Methods and compositions for detection of small RNAs

• US 8,962,253 B2
• Filed: 04/13/2010
• Issued: 02/24/2015
• 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): Horlick, Kenneth R.
• Assistant Examiner(s): THOMAS, DAVID C
• Application Number:
  US13/264,122
• Publication Number:
  US 20120164651A1
• Time in Patent Office: 1,778 Days
• Field of Search: None
• US Class Current: 435/6.12
• 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 one or more target RNAs in a sample, said method comprising:

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

end of said one or more target RNAs to produce one or more extended target polynucleotides;

b) circularizing said one or more extended target polynucleotides by ligating the 5′

-end of the one or more extended target polynucleotides to its 3′

- end;

c) synthesizing by rolling circle amplification complementary multimeric nucleic acids (MNAs) comprising multiple repeats of sequences that are complementary to said one or more circularized extended polynucleotides; and

d) detecting said MNAs, thereby detecting the presence of said one or more target RNAs in said sample.

2. The method of claim 1, wherein said target RNA is 10 or more nucleotides in length and comprises:

a) a 5′

-phosphate (5′

-p) or other 5′

-end group that is optionally converted to 5′

-p;

b) a 3′

-hydroxyl (3′

-OH) or other 3′

-end group that is optionally converted to 3′

-OH; and

c) a 2′

-OH or 2′

-OMe at its 3′

-terminal nucleotide.

3. The method of claim 1, wherein said adapter or linker oligonucleotide is 10 or more nucleotides in length and comprises:

a) RNA, or DNA, or a mix of DNA and RNA residues or chemical analogs thereof;

b) a sequence selected from the group consisting of a promoter for an RNA polymerase;

a sequence which is neither substantially complementary nor corresponds to any sequence expected to be present in said sample;

a Zip-code sequence;

a homopolynucleotide linker; and

a sequence corresponding to or encoding a linker sequence used for cloning and sequencing;

c) a 3′

-end group selected from a 3′

-OH and a 3′

-p;

d) a 5′

-group selected from a 5′

-p, a 5′

-App, and a 5′

-OH; and

e) a 2′

-group at the 3′

-terminal nucleotide selected from a 2′

-OH and a 2′

-OMe.

4. The method of claim 1, wherein said synthesizing comprises:

a) hybridizing said circularized extended target polynucleotide with an oligonucleotide RT primer that is complementary to said adapter; and

b) enzymatically extending said RT primer bound to said circularized extended target polynucleotide by reverse transcriptase.

5. The method of claim 1, wherein said MNAs are cRNA and said synthesizing comprises:

a) hybridizing an oligodeoxynucleotide to the adapter of said circularized extended target polynucleotide, wherein said oligodeoxynucleotide comprises one strand of a double-stranded promoter for an RNA polymerase and said adapter comprises the second strand of said promoter; and

b) synthesizing said cRNA by transcription using said polymerase.

6. The method of claim 1, wherein said detecting comprises sequencing.

7. The method of claim 6, wherein said detecting further comprises PCR amplification of MNAs before said sequencing using reverse and forward PCR primers that are specific to said adapter sequence.

8. The method of claim 7, wherein said forward and/or reverse PCR primers comprise at their 3′

ends sequences that are specific to said MNAs and at least one additional sequence at their 5′

ends selected from;

a) a sequence encoding a Zip-code sequence;

b) a sequence encoding cloning and/or sequencing linker or adapter; and

c) another adapter or linker sequence.

9. The method of claim 1, wherein said detecting comprises real-time qPCR amplification of said MNAs.

10. The method of claim 9, wherein said real-time qPCR amplification of MNAs is performed using a pair of forward and reverse PCR primers comprising sequences that are specific to said adapter sequence.

11. The method of claim 9, wherein said real-time qPCR amplification of MNAs is performed using a pair of forward and reverse PCR primers comprising sequences that are specific to a part of said adapter at their 5′

ends and to a part of said target sequences at their 3′

ends.

12. The method of claim 9, wherein said real-time qPCR of MNAs is performed using a pair of forward and reverse PCR primers comprising sequences that are:

a) substantially complementary to each other at their 5′

ends and can form a dimer with 3′

overhangs, wherein said 5′

overlap is ≧

2 nt shorter than said target sequences while said 3′

-end overhangs are ≧

1 nt long; and

b) complementary to the forward PCR primer is complementary to repeated target sequences with said multimeric cDNA; and

c) the reverse PCR primer is corresponding to repeated target sequences within said multimeric cDNA.

13. The method of claim 1, wherein said target RNA is 100 or fewer nucleotides in length.

14. The method of claim 1, wherein said target RNA is a miRNA.

15. The method of claim 1, wherein said one or more target RNAs comprise multiple target RNAs.

16. The method of claim 15, wherein said multiple target RNAs comprise different target RNAs.

17. The method of claim 15, wherein said multiple target RNAs comprise a 2′

OH at a 3′

end, 2′

-O-Me at a 3′

end, or a combination thereof.

18. The method of claim 1, further comprising determining whether said one or more target RNAs comprise a 2′

OH or 2′

-O-Me at a 3′

end of said one or more target RNAs.

19. The method of claim 1, wherein circularizing one or more extended target polynucleotides comprises use of one or more ligases.

20. The method of claim 19, wherein said one or more ligases is a RNA ligase, RNA/DNA ligase, or a combination thereof.

21. The method of claim 20, wherein the RNA ligase is a T4 RNA ligase.

22. The method of claim 20, wherein the RNA/DNA ligase is a CircLigase.

23. The method of claim 1, wherein the sample is derived from a cell.

24. The method of claim 1, wherein the sample comprises total cellular RNA or a cell lysate.