COMPOSITION AND METHODS FOR DETECTING ADENOSINE MODIFICATIONS

• US 20180245128A1
• Filed: 08/30/2016
• Published: 08/30/2018
• Est. Priority Date: 08/31/2015
• Status: Active Grant

• Current Assignee: University of Chicago
• Original Assignee: University of Chicago
• Inventors: He, Chuan, Chen, Kai, Dai, Qing
• Granted Patent: US 11,396,672 B2
• Time in Patent Office: Days
• CPC Class Codes: C12N 9/78 acting on carbon to nitrogen bonds other than peptide bonds (3.5)
• CPC Class Codes: C12Q 1/34 involving hydrolase
• CPC Class Codes: C12Q 1/6806 Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6827 for detection of mutation or polymorphism
• CPC Class Codes: C12Q 1/6858 Allele-specific amplification
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2521/539 Deaminase
• CPC Class Codes: C12Q 2525/117 incorporating modified base
• CPC Class Codes: C12Q 2537/164 Methylation detection other then bisulfite or methylation sensitive restriction endonucleases
• CPC Class Codes: C12Y 305/04002 Adenine deaminase (3.5.4.2)
• CPC Class Codes: G01N 2333/978 acting on carbon to nitrogen bonds other than peptide bonds (3.5)

Abstract

The current disclosure relates to methods, compositions and kits for detecting modified adenosine in a target RNA molecule. Aspects relate to a method for detecting modified adenosine in a target ribonucleic acid (RNA) comprising contacting the target RNA with an adenosine deaminase enzyme (adenosine deaminase, RNA-specific) to generate a target RNA with deaminated adenosines and sequencing the target RNA with deaminated adenosines; wherein the modified adenosine is detected when the nucleotide sequence is adenosine.

Claims

1. A method for detecting modified adenosine in a target ribonucleic acid (RNA) comprising contacting the target RNA with an adenosine deaminase enzyme to generate a target RNA with deaminated adenosines and sequencing the target RNA with deaminated adenosines;

wherein any modified adenosine in the target RNA is read as an adenosine in the sequencing of the target RNA with deaminated adenosines.

2. The method of claim 1, wherein the adenosine deaminase enzyme is ADAR (adenosine deaminase, RNA-specific).

3. The method of claim 1 or 2, wherein the method further comprises:

contacting target RNA with a demethylating enzyme;

contacting the demethylated target RNA with the adenosine deaminase enzyme to produce control RNA; and

sequencing the control RNA;

wherein any modified adenosine in the target RNA is read as a guanosine in the sequencing of the control RNA with deaminated adenosines.

4. The method of claim 3 further comprising comparing the sequence of the target RNA with deaminated adenosines to the sequence of the demethylated target RNA .

5. The method of claim 3 or 4, wherein the demethylating enzyme is an N⁶-methyladenosine-specific demethylating enzyme.

6. The method of any one of claims 3-6, wherein the demethylating enzyme is ALKBH5.

7. The method of any one of claims 1-6, wherein the target and/or demethylated target RNA is in a duplex with a complementary strand of RNA or DNA.

8. The method of claim 7, wherein the complementary strand is DNA.

9. The method of any one of claims 7-8, wherein the complementary strand comprises modified adenosine.

10. The method of any one of claims 1-9, wherein adenosine deaminase enzyme is from Drosophila.

11. The method of any one of claims 1-10, wherein the target RNA is mRNA, lncRNA, pri-microRNA, pre-piRNA, rRNA, tRNA, snoRNA, or snRNA.

12. The method of claim 11, wherein the target RNA is mRNA or lncRNA.

13. The method of any one of claims 1-12, wherein the method further comprises isolating RNA.

14. The method of any one of claims 1-13, wherein the method further comprises generating a nucleic acid strand that is complementary with the target and/or demethylated target RNA and hybridizing the complementary nucleic acid strand with the target RNA and/or demethylated target RNA.

15. The method of claim 14, wherein generating the nucleic acid strand that is complementary with the target and/or demethylated target RNAcomprises synthesis of a nucleic acid strand complementary to the target and/or demethylated target RNA.

16. The method of claim 15, wherein the synthesis of the nucleic acid strand comprises a synthesis reaction composition comprising adenosine triphosphate, wherein all of the adenosine triphosphate is modified adenosine triphosphate.

17. The method of any one of claims 1-16, wherein the modified adenosine is N⁶-methyladenosine.

18. The method of any one of claims 1-16, wherein the modified adenosine is N¹-methyladenosine.

19. The method of any one of claims 15-18, wherein the complementary nucleic acid strand is RNA.

20. The method of claim 15, wherein the complementary nucleic acid strand is DNA.

21. The method of any one of claims 14-19, wherein the method further comprises contacting the target and/or demethylated target RNAwith RNA replicase to synthesize a complementary RNA strand.

22. The method of claim 21, wherein the RNA replicase is Phi6.

23. The method of any one of claims 1-21, wherein the target RNA strand is immobilized on a solid support.

24. The method of any one of claims 1-23, wherein the target RNA strand is labeled.

25. The method of claim 24, wherein the label is a phosphothioate group.

26. The method of claim 25, wherein the target RNA is immobilized by reaction of the phosphothioate group with a thiol-reactive group.

27. The method of claim 26, wherein the thiol-reactive group is iodoacetamide, maleimide, or methanethiosulfonate.

28. The method of any one of claims 23-26, wherein the target RNA is fragmented prior to immobilization on the solid support.

29. The method of claim 28, wherein the RNA is fragmented into RNA molecules 50-300 nucleotides in length.

30. The method of claim 28, wherein the average RNA molecule fragment is 100 nucleotides.

31. The method of any one of claims 23 to 30, wherein the method comprises contacting the target and/or demethylated target RNA with RNA replicase to synthesize a complementary RNA strand that forms a duplex with the target RNA or demethylated target RNA.

32. The method of claim 31 wherein the method comprises denaturing of the RNA duplex to create mobilized complementary RNA.

33. The method of claim 32, wherein the method further comprises removal of the mobilized complementary RNA.

34. The method of any one of claims 23 to 33, wherein the method further comprises mobilizing the target RNA by separating it from the solid support.

35. The method of any one of claims 23 to 34, wherein the method comprises the steps of a) immobilizing the target RNA, b) contacting the target and/or demethylated target RNAwith RNA replicase to synthesize a complementary RNA strand that forms a duplex with the target RNA or demethylated target RNA, c) contacting the target RNA with an adenosine deaminase enzyme to generate a target RNA with deaminated adenosines, d) denaturing of the RNA duplex to create mobilized complementary RNA;

e) removal of the mobilized complementary RNA; and

f) sequencing of the target RNA.

36. The method of claim 35, wherein the method further comprises repeating steps b-e one or more times prior to step f.

37. The method of claim 36, wherein the steps are repeated at least four more times.

38. The method of any one of claims 1-38, wherein sequencing of the target RNA comprises construction of a library of nucleic acid molecules comprising the target RNA sequence.

39. The method of claim 15, wherein the method comprises synthesis of a complementary nucleic acid from a cDNA library.

40. The method of claim 39, wherein the complementary nucleic acid strand is RNA.

41. The method of any one of claim 14-15 or 20, wherein the method further comprises contacting the target and/or demethylated target RNA with reverse transcriptase to synthesize a complementary DNA strand.

42. The method of any one of claims 1-41, wherein the target RNA is 10-1000 nucleic acids in length.

43. The method of any one of claims 1-42, wherein the sequence of the target RNA is known.

44. The method of claim 43, wherein the method further comprises comparing the known sequence of the target RNA with the sequence of the target RNA with deaminated adenosines.

45. The method of any one of claims 1-44, wherein contacting the target and/or demethylated target RNA with the adenosine deaminase enzyme is done in the presence of GTP.

46. The method of claim 45, wherein the GTP is in a concentration of 0.5-5 mM.

47. The method of any one of claim 8-15, 20, or 39-46, wherein the method comprises contacting the RNA-DNA duplex with a DNA digesting agent prior to sequencing the RNA.

48. The method of claim 47, wherein the DNA digesting agent is a DNase.

49. The method of claim 47 or 48, wherein the method further comprises the steps of:

a) generating a DNA strand that is complementary to the target and/or demethylated target RNA and hybridizing the complementary DNA strand with the target and/or demethylated target RNA to generate an RNA-DNA duplex;

b) contacting the RNA-DNA duplex with the adenosine deaminase enzyme; and

c) contacting the RNA-DNA duplex with a DNA digesting agent;

wherein all the steps are done prior to target RNA sequencing.

50. The method of claim 49, wherein the method further comprises repeating steps a, b, and c one or more times.

51. The method of claim 50, wherein the steps are repeated at least two more times.

52. The method of claim 51, wherein the steps are repeated at least four more times.

53. The method of any one of claims 50-52, wherein the method further comprises step of strand separation after step b).

54. The method of any one of claims 50-53, wherein the method further comprises purification of the target and/or demethylated target RNAafter step c) and prior to target RNA sequencing.

55. The method of any one of claims 1-54, wherein the method further comprises:

providing a quantification RNA control comprising a known percentage of modified adenosine;

contacting the quantification control RNA with the adenosine deaminase enzyme;

and sequencing the deaminated quantification control RNA

56. The method of claim 55, wherein 0, 25, 50, 75, or 100% of the adenosine in the quantification RNA control is modified.

57. The method of claim 55, wherein 0, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100% of the adenosine in the quantification RNA control is modified.

58. A kit comprising:

an adenosine deaminase enzyme and instructions for detecting modified adenosines in target RNA.

59. The kit of claim 58 further comprising a control nucleic acid.

60. The kit of claim 59, wherein the control nucleic acid is a non-naturally occurring nucleic acid or non-widely present nucleic acid.

61. The kit of claim 59, wherein the control nucleic acid comprises a non-naturally occurring sequence.

62. The kit of claim 59, wherein the control nucleic acid comprises RNA or DNA comprising modified adenosines.

63. The kit of claim 62, wherein the percentage of adenosines that are modified in the control nucleic acid is known.

64. The kit of any one of claims 59-63, wherein the control nucleic acid comprises a duplex with a complementary nucleic acid strand.

65. The kit of claim 64, wherein the complementary nucleic acid strand is DNA or RNA with modified adenosines.

66. The kit of claim 64, wherein the complementary nucleic acid strand is RNA with modified adenosines.

67. The kit of any one of claims 58-66 further comprising an adenosine demethylase.

68. The kit of claim 67, wherein the adenosine demethylase is specific for N⁶-methyladenosine.

69. The kit of claim 67, wherein the adenosine demethylase is specific for N⁶-methyladenosine.

70. The kit of any one of claims 58-69, wherein the kit further comprises an adenosine deaminase enzyme reaction composition comprising GTP.

71. The kit of any one of claims 58-70, further comprising a DNAse.

72. The kit of any one of claims 58-71, further comprising a reverse transcriptase.

73. A method for detecting modified adenosine in a target ribonucleic acid (RNA) comprising contacting a double-stranded nucleic acid molecule comprising the target RNA with ADAR to generate a target RNA with deaminated adenosines and sequencing the target RNA with deaminated adenosines;

wherein the modified adenosine is detected when the sequence of the target RNA with deaminated adenosines is adenosine.

74. A method for detecting modified adenosine in a target ribonucleic acid (RNA) comprising:

a) providing a target RNA;

b) generating a DNA strand that is complementary with the target RNA and hybridizing the complementary DNA strand with the target RNA to generate a RNA-DNA duplex comprising the target RNA;

c) contacting the RNA-DNA duplex with ADAR to generate target RNA with deaminated adenosines; and

d) contacting the RNA-DNA duplex with a DNA digesting agent; and

e) sequencing the target RNA with the deaminated adenosines.

75. A method for detecting modified adenosine in a target ribonucleic acid (RNA) comprising:

a) providing a target RNA;

b) generating a DNA strand that is complementary with the target RNA and hybridizing the complementary DNA strand with the target RNA to generate a RNA-DNA duplex comprising the target RNA;

c) contacting the RNA-DNA duplex with ADAR to generate target RNA with deaminated adenosines; and

d) contacting the RNA-DNA duplex with a DNA digesting agent;

e) repeating steps b, c, and d one or more times; and

f) sequencing the target RNA with the deaminated adenosines.

76. A method for detecting modified adenosine in a target ribonucleic acid (RNA) comprising:

a) providing a target RNA;

b) generating an RNA strand with modified adenosine that is complementary with the target RNA to generate an RNA-RNA duplex comprising the target RNA;

c) contacting the RNA-RNA duplex with ADAR to generate target RNA with deaminated adenosines; and

d) sequencing the target RNA with the deaminated adenosines.