Altered cytidine deaminases and methods of use

Abstract

The present disclosure is concerned with modified proteins, methods, compositions, and kits for mapping of methylation status of nucleic acids, including 5-methylcytosine and 5-hydroxymethyl cytosine. In some embodiments, the modified proteins have been altered to increase protein stability. In some embodiment, the proteins selectively act on certain modified cytosines of target nucleic acids and include one or more substitution mutations that enhance the selectivity of the proteins for certain modified cytosines, optionally enhance the stability of the proteins, or optionally enhance both selectivity and stability. 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

Abstract This disclosure relates to modified proteins, methods, compositions, and kits for mapping the methylation state of nucleic acids, including 5-methylcytosine and 5-hydroxymethylcytosine. In some embodiments, the modified proteins are modified to improve their stability. In some embodiments, these proteins selectively act on certain modified cytosines of the target nucleic acid and contain one or more substitution mutations that enhance the protein's selectivity to certain modified cytosines, selectively enhance the protein's stability, or selectively enhance both selectivity and stability simultaneously. The invention also provides compositions and kits comprising one or more of said proteins, and methods for using one or more of said proteins.

Claims

Claims1. An altered cytidine deaminase (ACD) comprising one or more stability-enhancing alterations.

2. The ACD of claim 1, wherein the one or more stability-enhancing alteration is a substitution mutation, a deletion, an insertion, or a combination thereof.

3. The ACD of claim 1 or 2, wherein the one or more stability-enhancing alterations are selected from substitution mutations at a position functionally equivalent to Al 12X, A126X, A139X, A148X, A185X, A192X, A59X, A87X, C106X, C161X, C171X, C34X, D145X, D156X, D163X, D167X, D177X, D180X, D41X, D77X, D85X, E109X, E116X, E138X, E157X, E38X, G105X, G108X, G188X, G25X, G27X, H119X, Hl IX, H16X, H182X, H29X, H51LX, I17X, I26X, I89X, K47X, K60X, L135X, L62X, L78X, MMX, M48X, N117X, N196X, N21X, N42X, P80X, Q115X, Q141X, Q169X, Q184X, R111X, R123X, R189X, R39X, R74X, R91X, S103X, S183X, S187X, S20X, S45X, T118X, T164X, T19X, T31X, T93X, V110X, V79X, L12X, D14X, T19X, G27X, R28X, E38X, F54X, H56X, N57X, Y67X, L73X, D77X, S81X, Y90X, I96X, S97X, C101X, F102X, W104X, C106X, A107X, L114X, V120X, L122X, R128X, Y136X, M142X, A146X, K159X, C161X, L186X, a deletion selected from AM1-L12, AN61-G68, AW104-G105 , AQ195-N199 , or 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 wildtype amino acid at that position.

4. The ACD of any one of claims 1-3, 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.

5. The ACD of claim 4, wherein the ACD comprises l. thirteen or more stability -enhancing alterations, or m. fourteen or more stability-enhancing alterations.

6. The ACD of any one of claims 1-5, wherein the one or more stability enhancing alterations is a substitution mutation at a position functionally equivalent to a stability-enhancing mutation selected from Table 5.

7. The ACD of any one of the preceding claims, wherein the one or more stabilityenhancing alterations comprises a deletion selected from Table 6.

8. The ACD any one of the preceding claims, wherein the one or more stability-enhancing alterations comprises at least one substitution and at least one deletion, wherein the substitution mutation is at a position functionally equivalent to a stability-enhancing mutation selected from Table 5, and the deletion is a deletion selected from Table 6.

9. The ACD of any one of the preceding claims, wherein the one or more alterations comprises a deletion selected from Table 6 and one or more ancillary substitution mutations.

10. The ACD of claim 9, wherein the deletion and one or more ancillary substitution mutations of the ACD is AN61-G68 / A59P / K60R, AN61-G68 / A59L / R69Y, AN61- G68 / A59L / K60E / R69N, AN61-G68 / A59P / K60E / R69H, AN61-G68 / A59P / K60Q / R69H, AN61- G68 / A59L / R69N, AN61-G68 / R69D, AN61-G68 / A59L / K60R, AN61-G68 / A59P / K60G / R69L, AW104-G105 / F102R, AW104-G105 / S103N, AW104-G105 / F102R / S103N, AW104-G105 / A1- 12, AW104-G105 / A1-12 / F102R, AW104-G105 / A1-12 / S103N or AW104-G105 / Al- 12 / F102R / S103N.

11. The ACD of any one of the preceding claims, wherein the ACD comprises at least two or more mutations selected from the group consisting of I17T, T19Y, G25R / K, S45W, A59P, K60R, A61-68, R74L, A104, A105, G108A / C, A126C, C171A, and G188R, preferably at least 3- 14 mutations and the ACD has a greater stability than the wildtype cytidine deaminase.

12. The ACD of claim 11 or 12, wherein the ACD comprises a combination of stability mutations selected from:(a) I17T, T19Y, G25K, S45W, A59P, K60R, deletion of 61-68, R74L, deletion of G105, G108C, C171A, and G188R;(b) I17T, T19Y, G25K, S45W, A59P, K60R, deletion of 61-68, R74L, G108C, C171A, and G188R;(c) I17T, T19Y, G25K, S45W, A59P, K60R, deletion of 61-68, R74L, deletion of W104, deletion of G105, G108C, C171A, and G188R;(d) I17T, T19Y, G25K, S45W, A59P, K60R, deletion of 61-68, R74L, deletion of G105, G108C,(e) C171A, and G188R; I17T, T19Y, G25K, S45W, A59P, K60R, deletion of 61-68, R74L, G108C, C171A, and G188R; or(f) I17T, T19Y, G25K, S45W, A59P, K60R, deletion of 61-68, R74L, G108C, C171A, and G188R.

13. The ACD of claim 11 or 12, wherein the ACD further comprises one or more mutations additional mutations selected from Table 4 or Table 5, and / or one or more deletion mutations selected from Table 6.

14. The ACD of any one of the preceding claims, wherein the ACD comprises a combination of stability-enhancing alterations selected from Table 7 or 8.

15. The ACD of any one of claims 1-14, wherein the ACD is based on a member of the AID subfamily, the APOB EC 1 subfamily, the APOBEC2 subfamily, the APOBEC3A subfamily, the APOBEC3B subfamily, the APOBEC3C subfamily, the APOBEC3D subfamily, the APOBEC3F subfamily, the APOBEC3G subfamily, the APOBEC3H subfamily, or the APOBEC4 subfamily.

16. The ACD of claim 15, wherein the ACD is APOBECC3A (SEQ ID NO:3) comprising one or more stabilizing mutations.

17. The ACD of claim 16, wherein the cytidine deaminase comprises a ZDD motif H- [P / A / V]-E-X[23-28]-P-C-X[2-4]-C (SEQ ID NO: 12), wherein the ACD is a member of the APOBEC3A subfamily and comprises X[i6-26]-GRXXTXLCYXV-Xi5-GXXXN-Xi2-HAEXXF- X14-YXXTWXXSWSPC- X[2-4]-CA-X5-FL-X7-LXIXXXR(L / I)Y-X8-GLXXLXXXG-X5-M-X4- FXXCWXXFV-X6-FXPW-X13-LXXI- X[2-6] (SEQ ID NO: 14), or a combination thereof.

18. The ACD of any one of the preceding claims, wherein the ACD comprises wildtype activity.

19. The ACD any one of claims 1-17, wherein the ACD comprises one or more alteration resulting in 5mC-selective deaminase activity.

20. The ACD of claim 19, wherein the one or more alteration comprises (Tyr / Phe)130X substitution, wherein X is selected from A, G, F, H, Q, M, N, K, V, D, E, S, C, P or T, whereinthe position number designation is functionally equivalent to the position in a wildtype APOBEC3A (SEQ ID NO:3) which increases 5mC selectivity in the ACD.

21. The 5mC-selective ACD of claim 20, wherein the ACD further comprises Tyrl32X, wherein X is selected from R, H, K or Q, wherein the position number designation is functionally equivalent to the position in a wildtype APOBEC3A (SEQ ID NO:3).

22. The 5mC-selective ACD of claim 20 or 21, wherein the ACD comprises Y130A and Y132H, wherein the position number designation is functionally equivalent to the position in a wildtype APOBEC3A (SEQ ID NO:3).

23. The 5mC-selective ACD of any one of claims 19-22, wherein 5mC-selective ACD further comprises one or more selectivity-enhancing alteration.

24. The 5mC-selective ACD of claim 23, wherein the one or more selectivity-enhancing alteration comprises a substitution mutation at a position functionally equivalent to D133, wherein the position number designation is functionally equivalent to the position in a wildtype APOBEC3A (SEQ ID NO:3).

25. The 5mC-selective ACD of claim 24, wherein the substitution mutation at a position functionally equivalent to D133 is W or C.

26. The 5mC-selective ACD of any one of claims 20-25, wherein the one or more selectivityenhancing alteration comprises a substitution mutation at a position functionally equivalent to amino acids having proximity to the ACD active site.

27. The 5mC-selective ACD of one of claims 20-26, wherein the one or more selectivityenhancing alteration comprises a substitution mutation at a position functionally equivalent to amino acids that are expected to co-evolve with residues 57, 97, 98, 130, 130, 132, and / or 134, wherein the position number designation is functionally equivalent to the position in a wildtype APOBEC3A (SEQ ID NO:3)28. The 5mC-selective ACD of any one of claims 20-27, wherein the one or more selectivityenhancing alteration comprises a substitution mutation at a position functionally equivalent to amino acids based on the observation that substitution mutations at position 103 in an APOBEC3A protein affect selectivity, and deletion of residues 104-105 is beneficial to stability, wherein the position number designation is functionally equivalent to the position in a wildtype APOBEC3A (SEQ ID NO:3).

29. The 5mC-selective ACD of any one of claims 20-28, wherein the one or more selectivityenhancing alteration is selected from Table 2.

30. The 5mC-selective ACD of any one of claims 20-29, wherein the one or more selectivityenhancing alteration is selected from Table 3.

31. The 5mC-selective ACD of any one of claims 20-30, wherein the 5mC-selective ACD comprises Y130X, Y132X and D133X, wherein X is an amino acid substitution that enhances selectivity for 5mC.

32. The 5mC-selective ACD of claim 31, wherein the 5mC-selective ACD comprises Y130A, Y132H and D133W.

33. The ACD of any one of claims 1-32, wherein the ACD is a member of the APOBEC3A family and comprises and one of SEQ ID NO:68-77, SEQ ID NO: 116, or SEQ ID NO: 123.

34. The ACD of any one of claims 1-33, wherein the ACD is a dimer.

35. A polynucleotide encoding the ACD of any one of the preceding claims.

36. A composition comprising the ACD of any one of claims 1-34 and a buffer.

37. The composition of claim 36, wherein the composition further comprises a sample comprising DNA comprising at least one modified cytosine, wherein the modified cytosine is 5- methyl cytosine (5mC).

38. The composition of claim 37, wherein the DNA comprises single-stranded DNA.

39. The composition of claim 37 or 38, wherein the sample comprises genomic DNA or cell free DNA.

40. The composition of claim 39, wherein the genomic DNA is from a single cell or is a mixture from a plurality of cells.

41. A method compri sing : 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 ACD is the ACD of any one of claims 1-34; and processing the converted single-stranded DNA to produce a sequencing library.

42. The method of claim 41 or 43, wherein the method further comprises, before the providing, denaturing double-stranded DNA present in the sample to result in single-stranded DNA.

43. 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 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 claims 1-34; and converting the converted single-stranded DNA to a converted double-stranded DNA sequencing library.

44. The method of claim 43, wherein the processing comprises fragmentation or tagmentation of the double-stranded DNA and addition of a universal sequence to the doublestranded DNA fragments.

45. The method of claim 44, wherein the universal sequence is part of an adapter added to the double-stranded DNA fragments.

46. The method of claim 44, wherein the converting comprises amplifying the converted single-stranded DNA to be the converted double-stranded DNA.

47. The method of claim 41 or 43, wherein the sample is a biological sample.

48. The method of claim 47, wherein the biological sample comprises cell-free DNA.

49. The method of claim 47, wherein the biological sample comprises a fluid selected from blood or serum.

50. The method claim 47, wherein the sample comprises single cells or isolated nuclei.

51. The method of claim 47, wherein the biological sample comprises a tissue.

52. The method of claim 51, wherein the tissue comprises tumor tissue.

53. The method of claim 41 or 43, 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.

54. The method of claim 41 or 43, 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 converted double-stranded DNA 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 converted double-stranded DNA sequencing library.

55. 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 altered cytidine deaminase of any one of claims 1-34 to produce converted nucleic acids comprising at least one converted cytosine;(b) detecting the at least one converted cytosine in the converted nucleic acids of (a).

56. The method of claim 55, wherein the detecting comprises identifying thymidine nucleotides in the converted nucleic acid to determine the location of 5mC nucleotides in the target nucleic acid.

57. The method of any one of claims 55-56, wherein the detecting comprises sequencing the converted nucleic acids or hybridizing nucleic acid probes to the converted nucleic acids.

58. The method of any one of claims 55-57, wherein the detecting comprises sequencing the converted nucleic acids, the method further comprising:(c) comparing the sequence of the converted nucleic acids with an untreated reference sequence to determine which cytosines in the target nucleic acids are modified.

59. The method of any one of claims 55-58, wherein a predetermined sequence of the untreated reference sequence and a predetermined sequence of the converted nucleic acid are compared.

60. The method of any one of claims 55-59, wherein the predetermined sequence comprises a CpG island or a promoter.

61. The method of any one of claims 55-60, wherein the detecting comprises hybridizing the converted nucleic acids to the nucleic acid probes, optionally wherein the nucleic acid probes are present on an analyte array, the method further comprising sequencing the hybridized converted nucleic acids.

62. The method of any one of claims 55-60, wherein the detecting comprises hybridizing the converted nucleic acids to the nucleic acid probes, the method further comprising amplifying the converted nucleic acid, wherein the nucleic acid probes comprise two primers for amplification of a predetermined sequence, wherein the primers anneal to regions of converted nucleic acids comprising at least one converted cytosine with a greater affinity than to the regions of converted nucleic acids wherein at least one cytosine is not a converted cytosine, wherein the presence of an amplified product is indicative of a modified cytosine in the target nucleic acid.

63. The method of claim 55, wherein the detecting comprises hybridizing the converted nucleic acids to the nucleic acid probes, the method further comprising cleaving a single stranded DNA (ssDNA) reporter substrate by a CRISPR-based system, wherein the ssDNA reporter substrate comprises a fluorophore and a quencher, wherein the presence of fluorescence is indicative of a modified cytosine in the target nucleic acid.

64. The method of claim 63, wherein the CRISPR-based system comprises a guide RNA sequence that anneals to a predetermined sequence of a nucleic acid comprising at least one converted cytosine and anneals at lower affinity to the predetermined sequence of the nucleic acid when at least one cytosine is not a converted.

65. The method of claim 63, wherein the CRISPR-based system comprises CRISPR-Casl2.

66. The method of any one of claim 55-65, wherein the detecting comprises hybridizing the converted nucleic acids to the nucleic acid probes, wherein the converted nucleic acids are present in a fixed cell, wherein the nucleic acid probes comprise a fluorescent labeled probe, and wherein the nucleic acid probes anneal to a predetermined sequence of converted nucleic acids comprising at least one converted cytosine with a greater affinity than to the regions of converted nucleic acids wherein at least one cytosine is not a converted cytosine, wherein the presence of cell-associated fluorescence is indicative of a modified cytosine in the target nucleic acid.

67. The method of any one of claims 55-66, wherein the untreated reference sequence is a predetermined sequence.

68. The method of any one of claims 55-67, wherein the at least one modified cytosine is a 5mC or a 5hmC.

69. The method of any one of claims 55-68, further comprising providing the target nucleic acids, wherein the providing comprises preparing single stranded (ss) DNA from a sample comprising the target nucleic acids.

70. The method of claim 55, wherein the target nucleic acids are genomic DNA or cell free DNA.

71. The method of claim 70, wherein the genomic DNA is from a single cell or is a mixture from a plurality of cells.

72. The method of any one of claims 55-71, wherein the sequencing comprises processing the converted nucleic acids to produce a sequencing library.

73. The method of any one of claims 55-71, 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.

74. The method of any one of claims 55-72, wherein the target nucleic acids are obtained from a subject, wherein the detecting comprises obtaining a pattern of cytosine modification in the converted nucleic acids, the method further comprising comparing the pattern of cytosine modification in the converted nucleic acids with the pattern of cytosine modification in a reference nucleic acid.

75. The method of claim74, wherein the subject has or is at risk of having a disease or condition, wherein the reference nucleic acid is from a normal subject.

76. The method of claim 74, wherein the pattern of cytosine modification is linked in-cz5 to a coding region that is correlated with a disease or condition.

77. The method of claim 74, wherein the pattern of cytosine modification is linked in-cis to a coding region, wherein the coding region in the reference nucleic acid is transcriptionally activeor transcriptionally inactive, wherein the comparing further comprises 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.

78. The method of claim 74, wherein transcription of the coding region is correlated with a disease or condition.

79. The method of claim 74, wherein the subject has the disease or condition and is undergoing treatment for the disease or condition, the method further comprising determining if the treatment is correlated with a change in the pattern of cytosine modification in the subject.

80. The method of claim 74, wherein the subject previously had the disease or condition, the method further comprising comparing the pattern of cytosine modification in the subject with the pattern of cytosine modification in the subject when the subject had the disease or condition.

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