Methods and compositions for detection of small RNAS

• US 9,493,818 B2
• Filed: 08/25/2015
• Issued: 11/15/2016
• 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:
  US14/835,544
• Publication Number:
  US 20160115523A1
• Time in Patent Office: 448 Days
• Field of Search: None
• US Class Current: 1/1
• 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 an 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, multimeric nucleic acids (MNAs) comprising multiple repeats of sequences that are complementary to said one or more circularized extended polynucleotides; and

d) sequencing said MNAs or portions thereof.

2. The method of claim 1, wherein sequencing said MNAs or portions thereof comprises PCR amplifying sequences of said MNAs or portions thereof to produce one or more amplicons.

3. The method of claim 1, wherein said target RNAs comprise:

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.

4. 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.

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

end of said one or more target RNAs before said circularizing.

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

end of said one or more target RNAs before said circularizing.

7. The method of claim 1, wherein said adapter or linker oligonucleotide comprises a sequence encoding one or two linkers used for cloning and/or sequencing.

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

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

-end group is 5′

-p, further comprising converting the 5′

-p end group of said one or more target RNAs to a 5′

-App before ligating with a 3′

—

OH group of the adapter or linker oligonucleotide.

10. The method of claim 1, wherein said circularizing comprises contacting the one or more extended target polynucleotides with an RNA ligase.

11. The method of claim 1, wherein said circularization is accomplished by a splint-assisted ligation using a splint oligonucleotide complementary to both the 5′

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

-end sequence of the adapter, thereby selectively amplifying only the one or more target RNAs.

12. The method of claim 11, further comprising expression profiling of said one or more target RNAs by sequencing said MNAs or portions thereof.

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

14. 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 one or more target RNAs, and wherein said blocking group may be subsequently unblocked or modified to allow circularization to proceed.

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

-p, and wherein the 3′

-p is converted to 3′

—

OH by treatment with polynucleotide kinase before circularizing of the one or more extended target polynucleotides.

16. The method of claim 1, wherein said circularization of the one or more extended target polynucleotides is followed by degradation of non-circularized extended target polynucleotides by an exonuclease or mixture of exonucleases.

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

18. The method of claim 2, wherein said PCR amplifying is performed with a combo primer, wherein the combo primer comprises adapter-specific sequences and additional upstream (5′

-end) sequences used for cloning and/or sequencing.

19. The method of claim 2, wherein each of the one or more amplicons comprises a single copy of a sequence specific to the one or more target RNAs, wherein the single copy is flanked by sequences specific to said adapter or linker oligonucleotide sequences, wherein said one or more amplicons represents a sequencing library.

20. The method of claim 2, wherein each of the one or more amplicons comprises multiple repeats of a sequence specific to the one or more target RNAs, wherein the multiple repeats are flanked by sequences specific to the adapter or linker oligonucleotide sequences, and wherein said one or more amplicons represents a sequencing library.

21. The method of claim 1, wherein the method results in a preparation of sequencing libraries.

22. The methods of claim 1, wherein said sequencing is performed by using a next-generation sequencing technique.

23. The method of claim 1, wherein said sequencing comprises cloning said MNAs or portions thereof.

24. The method of claim 1, wherein said adapter or linker oligonucleotide comprises a restriction site.

25. The method of claim 1, wherein said one or more target RNAs comprise microRNAs.

26. The method of claim 1, wherein said sample is selected from:

biological samples, clinical samples, crude nucleic acid extract, total RNA extract, and fragments of RNAs whose length is limited by a method of purification.

27. The method of claim 1, further comprising purifying the one or more extended target polynucleotides before circularizing.