Methods and compositions for detection of small RNAs

• US 10,041,107 B2
• Filed: 10/06/2016
• Issued: 08/07/2018
• Est. Priority Date: 04/13/2009
• Status: Active Grant

• Current Assignee: Realseq Biosciences, Inc.
• Original Assignee: SomaGenics, Inc.
• Inventors: Kazakov, Sergei A., Kumar, Pavan, Johnston, Brian H.
• Primary Examiner(s): Thomas, David C
• Application Number:
  US15/287,254
• Publication Number:
  US 20170159106A1
• Time in Patent Office: 670 Days
• Field of Search: None
• 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 1/682 Signal amplification
• CPC Class Codes: C12Q 1/6844 Nucleic acid amplification reactions
• CPC Class Codes: C12Q 1/6865 Promoter-based amplification, e.g. nucleic acid sequence amplification [NASBA], self-sustained sequence replication [3SR] or transcription-based amplification system [TAS]
• CPC Class Codes: C12Q 2521/501 Ligase
• CPC Class Codes: C12Q 2525/143 incorporating a promoter sequence
• CPC Class Codes: C12Q 2525/207 siRNA, miRNA
• CPC Class Codes: C12Q 2525/313 Branched oligonucleotides
• CPC Class Codes: C12Q 2531/125 Rolling circle

Abstract

Currently, the circularization of small RNAs is broadly regarded as an obstacle in ligation-related assays and explicitly avoided while short lengths of linear RNA targets is broadly recognized as a factor limiting use of conventional primers in PCR-related assays. In contrast, the disclosed invention capitalizes on circularization of small RNA targets or their conjugates with oligonucleotide adapters. The circular RNA templates provide amplification of the target sequences via synthesis of multimer nucleic acids that can be either labeled for direct detection or subjected to PCR amplification and detection. Structure of small circular RNAs and corresponding multimeric nucleic acids provide certain advantages over current methods including flexibility in design of conventional RT and PCR primers as well as use of 5′-overlapping dimer-primers for efficient and sequence-specific amplification of short target sequences. Our invention also reduces number of steps and reagents while increasing sensitivity and accuracy of detection of small RNAs with both 2′OH and 2′-OMe at their 3′ ends. Our invention increase sensitivity and specificity of detection of microRNAs and other small RNAs with both 2′OH and 2′-OMe at their 3′ ends while allowing us to distinguish these two forms from each other.

Claims

1. A method of detecting the presence of at least one target RNA in a sample, said method comprising:

a) ligating an adapter or linker oligonucleotide to the 3′

or 5′

end of the at least one target RNA to produce at least one extended target polynucleotide;

b) circularizing the at least one extended target polynucleotide by ligating the 5′

-end of the at least one extended target polynucleotide to its 3′

-end to produce at least one circularized extended target polynucleotide;

c) synthesizing, by rolling circle amplification, at least one multimeric nucleic acid (MNA) comprising multiple repeats of sequences that are complementary to the at least one circularized extended target polynucleotide, wherein said synthesizing comprises;

i) hybridizing said at least one circularized extended target polynucleotide with an oligonucleotide reverse transcriptase (RT) primer that is complementary to a part of the extended target polynucleotide;

ii) enzymatically extending said oligonucleotide RT primer bound to said at least one circularized extended target polynucleotide with a DNA polymerase having reverse transcriptase activity to produce the at least one MNA; and

d) performing real-time qPCR amplification of the MNA or a portion thereof, thereby detecting the presence of said at least one target RNA in said sample.

2. The method of claim 1, wherein said at least one target RNA comprises:

a) 10 or more nucleotides,

b) a 5′

-phosphate (5′

-p), 5′

-hydroxyl (5′

-OH), 5′

-triphosphate, or 5′

-adenylated group (5′

-App);

c) a 3′

-hydroxyl (3′

-OH), 3′

-phosphate (3′

-p), or 2′

,3′

-cyclic phosphate; and

d) a 2′

-hydroxyl (2′

-OH) or 2′

-O-methyl (2′

-OMe) nucleotide at their 3′

-end.

3. The method of claim 1, wherein said adapter or linker oligonucleotide comprises:

a) RNA, DNA, chemical analogs thereof, or combinations thereof;

b) a 5′

-end group selected from a 5′

-OH, 5′

-p, or a 5′

-adenylated group (5′

-App);

c) a 3′

-end group selected from a 3′

-OH and 3′

-p; and

d) a 2′

-group selected from;

2′

-OH and 2′

-OMe on its 3′

terminal nucleotide.

4. The method of claim 1, wherein said adapter or linker oligonucleotide is ligated to the 5′

end of said at least one target RNA before said circularizing.

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

-end group is 5′

-p, comprising converting the 5′

-p end group of said at least one target RNA to a 5′

-App before ligating with a 3′

-OH group of the adapter or linker oligonucleotide.

6. The method of claim 1, wherein said adapter or linker oligonucleotide is ligated to the 3′

end of said at least one target RNA before said circularizing.

7. The method of claim 1, wherein said ligating or attaching an adaptor or linker oligonucleotide comprises contacting the adapter or linker oligonucleotide and said at least one target RNA with an RNA ligase.

8. The method of claim 1, wherein said circularizing comprises contacting the at least one extended target polynucleotide with an RNA ligase.

9. The method of claim 1, wherein said circularizing is accomplished by a splint-assisted ligation using a splint oligonucleotide complementary to both a first sequence at the 5′

-end of the one or more target RNAs and a second sequence at the 3′

-end of the adapter, thereby selectively amplifying only the at least one target RNA.

10. The method of claim 1, wherein said circularizing comprises splint-independent circularization.

11. The method of claim 1, wherein said circularizing is accomplished by a splint-assisted ligation using a splint oligonucleotide complementary to both a first sequence at an end of the at least one target RNA and a second sequence at an end of the adapter.

12. The method of claim 11, wherein said splint-assisted ligation is performed by using either an RNA or a DNA ligase, or by chemical ligation.

13. The method of claim 1, wherein said adapter or linker oligonucleotide has a temporary blocking group at an end to prevent adapter or linker circularization during the step of ligating of the adapter or linker oligonucleotide to said at least one target RNA, and wherein said temporary blocking group may be subsequently unblocked or modified to allow the circularizing to proceed.

14. The method of claim 13, wherein said temporary blocking group is 3′

-p, comprising converting the 3′

-p to 3′

-OH by contacting the at least one extended target polynucleotide with a polynucleotide kinase before circularizing.

15. The method of claim 1, wherein said circularization of the at least one extended target polynucleotide is followed by degradation of non-circularized extended target polynucleotides with an exonuclease or mixture of exonucleases.

16. The method of claim 1, wherein said synthesizing is performed using reverse transcriptase mutants lacking RNAse H activity.

17. The method of claim 1, wherein said oligonucleotide RT primer is a single universal RT primer specific to the adapter, thereby allowing simultaneously amplifying of multiple extended polynucleotide sequences, each of which corresponds to a different target RNA.

18. The method of claim 1, wherein said oligonucleotide RT primer is specific either to (i) the at least one target RNA or to (ii) both a part of said adapter at its 5′

end and to a part of said at least one target RNA.

19. The method of claim 11, wherein said splint oligonucleotide is used as the oligonucleotide RT primer for synthesizing the at least one MNA from the circularized extended target polynucleotide.

20. The method of claim 1, wherein said real-time qPCR amplification is performed using a pair of forward and reverse PCR primers comprising sequences that are specific to a 3′

portion of said target RNA and to a portion of said adapter at its 5′

end.

21. The method of claim 1, wherein said real-time qPCR amplification is performed using a pair of forward and reverse PCR primers, wherein the forward primer comprises a sequence that is specific to said target RNA and the reverse primer comprises a universal sequence that is specific to said adapter.

22. The method of claim 21, wherein said forward primer comprises a sequence that is specific to said target sequence at its 3′

end and to a part of said adapter at its 5′

end.

23. The method of claim 1, wherein said real-time qPCR amplification of the MNA is performed using a pair of universal forward and reverse PCR primers comprising sequences that are specific to said adapter sequences.

24. The method of claim 1, wherein said real-time qPCR amplification is performed with a combo primer comprising a first sequence at its 3′

end that hybridizes to said at least one MNA and a second sequence at its 5′

end that does not hybridize to said target RNA or adapter.