guo, M., et al. “Sequencing by avidity enables high accuracy with low reagent consumption.” Nat Biotechnol 42, 132-138 (2024). https://doi.org/10.1038/s41587-023-01750-7, which is incorporated by reference in its entirety. Reads using sequencing by avidity can be generated using instruments such as AVITI™ sequencing instruments from Element Biosciences (San Diego).
[000500] One example of SBS using an open flow cell and without using reversible terminators is disclosed in Almogy, G. (2022) “Cost-efficient whole genome-sequencing using novel mostly natural sequencing-by-synthesis chemistry and open fluidics platform” https://doi.Org/10. l 101/2022.05.29.493900, which is incorporation by reference in its entirety. Sequence reads using an open flow cell can be generated using instruments such as UG 100TM Sequencer from Ultima Genomics, Inc. (Fremont, CA).
[000501] Some SBS examples include detection of a proton released upon incorporation of a nucleotide into an extension product. For example, sequencing based on detection of released protons can use an electrical detector and associated techniques that are described in U.S. Pat. Nos. 8,262,900 B2, 7,948,015 B2, 8,349,167 B2, and U.S. Pat. Pub. 2010/0137143 Al, each of which is incorporated by reference in its entirety.
[000502] Sequence reads can be generated using instruments such as DNBSEQTM sequencing instruments from MGI Tech Co., Ltd. (Shenzhen, China) and as SURFSeq™, FASTASeq™, and GenoLab™ sequencing instruments from GeneMind Biosciences Co., Ltd. (Shenzhen, China).
[000503] Preferred embodiments include pyrosequencing techniques. Pyrosequencing detects the release of inorganic pyrophosphate (PPi) as particular nucleotides are incorporated into the nascent strand (Ronaghi, M., Karamohamed, S., Pettersson, B., Uhlen, M. and Nyren, P. (1996) "Real-time DNA sequencing using detection of pyrophosphate release." Analytical Biochemistry 242(1), 84-9; Ronaghi, M. (2001) "Pyrosequencing sheds light on DNA sequencing." Genome Res. 11(1), 3-11; Ronaghi, M., Uhlen, M. and Nyren, P. (1998) "A sequencing method based on real-time pyrophosphate." Science 281(5375), 363; U.S. Pat. Nos. 6,210,891; 6,258,568 and 6,274,320). In pyrosequencing, released PPi can be detected by being immediately converted to adenosine triphosphate (ATP) by ATP sulfurase, and the level of ATP generated is detected via luciferase-produced photons. The nucleic acids to be sequenced can be attached to features in an array and the array can be imaged to capture the chemiluminescent signals that are produced due to incorporation of a nucleotides at the features of the array. An image can be obtained after the array is treated with a particular nucleotide type (e.g., A, T, C or G). Images obtained after addition of each nucleotide type will differ with regard to which features in the array are detected. These differences in the image reflect the different sequence content of the features on the array. However, the relative locations of each feature will remain unchanged in the images. The images can be stored, processed and analyzed using the methods set forth herein. For example, images obtained after treatment of the array with each different nucleotide type can be handled in the same way as exemplified herein for images obtained from different detection channels for reversible terminator-based sequencing methods.
[000504] In another exemplary type of SBS, cycle sequencing is accomplished by stepwise addition of reversible terminator nucleotides containing, for example, a cleavable or photobleachable dye label as described, for example, in WO 04/018497 and U.S. Pat. No. 7,057,026. This approach is being commercialized by Solexa (now Illumina Inc.), and is also described in WO 91/06678 and WO 07/123,744. The availability of fluorescently-labeled terminators in which both the termination can be reversed and the fluorescent label cleaved facilitates efficient cyclic reversible termination (CRT) sequencing. Polymerases can also be co-engineered to efficiently incorporate and extend from these modified nucleotides. Examples nucleotides having a reversible termination property include modifications at the 3 '-OH of the nucleotide sugar moiety, such as a 3'-O-azidomethyl blocking group -CH2N3, a 3 '-OH acetal blocking group, or a 3 '-OH thiocarbamate blocking group (U.S. Patent No. 11,293,061; U.S. Published Patent Application No. 2022/0396832).
[000505] In some reversible terminator-based sequencing embodiments, the labels do not substantially inhibit extension under SBS reaction conditions. However, the detection labels can be removable, for example, by cleavage or degradation. Images can be captured following incorporation of labels into arrayed nucleic acid features. In particular embodiments, each cycle involves simultaneous delivery of four different nucleotide types to the array and each nucleotide type has a spectrally distinct label. Four images can then be obtained, each using a detection channel that is selective for one of the four different labels. Alternatively, different nucleotide types can be added sequentially and an image of the array can be obtained between each addition step. In such embodiments, each image will show nucleic acid features that have incorporated nucleotides of a particular type. Different features will be present or absent in the different images due the different sequence content of each feature. However, the relative position of the features will remain unchanged in the images. Images obtained from such reversible terminator- SBS methods can be stored, processed and analyzed as set forth herein. Following the image capture step, labels can be removed and reversible terminator moieties can be removed for subsequent cycles of nucleotide addition and detection. Removal of the labels after they have been detected in a particular cycle and prior to a subsequent cycle can provide the advantage of reducing background signal and crosstalk between cycles. Examples of useful labels and removal methods are set forth herein.
[000506] In particular embodiments some or all of the nucleotide monomers can include reversible terminators. In such embodiments, reversible terminators/cleavable fluorophores can include fluorophores linked to the ribose moiety via a 3' ester linkage (Metzker, Genome Res. 15: 1767-1776 (2005)). Other approaches have separated the terminator chemistry from the cleavage of the fluorescence label (Ruparel et al., Proc Natl Acad Sci USA 102: 5932-7 (2005)). Ruparel et al. described the development of reversible terminators that used a small 3' allyl group to block extension, but could easily be deblocked by a short treatment with a palladium catalyst. The fluorophore was attached to the base via a photocleavable linker that could easily be cleaved by a 30 second exposure to long wavelength UV light. Thus, either disulfide reduction or photocleavage can be used as a cleavable linker. Another approach to reversible termination is the use of natural termination that ensues after placement of a bulky dye on a dNTP. The presence of a charged bulky dye on the dNTP can act as an effective terminator through steric and/or electrostatic hindrance. The presence of one incorporation event prevents further incorporations unless the dye is removed. Cleavage of the dye removes the fluorophore and effectively reverses the termination. Examples of modified nucleotides are also described in U.S. Pat. Nos. 7,427,673, and 7,057,026.
[000507] Additional exemplary SBS systems and methods which can be used with the methods and systems described herein are described in U.S. Pub. Nos. 2007/0166705, 2006/0188901, 2006/0240439, 2006/0281109, 2012/0270305, and 2013/0260372, U.S. Pat. No. 7,057,026, PCT Publication No. WO 05/065814, U.S. Patent Application Publication No. 2005/0100900, and PCT Publication Nos. WO 06/064199 and WO 07/010,251.
[000508] Some embodiments can use detection of four different nucleotides using fewer than four different labels. For example, SBS can be performed using methods and systems described in the incorporated materials of U.S. Pub. No. 2013/0079232. As a first example, a pair of nucleotide types can be detected at the same wavelength, but distinguished based on a difference in intensity for one member of the pair compared to the other, or based on a change to one member of the pair (e.g., via chemical modification, photochemical modification or physical modification) that causes apparent signal to appear or disappear compared to the signal detected for the other member of the pair. As a second example, three of four different nucleotide types can be detected under particular conditions while a fourth nucleotide type lacks a label that is detectable under those conditions, or is minimally detected under those conditions (e.g., minimal detection due to background fluorescence, etc.). Incorporation of the first three nucleotide types into a nucleic acid can be determined based on presence of their respective signals and incorporation of the fourth nucleotide type into the nucleic acid can be determined based on absence or minimal detection of any signal. As a third example, one nucleotide type can include label(s) that are detected in two different channels, whereas other nucleotide types are detected In no more than one of the channels. The aforementioned three exemplary configurations are not considered mutually exclusive and can be used in various combinations. An exemplary embodiment that combines all three examples, is a fluorescent-based SBS method that uses a first nucleotide type that is detected in a first channel (e.g., dATP having a label that is detected in the first channel when excited by a first excitation wavelength), a second nucleotide type that is detected in a second channel (e.g., dCTP having a label that is detected in the second channel when excited by a second excitation wavelength), a third nucleotide type that is detected in both the first and the second channel (e.g., dTTP having at least one label that is detected in both channels when excited by the first and/or second excitation wavelength) and a fourth nucleotide type that lacks a label that is not, or minimally, detected in either channel (e.g., dGTP having no label). [000509] Further, as described in U.S. Pub. No. 2013/0079232, sequencing data can be obtained using a single channel. In such so-called one-dye sequencing approaches, the first nucleotide type is labeled but the label is removed after the first image is generated, and the second nucleotide type is labeled only after a first image is generated. The third nucleotide type retains its label in both the first and second images, and the fourth nucleotide type remains unlabeled in both images.
[000510] Some embodiments can use sequencing by ligation techniques. Such techniques use DNA ligase to incorporate oligonucleotides and identify the incorporation of such oligonucleotides. The oligonucleotides typically have different labels that are correlated with the identity of a particular nucleotide in a sequence to which the oligonucleotides hybridize. As with other SBS methods, images can be obtained following treatment of an array of nucleic acid features with the labeled sequencing reagents. Each image will show nucleic acid features that have incorporated labels of a particular type. Different features will be present or absent in the different images due the different sequence content of each feature, but the relative position of the features will remain unchanged in the images. Images obtained from ligation-based sequencing methods can be stored, processed and analyzed as set forth herein. Exemplary SBS systems and methods which can be utilized with the methods and systems described herein are described in U.S. Pat. Nos. 6,969,488, 6,172,218, and 6,306,597. [000511] Some embodiments can use nanopore sequencing (Deamer, D. W. & Akeson, M. "Nanopores and nucleic acids: prospects for ultrarapid sequencing." Trends Biotechnol. 18, 147-151 (2000); Deamer, D. and D. Branton, "Characterization of nucleic acids by nanopore analysis", Acc. Chem. Res. 35:817-825 (2002); Li, J., M. Gershow, D. Stein, E. Brandin, and J. A. Golovchenko, "DNA molecules and configurations in a solid-state nanopore microscope" Nat. Mater. 2:611-615 (2003)). In such embodiments, the modified target nucleic acid passes through a nanopore. The nanopore can be a synthetic pore or biological membrane protein, such as a-hemolysin. As the modified target nucleic acid passes through the nanopore, each base-pair can be identified by measuring fluctuations in the electrical conductance of the pore. (U.S. Pat. No. 7,001,792; Soni, G. V. & Meller, "A. Progress toward ultrafast DNA sequencing using solid-state nanopores." Clin. Chem. 53, 1996-2001 (2007); Healy, K. "Nanopore-based single-molecule DNA analysis." Nanomed. 2, 459-481 (2007); Cockroft, S. L., Chu, J., Amorin, M. & Ghadiri, M. R. "A single-molecule nanopore device detects DNA polymerase activity with single-nucleotide resolution." J. Am. Chem. Soc. 130, 818-820 (2008)). Data obtained from nanopore sequencing can be stored, processed and analyzed as set forth herein. In particular, the data can be treated as an image in accordance with the exemplary treatment of optical images and other images that is set forth herein.
[000512] Some embodiments can use methods involving the real-time monitoring of DNA polymerase activity. Nucleotide incorporations can be detected through fluorescence resonance energy transfer (FRET) interactions between a fluorophore-bearing polymerase and y-phosphate-labeled nucleotides as described, for example, in U.S. Pat. Nos. 7,329,492 and 7,211,414, or nucleotide incorporations can be detected with zero-mode waveguides as described, for example, in U.S. Pat. No. 7,315,019, and using fluorescent nucleotide analogs and engineered polymerases as described, for example, in U.S. Pat. No. 7,405,281 and U.S. Pub. No. 2008/0108082. The illumination can be restricted to a zeptoliter-scale volume around a surface-tethered polymerase such that incorporation of fluorescently labeled nucleotides can be observed with low background (Levene, M. J. et al. "Zero-mode waveguides for single-molecule analysis at high concentrations." Science 299, 682-686 (2003); Lundquist, P. M. et al. "Parallel confocal detection of single molecules in real time." Opt. Lett. 33, 1026-1028 (2008); Korlach, J. et al. "Selective aluminum passivation for targeted immobilization of single DNA polymerase molecules in zero-mode waveguide nano structures." Proc. Natl. Acad. Sci. USA 105, 1176-1181 (2008)). Images obtained from such methods can be stored, processed and analyzed as set forth herein. [000513] Some SBS embodiments include detection of a proton released upon incorporation of a nucleotide into an extension product. For example, sequencing based on detection of released protons can use an electrical detector and associated techniques that are commercially available from Ion Torrent (Guilford, CT, a Life Technologies subsidiary) or sequencing methods and systems described in U.S. Pub. Nos. 2009/0026082; 2009/0127589; 2010/0137143; and 2010/0282617. Methods set forth herein for amplifying target nucleic acids using kinetic exclusion can be readily applied to substrates used for detecting protons. More specifically, methods set forth herein can be used to produce clonal populations of amplicons that are used to detect protons.
[000514] The above SBS methods can be advantageously carried out in multiplex formats such that multiple different modified target nucleic acids are manipulated simultaneously. In particular embodiments, different modified target nucleic acids can be treated in a common reaction vessel or on a surface of a particular substrate. This allows convenient delivery of sequencing reagents, removal of unreacted reagents and detection of incorporation events in a multiplex manner. In embodiments using surface-bound target nucleic acids, the modified target nucleic acids can be in an array format. In an array format, the modified target nucleic acids can be typically bound to a surface in a spatially distinguishable manner. The modified target nucleic acids can be bound by direct covalent attachment, attachment to a bead or other particle or binding to a polymerase or other molecule that is attached to the surface. The array can include a single copy of a modified target nucleic acid at each site (also referred to as a feature) or multiple copies having the same sequence can be present at each site or feature. Multiple copies can be produced by amplification methods such as, bridge amplification or emulsion PCR as described in further detail herein.
[000515] The methods set forth herein can use arrays having features at any of a variety of densities including, for example, at least about 10 features/cm2, 100 features/ cm2, 500 features/ cm2, 1,000 features/ cm2, 5,000 features/ cm2, 10,000 features/ cm2, 50,000 features/ cm2, 100,000 features/ cm2, 1,000,000 features/ cm2, 5,000,000 features/ cm2, or higher.
[000516] An advantage of the methods set forth herein is that they provide for rapid and efficient detection of a plurality of features/cm2, in parallel. Accordingly, the present disclosure provides integrated systems capable of preparing and detecting nucleic acids using techniques known in the art such as those exemplified herein. Thus, an integrated system of the present disclosure can include fluidic components capable of delivering amplification reagents and/or sequencing reagents to one or more immobilized modified target nucleic acids, the system including components such as pumps, valves, reservoirs, fluidic lines and the like. An example of useful fluidic components includes a flow cell and a cartridge. A flow cell can be configured and/or used in an integrated system for detection of target nucleic acids. Exemplary flow cells are described, for example, in US Pat. No. 8,241,573 and US Pat. No. 8,951,781. As exemplified for flow cells, one or more of the fluidic components of an integrated system can be used for an amplification method and for a detection method. Taking a nucleic acid sequencing embodiment as an example, one or more of the fluidic components of an integrated system can be used for an amplification method set forth herein and for the delivery of sequencing reagents in a sequencing method such as those exemplified above. Alternatively, an integrated system can include separate fluidic systems to carry out amplification methods and to carry out detection methods. Examples of integrated sequencing systems that are capable of creating arrays of nucleic acids and also determining the sequence of the nucleic acids include, without limitation, the MiSeq™, HiSeq™, NextSeq™, MiniSeq™, NovaSeq™ and iSeq™ platforms (Illumina, Inc., San Diego, Calif.) and devices described in U.S. Pat. No. 8,951,781.
[000517] While the embodiments presented herein are generally described using a sequencing platform (such as a sequencing by synthesis platform) as a readout, one of ordinary skill in the art will recognize that nucleic acids modified by the ACDs presented herein can also be detected using any other suitable readout methodology. For example, the location and identity of modified cytosines can be assessed using a microarray. Any of a variety of analyte arrays (also referred to as “microarrays”) known in the art can be used in a method or system set forth herein. A typical array contains analytes, each having an individual probe or a population of probes. In the latter case, the population of probes at each analyte is typically homogenous having a single species of probe. For example, in the case of a nucleic acid array, each analyte can have multiple nucleic acid molecules each having a common sequence. However, in some implementations the populations at each analyte of an array can be heterogeneous. Similarly, protein arrays can have analytes with a single protein or a population of proteins typically, but not always, having the same amino acid sequence. The probes can be attached to the surface of an array for example, via covalent linkage of the probes to the surface or via non-covalent interaction(s) of the probes with the surface. In some implementations, probes, such as nucleic acid molecules, can be attached to a surface via a gel layer as described, for example, in U.S. patent application Ser. No. 13/784,368 and US Pat. App. Pub. No. 2011/0059865 Al.
[000518] Example arrays include, without limitation, a BeadChip Array available from Illumina, Inc. (San Diego, Calif.) or others such as those where probes are attached to beads that are present on a surface (e.g., beads in wells on a surface) such as those described in U.S. Pat. Nos. 6,266,459; 6,355,431; 6,770,441; 6,859,570; or 7,622,294; or PCT Publication No. WO 00/63437. Further examples of commercially available microarrays that can be used include, for example, an Infmium Methylation microarray, Affymetrix® GeneChip® microarray or other microarray synthesized in accordance with techniques sometimes referred to as VLSIPS™ (Very Large Scale Immobilized Polymer Synthesis) technologies. A spotted microarray can also be used in a method or system according to some implementations of the present disclosure. An example spotted microarray is a CodeLink™ Array available from Amersham Biosciences. Another microarray that is useful is one that is manufactured using inkjet printing methods such as SurePrint™ Technology available from Agilent Technologies.
[000519] In a specific embodiment, an ACD as presented herein can be used to convert 5- methyl cytosine (5mC) to thymidine (T) by deamination as described herein, such as by providing a sample of DNA suspected of including single-stranded DNA including at least one 5-methyl cytosine (5mC), ), at least one 5 -hydroxymethyl cytosine (5hmC), at least one 5-formyl cytosine (5fC), at least one 5-carboxy cytosine (5CaC), or a combination thereof; contacting the DNA with the ACD under conditions suitable for conversion of 5- methylcytosine (5mC) to thymidine (T) by deamination at a greater rate than conversion of cytosine (C) to uracil (U) by deamination, to result in converted single-stranded DNA, where 5mC, 5hmC, 5fC, and/or 5CaC are converted to T.
[000520] In a specific embodiment, an ACD of the present disclosure can be used to detect 5hmC as described herein, such as by providing a sample of DNA suspected of including single-stranded DNA that has at least one 5 -hydroxymethyl cytosine (5hmC); contacting the DNA with the ACD under conditions suitable for conversion of unmodified cytosine to uracil and 5mC to thymidine and no detectable conversion of 5hmC to 5hmU.
[000521] The converted single-stranded DNA can then be processed as needed to facilitate hybridization to a microarray. For example, the converted DNA can be amplified. Any one of a number of amplification methods as are known in the art can be performed. For example, whole-genome amplification or amplification using universal primers that hybridize to a common region in the converted DNA, such as an adaptor sequence, can be used. Additionally or alternatively, the converted DNA can be fragmented. Fragmentation can be performed prior to or following amplification, or in the absence of amplification. Any one of a number of fragmentation methods as are known in the art can be performed. As one example, fragmentation can be performed using an enzymatic process, such as a restriction endonuclease or other enzyme capable of cleaving the converted DNA. As another example, fragmentation can be performed using mechanical means, such as shearing using, for example a sonication device such as those supplied by Covaris. The fragmented converted DNA can then be precipitated and/or resuspended in a buffer suitable for hybridization to a microarray. Following hybridization, the methylation state of regions of interest, such as a specific CpG locus or loci, can be interrogated at specific locations on the microarray. Methods of preparing converted DNA for microarray analysis are known in the art. One example of such methods is described in the Methylation Protocol Guide for the Infmium HD Assay from Illumina (San Diego, CA). Whereas such a protocol guide may describe use of a microarray designed for interrogation of bisulfite-converted DNA, it will be understood that array features, specifically probe sequences, can be specifically designed for DNA that has not been bisulfite converted. As an example, a commercially available microarray such as the Infmium Methyl ationEPIC BeadChip (Illumina) is specifically designed to hybridize with DNA fragments with reduced complexity, as found in bisulfite converted DNA, where most if not all cytosines are converted to thymidine. Thus, for example, the same CpG sites can be interrogated in non-bisulfite-converted DNA by using a microarray including probes designed to hybridize to the same regions of native, non-bisulfite-converted DNA. One of skill in the art could readily obtain such a microarray. In one embodiment, a custom array could be designed using the manifest for an array such as the Infmium MethylationEPIC BeadChip, by using the “Forward Sequence” to identify a probe sequence including native DNA sequence that covers a similar or identical sequence region for the allele-specific probe sequences, which are designed to hybridize to DNA sequences where most or all cytosines have been converted to thymidine. Using such an array designed to hybridize to native (non- bisulfite-converted) DNA sequences, the methodologies and analysis methods described in the Methylation Protocol Guide for the Infmium HD Assay from Illumina (San Diego, CA) could be followed to identify methylated CpG sites in the sample DNA.
[000522] Compositions
[000523] The present disclosure also provides compositions that include an ACD described herein, a combination of an ACD described herein and a helicase described herein, or an ACD-helicase complex (e.g., a fusion protein) described herein. Examples of an ACD present in a composition include, but are not limited to, SEQ ID NO:23, SEQ ID NO:24, and the ACDs disclosed in Table 2, Table 3, and FIG. 9 (SEQ ID NO:479-517, SEQ ID NO:520- 605, SEQ ID NO:607-615, SEQ ID NO:635-954, SEQ ID NO:962-968). Examples of an ACD-helicase complex (e.g., a fusion protein) present in a composition include any of SEQ ID NO:23, SEQ ID NO:24, and the ACDs disclosed in Table 2, Table 3, and FIG. 9 (e.g., SEQ ID NO:479-517, SEQ ID N0:520-605, SEQ ID NO:607-615, SEQ ID NO:635-954, SEQ ID NO:962-968) fused to a helicase described herein, such as SEQ ID NO:427 or SEQ ID NO:429. The composition can include one or more additional other components in addition to the ACD. For example, the other component can include a single-stranded DNA, a double stranded DNA, or RNA substrate that includes, or is suspected of including, at least one modified cytosine, such as a 5-methyl cytosine, a 5 -hydroxymethyl cytosine, a 5-formyl cytosine (5fC), a 5-carboxy cytosine (5CaC), or a combination thereof. In one embodiment, the other component includes a single-stranded DNA, a double stranded DNA, or RNA substrate that includes, or is suspected of including, at least one a 5-methyl cytosine. In another example, a single-stranded DNA, a double stranded DNA, or RNA substrate can be one including one or more known modified cytosine, e.g., a single-stranded DNA, a double stranded DNA, or RNA substrate that can be used as a control to measure conversion efficiency. In another example, the other component can include a buffer, such as a buffer described herein, for example a citrate buffer, a sodium acetate buffer, or a Bis-Tris buffer, or HEPES. In another example, the other component can include a reductant, including but not limited to, DTT and/or TCEP, as well as Zn, or a denaturant, such as NaOH, DMSO, betaine, or a combination thereof.
[000524] A composition can include a polynucleotide encoding an ACD described herein or an ACD-helicase complex (e.g., a fusion protein) described herein. The polynucleotide can be present in a vector, such as a plasmid or virus vector. A vector that includes the polynucleotide can be present in a host cell, such as E. coli.
[000525] Cartridges
[000526] The present disclosure also provides cartridges for carrying out the methods disclosed herein. The cartridges can be configured for use with a sequencing instrument, such as an integrated sequencing system. In some embodiments, a cartridge for use with an sequencing system may include a chamber from which a composition (such as a composition that includes an ACD described herein) may be withdrawn or expelled for use in a method disclosed herein, (e.g., cluster generation, resynthesis, or both). A cartridge may include a releasably attached flow cell.
[000527] Kits
[000528] The present disclosure also provides kits for determining the methylation status of DNA or RNA. A kit includes at least one ACD described herein and one or more other components in a suitable packaging material in an amount sufficient for at least one reaction. Examples of other components include a positive control polynucleotide, such as a singlestranded DNA including one or more known modified cytosines for use in measuring conversion efficiency, or a negative control polynucleotide, such as a single-stranded DNA including unmodified cytosines. Another component can be a glucosyltransferase, such as T4-beta glucosyltransferase. Optionally, other reagents such as buffers and solutions needed to use the ACD and nucleotide solution are also included. Instructions for use of the packaged components are also typically included.
[000529] As used herein, the phrase "packaging material" refers to one or more physical structures used to house the contents of the kit. The packaging material is constructed by known methods, preferably to provide a sterile, contaminant-free environment. The packaging material has a label which indicates that the components can be used for determining the methylation status of DNA or RNA. In addition, the packaging material contains instructions indicating how the materials within the kit are employed to practice a reaction with an ACD. As used herein, the term "package" refers to a solid matrix or material such as glass, plastic, paper, foil, and the like, capable of holding within fixed limits the polypeptides. "Instructions for use" typically include a tangible expression describing the reagent concentration or at least one assay method parameter, such as the relative amounts of reagent and sample to be admixed, maintenance time periods for reagent/sample admixtures, temperature, buffer conditions, and the like.
[000530] The invention is defined in the claims. However, below there is provided a non- exhaustive listing of non-limiting exemplary aspects. Any one or more of the features of these aspects may be combined with any one or more features of another example, embodiment, or aspect described herein.
[000531] Exemplary Aspects
[000532] Aspect 1. An altered cytidine deaminase (ACD) comprising 5mC-selective deaminase activity, the ACD comprising at least one selectivity-enhancing alteration at a position functionally equivalent to amino acid 130, 131, 132, 133, 134, 135, or a combination thereof, in a wild-type APOBEC3A (SEQ ID NO:3), wherein the selectivity-enhancing alteration at amino acid 130, 131, 132, 133, 134, 135, or a combination thereof is a substitution mutation to any amino acid, preferably wherein the ACD comprises two or more selectivity-enhancing alterations at a position functionally equivalent to amino acid 130, 131, 132, 133, 134, 135, or a combination thereof. [000533] Aspect 2. The ACD of any of Aspects 1-18, wherein the selectivity-enhancing alteration is a substitution mutation, a deletion, or an insertion.
[000534] Aspect 3. The ACD of any of Aspects 1-18, wherein the amino acid sequence of amino acids 130, 131, 132, 133, and 134 is selected from SEQ ID NO:25-34 of FIG. 4B, SEQ ID NO:35-50 of Table 2, or the amino acids at positions 130, 131, 132, 133, and 134 in the sequences found in Figure 8 or 9.
[000535] Aspect 4. The ACD of any of Aspects 1-18, the ACD further comprising at least one selectivity-enhancing alteration at a position functionally equivalent to amino acid 102, 103, 104, 105, or a combination thereof in the wild-type APOBEC3A (SEQ ID NO:3).
[000536] Aspect 5. The ACD of any of Aspects 1-18, wherein the amino acid sequence of amino acids 102, 103, 104, 105, 130, 131, 132, 133, and 134 is selected from SEQ ID NOs: of Table 3.
[000537] Aspect 6. The ACD of any of Aspects 1-18, wherein the amino acid sequence of amino acids 102, 103, 104, 105, 130, 131, 132, 133, 134, and 135 is selected from SEQ ID NOs: of FIG. 8.
[000538] Aspect 7. The ACD of any of Aspects 1-18, wherein the amino acid sequence of amino acids 130, 131, 132, 133, and 134 is selected from SEQ ID NO: 23, SEQ ID NO:24, or the sequences described in FIG. 8 or FIG. 9 which demonstrate increased 5mC selectivity compared to wild-type (SEQ ID NO:3).
[000539] Aspect 8. The ACD of any of Aspects 1-18, wherein the amino acid sequence of amino acids 102, 103, 104, 105, 130, 131, 132, 133, and 134 is selected from SEQ ID NO: 23, SEQ ID NO:24, or the sequences described in FIG. 8 or FIG. 9 which demonstrate increased 5mC selectivity compared to wild-type (SEQ ID NO:3).
[000540] Aspect 9. The ACD of any of Aspects 1-18, wherein the amino acid sequence of amino acids 102, 103, 104, 105, 130, 131, 132, 133, and 134 is selected from
FIM A AHI YW, HLTG AHEYW, GLNG AHEYW, YKQY AGEYW, YIKL AVAHW, FIIA ALAHW, QTQY ARVKW, AYDG AHEYW, QTYG AHLYW, YYRM AHGYW, YLPA AKGFW, STNN AHIYW, QTRR AHQYW, YVND AHLYW, FSWG ANVHW, NIPA ARAYW, QTLG ANAFW, VYNA ARAYW, ETKH ALAHW, YVEG AHLYW, AQHG AHEYW, HLRG AHEYW, QTMH ANAFW, YYIA AGRHW, HLYG ARAYW, YIWG ARAYW, IRQY ANAFW, AQMG ARAYW , FSAA ADHWPL, FVPG AHARWL, and FVPA AKYPWL.
[000541] Aspect 10. The ACD of any of Aspects 1-18, further comprising two or more stability-enhancing alterations, preferably three or more stability-enhancing alterations.
[000542] Aspect 11. The ACD of any of Aspects 1-18, wherein the two or more stabilityenhancing alteration is a substitution mutation, a deletion, an insertion, or a combination thereof.
[000543] Aspect 12. The ACD of any of Aspects 1-18, wherein the two or more stabilityenhancing alterations are selected from substitution mutations at a position functionally equivalent to Hl IX, L12X, D14X, H16X, I17X, T19X, S20X, N21X, G25X, I26X, G27X, R28X, H29X, T31X, C34X, E38X, R39X, D41X, N42X, S45X, K47X, M48X, H51X, F54X, H56X, N57X, A59X, K60X, L62X, Y67X, L73X, R74X, D77X, L78X, V79X, P80X, S81X, D85X, A87X, I89X, Y90X, R91X, T93X, W94X, I96X, S97X, C101X, F102X, S103X, W104X, G105X, C106X, A107X, G108X, E109X, V110X, R111X, A112X, L114X, Q115X, E116X, N117X, T118X, H119X, V120X, L122X, R123X, R128X, L135X, Y136X, E138X, A139X, Q141X, M142X, D145X, A146X, A148X, D156X, E157X, K159X, C161X, D163X, T164X, D167X, Q169X, C171X, D177X, D180X, H182X, S183X, Q184X, A185X, L186X, S187X, G188X, R189X, A192X, N196X a deletion selected from AM1-L12, AE36- G53, AN61-G68, AW104-G105 , AQ195-N199, and AI26-G27, and combinations thereof, wherein the position number designation is functionally equivalent to the position in a wildtype APOBEC3A (SEQ ID NO:3) and X is an amino acid substitution different from the wild-type amino acid at that position, optionally wherein the stability enhancing mutations are selected from: R74L/C171A; R74L/T19Y; R74L/G25R; R74L/T19I/C171A;
R74L/T19L/C171A; R74L/T19Y/C171A/I17T; R74L/T19Y/C171A/G25A; R74L/T19Y/C171A/G25R/I17T; R74L /C171A/G25R/T19F; R74L/T19Y/C171A/G25D; R74L/T19Y/C171A/S45R; R74C/T19Y/C171A; R74L/C171A/T19F; R74L/C171A/T19W; R74L/T19Y/C171A/G108E; R74L/T19Y/C171A/G108D; R74L/T19Y/C171A/G108Q; R74L/T 19 Y/C 171 A/Gl 08 Y; R74L/T 19 Y/C 171 A/Gl 08H; R74L/T 19 Y/C 171 A/Gl 08L; R74L/T 19 Y/C 171 A/Gl 08K; R74L/T 19 Y/C 171 A/Gl 08R; R74L/T 19 Y/C 171 A/Al 26 V; R74L/T19Y/C171I; R74L/T19Y/C171A/G108M; R74L/T19Y/C171A/G108W;
R74L/T 19 Y/C 171 A/ A 126F ; R74L/T 19 Y/C 171 A/ A 1261; R74L/T 19 Y/C 171 A/ A 126L; R74L/T19Y/C171A; R74L/T19Y/C171A/S45W; R74L/T19Y/C171A/G25R;
R74L/T 19 Y/C 171 A/G25K; R74L/T 19 Y/C 171 A/Gl 88Q; R74L/T 19 Y/C 171 A/Gl 88 A; R74L/T 19 Y/C 171 A/Gl 88R; R74L/T 19 Y/C 171 A/Gl 08 A;
R74L/T19Y/C171A/G108A/G188R/G25K/S45W ; R74L/T19Y/C171A/G108C;
R74L/T 19 Y/C 171 A/Gl 08 A/Gl 88R/G25K/S45 W/117T;
R74L/T 19 Y/C 171 A/Gl 08 A/Gl 88R/G25K/S45 W/117T/A59P/K60R/A61-68;
R74L/T 19 Y/C 171 A/Gl 08C/G188R/G25K/S45 W/Il 7T;
R74L/T 19 Y/C 171 A/Gl 08C/G188R/G25K/S45 W/117T/A59P/K60R/A61 -68/A126C;
R74L/T 19 Y/C 171 A/Gl 08C/G188R/G25K/S45 W/117T/A59P/K60R/A61-68;
T 19 Y/R74L/C 171 A; 117T/T 19 Y/G25R/R74L/C 171 A;
T 19 Y/G25K/S45 WR74L/G108 A/C 171 A/Gl 88R;
117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/G108C/C 171 A/Gl 88R;
117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/G108C/A126C/C 171 A/Gl 88R; or 117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/AW 104-
G105/G108C/C 171 A/Gl 88R.
[000544] Aspect 13. The ACD of any of Aspects 1-18, wherein the ACD comprises: a. three or more stability-enhancing alterations, b. four or more stability— enhancing alterations, c. five or more stability-enhancing alterations, d. six or more stability-enhancing alterations, e. seven or more stability-enhancing alterations, f. eight or more stability-enhancing alterations, g. nine or more stability-enhancing alterations, h. ten or more stability-enhancing alterations, i. eleven or more stability-enhancing alterations, or k. twelve or more stabilityenhancing alterations.
[000545] Aspect 14. The ACD of any of Aspects 1-18, wherein the ACD comprises two or more stability mutations selected from I17T, T19Y, G25K, S45W, A59P, K60R, A61-68, R74L, G108A, G108C, C171A, G188R, and A104-105.
[000546] Aspect 15. The ACD of any of Aspects 1-18, wherein the ACD comprises a combination of stability mutations comprising T19Y, G25K, S45W, R74L, G108A, C171A, and G188R.
[000547] Aspect 16. The ACD of any of Aspects 1-18, wherein the ACD comprises a combination of stability mutations comprising I17T, T19Y, G25K, S45W, A59P, K60R, A61- 68, R74L, G108C, C171A, and G188R. [000548] Aspect 17. The ACD of any of Aspects 1-18, wherein the amino acid sequence of the ACD is selected from SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:479-517, SEQ ID N0:520-605, SEQ ID NO:607-615, SEQ ID NO:635-954, or SEQ ID NO:962-968.
[000549] Aspect 18. The ACD of any of Aspects 1-18, wherein the amino acid sequence of the ACD is selected from SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:523-567, SEQ ID NO:635-659, or SEQ ID NO:962-968.
[000550] Aspect 19. A polynucleotide encoding the ACD of any Aspects 1-18, or a composition comprising the ACD of any one of the preceding Aspects.
[000551] Aspect 20. The composition of Aspect 19, wherein the composition further comprises a sample comprising DNA comprising at least one 5-methyl cytosine (5mC).
[000552] Aspect 21. The composition of any of Aspects 20-23, wherein the DNA comprises single-stranded DNA or double-stranded DNA.
[000553] Aspect 22. The composition of any of Aspects 20-23, wherein the sample comprises genomic DNA or cell free DNA.
[000554] Aspect 23. The composition of any of Aspects 20-23, wherein the genomic DNA is from a single cell or is a mixture from a plurality of cells.
[000555] Aspect 24. A method comprising: providing a sample of DNA suspected of comprising single-stranded DNA comprising at least one 5-methyl cytosine (5mC); contacting the single-stranded DNA with an ACD under conditions suitable for conversion of 5 -methylcytosine (5mC) to thymidine (T) by deamination at a greater rate than conversion of cytosine (C) to uracil (U) by deamination, to result in converted single-stranded DNA, wherein the altered cytidine deaminase is the ACD of any one of Aspects 1-16; and processing the converted single-stranded DNA to produce a sequencing library.
[000556] Aspect 25. A method comprising: providing a sample of DNA suspected of comprising double-stranded DNA comprising at least one 5-methyl cytosine (5mC); processing the double-stranded DNA to produce a sequencing library; denaturing the sequencing library to result in a single-stranded DNA; contacting the single-stranded DNA with an ACD under conditions suitable for (i) conversion of 5 -methylcytosine (5mC) to thymidine (T) by deamination at a greater rate than conversion of cytosine (C) to uracil (U) by deamination, to result in converted single-stranded DNA, wherein the altered cytidine deaminase is the ACD of any one of Aspects 1-13; and converting the converted singlestranded DNA to a converted double-stranded DNA sequencing library.
[000557] Aspect 26. A method of detecting the location of a modified cytosine in a target nucleic acid, the method comprising: (a) contacting target nucleic acids suspected of comprising at least one modified cytosine with the ACD of any one of Aspects 1-13 to produce converted nucleic acids comprising at least one converted methyl cytosine; (b) detecting the at least one converted methyl cytosine in the converted nucleic acids of (a).
[000558] Aspect 27. The method of Aspect 26, wherein the detecting comprises identifying thymidine nucleotides in the converted nucleic acid to determine the location of 5mC nucleotides in the target nucleic acid.
[000559] Aspect 28. Use of the ACD of any one of Aspects 1-18 to identify a 5mC in a nucleic acid sequence by sequencing.
[000560] Aspect 29. A protein complex comprising an ACD of any of Aspects 1-18 and a helicase, wherein he ACD has activity on double stranded DNA.
[000561] Aspect 30. An altered cytidine deaminase (ACD) comprising 5mC-selective deaminase activity, the ACD comprising a Y130A and P134W mutation in a wild-type APOBEC3A (SEQ ID NO:3).
[000562] Aspect 31. The protein complex of Aspect 29 or the ACD of any of Aspects 30- 47, wherein the amino acid sequence of amino acids 130, 131, 132, 133, and 134 is selected from SEQ ID NO:25-34 of FIG. 4B, SEQ ID NO:35-50 of Table 2, or the amino acids at positions 130, 131, 132, 133, and 134 in the sequences found in Figure 8 or 9.
[000563] Aspect 32. The protein complex of Aspect 29 or the ACD of any of Aspects 30- 47, the ACD further comprising at least one selectivity-enhancing alteration at a position functionally equivalent to amino acid 102, 103, 104, 105, or a combination thereof in the wild-type APOBEC3A (SEQ ID NO:3).
[000564] Aspect 33. The protein complex of Aspect 29 or the ACD of any of Aspects 30- 47, the ACD further comprising at least two selectivity-enhancing alteration at a position functionally equivalent to amino acid 102, 103, 104, 105, or a combination thereof in the wild-type APOBEC3A (SEQ ID NO:3).
[000565] Aspect 34. The protein complex of Aspect 29 or the ACD of any of Aspects SO- 47, wherein the amino acid sequence of the ACD of amino acids 102, 103, 104, 105 is selected from the group consisting of: FIMA (SEQ ID NO:404); HLTG(SEQ ID NO:242); QVPA (SEQ ID NO:405); EFNQ (SEQ ID NO:243); QTDH (SEQ ID NO:244); GLNG (SEQ ID NO:245); YLP (SEQ ID NO:406); EMFA (SEQ ID NO:407); YKQY (SEQ ID NO:246); YVRL (SEQ ID NO:247); YIKL (SEQ ID NO:248); FILA ALAHW (SEQ ID NO:408); QTQY (SEQ ID NO:249); KTNN (SEQ ID NO:250); AYDG (SEQ ID NO:251); HLTG (SEQ ID NO:242); QTMH (SEQ ID NO:252); YVQR (SEQ ID NO:253); ELYA (SEQ ID NO:409):YVEN (SEQ ID NO:254); RMLA (SEQ ID NO:410); QTVG (SEQ ID NO:255); YVQD (SEQ ID NO:256) QTQY (SEQ ID NO:249); QTYG (SEQ ID NO:257_); YYRM AHGYW (SEQ ID NO:258): YLP A; STNN_(SEQ ID NO:259); QTRR (SEQ ID NO:260); AYEY (SEQ ID NO:261); YVND (SEQ ID NO:262); FSWG (SEQ ID NO:263); NIPA;YPFG (SEQ ID NO:264); QTLG_(SEQ ID NO:265); VYNA_; AFRA; ETKH (SEQ ID NO:266); YVEG (SEQ ID NO:267); QTMG_ (SEQ ID NO:268);
AQHG_(SEQ ID NO:269); HLRG (SEQ ID NO:270); QTMH (SEQ ID NO:252); YYIA_ (SEQ ID NO:YYIA); HLYG_ (SEQ ID NO:271); YMAG (SEQ ID NO:272); YIWG_ (SEQ ID NO:273); IRQY (SEQ ID NO:274); AQMG (SEQ ID NO:275); FVPG (SEQ
ID NO: 276); and FVPA, FVPG (SEQ ID NO:276), FTDG (SEQ ID NO:277), FVDG (SEQ ID NO: 278), FVPAG (SEQ ID NO:279), FVPLG (SEQ ID NO:280), FVPPG(SEQ ID NO:280), FVPAAG(SEQ ID NO:282), FVPLAG (SEQ ID NO:283), FVPPAG (SEQ ID NO:284), FVPASG (SEQ ID NO:285), FVPLSG (SEQ ID NO:286), FVPPSG (SEQ ID NO:287), FVPAFG (SEQ ID NO:288), FVPLFG (SEQ ID NO:289), FVPPFG (SEQ ID NO:289), FVPAQG (SEQ ID NO:290), FVPLQG (SEQ ID NO:291), FVPPQG (SEQ ID NO:292), FVPADG (SEQ ID NO:293), FVPLDG (SEQ ID NO:294), FVPPDG (SEQ ID NO:295), FVPAKG (SEQ ID NO:296), FVPLKG: (SEQ ID NO:297), and FVPPKG(SEQ ID NO:298).
[000566] Aspect 35. The protein complex of Aspect 29 or the ACD of any one of Aspects 29-34, wherein the amino acid sequence of amino acids 102, 103, 104, 105, 130, 131, 132, 133, 134, and 135 of the ACD is selected from the group consisting of: FIMA AHIYWL (SEQ ID NO:FIMA_53), HLTG AHEYWL (SEQ ID NO:242_545), QVPA AHDFWL (SEQ ID NO:QVPA 55), EFNQ ANVHWL (SEQ ID NO:243_56), QTDH AAEHWL (SEQ ID NO:244_57), GLNG AHEYWL (SEQ ID NO:245_54), YLPA ANGFWL (SEQ ID NO:YLPA 58), EMFA ANDFWL (SEQ ID NO:EMFA 59), YKQY AGEYWL (SEQ ID NO:246_60), YVRL AVPFWL (SEQ ID NO:247_61), YIKL AVAHWL (SEQ ID NO:248_62), FIIA ALAHWL (SEQ ID NO:FIIA 63), QTQY ARVKWL (SEQ ID NO:249_64), KTNN ASEHWL (SEQ ID NO:250_65), AYDG AHEYWL (SEQ ID NO:251_54), HLTG AVAYWL (SEQ ID NO:242_66), QTMH AHQYWL (SEQ ID NO:252_67), YVQR ANVYWL (SEQ ID NO:253_68), ELYA AHIYWL (SEQ ID NO:ELYA 53), YVEN ANGFWL (SEQ ID NO:254_58), RMLA AAEHWL (SEQ ID NO:RMLA 57), QTVG AKSHWL (SEQ ID NO:255_69), YVQD AHEYWL (SEQ ID NO:256_54), QTQY AHEYWL (SEQ ID NO:249_54), QTYG AHLYWL (SEQ ID NO:257_70), YYRM AHGYWL (SEQ ID NO:258_71), YLPA AKGFWL (SEQ ID NO:YLPA 72), STNN AHIYWL (SEQ ID NO:259_53), QTRR AHQYWL (SEQ ID NO:260_67), AYEY AHIYWL (SEQ ID NO:261_53), YVND AHLYWL (SEQ ID NO:262_70), FSWG ANVHWL (SEQ ID NO:263_56), NIPA ARAYWL (SEQ ID NO:NIPA 73), YPFG ANGFWL (SEQ ID NO:264_58), QTLG ANAFWL (SEQ ID NO:265_74), VYNA ARAYWL (SEQ ID NO:VYNA 73), AFRA ASKHWL (SEQ ID NO:AFRA 75), ETKH ALAHWL (SEQ ID NO:266_63), YVEG AHLYWL (SEQ ID NO:267_70), QTMG AHQYWL (SEQ ID NO:268_67), AQHG AHEYWL (SEQ ID NO:269_54), HLRG AHEYWL (SEQ ID NO:270_54), QTMH ANAFWL (SEQ ID NO:252_74), YYIA AGRHWL (SEQ ID NO:YYIA 76), HLYG ARAYWL (SEQ ID NO:271_73), YMAG AREYWL (SEQ ID NO:272_77), YIWG ARAYWL (SEQ ID NO:273_73), IRQY ANAFWL (SEQ ID NO:274_74), AQMG ARAYWL (SEQ ID NO:275_73), FSWG ADHWPL (SEQ ID NO:263_79), FSWG ADHWPL (SEQ ID NO:263_79), FSAA ADHWPL (SEQ ID NO:FS_79), FSWG ADHWTL (SEQ ID NO:263_80), FSWG DFDIYL (SEQ ID NO:263_81), FSWG TEQDTL (SEQ ID NO:263_82), FSWG YDTDGL (SEQ ID NO:263_83), FSWG PPISDL (SEQ ID NO:263_84), FSWG EITYHL (SEQ ID NO:263_85), FSWG MRQDVL (SEQ ID NO:263_86), FSWG VGVLML (SEQ ID NO:263_87), FSWG SDKDPL (SEQ ID NO:263_88), FSWG SDHDPL (SEQ ID NO:263_89), FSWG SDQDPL (SEQ ID NO:263_90), FSWG YQDGGL (SEQ ID NO:263_91), FSWG YDYDHL (SEQ ID NO:263_92), FSWG MHWHYL (SEQ ID NO:263_93), FSWG SDEYPL (SEQ ID NO:263_94), FSWG VQKEHL (SEQ ID NO:263_95), FSWG NMIYQL (SEQ ID NO:263_96), FSWG WTRNQL (SEQ ID NO:263_97), FSWG AAEYWL (SEQ ID NO:263_98), FSWG AAGHWL (SEQ ID NO:263_99), FSWG AGNIYL (SEQ ID N0:263_100), FSWG AHLKWL (SEQ ID NO:263_101), FSWG ANPHWL (SEQ ID NO:263_102), FSWG APAKYL (SEQ ID NO:263_103), FSWG APKRYL (SEQ ID NO:263_104), FSWG ARANWL (SEQ ID NO:263_105), FSWG ARVRWL (SEQ ID NO:263_106), FSWG ASTPWL (SEQ ID NO:263_107), FSWG AVVKWL (SEQ ID NO:263_108), FSWG FPTDHL (SEQ ID NO:263_109), FSWG FQESKL (SEQ ID NO:263_110), FSWG FTNELL (SEQ ID NO:263_111), FSWG HHDTNL (SEQ ID NO:263_112), FSWG LSANEL (SEQ ID NO:263_113), FSWG PMLERL (SEQ ID NO:263_114), FSWG QRSQRL (SEQ ID NO:263_115), FSWG SPGEHL (SEQ ID NO:263_116), FSWG VVMQDL (SEQ ID NO:263_117), FSWG YDAGHL (SEQ ID NO:263_118), FSWG YTIDQL (SEQ ID NO:263_121), FSWG YTMERL (SEQ ID NO:263_122), FSWG AKPHWL (SEQ ID NO:263_123), FSWG AHGYWL (SEQ ID NO:263_71), FSWG AGRFWL (SEQ ID NO:263_124), FSWG AHEYWL (SEQ ID NO:263_54), FVPG AKYPWL (SEQ ID NO:276_125), FTDG AKYPWL (SEQ ID NO:277_125), FVDG AKYPWL (SEQ ID NO:278_125), FVPG AVVKWL (SEQ ID NO:276_108), FTDG AVVKWL (SEQ ID NO:277_108), FVDG AVVKWL (SEQ ID NO:278_108), FVPG ANPHWL (SEQ ID NO:276_102), FTDG ANPHWL (SEQ ID NO:277_102), FVDG ANPHWL (SEQ ID NO:278_102), FVPG AKSHWL (SEQ ID NO:276_69), FTDG AKSHWL (SEQ ID NO:277_69), FVDG AKSHWL (SEQ ID NO:278_69), FVPG AHARWL (SEQ ID NO:276_126), FTDG AHARWL (SEQ ID NO:277_126), FVDG AHARWL (SEQ ID NO:278_126), FVPG AHVVWL (SEQ ID NO:276_127), FTDG AHVVWL (SEQ ID NO:277_127), FVDG AHVVWL (SEQ ID NO:278_127), FVPG ASTPWL (SEQ ID NO:276_107), FTDG ASTPWL (SEQ ID NO:277_107), FVDG ASTPWL (SEQ ID NO:278_107), FVPG AHLKWL (SEQ ID NO:276_101), FTDG AHLKWL (SEQ ID NO:277_101), FVDG AHLKWL (SEQ ID NO:278_101), FQAG AKSHWL (SEQ ID NO:FQAG 69), FQAG AHARWL (SEQ ID NO:FQAG 126), FQAG AKYPWL (SEQ ID NO:FQAG _125), FQAG AVVKWL (SEQ ID NO:FQAG _108), FQAG ANPHWL (SEQ ID NO:FQAG _102), FQAG AHVVWL (SEQ ID NO:FQAG 127), FQAG ASTPWL (SEQ ID NO:FQAG 107), FQAG AHKLML (SEQ ID NO:FQAG 128), FSAG AKSHWL (SEQ ID NO:FSAG 69), FSAG AHARWL (SEQ ID NO:FSAG _126), FSAG AKYPWL (SEQ ID NO:FSAG 125), FSAG AVVKWL (SEQ ID NOE SAG 108), FSAG ANPHWL (SEQ ID NOE SAG 102), FSAG AHVVWL (SEQ ID NOE SAG 127), FSAG ASTPWL (SEQ ID NOE SAG 107), FSAG AHKLML (SEQ ID NOESAG 128), FSWG AGEYWL (SEQ ID NO:263_60), FSWG AGPFWL (SEQ ID NO:263_129), FSWG AHEYWL (SEQ ID NO:263_54), FSWG AHGYWL (SEQ ID NO:263_71), FSWG AHMHWL (SEQ ID NO:263_130), FSWG AHPHWL (SEQ ID NO:263_131), FSWG AHRIWL (SEQ ID NO:263_132), FSWG AKEFWL (SEQ ID NO:263_133), FSWG ALPFWL (SEQ ID NO:263_134), FSWG ANGFWL (SEQ ID NO:263_58), FSWG ARAPWL (SEQ ID NO:263_135), FSWG AVAHWL (SEQ ID NO:263_62), FSWG AGRFWL (SEQ ID NO:263_124), FSWG AHMIWL (SEQ ID NO:263_136), FSWG AHRVWL (SEQ ID NO:263_137), FSWG AHTPWL (SEQ ID NO:263_138), FSWG AKSHWL (SEQ ID NO:263_69), FSWG AKTHWL (SEQ ID NO:263_139), FSWG APPHYL (SEQ ID NO:263_140), FSWG IYMEDL (SEQ ID NO:263_141), FSWG AKFHWL (SEQ ID NO:263_142), FSWG AQSYWL (SEQ ID NO:263_143), FSWG AAQYWL (SEQ ID NO:263_144), FSWG AHLYWL (SEQ ID NO:263_70), FSWG AKVVWL (SEQ ID NO:263_145), FSWG YTNEHL (SEQ ID NO:263_146), FSWG VEGTYL (SEQ ID NO:263_147), FSWG HPWQAL (SEQ ID NO:263_148), FSWG MTWAWL (SEQ ID NO:263_149), FSWG MRGYWL (SEQ ID NO:263_150), FSWG AHTHWL (SEQ ID NO:263_151), FSWG AHQYWL (SEQ ID NO:263_67), FSWG AHIYWL (SEQ ID NO:263_53), FSWG AKSKWL (SEQ ID NO:263_152), FSWG ARPNWL (SEQ ID NO:263_153), FSWG ARQFWL (SEQ ID NO:263_154), FSWG AHAHWL (SEQ ID NO:263_155), FSWG ANPSWL (SEQ ID NO:263_156), FSWG ANIIWL (SEQ ID NO:263_157), FSWG AHDHWL (SEQ ID NO:263_158), FSWG APTKYL (SEQ ID NO:263_158), FSWG ASAHWL (SEQ ID NO:263_160), FSWG ARAYWL (SEQ ID NO:263_73), FSWG AHRGWL (SEQ ID NO:263_161), FSWG AHTKWL (SEQ ID NO:263_162), FSWG AKPHWL (SEQ ID NO:263_123), FSWG SAPFWL (SEQ ID NO:263_163), FSWG VARHWL (SEQ ID NO:263_164), AVPG AKYPWL (SEQ ID NO:AVPG 125), FVPA AKYPWL (SEQ ID NO EVP A 125), AVPA AKYPWL (SEQ ID NO: AVPA_125), FVPAG AKYPWL (SEQ ID NO:279_125), FVPLG AKYPWL (SEQ ID NO:280_125), FVPPG AKYPWL (SEQ ID NO:281_125), FVPAAG AKYPWL (SEQ ID NO:282_125), FVPLAG AKYPWL (SEQ ID NO:283_125), FVPPAG AKYPWL (SEQ ID NO:284_125), FVPASG AKYPWL (SEQ ID NO:285_125), FVPLSG AKYPWL (SEQ ID NO:286_125), FVPPSG AKYPWL (SEQ ID NO:287_125), FVPAFG AKYPWL (SEQ ID NO:288_125), FVPLFG AKYPWL (SEQ ID NO:289_125), FVPPFG AKYPWL (SEQ ID NO:290_125), FVPAQG AKYPWL (SEQ ID NO:291_125), FVPLQG AKYPWL (SEQ ID NO:292_125), FVPPQG AKYPWL (SEQ ID NO:293_125), FVPADG AKYPWL (SEQ ID NO:294_125), FVPLDG AKYPWL (SEQ ID NO:295_125), FVPPDG AKYPWL (SEQ ID NO:296_125), FVPAKG AKYPWL (SEQ ID NO:297_125), FVPLKG AKYPWL (SEQ ID NO:298_125), FVPPKG AKYPWL (SEQ ID NO:299_125), YLIA ARAYWL (SEQ ID NO:YLIA 73), RYAA AHQYWL (SEQ ID NO:RYAA 67), QTRN AHLYWL (SEQ ID N0:300_70), LFWA ARAYWL (SEQ ID NO:LFWA 73), AVQA ARAYWL (SEQ ID NO:AVQA 73), RYAA ALPFWL (SEQ ID NO:RYAA 134), ETKD AHQYWL (SEQ ID NO:301_67), ASQN ARAYWL (SEQ ID NO:302_73), LFWA AHEYWL (SEQ ID NO:LFWA 54), RYAA ARAYWL (SEQ ID NO:RYAA 73), KIEG AHQYWL (SEQ ID NO:303_67), LRNG AHIYWL (SEQ ID NO:304_53), IRYA ARAYWL (SEQ ID NO:IRYA 73), STMG AHIYWL (SEQ ID NO:305_53), STMG AHGYWL (SEQ ID NO:305_71), STQH AHGYWL (SEQ ID NO:306_71), IRYA AHIYWL (SEQ ID NO:IRYA 53), KIEG AHGYWL (SEQ ID NO:303_71), YLIA AHEYWL (SEQ ID NO:YLIA 54), VLYA AHGYWL (SEQ ID NO:VLYA 71), RHYA AHEYWL (SEQ ID NO:RHYA 54), TTMG AHIYWL (SEQ ID NO:307_53), TTMG AHGYWL (SEQ ID NO:307_71), TTMG ARAYWL (SEQ ID NO:307_73), FMHA ARAYWL (SEQ ID NO:FMHA 73), QFPA ARAYWL (SEQ ID NO:QFPA 73), RRAG AHEYWL (SEQ ID NO:308_54), RRPA ARAYWL (SEQ ID NO:RRPA 73), RIYA ARAYWL (SEQ ID NO:RIYA 73), RIYA AHEYWL (SEQ ID NO:RIYA 54), YTVG AHQYWL (SEQ ID NO:309_67), YYIA ARAYWL (SEQ ID NO:YYIA 73), ATYR ARAYWL (SEQ ID NO:310_73), RMLA AHEYWL (SEQ ID NO:RMLA 54), VLYA ARAYWL (SEQ ID NO:VLYA 73), LRNG ARAYWL (SEQ ID NO:304_73), RMLA ARAYWL (SEQ ID NO:RMLA 73), KIEG ARAYWL (SEQ ID NO:303_73), AVQA AHQYWL (SEQ ID NO: AVQA 67), QTNG ARAYWL (SEQ ID NO:311 73), QTNG AHEYWL (SEQ ID NO:311 54), KIEG AHEYWL (SEQ ID NO:303_54), AVQA AHGYWL (SEQ ID NO: AVQA 71), QVWA ARAYWL (SEQ ID NO:QVWA 73), RRPA AHEYWL (SEQ ID NO:RRPA 54), YTVG ARAYWL (SEQ ID NO:309_73), RYAA AHEYWL (SEQ ID NO:RYAA 54).
[000567] Aspect 36. The protein complex of Aspect 29 or the ACD of any of Aspects 30- 47, wherein the ACD comprises SEQ ID NO: 23, 24, SEQ ID NO: 479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 489, 490, 491, 492, 493, 494, 495, 496, 497, 498,
499, 500, 501, 502, 503, 504, 505, 506, 507, 508, 509, 510, 511, 512, 513, 514,
515, 516, 517, 520, 521, 523, 524, 525, 526, 527, 528, 529, 530, 531, 532, 533,
534, 535, 536, 537, 538, 539, 540, 541, 543, 544, 545, 546, 547, 548, 549, 550,
551, 552, 553, 554, 555, 556, 557, 558, 559, 560, 561, 562, 563, 564, 565, 566,
567, 568, 569, 570, 571, 572, 573, 574, 575, 576, 577, 578, 579, 580, 581, 582, 583, 584, 585, 586, 587, 588, 589, 590, 591, 592, 593, 594, 595, 596, 597, 598,
599, 600, 601, 602, 603, 604, 605, 607, 608, 609, 610, 611, 612, 613, 614, 615,
635, 637, 638, 639, 640, 641, 642, 643, 644, 645, 646, 647, 648, 649, 650, 651,
652, 653, 654, 655, 656, 657, 658, 659, 660, 661, 662, 663, 664, 665, 666, 667,
668, 669, 670, 671, 672, 673, 674, 675, 676, 677, 678, 679, 680, 681, 682, 683,
684, 685, 686, 687, 688, 689, 690, 691, 692, 693, 694, 695, 696, 697, 698, 699,
700, 701, 702, 703, 704, 705, 706, 707, 708, 709, 710, 711, 712, 713, 714, 715,
716, 717, 718, 719, 720, 721, 722, 723, 724, 725, 726, 727, 728, 729, 730, 731,
732, 733, 734, 735, 736, 737, 738, 739, 740, 741, 742, 743, 744, 745, 746, 747,
748, 749, 750, 751, 752, 753, 754, 755, 756, 757, 758, 759, 760, 761, 762, 763,
764, 765, 766, 767, 768, 769, 770, 771, 772, 773, 774, 775, 776, 777, 778, 779,
780, 781, 782, 783, 784, 785, 786, 787, 788, 789, 790, 791, 792, 793, 794, 795,
796, 797, 798, 799, 800, 801, 802, 803, 804, 805, 806, 807, 808, 809, 810, 811,
812, 813, 814, 815, 816, 817, 818, 819, 820, 821, 822, 823, 824, 825, 826, 827,
828, 829, 830, 831, 832, 833, 834, 835, 836, 837, 838, 839, 840, 841, 842, 843,
844, 845, 846, 847, 848, 849, 850, 851, 852, 853, 854, 855, 856, 857, 858, 859,
860, 861, 862, 863, 864, 865, 866, 867, 868, 869, 870, 871, 872, 873, 874, 875,
876, 877, 878, 879, 880, 881, 882, 883, 884, 885, 886, 887, 888, 889, 890, 891,
892, 893, 894, 895, 896, 897, 898, 899, 900, 901, 902, 903, 904, 905, 906, 907,
908, 909, 910, 911, 912, 913, 914, 915, 916, 917, 918, 919, 920, 921, 922, 923,
924, 925, 926, 927, 928, 929, 930, 931, 932, 933, 934, 935, 936, 937, 938, 939,
940, 941, 942, 943, 944, 945, 946, 947, 948, 950, 951, 952, 953, 954, 955, 956,
957, 958, 959, 960, and 961.
[000568] Aspect 37. The protein complex of Aspect 29 or the ACD of any of Aspects 30 47, wherein the amino acid sequence of amino acids 102, 103, 104, 105, 130, 131, 132, 133 and 134 of the ACD are selected from the group consisting of: FIMD AHIYW (SEQ ID NO:404_26); HLTG AHEYW (SEQ ID NO:242_27); QVPA AHDFW (SEQ ID NO:405_32); EFNQ ANVHW (SEQ ID NO:243_411); QTDH AAEHW (SEQ ID NO:244_412); GLNG AHEYW ((SEQ ID NO:245_27); YLPA ANGFW (SEQ ID NO:406_29); EMFA ANDFW (SEQ ID NO:407_413); YKQY AGEYW (SEQ ID NO:246_414); YVRL AVPFW (SEQ ID NO:247_415); YIKL AVAHW (SEQ ID NO:248_25); FILA ALAHW (SEQ ID NO:408_416); QTQY ARVKW (SEQ ID NO:249_37); KTNN ASEHW (SEQ ID NO:250_417); AYDG AHEYW (SEQ ID NO:251_27); HLTG AVAYW (SEQ ID NO:242_418); QTMH AHQYW (SEQ ID NO:252_419); YVQR ANVYW (SEQ ID NO:253_420); ELYA AHIYW (SEQ ID NO:409_26):YVEN_ANGFW (SEQ ID NO:254_29); RMLA AAEHW (SEQ ID NO:410_412); QTVG AKSHW (SEQ ID NO:255_30); YVQD AHEYW (SEQ ID NO:256_27) QTQY AHEYW (SEQ ID NO:249_27); QTYG AHLYW (SEQ ID NO:257_31); YYRM AHGYW (SEQ ID NO:258_422): YLPA AKGFW (SEQ ID NO:(YLPA_421); STNN AHIYW (SEQ ID NO:259_26) QTRR AHQYW (SEQ ID NO:260_419); AYEY AHIYW (SEQ ID NO:261_26); YVND AHLYW (SEQ ID NO:262_31); FSWG ANVHW (SEQ ID NO:263_411); NIPA ARAYW (SEQ ID NO:NIPA_28);YPFG_ANGFW (SEQ ID NO:264_29); QTLG ANAFW (SEQ ID NO:265_423); VYNA_ARAYW(SEQ ID NO:VYNA_28); AFRA ASKHW (SEQ ID NO:AFRA_426); ETKH ALAHW (SEQ ID NO:266_416); YVEG AHLYW (SEQ ID NO:267_31); QTMG AHQYW (SEQ ID NO:268_419); AQHG AHEYW (SEQ ID NO:269_27); HLRG AHEYW (SEQ ID NO:270_27); QTMH ANAFW (SEQ ID NO:252_423); YYIA AGRHW (SEQ ID NO:YYIA_424); HLYG ARAYW (SEQ ID NO:271_28); YMAG AREYW (SEQ ID NO:272_425); YIWG ARAYW (SEQ ID NO:273_28); IRQY ANAFW (SEQ ID NO:274_423); AQMG ARAYW (SEQ ID NO:275_28); FVPG AHARW (SEQ ID NO: 276 42); and FVPA AKYPW (SEQ ID NO:FVPA_125).
[000569] Aspect 38. The protein complex of Aspect 29 or the ACD of any of Aspects 30- 47, wherein the amino acid sequence of amino acids 102, 103, 104, 105, 130, 131, 132, 133, and 134 of the ACD is selected from the group consisting of: FIMA AHIYW, HLTG AHEYW, GLNG AHEYW, YKQY AGEYW, YIKL AVAHW, FIIA ALAHW, QTQY ARVKW, AYDG AHEYW, QTYG AHLYW, YYRM AHGYW, YLPA AKGFW, STNN AHIYW, QTRR AHQYW, YVND AHLYW, FSWG ANVHW, NIPA ARAYW, QTLG ANAFW, VYNA ARAYW, ETKH ALAHW, YVEG AHLYW, AQHG AHEYW, HLRG AHEYW, QTMH ANAFW, YYIA AGRHW, HLYG ARAYW, YIWG ARAYW, IRQY ANAFW, AQMG ARAYW , FSAA ADHWPL, FVPG AHARWL, and FVPA AKYPWL.
[000570] Aspect 39. The protein complex of Aspect 29 or the ACD of any of Aspects 30- 47, further comprising two or more stability-enhancing alteration, preferably three or more stability-enhancing alterations. [000571] Aspect 40. The protein complex of Aspect 29 or the ACD of any of Aspects SO- 47, wherein the one or more stability-enhancing alteration is a substitution mutation, a deletion, an insertion, or a combination thereof.
[000572] Aspect 41. The protein complex of Aspect 29 or the ACD of any of Aspects SO- 47, wherein the two or more stability-enhancing alterations are selected from substitution mutations at a position functionally equivalent to Hl IX, L12X, D14X, H16X, I17X, T19X, S20X, N21X, G25X, I26X, G27X, R28X, H29X, T31X, C34X, E38X, R39X, D41X, N42X, S45X, K47X, M48X, H51X, F54X, H56X, N57X, A59X, K60X, L62X, Y67X, L73X, R74X, D77X, L78X, V79X, P80X, S81X, D85X, A87X, I89X, Y90X, R91X, T93X, W94X, I96X, S97X, C101X, F102X, S103X, W104X, G105X, C106X, A107X, G108X, E109X, V110X, R11 IX, Al 12X, LI 14X, QI 15X, El 16X, N117X, T118X, Hl 19X, V120X, L122X, R123X, R128X, L135X, Y136X, E138X, A139X, Q141X, M142X, D145X, A146X, A148X, D156X, E157X, K159X, C161X, D163X, T164X, D167X, Q169X, C171X, D177X, D180X, H182X, S183X, Q184X, A185X, L186X, S187X, G188X, R189X, A192X, N196X a deletion selected from AM1-L12, AE36-G53, AN61-G68, AW104-G105 , AQ195-N199, and AI26- G27, and combinations thereof, wherein the position number designation is functionally equivalent to the position in a wild-type APOBEC3A (SEQ ID NO:3) and X is an amino acid substitution different from the wild-type amino acid at that position.
[000573] Aspect 42. The protein complex of Aspect 29 or the ACD of any of Aspects 30- 47, wherein the ACD comprises: a. three or more stability-enhancing alterations, b. four or more stability— enhancing alterations, c. five or more stability-enhancing alterations, d. six or more stability-enhancing alterations, e. seven or more stability-enhancing alterations, f. eight or more stability-enhancing alterations, g. nine or more stability-enhancing alterations, h. ten or more stability-enhancing alterations, i. eleven or more stability-enhancing alterations, or k. twelve or more stability-enhancing alterations.
[000574] Aspect 43. The protein complex of Aspect 29 or the ACD of any of Aspects 30- 47, wherein the ACD comprises two or more stability mutations selected from I17T, T19Y, G25K, S45W, A59P, K60R, A61-68, R74L, G108A, G108C, C171A, G188R, and A104-105.
[000575] Aspect 44. The protein complex of Aspect 29 or the ACD of any of Aspects 30- 47, wherein the ACD comprises stability mutations selected from the group consisting of: R74L/C171A; R74L/T19Y; R74L/G25R; R74L/T19I/C171A; R74L/T19L/C171A; R74L/T 19 Y/C 171 A/Il 7T; R74L/T 19 Y/C 171 A/G25 A; R74L/T 19 Y/C 171 A/G25R/I17T;
R74L /C171A/G25R/T19F; R74L/T19Y/C171A/G25D; R74L/T19Y/C171A/S45R; R74C/T19Y/C171A; R74L/C171A/T19F; R74L/C171A/T19W; R74L/T19Y/C171A/G108E; R74L/T 19 Y/C 171 A/Gl 08D; R74L/T 19 Y/C 171 A/Gl 08Q; R74L/T 19 Y/C 171 A/Gl 08 Y;
R74L/T 19 Y/C 171 A/Gl 08H; R74L/T 19 Y/C 171 A/Gl 08L; R74L/T 19 Y/C 171 A/Gl 08K; R74L/T19Y/C171A/G108R; R74L/T19Y/C171A/A126V; R74L/T19Y/C171I;
R74L/T 19 Y/C 171 A/Gl 08M; R74L/T 19 Y/C 171 A/Gl 08W; R74L/T 19 Y/C 171 A/Al 26F;
R74L/T19Y/C171A/A126I; R74L/T19Y/C171A/A126L; R74L/T19Y/C171A;
R74L/T19Y/C171A/S45W; R74L/T19Y/C171A/G25R; R74L/T19Y/C171A/G25K;
R74L/T 19 Y/C 171 A/Gl 88Q; R74L/T 19 Y/C 171 A/Gl 88 A; R74L/T 19 Y/C 171 A/Gl 88R; R74L/T19Y/C171A/G108A; R74L/T19Y/C171A/G108A/G188R/G25K/S45W ;
R74L/T 19 Y/C 171 A/Gl 08C; R74L/T 19 Y/C 171 A/Gl 08 A/Gl 88R/G25K/S45 W/117T;
R74L/T 19 Y/C 171 A/Gl 08 A/Gl 88R/G25K/S45 W/117T/A59P/K60R/A61-68;
R74L/T 19 Y/C 171 A/Gl 08C/G188R/G25K/S45 W/Il 7T;
R74L/T 19 Y/C 171 A/Gl 08C/G188R/G25K/S45 W/117T/A59P/K60R/A61 -68/A126C;
R74L/T 19 Y/C 171 A/Gl 08C/G188R/G25K/S45 W/117T/A59P/K60R/A61-68;
T 19 Y/R74L/C 171 A; 117T/T 19 Y/G25R/R74L/C 171 A;
T 19 Y/G25K/S45 WR74L/G108 A/C 171 A/Gl 88R;
117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/G108C/C 171 A/Gl 88R;
117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/G108C/A126C/C 171 A/Gl 88R; and
117T/T 19 Y/G25K/S45 W/A59P/K60R/AN61 -G68/R74L/AW 104-
G105/G108C/C 171 A/Gl 88R.
[000576] Aspect 45. The protein complex of Aspect 29 or the ACD of any of Aspects SO- 47, wherein the ACD comprises a combination of stability mutations comprising T19Y, G25K, S45W, R74L, G108A, C171A, and G188R.
[000577] Aspect 46. The protein complex of Aspect 29 or the ACD of any of Aspects SO- 47, wherein the ACD comprises a combination of stability mutations comprising I17T, T19Y, G25K, S45W, A59P, K60R, A61-68, R74L, G108C, C171A, and G188R.
[000578] Aspect 47. The protein complex of Aspect 29 or the ACD of any of Aspects SO- 47, wherein the amino acid sequence of the ACD is selected from SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:479-517, SEQ ID N0:520-605, SEQ ID NO: 607-615, SEQ ID NO:635- 954, or SEQ ID NO: 962-968. [000579] Aspect 48. An ACD of any one of the preceding Aspects, wherein the amino acid sequence of the ACD is selected from SEQ ID NO:23 or SEQ ID NO:24.
[000580] Aspect 49. A polynucleotide encoding the ACD of any of Aspects 29-48.
[000581] Aspect 50. A composition comprising the ACD of any of Aspects 29-49.
[000582] Aspect 51. The composition of any of Aspects 50-54, wherein the composition further comprises a sample comprising DNA comprising at least one modified cytosine, wherein the modified cytosine is 5-methyl cytosine (5mC).
[000583] Aspect 52. The composition of any of Aspects 50-54, wherein the DNA comprises single-stranded DNA.
[000584] Aspect 53. The composition of any of Aspects 50-54, wherein the sample comprises genomic DNA or cell free DNA.
[000585] Aspect 54. The composition of any of Aspects 50-54, wherein the genomic DNA is from a single cell or is a mixture from a plurality of cells.
[000586] Aspect 55. A method comprising: providing a sample of DNA suspected of comprising single-stranded DNA comprising at least one 5-methyl cytosine (5mC); contacting the single-stranded DNA with an ACD under conditions suitable for conversion of 5 -methylcytosine (5mC) to thymidine (T) by deamination at a greater rate than conversion of cytosine (C) to uracil (U) by deamination, to result in converted single-stranded DNA, wherein the altered cytidine deaminase is the ACD of any one of Aspects 29-49; and processing the converted single-stranded DNA to produce a sequencing library.
[000587] Aspect 56. A method comprising: providing a sample of DNA suspected of comprising double-stranded DNA comprising at least one 5-methyl cytosine (5mC); processing the double-stranded DNA by denaturing to result in a single-stranded DNA; contacting the single-stranded DNA with an ACD under conditions suitable for (i) conversion of 5-methylcytosine (5mC) to thymidine (T) by deamination at a greater rate than conversion of cytosine (C) to uracil (U) by deamination to result in converted single-stranded DNA, wherein the altered cytidine deaminase is the ACD of any one of Aspects 29-49; and converting the converted single-stranded DNA to a converted double-stranded DNA sequencing library. [000588]
[000589] Aspect 57. A method of detecting the location of a modified cytosine in a target nucleic acid, the method comprising: (a) contacting target nucleic acids suspected of comprising at least one modified cytosine with the ACD of any one of Aspects 29-49 to produce converted nucleic acids comprising at least one converted methyl cytosine; (b) detecting the at least one converted methyl cytosine in the converted nucleic acids of (a).
[000590] Aspect 58. The method of any of Aspects 57-59, wherein the detecting comprises identifying thymidine nucleotides in the converted nucleic acid to determine the location of 5mC nucleotides in the target nucleic acid.
[000591] Aspect 59. The method of any of Aspects 57-59, the method further comprising: detecting one or more single variant nucleotides within the sequence in addition to at least one 5mC.
[000592] Aspect 60. Use of the ACD of any one of Aspects 29-48 to identify a 5mC in a nucleic acid sequence by sequencing.
[000593] Aspect 61. A helicase-ACD complex comprising a helicase attached to an ACD of any one of Aspects 1-18 or 30-47.
[000594] Aspect 62. The helicase-ACD complex of and of Aspects 61-81, wherein the ACD and the helicase are attached to eachother.
[000595] Aspect 63. The helicase-ACD complex of and of Aspects 61-81, wherein the complex comprises the helicase attached to the N-terminus.
[000596] Aspect 64. The helicase-ACD complex of and of Aspects 61-81, wherein the complex comprises the helicase attached to the C-terminus.
[000597] Aspect 65. The helicase-ACD complex of and of Aspects 61-81, wherein the cytidine deaminase and the helicase comprise a fusion protein.
[000598] Aspect 66. The helicase-ACD complex of and of Aspects 61-81, wherein the fusion protein comprises a linker between the helicase and ACD. [000599] Aspect 67. The helicase-ACD complex of and of Aspects 61-81, wherein the fusion protein comprises the an amino acid sequence helicase-linker-ACD from N-terminus to C-terminus.
[000600] Aspect 68. The helicase- ACD complex of and of Aspects 61-81, wherein the cytidine deaminase and the helicase are biochemically conjugated.
[000601] Aspect 69. The helicase- ACD complex of and of Aspects 61-81, wherein the helicase unwinds double-stranded DNA (dsDNA).
[000602] Aspect 70. The helicase- ACD complex of and of Aspects 61-81, wherein the helicase is a member of helicase Superfamily 1, Superfamily 2, Superfamily 3, or Family 4.
[000603] Aspect 71. The helicase- ACD complex of and of Aspects 61-81, wherein the helicase comprises Walker A motif (SEQ ID NO:68), a Walker B motif (SEQ ID NO:69), or both.
[000604] Aspect 72. The helicase- ACD complex of and of Aspects 61-81, wherein the helicase in selected from the group consisting of SEQ ID NO: 427-478 and SEQ ID NO:964- 966.
[000605] Aspect 73. The helicase- ACD complex of and of Aspects 61-81, wherein the protein complex converts 5mC to T by deamination in dsDNA at a greater rate than a comparable protein complex without a helicase.
[000606] Aspect 74. The helicase- ACD complex of and of Aspects 61-81, wherein the helicase is selected from SEQ ID NO:427-478, preferably wherein the helicase is CaTe (SEQ ID NO: 427.
[000607] Aspect 75. The helicase-ACD complex of and of Aspects 61-81, wherein the fusion protein comprises a linker comprising glycine and serines and the linker comprises from 5 to 40 amino acids in length.
[000608] Aspect 76. The helicase-ACD complex of and of Aspects 61-81, wherein the linker is from 5-15 amino acids in length.
[000609] Aspect 77. The helicase-ACD complex of and of Aspects 61-81, wherein the linker is selected from any one of SEQ ID NO: 967-995. [000610] Aspect 78. The helicase-ACD complex of and of Aspects 61-81, wherein the fusion protein comprising from Helicase-linker-ACD, wherein the C terminus of the helicase is connected to the N terminus of the ACD by the linker.
[000611] Aspect 79. The helicase-ACD complex of and of Aspects 61-81, wherein the fusion protein comprises a amino acid sequence selected from the group consisting of SEQ ID NO:996-1116.
[000612] Aspect 80. The helicase-ACD complex of and of Aspects 61-81, wherein the fusion protein comprises an amino acid sequence selected from the group consisting of SEQ ID NO:996- 1006.
[000613] Aspect 81. The helicase-ACD complex of and of Aspects 61-81, wherein the fusion protein comprises an amino acid sequence selected from SEQ ID NO:996 and 997.
[000614] Aspect 82. A composition comprising: the protein complex of any one of Aspects 60-80; and a DNA sample comprising double-stranded DNA comprising at least one 5mC.
[000615] Aspect 83. The composition of any of Aspects 82-84, wherein the sample comprises genomic DNA.
[000616] Aspect 84. The composition of any of Aspects 82-82, wherein the genomic DNA is from a single cell or is a mixture from a plurality of cells.
[000617] Aspect 85. A method comprising: providing a sample of DNA suspected of comprising dsDNA comprising at least one 5-methyl cytosine (5mC); contacting the sample with the protein complex of any one of Aspects 60-80 under conditions suitable for conversion of 5 methylcytosine (5mC) to thymidine (T) to result in converted dsDNA, wherein 5mC is converted to T; and processing the converted dsDNA to produce a sequencing library.
[000618] Aspect 86. The method of any of Aspects 85-98, further comprising: providing a surface comprising a plurality of amplification sites, wherein the amplification sites comprise at least two populations of attached single-stranded capture oligonucleotides having a free 3' end; and contacting the surface comprising amplification sites with the sequencing library under conditions suitable to produce a plurality of amplification sites that each comprise a clonal population of amplicons from an individual member of the sequencing library.
[000619] Aspect 87. The method of any of Aspects 85-98, wherein the method is performed without an amplification step.
[000620] Aspect 88. The method of any of Aspects 85-98, wherein the dsDNA is adhered to a surface prior to treatment with the protein complex.
[000621] Aspect 89. The method of any of Aspects 85-98, wherein the surface is a flow cell surface.
[000622] Aspect 90. The method of any of Aspects 85-98, wherein the dsDNA undergoes library preparation on the flow cell and is subsequently sequenced on the same flow cell.
[000623] Aspect 91. The method of any of Aspects 85-98, wherein the sample is a biological sample.
[000624] Aspect 92. The method of any of Aspects 85-98, wherein the biological sample comprises cell-free DNA.
[000625] Aspect 93. The method of any of Aspects 85-98, wherein the biological sample comprises a fluid selected from blood or serum.
[000626] Aspect 94. The method of any of Aspects 85-98, wherein the sample comprises single cells or isolated nuclei.
[000627] Aspect 95. The method of any of Aspects 85-98, wherein the biological sample comprises a tissue.
[000628] Aspect 96. The method of any of Aspects 85-98, wherein the tissue comprises tumor tissue.
[000629] Aspect 97. The method of any of Aspects 85-98, further comprising determining the sequence of at least a portion of the library, the method further comprising: comparing the sequence of the at least a portion of the library with an untreated reference sequence to determine at least one 5mC present in the sample of DNA. [000630] Aspect 98. The method of any of Aspects 85-98, further comprising detecting a variant sequence at a position in the sequence, wherein the variant is a single nucleotide variant (SNV) and not a sequencing error.
[000631] Aspect 99. A kit for producing a DNA library for sequencing, the kit comprising the ACD of any one of Aspects 1-18 or 29-48; adaptors for adding adaptors comprising sequencing primer binding sites, suitable buffers and instructions for use in preparing a DNA library. .
[000632] Aspect 100. The kit of any of Aspects 99-104, wherein the kit further comprises an enzyme for fragmenting the DNA prior to adaptor addition.
[000633] Aspect 101. The kit of any of Aspects 99-104, further comprises labeled nucleotides for sequencing.
[000634] Aspect 102. The kit of any of Aspects 99-104, further comprising a flow cell. Aspect 103. The kit of any of Aspects 99-104, wherein he flow cell comprises a substrate having depressions separated by interstitial regions; first and second primers immobilized within each of the depressions; and first and second transposome complexes immobilized within each of the depressions, over the interstitial regions, or both within each of the depressions and over the interstitial regions; and an extension mix.
[000635] Aspect 104. A kit, comprising: a flow cell including: a substrate having depressions separated by interstitial regions; first and second primers immobilized within each of the depressions; and first and second transposome complexes immobilized within each of the depressions, over the interstitial regions, or both within each of the depressions and over the interstitial regions; an extension mix; and an enzymatic methylation conversion mix comprising the ACD or ACD-helicase complex and a buffer solution.
[000636] Aspect 105. A method, comprising: initiating tagmentation of a DNA sample using first and second transposome complexes asymmetrically attached in a flow cell, thereby forming partially adapted hybridized fragments including a first partially adapted DNA fragment that is immobilized, at its 5’ end, to a substrate of the flow cell, and a second partially adapted DNA fragment that is removably attached to the substrate; removing a transposase enzyme from each of the first and second transposome complexes; initiating an extension reaction of the partially adapted hybridized fragments to form fully adapted hybridized fragments; initiating enzymatic methylation conversion of the first fully adapted DNA fragment using the ACD-helicase complex of any one of Aspects 29-49.
[000637] Aspect 106. An altered cytidine deaminase (ACD) comprising 5mC-selective deaminase activity, the ACD comprising selectivity-enhancing alterations at positions functionally equivalent to amino acid Y130 and P134 in a wild-type APOBEC3A (SEQ ID NO:3).
[000638] Aspect 107. The ACD of any of Aspects 106-129, wherein the selectivityenhancing alterations are substitution mutations, wherein the substitution mutation at Y130 is alanine or histidine and the substitution mutation at Pl 34 is tryptophan or tyrosine, , optionally wherein the substitution mutation at Y130 is alanine and the substitution mutation at Pl 34 is tryptophan.
[000639] Aspect 108. The ACD of any of Aspects 106-129, further comprising one or more selectivity-enhancing substitution mutations at positions functionally equivalent to amino acid D131, Y132, and D133 in the wild-type APOBEC3A (SEQ ID NO:3), wherein the substitution mutations are D131X where X is any amino acid except aspartic acid, Y132X where X is any amino acid except tyrosine, and D133X where X is any amino acid except aspartic acid.
[000640] Aspect 109. The ACD of any of Aspects 106-129, wherein the substitution mutation at D131 is histidine, arginine, lysine, proline, glycine, asparagine, alanine, or valine.
[000641] Aspect 110. The ACD of any of Aspects 106-129, wherein the substitution mutation at DI 32 is alanine, proline, aspartic acid, glutamic acid, glycine, threonine, valine, glutamine, or arginine.
[000642] Aspect 11 l.The ACD of any of Aspects 106-129, wherein the substitution mutation at D133 is histidine, tyrosine, phenylalanine, threonine, isoleucine, or valine.
[000643] Aspect 112. The ACD of any of Aspects 106-129, wherein the substitution mutation at D131 is histidine or lysine, wherein the substitution mutation at Y132 is alanine or tyrosine, and the substitution mutation at D133 is arginine or proline.
[000644] Aspect 113. The ACD of any of Aspects 106-129, the ACD further comprising selectivity-enhancing substitution alterations at one or more of positions functionally equivalent to amino acid D131, Y132, and D133 in a wild-type APOBEC3A (SEQ ID NO:3), wherein the amino acid sequence of Y130, D131, Y132, D133, and P134 is selected from SEQ ID NO: 125, SEQ ID NO: 126, or the sequences described in Table 2, Table 3, FIG. 4B, FIG. 8, or FIG. 9 which demonstrate increased 5mC selectivity compared to wild-type (SEQ ID N0:3).
[000645] Aspect 114. The ACD of any of Aspects 106-129, the ACD further comprising a selectivity-enhancing alteration at L135 in the wild-type APOBEC3A (SEQ ID NO:3), one or more of positions functionally equivalent to amino acid Y130, D131, Y132, D133, P134, and L135 in a wild-type APOBEC3A (SEQ ID NO:3), wherein the amino acid sequence of Y130, D131, Y132, D133, P134, and L135 is selected from SEQ ID NO: 125, SEQ ID NO: 126, or the sequences described in FIG. 4B, FIG. 8, or FIG. 9 which demonstrate increased 5mC selectivity compared to wild-type (SEQ ID NO:3).
[000646] Aspect 115. The ACD of any of Aspects 106-129, wherein the ACD further comprises selectivity-enhancing alterations at positions functionally equivalent to amino acid S103 and W104 in the wild-type APOBEC3A (SEQ ID NO:3), wherein the selectivityenhancing alterations are substitution mutations, and the substitution mutations are S103X where X is any amino acid except serine and W104 where X is any amino acid except tryptophan.
[000647] Aspect 116. The ACD of any of Aspects 106-129, wherein the substitution mutation at SI 03 is aspartic acid, histidine, threonine, or valine, and wherein the substitution mutation at amino acid W104 is alanine, aspartic acid, methionine, or glutamic acid.
[000648] Aspect 117. The ACD of any of Aspects 106-129, further comprising a selectivity-enhancing alteration at amino acid G105, wherein the selectivity-enhancing alteration is a deletion of G105.
[000649] Aspect 118. The ACD of any of Aspects 106-129, the ACD comprising selectivity-enhancing alterations at one or more of positions functionally equivalent to amino acid F102, S103, W104, G105, Y130, D131, Y132, D133, and P134 in a wild-type APOBEC3A (SEQ ID NO:3), wherein the amino acid sequence of F102, S103, W104, G105, Y130, D131, Y132, D133, and P134 is selected from SEQ ID NO: 23, SEQ ID NO:24, or the sequences described in FIG. 8 or FIG. 9 which demonstrate increased 5mC selectivity compared to wild-type (SEQ ID NO:3). [000650] Aspect 119. The ACD of any of Aspects 106-129, the ACD further comprising a selectivity-enhancing alteration at L135 in the wild-type APOBEC3A (SEQ ID NO:3), wherein the amino acid sequence of F102, S103, W104, G105, Y130, D131, Y132, D133, P134, and L135 is selected from SEQ ID NO: 23, SEQ ID NO:24, or the sequences described in FIG. 8 or FIG. 9 which demonstrate increased 5mC selectivity compared to wild-type (SEQ ID NO:3).
[000651] Aspect 120. The ACD of any of Aspects 106-129, further comprising two or more stability-enhancing alterations.
[000652] Aspect 121. The ACD of any of Aspects 106-129, wherein the two or more stability-enhancing alteration is a substitution mutation, a deletion, an insertion, or a combination thereof.
[000653] Aspect 122. The ACD of any of Aspects 106-129, wherein the ACD comprises two or more stability-enhancing alterations are at a position functionally equivalent to amino acids selected from 117, T19, G25, S45, A59, K60, N61, L62, L63, C64, G65, F66, Y67, G68, R74, W104, G105, G108, G108, C171, and G188 in the wild-type APOBEC3A (SEQ ID NO:3).
[000654]
[000655] Aspect 123. The ACD of any of Aspects 106-129, wherein the at least two stability-enhancing alterations are (i) a substitution mutation, wherein the substitution mutation at 117 is threonine, at T19 is tyrosine, at G25 is lysine or arginine, at S45 is tryptophan, at A59 is proline, at K60 is arginine, at R74 is leucine, at G108 is alanine, at G108 is cytosine or alanine, at C171 is alanine, at G188 is arginine, (ii) a deletion, wherein the deletion is a deletion of W104 and G105R, a deletion of K60-G68, or (iii) any combination thereof.
[000656] Aspect 124. The ACD of any of Aspects 106-129, wherein the ACD comprises stability-enhancing alterations comprising I17T, T19Y, G25K or G25R, S45W, A59P, K60R, deletion of K61-G68, R74L, G108C or G108A, C171A, and G188R.
[000657] Aspect 125. The ACD of any of Aspects 106-129, wherein the ACD comprises (i) stability-enhancing alterations comprising I17T, T19Y, G25K, S45W, A59P, K60R, deletion of K61-G68, R74L, G108C, C171A, and G188R, and (ii) selectivity-enhancing alterations comprising either Y130A, D131H, Y132A, D133R, and P134W, or Y130A, D131K, D133P, and P134W.
[000658] Aspect 126. The ACD of any of Aspects 106-129, wherein the ACD comprises stability-enhancing alterations comprising I17T, T19Y, G25K, S45W, A59P, K60R, deletion of K61-G68, R74L, G108C, C171A, and G188R, and selectivity-enhancing alterations comprising Y130A, D131H, Y132A, D133R, and P134W, and further comprises selectivityenhancing alterations comprising SI 03V and W104P.
[000659] Aspect 127. The ACD of any of Aspects 106-129, wherein the ACD comprises SEQ ID NO:23, or an amino acid sequence having at least 80% identity to SEQ ID NO:23.
[000660] Aspect 128. The ACD of any of Aspects 106-129, wherein the ACD comprises stability-enhancing alterations comprising I17T, T19Y, G25K, S45W, A59P, K60R, deletion of K61-G68, R74L, G108C, C171A, and G188R, and selectivity-enhancing alterations comprising Y130A, D131K, D133P, and P134W, and further comprises selectivityenhancing alterations comprising SI 03V, W104P, and a deletion of G105.
[000661] Aspect 129. The ACD of any of Aspects 106-129, wherein the ACD comprises SEQ ID NO:24, or an amino acid sequence having at least 80% identity to SEQ ID NO:24.
[000662] Aspect 130. An ACD-helicase complex comprising a helicase attached to an ACD of any of Aspects 106-129.
[000663] Aspect 131. The ACD-helicase complex of any of Aspects 130-133, wherein the ACD and the helicase are a fusion protein.
[000664] Aspect 132. The ACD-helicase complex of any of Aspects 130-133, wherein the fusion protein comprises a linker.
[000665] Aspect 133. The ACD-helicase complex of any of Aspects 130-133, wherein the cytidine deaminase and the helicase are conjugated.
[000666] Aspect 134. A method for deaminating a 5 -methyl cytosine, comprising: providing a sample of DNA suspected of comprising single-stranded DNA comprising at least one 5-methyl cytosine (5mC); and contacting the single-stranded DNA with an ACD under conditions suitable for conversion of 5-methylcytosine (5mC) to thymidine (T) by deamination at a greater rate than conversion of cytosine (C) to uracil (U) by deamination, to result in converted single-stranded DNA, wherein the altered cytidine deaminase is the ACD of any of Aspects 1-18, 30-47, or 106-129.
[000667] Aspect 135. A method for deaminating a 5 -methyl cytosine, comprising: providing a sample of DNA suspected of comprising double-stranded DNA comprising at least one 5-methyl cytosine (5mC); processing the double-stranded DNA to produce a sequencing library; denaturing the sequencing library to result in a single-stranded DNA; contacting the single-stranded DNA with an ACD under conditions suitable for conversion of 5-methylcytosine (5mC) to thymidine (T) by deamination at a greater rate than conversion of cytosine (C) to uracil (U) by deamination, to result in converted single-stranded DNA, wherein the altered cytidine deaminase is the ACD of any of Aspects 1-18, 30-47, or 106-129.
[000668] Aspect 136. The method of Aspect 134-135, further comprising converting the converted single-stranded DNA to a converted double-stranded DNA sequencing library.
[000669] Aspect 137. A method for deaminating a 5-methyl cytosine, comprising: providing a sample of DNA suspected of comprising double-stranded DNA comprising at least one 5-methyl cytosine (5mC); contacting the double-stranded DNA with an ACD -helicase complex under conditions suitable for (i) transient conversion of at least a portion of the double-stranded DNA to single-stranded DNA, and (ii) conversion of 5- methylcytosine (5mC) to thymidine (T) by deamination at a greater rate than conversion of cytosine (C) to uracil (U) by deamination, to result in converted double-stranded DNA, wherein the altered cytidine deaminase is the ACD-helicase complex of any of Aspects 61-81 or 130-133; and processing the converted double-stranded DNA to produce a sequencing library.
[000670] Aspect 138. A method of detecting the location of a modified cytosine in a target nucleic acid, the method comprising: (a) contacting target nucleic acids suspected of comprising at least one modified cytosine with the ACD of any of Aspects 61-81 or 130- 133to produce converted nucleic acids comprising at least one converted methyl cytosine; (b) detecting the at least one converted methyl cytosine in the converted nucleic acids of (a). [000671] Aspect 139. A composition comprising the ACD of any of Aspects 1-18, SO- 47, or 106-129 1 or the ACD-helicase complex of any one of Aspects 25-28 and a suitable buffer.
[000672] Aspect 140. A polynucleotide encoding the ACD of any of Aspects 1-18, SO- 47, 106-125, or the ACD-helicase fusion protein of any of Aspects 61-81 or 130-133 capable of expressing the ACD or ACD-helicase fusion.
[000673] EXAMPLES
[000674] The present disclosure is illustrated by the following examples. It is to be understood that the particular examples, materials, amounts, and procedures are to be interpreted broadly in accordance with the scope and spirit of the disclosure as set forth herein.
[000675] Example 1
[000676] Screen to identify mutants with increased specificity for 5mC
[000677] Altered cytidine deaminases (ACDs) having a selectivity-enhancing alteration, for instance one or more substitution mutations, one or more deletions, one or more insertions, or combinations thereof, of amino acids at positions functionally equivalent to amino acids in SEQ ID NO:3 were produced in specific backbones (e.g., ScK (SEQ ID NO: 14), Scl (SEQ ID NO: 17), or ScC (SEQ ID NO: 19) using standard methods. Screens were then conducted to identify proteins with increased specificity for 5mC compared to the parent amino acid sequence before adding the substitution mutations, deletions, and/or insertions.
[000678] In an initial screen, ACDs with increased 5mC specificity were enriched and sequenced (FIG. 4). The diversity at positions Y130 to P134 before the assay and after two rounds of enrichment is shown at FIG. 4A. The most prevalent 10 identified sets of mutations are shown at FIG. 4B.
[000679] A selection of the best mutants based on the enrichment data or randomly picked colonies were tested, where the results can be represented as a selectivity plot (FIG. 5A and 5B). Even though our previous mutant ACD with substitution mutations Y130A/Y132H/D133W (SEQ ID NO:13) showed high 5mC selectivity, it was realized that there was an unexpected and surprising level of plasticity to the active site and there are many solutions for a wild-type APOBEC3 A, e.g., SEQ ID NO:3, to have increased 5mC selectivity. In most of the mutants identified in this experiment, Y 130 was mostly fixed on alanine and P134 was mostly mutated to tryptophan. However, positions D 131, Y132, D133 could be modified to many different combinations and still preserve the 5mC selectivity (e.g., Table 2). Accordingly, these types of mutations can be referred to as AxxxW mutations. [000680] Table 2. Selectivity of Exemplary AxxxW mutants.
Table 2: Mutants at Positions 130-134a
[000681] Table 2 is exemplary examples. Further examples of mutations that had increased 5mC selectivity with the AxxxW (SEQ ID NO:51) mutation can be found in FIG. 8 and FIG. 9 that were tested as above.
[000682] Various mutations at position SI 03/ W104 were screened, and mutations were identified that appeared to be potentially beneficial for increased 5mC selectivity of an ACD with stability-enhancing alterations (FIG. 6A). The S103V/W104P mutations were identified as beneficial and combined with some of the AxxxW (SEQ ID NO:51) mutants in different sets of ACDs having substitution mutations that increase stability. For example, the ACD with substitution mutations S103V/W104P/D133R/P134W added to Y130A/D131H/Y132A showed high selectivity toward 5mC in this experiment (FIG. 6B). Moreover, based on these results we tested the positions F102/S103/W104/G105 in combination with AxxxW (SEQ ID NO: 51) mutants and found many mutants with selectivity that showed even greater improvement over a mutant, ScK, that was previously identified as showing high 5mC selectivity (FIG. 6C, Table 3).
[000683] Further, various mutations and deletions at positions 102-105 were screened and mutations identified that have beneficial for increased 5mC selectivity, particularly when the muations at positions 102, 103, 104 or 105 are combined with mutations or deletions in AxxxW for positions 130-134 and optionally one or more stability mutations described herein.
[000684] Table 3. Selectivity of some exemplary AxxxW with 102-105 mutants from FIG.
9.
Table 3: Mutants at Positions 102-105 and 130-134
"A" refers to deletion of one amino acid.
[000685] All mutations found in Figure 9 (e.g., SEQ ID NO:479-968) were tested in the same manner as the examples in Table 3 and increased 5mC is noted in the Table as a (+); specific data is not shown for simplicity.
[000686] Some mutants were evaluated by comparing the deamination of a fully unmethylated target (Lambda DNA) and a methylated target (CG methylated pUC19) by plotting on a selectivity plot (FIG. 7A-C).
[000687] EXAMPLE 2
[000688] This example demonstrates a one-step enzymatic detection of 5mC on a single base resolution on double stranded DNA. The above example showed that it is possible to engineer APOBEC3 A into 5mC specific enzyme that works mainly on single- stranded DNA, which complicates the workflow as it requires denaturation steps which may not be compatible with the deamination reaction under some conditions, for example, on-board a flow cell. Furthermore, the deamination activity of APOBEC3 A is hindered by its sensitivity to secondary structure regions.
[000689] Here we demonstrate that we can eliminate denaturation step in the workflow by using our 5mC selective mutants (e.g. ACD described herein) fused to helicase. We have developed a fusion protein of the 5mC selective altered cytidine deaminase to helicases (using certain linkers between the protein, e.g., GS or HIS) allowed for the development of a soluble protein (FIG. 10) that had 5-mC specific activity on double stranded DNA (FIG. 14 and FIG. 15). Furthermore, ACD-helicase fusion is active and able to unwind dsDNA while maintaining selective 5mC deaminase activity (FIG.12). This fusion enzyme thus is further improvement that allows for some applications where removing the denaturation would hinder the assay or require an extra washing step which would further reduce the viable DNA for running on the assay, for example, for the direct on-flow cell deamination for methylation detection as well as PCR-free one-step enzymatic detection.
[000690] DNA methylation (5mC) is a crucial epigenetic modification in eukaryotes, playing an essential role in gene expression regulation. Accurately quantifying 5mC at singlebase resolution is key to understanding its genomic impact. However, existing mapping techniques have notable drawbacks. For instance, bisulfite sequencing, the gold-standard method, exposes DNA to harsh chemical conditions, leading to degradation. Additionally, the conversion of unmethylated cytosines to uracil reduces sequence complexity, restricting analysis to a three-base resolution. More recently, enzymatic methods such as EM-seq, which utilize the AP0BEC3 A deaminase, have been developed as alternatives to bisulfite- based approaches. These methods offer the advantage of preserving DNA integrity, enabling analysis with lower DNA input. However, they require multiple enzymatic reactions along with a denaturation step, as AP0BEC3 A functions only on single-stranded DNA.
[000691] Using our engineered APOBEC3 A described above (that preferentially deaminates only methylated C into T), we made ACD-helicase fusions. This method has an advantage of reducing the complexity of the assay by being able to use a single enzymatic reaction without reducing DNA complexity. Unlike the assays using ACD alone, which requires adenaturation step for the APOBEC3 A derived ACDs to be able to act on the ssDNAthe ACD-helicase complexes or fusions allows for a reduced or eliminated denaturation step as it can work on dsDNA, simplifying the assay and also allowing the assay to work in methods that may not be compatible with a denaturation step. Moreover, the current workflow for the direct on-flow cell deamination requires adding 2 additional steps: extension and denaturation before deamination. Not only is the workflow for this solution more complicated, but adding sodium hydroxide/ DMSO or using high temperature for DNA denaturation can be detrimental to the flow cell. Thus this ACD-fusion elevates this issue by reducing or eliminating denaturation, thus not harming the flow cell for further steps in the sequencing methods downstream from library preparation.
[000692] In this invention, we show that it is possible to deaminate dsDNA with 5mC selectivity by using ACD fusion with helicases. Not to be bound by any theory, but the inventors envision that there are other methods to conjugate the ACD to helicase, and also that the linker that is required can be any flexible linker that allows for the helicase and ACD to maintain activity. The description below are just exemplary and not to be limiting to this invention.
[000693] Suitable Helicase- ACD fusions are found in Table 4:
Xaa is a linker described in the description, including, e.g., GS linkers (e.g., 5-30 aa GS linkers (SEQ ID NO: 967-995) or rigid linkers, e.g., Helix (SEQ ID NO:995) “/” denoted different ACD that may be attached to the helicase noted
Section 1: Expression and purification of Exemplary ACD helicase fusion constructs [000694] First, we evaluated whether a few different protein fusions of ACD and helicase could be efficiently expressed and purified using a bacterial expression system (Figure 10). The fusion proteins were expressed in E. coli BL21 (DE3) cells and purified via Ni-NTA affinity chromatography, followed by size-exclusion chromatography.
The Helicase CaTe connected to ACD through GS linker demonstrated the higher expression levels than the CaTe connected via a Helix linker and was readily purified (Figure 10c) (e.g., SEQ ID NO:962; 963) and is demonstrated to have dsDNA deaminase activity.
Section 2: DNA deaminase activity of ACD-helicase fusion constructs on ssDNA [000695] After protein purification, we verified whether the constructs retained deamination activity using a gel-based assay with the restriction enzyme Swal. In the assay deamination of 5mC into T results in a mismatched DNA substrate that can be then cleaved by Swal and the result can be visualized on TBE-Urea gel (FIG. I la). The experiment confirmed that ACD fusion with helicase is still active and able to deaminate single stranded 5mC oligo (FIG. 1 lb).
[000696] Next, we tested the proteins for deamination activity and selectivity towards 5mC using oligo NGS based assay. The substrate ssDNA oligo contains 17 unmethylated cytosines and 16 methylated C sites. Deamination by ACD results in conversion of unmethylated C residues to uracil or conversion of mC to thymine. These deamination events can be then read out as CT mutations via sequencing (FIG. 12a). Furthermore, using this assay we investigated whether the buffer conditions required for helicase activity, including the presence of ATP or Mg2, were compatible with 5mC-deaminase activity. All fusion constructs exhibited deamination activity on ssDNA with selectivity towards 5mC, and the addition of ATP or Mg2 did not inhibit the reaction; instead, it enhanced it (FIG. 12b).
Section 3: Helicase activity of ACD-helicase fusion constructs
[000697] Next, we tested the activity of helicase in the fusion proteins using plate reader assay. The assay utilizes a 5 '-F AM-labeled oligonucleotide, where the complementary strand is paired with a quencher to suppress fluorescence. Upon helicase activity, the enzyme unwinds the double-stranded DNA, displacing the quencher-containing strand and replacing it with a competitor oligo that does not have the quencher. This replacement alleviates the quenching effect, leading to a noticeable increase in FAM fluorescence, which can be measured (FIG. 13a). The helicase fusion proteins exhibited slightly lower activity compared to the commercially available UvrD helicase (NEB), but it remained functional (FIG. 13b). Section 4: Deamination of double stranded DNA with ACD-helicase fusion constructs [000698] Upon confirmation of both helicase and deaminase activity of fusion proteins we tested the activity of the new constructs on model dsDNA by NGS oligo assay. Conceptually it is the same oligo that was used for single stranded DNA activity, but before deamination the oligo is hybridized to reverse complement strand in excess. Remarkably, ACD-helicase fusion proteins especially CaTe-GS-ACD showed to be able to deaminate dsDNA while retaining the specificity towards 5mC (FIG. 14) if the reaction buffer contained ATP and Mg. [000699] We next tested dsDNA deamination activity with a more complex substrate. For this, we mixed non-methylated DNA (lambda DNA) with fully methylated control (pUC19 enzymatically methylated with M.SssI Methyltransferase). Similarly, to what we observed in NGS oligo assay, ACD on its own is not able to deaminate dsDNA and requires denaturation step first to be active. However, ACD-helicase fusion constructs show high activity on dsDNA with very similar selectivity that was observed for ACD on ssDNA on its own (FIG. 15).
Section 5: Direct on-flow cell deamination for methylation detection
[000700] Another way we envision mapping 5-methylcytosine (5mC) is through direct deamination and detection on-flow cell. Some of the initial testing of ACDs directly on flow cell showed low percentage of cluster passing internal quality filter (%PF). We demonstrate that the use of the helicase- ACD with a workflow having sodium hydroxide for denaturation resulted in effective conversion of deaminated DNA and an improvement in the PF% values when a wash step was introduced following the deamination reaction. This suggests that the observed decrease in PF% is not due to the denaturation conditions or the high temperature during deamination, but rather to the binding of ACD protein to the DNA post-deamination (FIG. 16 and FIG. 17).
[000701] Furthermore, we tested if ACD deamination is compatible with SolutionTag workflow for on-flow cell library prep on NovaseqX. To do that first we use aBot to help with adding reagents for Tn5 DNA fragmentation, extension and deamination steps. After that, the flow cell was moved to Novaseq instrument for clustering and sequencing. The initial results show very good positive control (pUC19) conversion with low negative control conversion (lambda DNA). (FIG. 17) for the Helicase- ACD fusion protein, showing that we are able to our fusion is able to deaminate double stranded DNA.
[000702] The complete disclosure of all patents, patent applications, and publications, and electronically available material (including, for instance, nucleotide sequence submissions in, e.g., GenBank and RefSeq, and amino acid sequence submissions in, e.g., SwissProt, PIR, PRF, PDB, and translations from annotated coding regions in GenBank and RefSeq) cited herein are incorporated by reference in their entirety. Supplementary materials referenced in publications (such as supplementary tables, supplementary figures, supplementary materials and methods, and/or supplementary experimental data) are likewise incorporated by reference in their entirety. In the event that any inconsistency exists between the disclosure of the present application and the disclosure(s) of any document incorporated herein by reference, the disclosure of the present application shall govern. The foregoing detailed description and examples have been given for clarity of understanding only. No unnecessary limitations are to be understood therefrom. The disclosure is not limited to the exact details shown and described, for variations obvious to one skilled in the art will be included within the disclosure defined by the claims.
[000703] Unless otherwise indicated, all numbers expressing quantities of components, molecular weights, and so forth used in the specification and claims are to be understood as being modified in all instances by the term "about." Accordingly, unless otherwise indicated to the contrary, the numerical parameters set forth in the specification and claims are approximations that may vary depending upon the desired properties sought to be obtained by the present disclosure. At the very least, and not as an attempt to limit the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. [000704] Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the disclosure are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. All numerical values, however, inherently contain a range necessarily resulting from the standard deviation found in their respective testing measurements.
[000705] All headings are for the convenience of the reader and should not be used to limit the meaning of the text that follows the heading, unless so specified.

Claims

1. An altered cytidine deaminase (ACD) comprising 5mC-selective deaminase activity, the ACD comprising selectivity-enhancing alterations at positions functionally equivalent to amino acid Y130 and P134 in a wild-type AP0BEC3A (SEQ ID NO:3).
2. The ACD of claim 1, wherein the selectivity-enhancing alterations are substitution mutations, wherein the substitution mutation at Y130 is alanine or histidine and the substitution mutation at Pl 34 is tryptophan or tyrosine, optionally wherein the substitution mutation at Y130 is alanine and the substitution mutation at P134 is tryptophan.
3. The ACD of claim 1, further comprising one or more selectivity-enhancing substitution mutations at positions functionally equivalent to amino acid D131, Y132, and D133 in the wild-type AP0BEC3 A (SEQ ID NO:3), wherein the substitution mutations are D13 IX where X is any amino acid except aspartic acid, Y132X where X is any amino acid except tyrosine, and D133X where X is any amino acid except aspartic acid.
4. The ACD of claim 2, wherein the substitution mutation at DI 31 is histidine, arginine, lysine, proline, glycine, asparagine, alanine, or valine.
5. The ACD of claim 2, wherein the substitution mutation at DI 32 is alanine, proline, aspartic acid, glutamic acid, glycine, threonine, valine, glutamine, or arginine.
6. The ACD of claim 2, wherein the substitution mutation at D133 is histidine, tyrosine, phenylalanine, threonine, isoleucine, or valine.
7. The ACD of claim 2, wherein the substitution mutation at D131 is histidine or lysine, wherein the substitution mutation at Y132 is alanine or tyrosine, and the substitution mutation at D133 is arginine or proline.
8. The ACD of claim 1, the ACD further comprising selectivity-enhancing substitution alterations at one or more of positions functionally equivalent to amino acid D131, Y132, and D133 in a wild-type AP0BEC3A (SEQ ID NO:3), wherein the amino acid sequence of Y130, D131, Y132, D133, and P134 is selected from SEQ ID NO: 125, SEQ ID NO: 126, or the sequences described in Table 2, Table 3, FIG. 4B, FIG. 8, or FIG. 9 which demonstrate increased 5mC selectivity compared to wild-type (SEQ ID NO:3).
9. The ACD of claim 7, the ACD further comprising a selectivity-enhancing alteration at L135 in the wild-type AP0BEC3A (SEQ ID NO:3), one or more of positions functionally equivalent to amino acid Y130, D131, Y132, D133, P134, and L135 in a wild-type AP0BEC3A (SEQ ID NO:3), wherein the amino acid sequence of Y130, D131, Y132, D133, P134, and L135 is selected from SEQ ID NO: 125, SEQ ID NO: 126, or the sequences described in FIG. 4B, FIG. 8, or FIG. 9 which demonstrate increased 5mC selectivity compared to wild-type (SEQ ID NO:3).
10. The ACD of claim 1 or 2, wherein the ACD further comprises selectivity-enhancing alterations at positions functionally equivalent to amino acid SI 03 and W 104 in the wild-type APOBEC3A (SEQ ID NO:3), wherein the selectivity-enhancing alterations are substitution mutations, and the substitution mutations are S103X where X is any amino acid except serine and W 104 where X is any amino acid except tryptophan.
11. The ACD of claim 10, wherein the substitution mutation at SI 03 is aspartic acid, histidine, threonine, or valine, and wherein the substitution mutation at amino acid W104 is alanine, aspartic acid, methionine, or glutamic acid.
12. The ACD of claim 10, further comprising a selectivity-enhancing alteration at amino acid G105, wherein the selectivity-enhancing alteration is a deletion of G105.
13. The ACD of claim 10 or 11, the ACD comprising selectivity-enhancing alterations at one or more of positions functionally equivalent to amino acid F102, S103, W104, G105, Y130, D131, Y132, D133, and P134 in a wild-type APOBEC3A (SEQ ID NO:3), wherein the amino acid sequence of F102, S103, W104, G105, Y130, D131, Y132, D133, and P134 is selected from SEQ ID NO: 23, SEQ ID NO:24, or the sequences described in FIG. 8 or FIG.
9 which demonstrate increased 5mC selectivity compared to wild-type (SEQ ID NO:3).
14. The ACD of claim 13, the ACD further comprising a selectivity-enhancing alteration at L135 in the wild-type APOBEC3A (SEQ ID NO:3), wherein the amino acid sequence of F102, S103, W104, G105, Y130, D131, Y132, D133, P134, and L135 is selected from SEQ ID NO: 23, SEQ ID NO:24, or the sequences described in FIG. 8 or FIG. 9 which demonstrate increased 5mC selectivity compared to wild-type (SEQ ID NO:3).
15. The ACD of one of claims 1-9, 11, 12, or 14, further comprising two or more stabilityenhancing alterations.
16. The ACD of claim 15, wherein the two or more stability-enhancing alteration is a substitution mutation, a deletion, an insertion, or a combination thereof.
17. The ACD of claim 16, wherein the ACD comprises two or more stability-enhancing alterations are at a position functionally equivalent to amino acids selected from 117, T 19, G25, S45, A59, K60, N61, L62, L63, C64, G65, F66, Y67, G68, R74, W104, G105, G108, G108, C171, and G188 in the wild-type AP0BEC3A (SEQ ID NO:3).
18. The ACD of claim 17, wherein the at least two stability-enhancing alterations are (i) a substitution mutation, wherein the substitution mutation at 117 is threonine, at T19 is tyrosine, at G25 is lysine or arginine, at S45 is tryptophan, at A59 is proline, at K60 is arginine, at R74 is leucine, at G108 is alanine, at G108 is cytosine or alanine, at C171 is alanine, at G188 is arginine, (ii) a deletion, wherein the deletion is a deletion of W104 and G105R, a deletion of K60-G68, or (iii) any combination thereof.
19. The ACD of claim 18, wherein the ACD comprises stability-enhancing alterations comprising I17T, T19Y, G25K or G25R, S45W, A59P, K60R, deletion of K61-G68, R74L, G108C or G108A, C171A, and G188R.
20. The ACD of claim 19, wherein the ACD comprises (i) stability-enhancing alterations comprising I17T, T19Y, G25K, S45W, A59P, K60R, deletion of K61-G68, R74L, G108C, C171A, and G188R, and (ii) selectivity-enhancing alterations comprising either Y130A, D131H, Y132A, D133R, and P134W, or Y130A, D131K, D133P, and P134W.
21. The ACD of claim 20, wherein the ACD comprises stability-enhancing alterations comprising I17T, T19Y, G25K, S45W, A59P, K60R, deletion of K61-G68, R74L, G108C, C171A, and G188R, and selectivity-enhancing alterations comprising Y130A, D131H, Y132A, D133R, and P134W, and further comprises selectivity-enhancing alterations comprising S103V and W104P.
22. The ACD of claim 20, wherein the ACD comprises SEQ ID NO:23, or an amino acid sequence having at least 80% identity to SEQ ID NO:23.
23. The ACD of claim 20, wherein the ACD comprises stability-enhancing alterations comprising I17T, T19Y, G25K, S45W, A59P, K60R, deletion of K61-G68, R74L, G108C, C171A, and G188R, and selectivity-enhancing alterations comprising Y130A, D131K, D133P, and P134W, and further comprises selectivity-enhancing alterations comprising S103V, W104P, and a deletion of G105.
24. The ACD of claim 23, wherein the ACD comprises SEQ ID NO:24, or an amino acid sequence having at least 80% identity to SEQ ID NO:24.
25. An ACD-helicase complex comprising a helicase attached to an ACD of claim 1.
26. The ACD-helicase complex of claim 25, wherein the ACD and the helicase are a fusion protein.
27. The ACD-helicase complex of claim 26, wherein the fusion protein comprises a linker.
28. The ACD-helicase complex of claim 26, wherein the cytidine deaminase and the helicase are conjugated.
29. A method for deaminating a 5-methyl cytosine, comprising: providing a sample of DNA suspected of comprising single-stranded DNA comprising at least one 5-methyl cytosine (5mC); and contacting the single-stranded DNA with an ACD under conditions suitable for conversion of 5 -methylcytosine (5mC) to thymidine (T) by deamination at a greater rate than conversion of cytosine (C) to uracil (U) by deamination, to result in converted single-stranded DNA, wherein the altered cytidine deaminase is the ACD of claim 1.
30. A method for deaminating a 5-methyl cytosine, comprising: providing a sample of DNA suspected of comprising double-stranded DNA comprising at least one 5-methyl cytosine (5mC); processing the double-stranded DNA to produce a sequencing library; denaturing the sequencing library to result in a single-stranded DNA; contacting the single-stranded DNA with an ACD under conditions suitable for conversion of 5 -methylcytosine (5mC) to thymidine (T) by deamination at a greater rate than conversion of cytosine (C) to uracil (U) by deamination, to result in converted single-stranded DNA, wherein the altered cytidine deaminase is the ACD of claim 1.
31. The method of claim 29 or 30, further comprising converting the converted singlestranded DNA to a converted double-stranded DNA sequencing library.
32. A method for deaminating a 5-methyl cytosine, comprising: providing a sample of DNA suspected of comprising double-stranded DNA comprising at least one 5-methyl cytosine (5mC); contacting the double-stranded DNA with an ACD -helicase complex under conditions suitable for (i) transient conversion of at least a portion of the double-stranded DNA to single-stranded DNA, and (ii) conversion of 5-methylcytosine (5mC) to thymidine (T) by deamination at a greater rate than conversion of cytosine (C) to uracil (U) by deamination, to result in converted double-stranded DNA, wherein the altered cytidine deaminase is the ACD-helicase complex of any one of claims 25- 28; and processing the converted double-stranded DNA to produce a sequencing library.
32. A method of detecting the location of a modified cytosine in a target nucleic acid, the method comprising:
(a) contacting target nucleic acids suspected of comprising at least one modified cytosine with the ACD of any one of claims 25-28 to produce converted nucleic acids comprising at least one converted methyl cytosine;
(b) detecting the at least one converted methyl cytosine in the converted nucleic acids of (a).
33. A composition comprising the ACD of claim 1 or the ACD-helicase complex of any one of claims 25-28 and a suitable buffer.
34. A polynucleotide encoding the ACD of claim 1 or the ACD-helicase fusion protein of any one of claims 26-28 capable of expressing the ACD or ACD-helicase fusion.
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