DETECTION OF MATURE SMALL RNA MOLECULES

• US 20080194416A1
• Filed: 02/08/2007
• Published: 08/14/2008
• Est. Priority Date: 02/08/2007
• Status: Abandoned Application

• Current Assignee: Sigma-Aldrich Company (Sigma-Aldrich Corporation)
• Original Assignee: Sigma Aldrich
• Inventors: Chen, Fuqiang
• Application Number:
  US11/672,591
• Publication Number:
  US 20080194416A1
• Time in Patent Office: Days
• US Class Current: 506/9
• CPC Class Codes: C12Q 1/6816 characterised by the detection means C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 2525/207 siRNA, miRNA
• CPC Class Codes: C12Q 2561/125 Ligase Detection Reaction [LDR]

Abstract

Methods, compositions, and kits for detecting mature small RNAs are provided herein. The methods comprise ligating at least one linker to a mature small RNA in the presence of a complementary ligation template to generate a ligation product. The ligation product is a hybrid molecule comprising the linker and the mature small RNA.

Claims

1. A method for detecting a mature small RNA, the method comprising:

a. providing a sample comprising a mature small RNA whose sequence is known;

b. ligating a 5′

linker to the 5′

end of the mature small RNA in the presence of a complementary ligation template that spans the ligation junction, whereby a ligation product is formed, the ligation product comprising the 5′

linker and the mature small RNA; and

,

c. assaying the ligation product, such that the mature small RNA is detected.

2. The method of claim 1, wherein the mature small RNA is selected from the group consisting of a microRNA (miRNA), a short interfering RNA (siRNA), a repeat associated siRNA (rasiRNA), a transacting siRNA (tasiRNA), a Piwi interacting RNA (piRNA), and a 21-U RNA.

3. The method of claim 1, wherein the sample comprising a mature small RNA is selected from the group consisting of an isolated preparation of RNA, a cellular extract, an intact cell, an in vitro transcription reaction, and a chemical synthesis.

4. The method of claim 1, wherein the 5′

linker is selected from the group consisting of a DNA polynucleotide and a chimeric DNA-RNA polynucleotide having at least one ribonucleotide at the 3′

end.

5. The method of claim 4, wherein the linker further comprises a stem-loop structure, with the 5′

end forming part of the stem and the 3′

end forming a single-stranded overhang.

6. The method of claim 1, wherein the complementary ligation template comprises a 3′

region that hybridizes under stringent conditions to the 3′

end of the linker and a 5′

region that hybridizes under stringent conditions to the 5′

end of the mature small RNA.

7. The method of claim 6, wherein the ligation template is selected from the group consisting of a RNA oligonucleotide, a chimeric DNA-RNA oligonucleotide, a DNA oligonucleotide, and a DNA oligonucleotide comprising at least one PCR blocker.

8. The method of claim 7, wherein the PCR blocker is selected from the group consisting of a dideoxynucleotide, an amine group, a methyl group, a phosphate group, and carbon spacers.

9. The method of claim 1, wherein the ligation is catalyzed by a ligase, the ligase is a template-dependent ligase selected from the group consisting of T4 DNA ligase, vaccinia DNA ligase, and mammalian DNA ligases.

10. The method of claim 1, wherein the mature small RNA is a microRNA, the 5′

linker is a chimeric DNA-RNA polynucleotide of about 90 nucleotides that forms a stem loop structure, the complementary ligation template is an RNA oligonucleotide of about 14 nucleotides, and the ligase is T4 DNA ligase.

11. The method of claim 1, wherein the ligation product is assayed by a method selected from the group consisting of an amplification method and a hybridization method.

12. The method of claim 11, wherein the amplification method is selected from the group consisting of quantitative real-time PCR, quantitative end-point PCR, and standard PCR

13. The method of claim 11, wherein the hybridization method is selected from the group consisting of nucleic acid microarray, nucleic acid-coupled microsphere array, and branched DNA technology.

14. The method of claim 12, wherein the PCR method comprises a forward PCR primer that corresponds to a sequence of the 5′

linker and a reverse PCR primer that is complementary to a portion of the small RNA.

15. The method of claim 14, wherein the PCR primers comprise at least one modified nucleotide, the modified nucleotide comprises a locked nucleic acid (LNA).

16. The method of claim 1, wherein step (b) is performed in the presence of a plurality of complementary ligation templates, rather than a single ligation template, such that a plurality of mature small RNAs is detected.

17. The method of claim 16, wherein the 5′

end of each of the ligation templates is complementary to the 5′

end of a discrete mature small RNA in the plurality of mature small RNAs.

18. A method for detecting a population of mature small RNAs selected from the group consisting of known mature small RNAs and unknown mature small RNAs, the method comprising:

a. providing a sample comprising a population of mature small RNAs;

b. ligating a first linker to one end of a mature small RNA in the population of mature small RNAs in the presence of a first semi-degenerate ligation template that spans the first ligation junction, whereby a plurality of first ligation products is formed, each first ligation product comprising a first linker and a mature small RNA;

c. ligating a second linker to the small RNA end of a ligation product in the plurality of first ligation products in the presence of a second semi-degenerate ligation template that spans the second ligation junction, whereby a plurality of second ligation products is formed, each second ligation product comprising a first linker, a mature small RNA, and a second linker; and

,

d. assaying the plurality of second ligation products, such that a population of mature small RNAs is detected.

19. The method of claim 18, wherein steps (b) and (c) are performed simultaneously.

20. The method of claim 18, wherein steps (b) and (c) are performed sequentially.

21. The method of claim 19, wherein the second ligation products are separated from the reaction materials before step (d).

22. The method of claim 20, wherein the first ligation products are separated from the reaction materials before step (c), and the second ligation products are separated from the reaction materials before step (d).

23. The method of claim 18, wherein the sample comprising a population of mature small RNAs is selected from the group consisting of an isolated preparation of RNA, a cellular extract, and an intact cell.

24. The method of claim 18, wherein the first linker is a 5′

linker, the first semi-degenerate ligation template is a 5′

semi-degenerate ligation template that spans the 5′

ligation junction, the second linker is a 3′

linker, and the second semi-degenerate ligation template is a 3′

semi-degenerate ligation template that spans the 3′

ligation junction.

25. The method of claim 18, wherein the first linker is a 3′

linker, the first semi-degenerate ligation template is a 3′

semi-degenerate ligation template that spans the 3′

ligation junction, the second linker is a 5′

linker, and the second semi-degenerate ligation template is a 5′

semi-degenerate ligation template that spans the 5′

ligation junction.

26. The method of claim 18, wherein the linkers and semi-degenerate ligation templates are selected from the group consisting of a RNA oligonucleotide, a DNA oligonucleotide, and a chimeric RNA-DNA oligonucleotide.

27. The method of claim 18, wherein the 5′

semi-degenerate ligation template comprises a 3′

region that hybridizes under stringent conditions to the 3′

end of the 5′

linker, and a degenerate 5′

region comprising a random mix of nucleotides, such that each ligation template hybridizes with the 5′

end of a discrete small RNA in the population of small RNAs.

28. The method of claim 18, wherein the 3′

semi-degenerate ligation template comprises a 5′

region that hybridizes under stringent conditions to the 5′

end of the 3′

linker, and a degenerate 3′

region comprising a random mix of nucleotides, such that each ligation template hybridizes with the 3′

end of a discrete small RNA in the population of small RNAs.

29. The method of claim 18, wherein the 5′

and 3′

semi-degenerate ligation templates further comprise a separation tag selected from the group consisting of biotin, digoxigenin, a fluorophore, and a magnetic particle.

30. The method of claim 18, wherein the 31 linker comprises a 5′

phosphate group.

31. The method of claim 18, wherein the 5′

linker further comprises a stem-loop structure, with the 5′

end forming part of the stem and the 3′

end forming a single-stranded overhang, and the 3′

linker further comprises a stem-loop structure, with the 3′

end forming part of the stem and the 5′

end forming a single-stranded overhang.

32. The method of claim 18, wherein the ligation reactions are catalyzed by a ligase, the ligase is a template-dependent ligase selected from the group consisting of T4 DNA ligase, vaccinia DNA ligase, and mammalian DNA ligases.

33. The method of claim 18, wherein the plurality of second ligation products is assayed by converting the ligation products into DNA copies and amplifying the DNA copies.

34. The method of claim 33, wherein the DNA copies are generated using a method comprising a reverse transcriptase and a reverse primer that is complementary to a region of the 3′

linker.

35. The method of claim 33, wherein the DNA copies are amplified using a PCR method comprising a forward PCR primer that corresponds to a sequence of the 5′

linker and a reverse PCR primer that is complementary to a sequence of the 3′

linker.

36. The method of claim 33, wherein the DNA copies are amplified using a transcription method comprising at least one RNA polymerase, provided that the 5′

linker and the 3′

linker each further comprise a RNA polymerase promoter sequence in the proper orientation.

37. A kit for detecting a mature small RNA whose sequence is known, the kit comprising a 5′

linker for ligating to the 5′

end of the mature small RNA, a ligation template that is complementary to the junction between the 5′

linker and the mature small RNA, a ligase, and instructions for using the kit.

38. The kit of claim 37, wherein the 5′

linker is selected from the group consisting of a DNA polynucleotide and a chimeric DNA-RNA polynucleotide having at least one ribonucleotide at the 3′

end.

39. The kit of claim 38, wherein the 5′

linker further comprises a stem-loop structure, with the 5′

end forming part of the stem and the 3′

end forming a single-stranded overhang.

40. The kit of claim 37, wherein the complementary ligation template comprises a 3′

region that hybridizes under stringent conditions to the 3′

end of the 5′

linker and a 5′

region that hybridizes under stringent conditions to the 5′

end of the mature small RNA

41. The kit of claim 37, wherein the complementary ligation template is selected from the group consisting of a RNA oligonucleotide, a chimeric DNA-RNA oligonucleotide, a DNA oligonucleotide, and a DNA oligonucleotide comprising at least one PCR blocker, wherein the PCR blocker is selected from the group consisting of a dideoxynucleotide, an amine group, a methyl group, a phosphate group, and carbon spacers.

42. The kit of claim 37, wherein the ligase is a template-dependent ligase selected from the group consisting of T4 DNA ligase, vaccinia DNA ligase, and mammalian DNA ligases.

43. The kit of claim 37 further comprising a forward PCR primer that corresponds to a sequence of the 5′

linker, a reverse PCR primer that is complementary to a portion of the small RNA, and a set of reagents for quantitative PCR.

44. The kit of claim 37, further comprising a plurality of complementary ligation templates for the detection of a plurality of mature small RNAs whose sequences are known, wherein the 5′

end of each of the ligation templates is complementary to the 5′

end of a discrete small RNA in the plurality of mature small RNAs.

45. A kit for detecting a population of mature small RNAs selected from the group consisting of known small RNAs and unknown small RNAs, the kit comprising a 5′

linker for ligating to the 5′

end of a mature small RNA, a 3′

linker for ligating to the 3′

end of a mature small RNA, a 5′

semi-degenerate ligation template that is complementary to the 5′

ligation junction, a 3′

semi-degenerate complementary ligation template that is complementary to the 3′

ligation junction, a ligase, and instructions for using the kit.

46. The kit of claim 45, wherein the 5′

semi-degenerate ligation template comprises a 3′

region that hybridizes under stringent conditions to the 3′

end of the 5′

linker, and a degenerate 5′

region comprising a random mix of nucleotides, such that each semi-degenerate ligation template hybridizes with the 5′

end of a discrete mature small RNA in the population of mature small RNAs.

47. The kit of claim 45, wherein the 3′

semi-degenerate ligation template comprises a 5′

region that hybridizes under stringent conditions to the 5′

end of the 3′

linker, and a degenerate 3′

end sequence comprising a random mix of nucleotides, such that each semi-degenerate ligation template hybridizes with the 3′

end of a discrete mature small RNA in the population of mature small RNAs.

48. The kit of claim 45, wherein the linkers and semi-degenerate ligation templates are selected from the group consisting of a RNA oligonucleotide, a DNA oligonucleotide, and a chimeric RNA-DNA oligonucleotide.

49. The kit of claim 45, wherein the 5′

and 3′

semi-degenerate ligation templates further comprise a separation tag selected from the group consisting of biotin, digoxigenin, a fluorophore, and a magnetic particle.

50. The kit of claim 45, wherein the 3′

linker comprises a 5′

phosphate group.

51. The kit of claim 45, wherein the 5′

linker further comprises a stem-loop structure, with the 5′

end forming part of the stem and the 3′

end forming a single-stranded overhang, and the 3′

linker further comprises a stem-loop structure, with the 3′

end forming part of the stem and the 5′

end forming a single-stranded overhang.

52. The kit of claim 45, wherein the ligase is a template-dependent ligase selected from the group consisting of T4 DNA ligase, vaccinia DNA ligase, and mammalian DNA ligases.

53. The kit of claim 45 further comprising a reverse transcriptase and a reverse primer that is complementary to a sequence of the 3′

linker.

54. The kit of claim 45 further comprising a forward PCR primer that corresponds to a sequence of the 5′

linker, a reverse PCR primer that is complementary to a portion of the 3′

linker, and a set of reagents for quantitative PCR.