METHOD OF ADAPTOR-DIMER SUBTRACTION USING A CRISPR CAS6 PROTEIN

• US 20130330778A1
• Filed: 05/15/2013
• Published: 12/12/2013
• Est. Priority Date: 06/11/2012
• Status: Active Grant

• Current Assignee: Agilent Technologies, Inc.
• Original Assignee: Agilent Technologies, Inc.
• Inventors: Zeiner, Gusti, Bruhn, Laurakay
• Granted Patent: US 9,102,936 B2
• Time in Patent Office: Days
• US Class Current: 435/91.21
• CPC Class Codes: C12N 15/1096 cDNA Synthesis; Subtracted cDNA library construction, e.g. RT, RT-PCR

Abstract

A method of processing a target RNA is provided. In certain embodiments, this method comprises: contacting the products of an RNA ligase-mediated ligation reaction with an CAS6 protein, wherein: (i) the RNA ligase-mediated ligation reaction comprises: a target RNA, an RNA ligase, and first and second adaptors that can ligate together to produce an adaptor dimer that contains a CRISPR stem loop; and (ii) the CAS6 protein recognizes the CRISPR stem loop; thereby preventing the adaptor dimer from being reverse transcribed.

Claims

1. A method of processing a target RNA, comprising:

contacting the products of an RNA ligase-mediated ligation reaction with a CAS6 protein, wherein;

(i) said RNA ligase-mediated ligation reaction comprises;

a target RNA, an RNA ligase, and first and second adaptors that can ligate together to produce an adaptor dimer that contains a CRISPR stem loop; and

(ii) said CAS6 protein recognizes said CRISPR stem loop;

thereby preventing said adaptor dimer from being reverse transcribed.

2. The method of claim 1, wherein the amino acid sequence of said CAS6 protein is at least 80% identical to the amino acid sequence of a wild-type CAS6 protein.

3. The method of claim 1, wherein the amino acid sequence of said CAS6 protein is at least 95% identical to the amino acid sequence of the Csy4 protein from Pseudomonas aeruginosa.

4. The method of claim 3, wherein said CRISPR stem loop has the sequence CUGCCGUAUAGGCAGC (SEQ ID NO:

1).

5. The method of claim 1, wherein said CAS6 protein prevents said adaptor dimer from being reverse transcribed by cleaving said adaptor dimer.

6. The method of claim 1, wherein said CAS6 protein is mutated relative to a wild type sequence so that it is unable to cleave said adaptor dimer, and said CAS6 protein blocks said adaptor dimer from being reverse transcribed by binding to said adaptor dimer with high-affinity.

7. The method of claim 6, wherein said CAS protein has a mutation at His29.

8. The method of claim 1, wherein said first and second adaptors have the same nucleotide sequence.

9. The method of claim 1, wherein said first and second oligonucleotides have different sequences.

10. The method of claim 1, wherein said RNA ligase is eukaryotic tRNA ligase or a bacteriophage RNA ligase.

11. The method of claim 1, wherein said adaptors comprising a 5′

-PO, 5′

-OH and/or a 3′

terminus comprising either a 2′

-PO group and 3′

-OH group or a 2′

,3′

-cyclic phosphate group.

12. The method of claim 1, wherein said target RNA is an unfragmented short RNA molecule that comprises a 5′

phosphate group and a 3′

OH group.

13. The method of claim 12, wherein said short RNA molecule is a short interfering RNA (siRNA), microRNA (miRNA), tiny non-coding RNA (tncRNA), small modulatory RNA (smRNA) or piwi-interacting small RNA (piRNAs).

14. The method of claim 1, wherein said target RNA is a fragment of a long RNA molecule, wherein said fragment comprises a 5′

-OH group and a 2′

-PO group and a 3′

OH group or a 2′

-3′

-cyclic phosphate group.

15. The method of claim 1, further comprising contacting said products with a reverse transcriptase, a primer that hybridizes to an adaptor that hybridizes to at least one of said first and second adaptors, and nucleotides, thereby reverse transcribing at least part of any adaptor-ligated target RNA that are produced by ligation of said first and second adaptors to said target into cDNA.

16. A kit for making cDNA from a target RNA comprising:

an RNA ligase,

first and second adaptors that produce an adaptor dimer that contains a CRISPR stem loop when they are directly ligated together;

a CAS6 protein that recognizes said CRISPR stem loop.

17. The kit of claim 16, further comprising a reverse transcriptase.

18. A composition of matter comprising:

first and second adaptors that produce an adaptor dimer that contains a CRISPR stem loop when they are directly ligated together;

said an adaptor dimer; and

a CAS6 protein that recognizes said CRISPR stem loop.

19. The composition of matter of claim 18, further comprising a reverse transcriptase.