Mapping Cytosine Modifications

• US 20160138079A1
• Filed: 01/12/2016
• Published: 05/19/2016
• Est. Priority Date: 03/15/2012
• Status: Active Grant

• Current Assignee: New England Biolabs Incorporated
• Original Assignee: New England Biolabs Incorporated
• Inventors: Guan, Shengxi, Dai, Nan, Zhu, Zhenyu, Correa, Ivan R. Jr., Quimby, Aine, Borgaro, Janine
• Granted Patent: US 10,081,827 B2
• Time in Patent Office: Days
• US Class Current: 1/1
• CPC Class Codes: C12N 9/0069 acting on single donors with incorporation of molecular oxygen, i.e. oxygenases (1.13)
• CPC Class Codes: C12N 9/0071 acting on paired donors with incorporation of molecular oxygen (1.14)
• CPC Class Codes: C12N 9/1048 Glycosyltransferases (2.4)
• CPC Class Codes: C12N 9/1051 Hexosyltransferases (2.4.1)
• CPC Class Codes: C12N 9/22 Ribonucleases RNAses, DNAses catalytic nucleic acids C12N15/113
• CPC Class Codes: C12P 19/18 produced by the action of a glycosyl transferase, e.g. alpha-, beta- or gamma-cyclodextrins
• CPC Class Codes: C12P 19/30 Nucleotides
• CPC Class Codes: C12P 19/34 Polynucleotides, e.g. nucleic acids, oligoribonucleotides
• CPC Class Codes: C12Q 1/68 involving nucleic acids
• CPC Class Codes: C12Q 1/6827 for detection of mutation or polymorphism
• CPC Class Codes: C12Q 2521/301 Endonuclease
• CPC Class Codes: C12Q 2521/307 Single strand endonuclease
• CPC Class Codes: C12Q 2523/115 oxidising agents
• CPC Class Codes: C12Q 2525/101 incorporating non-naturally occurring nucleotides, e.g. inosine
• CPC Class Codes: C12Q 2527/125 Specific component of sample, medium or buffer
• CPC Class Codes: C12Q 2537/164 Methylation detection other then bisulfite or methylation sensitive restriction endonucleases
• CPC Class Codes: C12Q 2563/131 the label being a member of a cognate binding pair, i.e. extends to antibodies, haptens, avidin
• CPC Class Codes: C12Q 2565/501 being an array of oligonucleotides
• CPC Class Codes: C12Y 114/11 with 2-oxoglutarate as one donor, and incorporation of one atom each of oxygen into both donors (1.14.11)

Abstract

Methods, compositions and kits for selectively altering and detecting modified cytosine residues are provided.

Claims

1. A preparation comprising a reducing agent;

a glycosyltransferase; and

and a UDP derivative selected from the group consisting of UDP-glucose and UDP-azidoglucose.

2. A preparation according to claim 1, wherein the reducing agent is NaBH₄.

3. A preparation according to claim 1 or 2, further comprising an endonuclease.

4. A preparation according to claim 3, wherein the endonuclease has an amino acid sequence at least 95% identical to AbaSI.

5. A preparation according to claim 3, wherein the endonuclease has an amino acid sequence at least 95% identical to an enzyme selected from the group consisting of AbaAI, AbaCI, AbaDI, AbaTI, AbaUI, AcaPI, and PxyI.

15. A preparation according to any one of claims 1-14, further comprising a nucleic acid.

16. A preparation according to claim 15, wherein the nucleic acid comprises 5-hydroxymethylcytosine.

17. A preparation according to claim 15 or 16, wherein the nucleic acid comprises glucosyl-5-hydroxymethylcytosine.

18. A preparation according to any one of claims 15-17, wherein the nucleic acid comprises β

-glucosaminyloxy-5-methylcytosine.

19. A preparation according to any one of claims 15-18, wherein the nucleic acid comprises 5-formylcytosine.

20. A preparation according to any one of claims 15-19, wherein the nucleic acid comprises 5-carboxycytosine.

21. A preparation according to any one of claims 15-20, wherein the nucleic acid comprises 5-methylcytosine.

22. A preparation according to any one of claims 15-21, wherein the nucleic acid comprises DNA.

23. A preparation according to any one of claims 15-22, wherein the nucleic acid comprises RNA.

6. A preparation comprising:

a β

-glycosyltransferase;

UDP-glucosamine, and an endonuclease having an amino acid sequence at least 95% identical to an enzyme selected from the group consisting of AbaSI, AbaAI, AbaCI, AbaDI, AbaTI, AbaUI, AcaPI, and PxyI.

7. A preparation comprising:

(i) a glycosyltransferase;

(ii) an endonuclease having an amino acid sequence at least 95% identical to an enzyme selected from the group consisting of AbaSI, AbaAI, AbaCI, AbaDI, AbaTI, AbaUI, AcaPI, and PxyI UDP-glucose;

(iii) UDP-glucose; and

(iv) an oxidizing agent.

8. A preparation according to claim 7, comprising UDP-glucosamine.

9. A preparation according to claim 7, comprising UDP-glucose and an oxidizing agent.

10. A preparation according to claim 9, wherein the oxidizing agent is a methylcytosine oxygenase.

11. A preparation according to claim 10, wherein the methylcytosine oxygenase is a TET enzyme.

12. A preparation according to claim 10, wherein the methylcytosine oxygenase is mYOX1.

13. A preparation according to claim 9, wherein the oxidizing agent is an inorganic oxidizing agent.

14. A preparation according to claim 13, wherein the oxidizing agent is KRuO₄.

24. A method for selectively altering modified nucleotides in a nucleic acid containing modified nucleotides, comprising:

reacting a first subset of the modified nucleotides in the nucleic acid with UDP-glucosamine in the presence of a β

-glycosyltransferase to convert hydroxymethylated nucleotides in the nucleic acid to glucosaminylated nucleotides, and

subsequently reacting the nucleic acid with (i) a reducing agent (ii) a UDP derivative selected from the group consisting of UDP-glucose and UDP-azidoglucose to convert a second subset of nucleotides in the nucleic acid to glucosylated or azidoglucosylated nucleotides.

25. A method according to claim 24, wherein the reaction with the UDP derivative is catalyzed by a glycosyltransferase.

26. A method according to claim 24 or 25, wherein the second subset of nucleotides comprises 5-formylcytosine.

27. A method according to claim 26, comprising differentiating pre-existing 5-hydroxymethylcytosine from newly formed 5-hydroxymethylcytosine in a nucleic acid where 5-formylcytosine is a 5-hydroxymethylcytosine precursor.

28. A method according to any one of claims 24-26, wherein the reducing agent is NaBH₄.

29. A method according to any one of claims 24-26, wherein the second subset of nucleotides comprises 5-carboxycytosine.

30. A method according to any one of claims 24-29, further comprising cleaving the nucleic acid with an endonuclease specific for the glucosylated or azidoglucosylated nucleotides and not for glucosaminylated nucleotides.

31. A method according to claim 30, comprising ligating an adapter to the cleaved end of the nucleic acid.

32. A method according to claim 30 or 31, wherein the endonuclease is AbaSI.

33. A method according to claim 30 or 31, wherein the endonuclease is selected from the group consisting of AbaAI, AbaCI, AbaDI, AbaTI, AbaUI, AcaPI, and PxyI.

34. A method according to any one of claims 24-33, wherein the UDP derivative is UDP-glucose.

35. A method according to any one of claims 24-34, wherein the UDP derivative is UDP-azidoglucose.

36. A method according to claim 35, wherein the UDP-azidoglucose is UDP-6-azidoglucose, converting a 5-formylcytosine or 5-carboxycytosine in the nucleic acid to a β

-6-azide-glucosyl-5-hydroxymethylcytosine.

37. A method according to claim 35, further comprising labeling the β

-6-azide-glucosyl-5-hydroxymethylcytosine with biotin.

38. A method comprising placing off and on switches for endonuclease cleavage of a nucleic acid containing modified nucleotides by alternatively reacting the nucleic acid with a UDP-glucosamine substrate and a UDP-glucose substrate.

39. A method for differentiating 5-(β

-glucosyloxymethyl)cytosine from 5-(β

-2-glucosaminyloxymethyl)cytosine in a nucleic acid, comprising;

reacting the nucleic acid with an endonuclease capable of cleaving a nucleic acid at a glucosylated nucleotide but not at an glucosaminylated nucleotide.

40. A method according to claim 39, wherein the endonuclease has an amino acid sequence at least 95% identical to AbaSI.

41. A method according to claim 39, wherein the endonuclease has an amino acid sequence at least 95% identical to an enzyme selected from the group consisting of AbaAI, AbaCI, AbaDI, AbaTI, AbaUI, AcaPI, and PxyI.

42. A method for selectively altering modified nucleotides in a nucleic acid containing modified nucleotides, comprising:

reacting a first subset of the modified nucleotides in the nucleic acid with UDP-glucosamine in the presence of a glycosyltransferase to convert hydroxymethylated nucleotides in the nucleic acid to glucosaminylated nucleotides, and

subsequently reacting the nucleic acid with an oxidizing agent and UDP-glucose to convert a second subset of modified nucleotides in the nucleic acid to glucosylated nucleotides.

43. A method according to claim 42, wherein the reaction with UDP-glucose is catalyzed by a glycosyltransferase.

44. A method according to claim 42, further comprising cleaving the nucleic acid with an endonuclease specific for the glucosylated nucleotides and not for glucosaminylated nucleotides.

45. A method according to claim 44, wherein the endonuclease has an amino acid sequence at least 95% identical to AbaSI.

46. A method according to claim 44, wherein the endonuclease has an amino acid sequence at least 95% identical to an enzyme selected from the group consisting of AbaAI, AbaCI, AbaDI, AbaTI, AbaUI, AcaPI, and PxyI.

47. A method according to any one of claims 44-46, comprising ligating an adapter to the cleaved end of the nucleic acid.

48. A method according to any one of claims 44-46, wherein the modified nucleotides of the second subset are 5-methylcytosines and the oxidizing agent is a methylcytosine oxygenase.

49. A method according to claim 48, wherein the methylcytosine oxygenase is a TET enzyme.

50. A method according to claim 48, wherein the methylcytosine oxygenase is mYOX1.

51. A method according to any one of claims 42-47, wherein the modified nucleotides of the second subset are 5-methylcytosines and the oxidizing agent is an inorganic oxidizing agent.

52. A method according to claim 51, wherein the oxidizing agent is KRuO₄.

53. A method according to any of claims 42 through 52, comprising differentiating pre-existing 5-hydroxymethylcytosine from newly formed 5-hydroxymethylcytosine in a nucleic acid where 5-methylcytosine is a precursor of 5-hydroxymethylcytosine.

54. A method for differentiating a 5-methylcytosine from 5-hydroxymethylcytosine in a genome or genome fragment, comprising:

(a) reacting the isolated genome or genome fragment containing 5-methylcytosine and 5-hydroxymethylcytosine with (i) UDP-glucose or UDP-glucosamine in the presence of a glycosyltransferase catalyzing transfer of glucose or glucosamine to the 5-hydroxymethylcytosine;

(ii) TET or mYOX1 to convert 5-methylcytosine to 5-hydroxymethylcytosine and (iii) UDP-glucose or UDP-glucosamine in the presence of a glycosyltransferase catalyzing transfer of glucose or glucosamine to the 5-hydroxymethylcytosine generated by TET or mYOX1;

(b) cleaving the glucosylated template with a modification-dependent endonuclease that recognizes at least one of the modified nucleotides; and

(c) differentiating the 5-methylcytosine from the 5-hydroxymethylcytosine by an altered cleavage pattern.

55. A method according to claim 54, wherein exposure of the isolated genome or genome fragment from step (a)(i) is followed by simulataneous exposure to TET or mYOX1 and UDP-glucose or UDP-glucosamine in the presence of a glycosyltransferase, such that steps (a)(ii) and (a)(iii) are performed concurrently, glycosylating 5-hydroxymethylcytosine as it is being generated.

56. A method for differentiating a 5-methylcytosine from one or more of its oxidation products in a genome or genome fragment containing 5-methylcytosine and 5-hydroxymethylcytosine, comprising:

(a) reacting the isolated genome or genome fragment with UDP-2-glucosamine in the presence of a β

-glycosyltransferase catalyzing the transfer of 2-glucosamine to the 5-hydroxymethylcytosine;

(b) reacting the isolated genome or genome fragment from (a) with mYOX1 or TET or a chemical oxidizing agent and optionally with a reducing agent;

(c) cleaving the template with a modification dependent endonuclease that is capable of selectively cleaving a 5-hydroxymethylcytosine and not a 5-glucosaminated hydroxymethylcytosine; and

(d) differentiating the 5-methylcytosine from one or more of its oxidation products by an altered cleavage pattern.

57. A kit comprising a reducing agent;

a glycosyltransferase; and

a UDP derivative selected from the group consisting of UDP-glucose and UDP-azidoglucose.

58. A kit according to claim 57, wherein the preparation comprises a reducing agent.

59. A kit according to claim 58, wherein the reducing agent is NaBH₄.

60. A kit according to any one of claims 57-59, wherein the preparation comprises an oxidizing agent.

61. A kit according to any one of claims 57-60, wherein the UDP-derivative is UDP-glucose.

62. A kit according to any one of claims 57-60, wherein the UDP derivative is UDP-azidoglucose.

63. A kit according to claim 62, wherein the UDP-azidoglucose is UDP-6-azidoglucose.

64. A kit according to claim 62 or 63, further comprising UDP-glucose.

65. A kit according to any one of claims 57-64, wherein the glycosyltransferase is a β

-glycosyltransferase.

66. A kit according to any one of claims 57-65, further comprising UDP-glucosamine.

67. A kit according to any one of claims 57-66, further comprising a restriction endonuclease.

68. A kit according to claim 67, wherein the restriction endonuclease has an amino acid sequence at least 95% identical to AbaSI.

69. A kit according to claim 67, wherein the restriction endonuclease has an amino acid sequence at least 95% identical to an enzyme selected from the group consisting of AbaAI, AbaCI, AbaDI, AbaTI, AbaUI, AcaPI, and PxyI.

74. A kit according to claim 60, 70, or 73, wherein the oxidizing agent is a methylcytosine oxygenase.

75. A kit according to claim 74, wherein the methylcytosine oxygenase is a TET enzyme.

76. A kit according to claim 74, wherein the methylcytosine oxygenase is mYOX1.

77. A kit according to claim 60, 70, or 73, wherein the oxidizing agent is an inorganic oxidizing agent.

78. A kit according to claim 77, wherein the oxidizing agent is KRuO₄.

79. A kit according to any one of claims 57-78, further comprising a nucleic acid.

80. A kit according to claim 79, wherein the nucleic acid comprises 5-hydroxymethylcytosine.

81. A kit according to claim 79 or 80, wherein the nucleic acid comprises glucosyl-5-hydroxymethylcytosine.

82. A kit according to any one of claims 76-78, wherein the nucleic acid comprises β

-glucosaminyloxy-5-methylcytosine.

83. A kit according to any one of claims 76-79, wherein the nucleic acid comprises 5-formylcytosine.

84. A kit according to any one of claims 76-80, wherein the nucleic acid comprises 5-carboxycytosine.

70. A kit comprising:

a β

-glycosyltransferase;

UDP-glucosamine;

UDP-glucose; and

an oxidizing agent.

71. A kit comprising:

(i) a glycosyltransferase;

(ii) an endonuclease having an amino acid sequence at least 95% identical to an enzyme selected from the group consisting of AbaSI, AbaAI, AbaCI, AbaDI, AbaTI, AbaUI, AcaPI, and PxyI UDP-glucose; and

(iii) (a) UDP-glucose and an oxidizing agent or (b) UDP-glucosamine.

72. A kit according to claim 71, comprising UDP-glucosamine.

73. A kit according to claim 71 or 72, comprising UDP-glucose and an oxidizing agent.

85. A method for selectively altering modified nucleotides in a nucleic acid containing modified nucleotides, comprising:

i. reacting a first subset of the modified nucleotides in the nucleic acid with UDP-glucosamine in the presence of a β

-glycosyltransferase to convert hydroxymethylated nucleotides in the nucleic acid to glucosaminylated nucleotides, and

ii. subsequently reacting the nucleic acid with (i) a reducing agent (ii) a UDP derivative selected from the group consisting of UDP-glucose and UDP-azidoglucose to convert a second subset of nucleotides in the nucleic acid to glucosylated or azidoglucosylated nucleotides.

86. A method according to claim 85, wherein the reaction with the UDP derivative is catalyzed by a glycosyltransferase.

87. A method according to claim 85, wherein the second subset of nucleotides comprises 5-formylcytosine.

88. A method according to claim 85, further comprising cleaving the nucleic acid with an endonuclease specific for the glucosylated or azidoglucosylated nucleotides and not for glucosaminylated nucleotides.

89. A method for differentiating 5-β

-glucosyloxymethyl)cytosine from 5-(β

-2-glucosaminyloxymethyl)cytosine in a nucleic acid, comprising;

reacting the nucleic acid with an endonuclease capable of cleaving a nucleic acid at a glucosylated nucleotide but not at an glucosaminylated nucleotide.

90. A preparation comprising a reducing agent;

a glycosyltransferase; and

and a UDP derivative selected from the group consisting of UDP-glucose and UDP-azidoglucose.

91. A preparation according to claim 90, further comprising an endonuclease.

92. A preparation according to claim 90, wherein the endonuclease has an amino acid sequence at least 95% identical to an enzyme selected from the group consisting of AbaSI, AbaAI, AbaCI, AbaDI, AbaTI, AbaUI, AcaPI, and PxyI.

93. A preparation according to claim 90, further comprising a nucleic acid.

94. A preparation comprising:

a β

-glycosyltransferase;

UDP-glucosamine, and an endonuclease having an amino acid sequence at least 95% identical to an enzyme selected from the group consisting of AbaSI, AbaAI, AbaCI, AbaDI, AbaTI, AbaUI, AcaPI, and PxyI.

95. A kit comprising a reducing agent;

a glycosyltransferase; and

a UDP derivative selected from the group consisting of UDP-glucose and UDP-azidoglucose.

96. A kit according to any claim 95, further comprising an oxidizing agent.

97. A kit according to claim 95, wherein the glycosyltransferase is a β

-glycosyltransferase.

98. A kit according to claim 95, further comprising UDP-glucosamine.

99. A kit according to claim 95, further comprising a restriction endonuclease.

100. A kit according to claim 95, further comprising a nucleic acid.

101. A kit comprising:

a β

-glycosyltransferase;

UDP-glucosamine;

UDP-glucose; and

an oxidizing agent.

102. A kit according to claim 101, further comprising UDP-glucosamine.

103. A kit according to claim 101, wherein the oxidizing agent is a methylcytosine oxygenase.

104. A kit according to claim 101, wherein the methylcytosine oxygenase is a TET enzyme.

105. A kit according to claim 101, wherein the methylcytosine oxygenase is mYOX1.

106. A kit according to claim 101, wherein the oxidizing agent is an inorganic oxidizing agent.

107. A kit according to claim 106, wherein the oxidizing agent is KRuO₄.

108. A kit according to claim 101, further comprising a nucleic acid.