Methods of constructing small RNA libraries and their use for expression profiling of target RNAs

• US 9,816,130 B2
• Filed: 12/21/2012
• Issued: 11/14/2017
• Est. Priority Date: 12/22/2011
• Status: Active Grant

• Current Assignee: Realseq Biosciences, Inc.
• Original Assignee: SomaGenics, Inc.
• Inventors: Kazakov, Sergei A.
• Primary Examiner(s): Weiler, Karen S
• Application Number:
  US14/367,200
• Publication Number:
  US 20150051099A1
• Time in Patent Office: 1,789 Days
• Field of Search: None
• CPC Class Codes: C12N 15/1093 General methods of preparing gene libraries, not provided for in other subgroups
• CPC Class Codes: C12N 15/1096 cDNA Synthesis; Subtracted cDNA library construction, e.g. RT, RT-PCR
• CPC Class Codes: C12Q 1/6813 Hybridisation assays
• CPC Class Codes: C12Q 1/6837 using probe arrays or probe chips C12Q1/6874 takes precedence
• CPC Class Codes: C12Q 1/6846 Common amplification features
• CPC Class Codes: C12Q 1/6853 using modified primers or templates
• CPC Class Codes: C12Q 1/6855 Ligating adaptors
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2525/186 incorporating a non-extendable or blocking moiety
• CPC Class Codes: C12Q 2525/191 incorporating an adaptor
• CPC Class Codes: C12Q 2525/207 siRNA, miRNA
• CPC Class Codes: C12Q 2525/301 Hairpin oligonucleotides
• CPC Class Codes: C12Q 2525/307 Circular oligonucleotides
• CPC Class Codes: C12Q 2539/101 Subtraction analysis

Abstract

Methods, compositions, and kits comprising target-specific oligonucleotides (TSOs) are disclosed herein. Methods, compositions, and kits comprising target-specific oligonucleotides (TSOs) can be used to attach adapters and/or linkers to target RNAs. Methods, compositions, and kits comprising target-specific oligonucleotides (TSOs) can be used in reactions, including, but not limited to, ligation reactions, amplification reactions, and sequencing reactions. Additionally, methods, compositions, and kits comprising target-specific oligonucleotides (TSOs) can be used for reducing and/or preventing the formation of secondary structures in target RNAs. These methods, compositions, and kits can also find use in a number of applications, for example, any application that benefits from stabilizing primary RNA structure, such as detecting and quantifying target RNAs in a sample, in the construction of small RNA libraries, in microarray and RT-qPCR applications, etc.

Claims

1. A method for detecting and quantifying an amount of a target RNA in a sample, the method comprising:

a) hybridizing the target RNA with a target-specific oligonucleotide (TSO) to produce a TSO-hybridized target RNA;

wherein the TSO is

(i) shorter than the target RNA, or as long as or shorter than the target RNA wherein the target RNA is a miRNA; and

(ii) substantially complementary to the target RNA sequence;

b) ligating a 5′

adapter to a 5′

end of the TSO-hybridized target RNA and/or ligating a 3′

adapter to a 3′

end of the TSO-hybridized target RNA, wherein the ligating comprises performing a splint-independent ligation to produce an adapter-ligated TSO-hybridized target RNA, and reverse transcribing the adapter-ligated TSO-hybridized target RNA, or a portion thereof, to produce a reverse transcript thereof;

c) enzymatically amplifying the reverse transcript, or a portion thereof, to produce an amplified polynucleotide comprising a sequence corresponding to or complementary to the target RNA sequence; and

d) quantitatively detecting an amount of the amplified polynucleotide, wherein the amount of the amplified polynucleotide correlates with the amount of the target RNA,

wherein the TSO is not immobilized in any step of the method.

2. The method of claim 1, wherein the hybridizing of the target RNA with the TSO inhibits or promotes ligating oligonucleotide adapter(s) to the ends of the target RNA.

3. The method of claim 1, comprising ligating the 5′

adapter to a 5′

end of each of a plurality of TSO-hybridized target RNAs to produce a plurality of 5′

-end adapter-ligated TSO-hybridized target RNAs.

4. The method of claim 3, wherein the 5′

oligonucleotide adaptor comprises a 5′

-end group selected from a 5′

-phosphate and a 5′

-hydroxyl; and

wherein the 5′

oligonucleotide adaptor is a 5′

-hydroxyl, the method further comprises phosphorylating the 5′

-hydroxyl after the ligating.

5. The method of claim 1, comprising ligating the 3′

adapter to a 3′

end of each of a plurality of TSO-hybridized target RNAs to produce a plurality of 3′

-end adapter-ligated TSO-hybridized target RNAs.

6. The method of claim 5 wherein the 3′

adapter comprises a 5′

-end group that is a 5′

-phosphate or a 5′

, 5′

-adenyl pyrophosphoryl group.

7. The method of claim 1, comprising ligating the 5′

adapter to the 5′

-end of the TSO-hybridized target RNA and ligating the 3′

adapter to the 3′

-end of the TSO-hybridized target RNA to produce a 5′

- and 3′

-end adapter-ligated TSO-hybridized target RNA, wherein ligating the 5′

adapter occurs before ligating the 3′

adapter;

ligating the 5′

adapter occurs after ligating the 3′

adapter;

or ligating the 5′

adapter and ligating the 3′

adapter occur simultaneously.

8. The method of claim 1, wherein the 3′

- and 5′

-adapters are contained within a single oligonucleotide, which is ligated to the 5′

or 3′

end of the target RNA.

9. The method of claim 1, wherein the ligating comprises adding to the TSO-hybridized target RNA an RNA ligase selected from the group consisting of:

T4 RNA ligase 1 (Rnl1), T4 RNA ligase 2 (Rnl2), and a truncated and/or mutated derivative of T4 RNA ligase 2.

10. The method of claim 1, wherein the 3′

-adapter and/or the 5′

-adapter comprise a sequence used for amplification and/or sequencing.

11. The method of claim 1, further comprising circularizing a non-target RNA in the sample after hybridizing the TSO and the target RNA, but prior to the ligating the TSO-hybridized target RNA, wherein the circularizing prevents ligating of the non-target RNA.

12. The method of claim 1, further comprising circularizing the adapter-ligated TSO-hybridized target RNA prior to the reverse transcribing.

13. The method of claim 1, wherein the quantitatively detecting comprises next generation sequencing.

14. The method of claim 1, comprising purifying the adapter-ligated TSO-hybridized target RNA, the reverse transcript thereof and/or amplified polynucleotide prior to detecting the amplified polynucleotide, wherein the purifying of the products comprises size-dependent separation of at least one of the adapter-ligated TSO-hybridized target RNA, reverse transcript thereof, and the amplified polynucleotide by gel- or capillary electrophoresis.

15. The method of claim 1, wherein the TSO comprises a hapten attached via a non-nucleotide or oligonucleotide linker.

16. The method of claim 1, wherein the target RNA is selected from the group consisting of:

a microRNA, a pre-miRNA, a non-coding RNA, fragments of the non-coding RNA, a coding RNA, and fragments of the coding RNA.

17. The method of claim 16, wherein the target RNA is about 15 to 150 nucleotides in length.

18. The method of claim 1, wherein the sample is selected from the group consisting of:

a tissue extract, a cell extract, a cell lysate, a tissue lysate, an extracellular bio-fluid extract, and an extracellular bio-fluid lysate.

19. The method of claim 1, wherein the TSO comprises one or more ribonucleotides and/or deoxyribonucleotides, wherein the one or more ribonucleotides and/or deoxyribonucleotides has a modification selected from the group consisting:

a 2′

-OMe, a 2′

-fluoro, a locked nucleic acid (LNA), an abasic site, a non-nucleotide linker, and combinations thereof.

20. The method of claim 1, wherein the TSO comprises a blocking group at (a) its 3′

-end, which prevents ligation to or extension of its 3′

end, and/or (b) its 5′

end, which prevents ligation and/or phosphorylation at its 5′

end.