Compositions and methods for detecting small RNAs, and uses thereof

• US 8,399,222 B2
• Filed: 11/25/2009
• Issued: 03/19/2013
• Est. Priority Date: 11/25/2008
• Status: Expired due to Fees

• Current Assignee: Gen-Probe Incorporated (Hologic Incorporated)
• Original Assignee: Gen-Probe Incorporated (Hologic Incorporated)
• Inventors: Siva, Amara, Reynolds, Mark Alan
• Primary Examiner(s): Strzelecka, Teresa E
• Assistant Examiner(s): Pande, Suchira
• Application Number:
  US12/625,609
• Publication Number:
  US 20100129822A1
• Time in Patent Office: 1,210 Days
• Field of Search: None
• US Class Current: 435/91.2
• CPC Class Codes: C12Q 1/6853 using modified primers or templates
• CPC Class Codes: C12Q 1/6876 Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
• CPC Class Codes: C12Q 2525/301 Hairpin oligonucleotides
• CPC Class Codes: C12Q 2537/143 Multiplexing, i.e. use of multiple primers or probes in a single reaction, usually for simultaneously analyse of multiple analysis
• CPC Class Codes: C12Q 2565/525 characterised by the capture oligonucleotide being double stranded
• CPC Class Codes: C12Q 2565/537 characterised by the capture oligonucleotide acting as a primer
• CPC Class Codes: C12Q 2600/178 miRNA, siRNA or ncRNA

Abstract

Compositions and methods are provided for the detection of small RNA target nucleic acids, preferably miRNA target nucleic acids, wherein the compositions and methods provide for sensitive and specific detection of the target nucleic acids. The compositions and methods include using one or more of a first amplification oligomer that is preferably an extender primer, a target capture oligomer that is preferably at least partially double stranded, a promoter primer/provider, a reverse primer that is preferably a universal primer and a detection probe. The compositions and methods are useful for diagnostics, prognostics, monitoring the effectiveness of treatment and/or determining a treatment.

Claims

1. A multiplex amplification method for specifically detecting the presence of target nucleic acids in a sample comprising the steps of:

(a) providing a target capture probe for capturing a miR-221 target nucleic acid and a target capture probe for capturing a miR-182 target nucleic acid, wherein the target capture probes are each made up of an internal target hybridizing sequence flanked by a capture region and a terminal region, wherein the terminal region binds to the capture region to form a double-stranded stem portion of the hairpin structure, wherein the target hybridizing region forms a single-stranded loop portion of the hairpin structure, and wherein the target hybridizing sequence of the miR-221 target capture probe is substantially complementary to all or a portion of a miR-221 sequence and the target hybridizing sequence of the miR-182 target capture probe is substantially complementary to all or a portion of a miR-182 sequence, wherein the target capture probe that specifically hybridizes to a miR-221 target nucleic acid has a nucleotide sequence that is at least 95% identical to SEQ ID NO;

24, and wherein the target capture probe that specifically hybridizes to a miR-182 target nucleic acid has a nucleotide sequence that is at least 95% identical to SEQ ID NO;

23,

(b) mixing a sample suspected of containing a miR-221 target nucleic acid and a miR-182 target nucleic acid with the capture probes,

(c) specifically hybridizing the target hybridizing sequence of the capture probes to a target sequence in their respective target nucleic acids,

(d) binding the capture regions to an immobilized probe attached to a capture support by binding together members of a specific binding pair, thereby forming a capture hybrid made up of the miR-221 target nucleic acid, the capture probe and the immobilized probe attached to the capture support, and a capture hybrid made up of the miR-182 target nucleic acid, the capture probe, and the immobilized probe attached to the capture support,

(e) separating the capture hybrids from other components in the sample,

(f) releasing the miR-221 and miR-182 target nucleic acids from the capture hybrids and providing a set of conditions wherein the target capture probes are partially double-stranded hairpin structures,

(g) providing to the mixture comprising the released miR-221 and miR-182 target nucleic acids and the target capture probes a plurality of amplification oligomers, the plurality comprising

(i) at least a first amplification oligomer and a promoter-based oligomer for specifically hybridizing to a miR-221 target nucleic acid or cDNA strand thereof, wherein the first amplification oligomer comprises a target hybridizing sequence, a portion of which is substantially identical to a portion of the target hybridizing region of the miR-221 target capture probe, and wherein the promoter-based oligomer comprises a target hybridizing sequence, a portion of which is substantially complementary to a portion of the target hybridizing region of the miR-221 target capture probe, and

(ii) at least a first amplification oligomer and a promoter-based oligomer for hybridizing to a miR-182 target nucleic acid or cDNA strand thereof, wherein the first amplification oligomer comprises a target hybridizing sequence, a portion of which is substantially identical to a portion of the target hybridizing region of the miR-182 target capture probe, and wherein the promoter-based oligomer comprises a target hybridizing sequence, a portion of which is substantially complementary to a portion of the target hybridizing region of the miR-182 target capture probe;

(h) providing a set of conditions wherein the first amplification oligomers hybridizes to their respective target nucleic acids,

(i) performing an amplification reaction using the plurality of amplification oligomers to generate amplification products from the miR-221 and miR-182 target nucleic acids; and

(j) detecting the amplification products to indicate presence of miR-221 target nucleic acid, miR-182 target nucleic acid or both in the sample.

2. The method of claim 1, wherein binding the capture region to the immobilized probe results from hybridizing complementary sequences of the capture region and the immobilized probe.

3. The method of claim 1, wherein the capture region is a nucleotide sequence selected from the group consisting of:

dT_{(0-3)A(10-30)}.

4. The method of claim 1, wherein the capture region is dA₍₁₂₎.

5. The method of claim 1, wherein releasing the miR-221 and miR-182 target nucleic acids from the capture hybrids further releases the capture probes from the immobilized probes.

6. The method of claim 1, wherein the target capture probe that specifically hybridizes to a miR-221 target nucleic acid is SEQ ID NO:

24.

7. The method of claim 1, wherein the target capture probe that specifically hybridizes to a miR-182 target nucleic acid is SEQ ID NO:

23.

8. The method of claim 1, wherein the miR-221 first amplification oligomer has an extension nucleic acid sequence that is at least 95% identical to SEQ ID NO:

49.

12. The method of claim 1, wherein the first amplification oligomer is SEQ ID NO:

17.

16. The method of claim 1, wherein the first amplification oligomer is SEQ ID NO:

16.

17. The method of claim 12, wherein the first amplification oligomer that specifically hybridizes to miR-182 is an extender primer comprising a 3′

target hybridizing sequence and a 5′

extension nucleic acid sequence, wherein the 3′

target hybridizing sequence is substantially complementary to about 9 or more contiguous nucleic acids at the 3′

end of SEQ ID NO;

41.

18. The method of claim 1, wherein the promoter based oligomer that specifically hybridizes to a complement of the miR-221 target nucleic acid comprises a target hybridizing sequence that is substantially complementary to 9 or more contiguous nucleic acids on 3′

end of the miR-221 target capture probe target hybridizing region.

19. The method of claim 1, wherein the promoter based amplification oligomer that specifically hybridizes to a complement of the miR-221 target nucleic acid is SEQ ID NO:

11.

20. The method of claim 19, wherein the promoter based oligomer that specifically hybridizes to a complement of the miR-221 target nucleic acid comprises a target hybridizing sequence that is substantially complementary to 9 or more contiguous nucleic acids on 3′

end of the miR-221 target capture probe target hybridizing region.

21. The method of claim 1, wherein the promoter based oligomer that specifically hybridizes to a complement of the miR-182 target nucleic acid comprises a target hybridizing sequence that is substantially complementary to 9 or more contiguous nucleic acids on 3′

end of the miR-182 target capture probe target hybridizing region.

22. The method of claim 1, wherein the promoter based amplification oligomer that specifically hybridizes to a complement of the miR-182 target nucleic acid is SEQ ID NO:

10.

23. The method of claim 22, wherein the promoter based oligomer that specifically hybridizes to a complement of the miR-221 target nucleic acid comprises a target hybridizing sequence that is substantially complementary to 9 or more contiguous nucleic acids on 3′

end of the miR-221 target capture probe target hybridizing region.

24. The method of claim 1, further comprising a reverse primer oligomer.

25. The method of claim 1, wherein the first amplification oligomer that specifically hybridizes to miR-221 is an extender primer comprising a 3′

target hybridizing sequence and a 5′

extension nucleic acid sequence, and wherein at or after step (g) a reverse primer is provided.

26. The method of claim 25, wherein the reverse primer is configured to hybridize to a portion of the complement of the extension nucleic acid.

27. The method of claim 1, wherein the first amplification oligomer that specifically hybridizes to miR-182 is an extender primer comprising a 3′

target hybridizing sequence and a 5′

extension nucleic acid sequence, and wherein at or after step (g) a reverse primer is provided.

28. The method of claim 27, wherein the reverse primer is configured to hybridize to a portion of the complement of the extension nucleic acid.

29. The method of claim 25, wherein the first amplification oligomer that specifically hybridizes to miR-182 is an extender primer comprising a 3′

target hybridizing sequence and a 5′

extension nucleic acid sequence, and wherein at or after step (g) a reverse primer is provided.

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

extension nucleic acid sequences for both the miR-221 extender primer and the miR-182 extender primer are substantially identical and the reverse primer is a universal primer configured to hybridize to a portion of the complement of the extension nucleic acid.

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

extension nucleic acid sequences for both the miR-221 extender primer and the miR-182 extender primer comprise a complement of a reverse primer target sequence.

32. The method of claim 31, wherein the complement reverse primer target sequences on the miR-221 extender primer and the miR-182 extender primer are substantially identical, and wherein the reverse primer is a universal primer.

33. The method of claim 24, wherein the reverse primer is SEQ ID NO:

20.

34. The method of claim 30, wherein the reverse primer is SEQ ID NO:

20.

35. The method of claim 32, wherein the reverse primer is SEQ ID NO:

20.

36. The method of claim 1, wherein the amplification reaction is performed in the presence of target capture probe and wherein the amplification reaction conditions are configured to maintain the target capture probe in a hairpin formation.

37. The method of claim 1, wherein the amplification reaction is an isothermal amplification reaction.

38. The method of claim 1, wherein amplification products are detected using a detection probe oligomer.

39. The method of claim 38, wherein the detection probe oligomer is a linear detection probe.

40. The method of claim 38, wherein the detection probe oligomer is a hairpin detection probe.

41. The method of claim 38, wherein the detection probe oligomer is labeled.

42. The method of claim 38, wherein the detection probe oligomer is dual labeled.

43. The method of claim 1, wherein the detection of the target nucleic acid is a real-time detection.

9. The method of 1, wherein the first amplification oligomer that specifically hybridizes to miR-221 is an extender primer comprising a 3′

target hybridizing sequence and a 5′

extension nucleic acid sequence, wherein the 3′

target hybridizing sequence is substantially identical to about 9 or more contiguous nucleic acids at the 5′

end of the target hybridizing sequence of the miR-221 target capture probe.

10. The method of claim 9, wherein the miR-221 target capture probe is SEQ ID NO:

24.

11. The method of claim 9, wherein the miR-221 first amplification oligomer has an extension nucleic acid sequence that is at least 95% identical to SEQ ID NO:

49.

13. The method of 1, wherein the first amplification oligomer that specifically hybridizes to miR-182 is an extender primer comprising a 3′

target hybridizing sequence and a 5′

extension nucleic acid sequence, wherein the 3′

target hybridizing sequence is substantially identical to about 9 or more contiguous nucleic acids at the 5′

end of the target hybridizing sequence of the miR-182 target capture probe.

14. The method of claim 13, wherein the miR-182 target capture probe is SEQ ID NO:

23.

15. The method of claim 13, wherein the miR-182 first amplification oligomer has an extension nucleic acid sequence that is at least 95% identical to SEQ ID NO:

49.

44. A method of detecting the presence of a target nucleic acid in a sample comprising the steps of:

(a) providing a target capture probe that forms under hybridizing conditions a partially double-stranded hairpin structure made up of an internal target hybridizing sequence flanked by a capture region and a terminal region, wherein the terminal region binds to the capture region to form a double-stranded stem portion of the hairpin structure, wherein the target hybridizing region forms a single-stranded loop portion of the hairpin structure, and wherein the target capture probe specifically hybridizes to a miR-221 target nucleic acid and has a nucleotide sequence that is at least 95% identical to SEQ ID NO;

24,

(b) mixing a sample suspected of containing a miR-221 target nucleic acid with the capture probe,

(c) specifically hybridizing the target hybridizing sequence of the capture probe to a target sequence in the miR-221 target nucleic acid,

(d) binding the capture region to an immobilized probe attached to a capture support by binding together members of a specific binding pair, thereby forming a capture hybrid made up of the miR-221 target nucleic acid, the capture probe, and the immobilized probe attached to the capture support,

(e) separating the capture hybrid attached to the capture support from other components in the sample,

(f) releasing the miR-221 target nucleic acid from the capture hybrid,

(g) providing to the mixture comprising the released miR-221 target nucleic acid and the target capture probe at least a first amplification oligomer comprising a target hybridizing sequence, a portion of which is substantially identical to a portion of the target hybridizing region of the target capture probe;

(h) providing a set of conditions wherein the first amplification oligomer hybridizes to the miR-221 target nucleic acid and an amplification reaction takes place, and wherein the target capture probe is in a partially double stranded hairpin formation,

(i) generating a detectable amplification product in an amplification reaction wherein the first amplification oligomer generates a cDNA strand from the miR-221 target nucleic acid; and

(j) detecting the amplification product to indicate presence of miR-221 target nucleic acid in the sample.

45. A method of detecting the presence of a target nucleic acid in a sample comprising the steps of:

(a) providing a target capture probe that forms under hybridizing conditions a partially double-stranded hairpin structure made up of an internal target hybridizing sequence flanked by a capture region and a terminal region, wherein the terminal region binds to the capture region to form a double-stranded stem portion of the hairpin structure, wherein the target hybridizing region forms a single-stranded loop portion of the hairpin structure, and wherein the target capture probe specifically hybridizes to a miR-221 target nucleic acid and has a nucleotide sequence that is at least 95% identical to SEQ ID NO;

24,

(b) mixing a sample suspected of containing a miR-221 target nucleic acid with the capture probe,

(c) specifically hybridizing the target hybridizing sequence of the capture probe to a target sequence in the miR-221 target nucleic acid,

(d) binding the capture region to an immobilized probe attached to a capture support by binding together members of a specific binding pair, thereby forming a capture hybrid made up of the miR-221 target nucleic acid, the capture probe, and the immobilized probe attached to the capture support,

(e) separating the capture hybrid attached to the capture support from other components in the sample,

(f) releasing the miR-221 target nucleic acid from the capture hybrid and providing a set of conditions wherein the target capture probe is a partially double-stranded hairpin structure,

(g) providing to the mixture comprising the released miR-221 target nucleic acid and the target capture probe a plurality of amplification oligomers, the plurality comprising at least a first amplification oligomer and a promoter-based oligomer, wherein the first amplification oligomer comprises a target hybridizing sequence, a portion of which is substantially identical to a portion of the target hybridizing region of the target capture probe, and wherein the promoter-based oligomer comprises a target hybridizing sequence, a portion of which is substantially complementary to a portion of the target hybridizing region of the target capture probe;

(h) providing a set of conditions wherein the first amplification oligomer hybridizes to the miR-221 target nucleic acid in the presence of the hairpin capture probe,

(i) performing an amplification reaction using the plurality of amplification oligomers to generate amplification product from the miR-221 target nucleic acid; and

(j) detecting the amplification product to indicate presence of miR-221 target nucleic acid in the sample.