USE OF DNAZYMES FOR ANALYSIS OF AN RNA SAMPLE

• US 20130123129A1
• Filed: 11/01/2012
• Published: 05/16/2013
• Est. Priority Date: 11/11/2011
• Status: Active Grant

• Current Assignee: Agilent Technologies, Inc.
• Original Assignee: Agilent Technologies, Inc.
• Inventors: Zeiner, Gusti, Ach, Robert A.
• Granted Patent: US 8,815,782 B2
• Time in Patent Office: Days
• US Class Current: 506/9
• CPC Class Codes: C12N 15/111 General methods applicable to biologically active non-coding nucleic acids
• CPC Class Codes: C12N 15/113 Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing when used in plants C12N15/8218
• CPC Class Codes: C12N 2310/127 DNAzymes
• CPC Class Codes: C12N 2320/10 in screening processes
• CPC Class Codes: C12P 19/34 Polynucleotides, e.g. nucleic acids, oligoribonucleotides
• 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 2521/107 RNA dependent DNA polymerase,(i.e. reverse transcriptase)
• CPC Class Codes: C12Q 2521/119 RNA polymerase
• CPC Class Codes: C12Q 2521/345 DNAzyme
• CPC Class Codes: C12Q 2521/501 Ligase
• CPC Class Codes: C12Q 2565/501 being an array of oligonucleotides

Abstract

Provided herein is method comprising: contacting an initial RNA sample containing a population of different RNA molecules with a divalent cation and a set of DNAzymes that are designed to cleave multiple target RNAs in the initial sample, thereby producing a product RNA sample that comprises: a) uncleaved RNA molecules and b) cleaved RNA fragments that contain a 2′,3′-cyclic-phosphate and a 5′ hydroxyl as the result of DNAzyme cleavage.

Claims

1. A method comprising:

contacting an initial RNA sample containing a population of different RNA molecules with a divalent cation and a set of DNAzymes that are designed to cleave multiple target RNAs in said initial sample, thereby producing a product RNA sample that comprises;

a) uncleaved RNA molecules and b) cleaved RNA fragments that contain a 2′

,3′

-cyclic-phosphate and a 5′

hydroxyl as the result of DNAzyme cleavage.

2. The method of claim 1, wherein said DNAzymes are 10-23 DNAzymes.

3. The method of claim 1, wherein contacting is done by cycling through denaturation and renaturation temperatures to maximize cleavage by said DNAzyme.

4. The method of claim 1, wherein said set of DNAzymes comprises at least 100 different DNAzymes that specifically target different RNAs.

5. The method of claim 1, further comprising ligating a first adaptor onto the 3′

ends of at least some of said cleaved RNA fragments using a 2′

,3′

-cyclic phosphate-specific RNA ligase, thereby producing a ligated sample comprising ligation products.

6. The method of claim 5, wherein said first adaptor comprises a 5′

-phosphate or 5′

-hydroxyl and said ligase is a eukarytic tRNA ligase.

7. The method of claim 5, wherein said first adaptor comprises a 5′

-hydroxyl and said ligase is an RtcB ligase.

8. The method of claim 5, wherein said first adaptor is labeled, and the method produces a labeled RNA sample.

9. The method of claim 8, wherein said method further comprises:

a) hybridizing said labeled RNA sample to an array;

b) reading said array to provide results, and c) analyzing said results to obtain information on said initial sample.

10. The method of claim 5, wherein the first adaptor comprises an RNA polymerase promoter and said method comprises:

a) making double stranded cDNA from said product RNA sample using a primer that hybridizes with said first adaptor and a reverse transcriptase, and b) transcribing the cDNA using an RNA polymerase in the presence of labeled a rNTP, thereby making labeled RNA.

11. The method of claim 5, wherein said method comprises:

a) making cDNA from said product RNA sample using a primer that hybridizes with said first adaptor and a reverse transcriptase;

b) adding a second adaptor to said cDNA at the opposite end to said first adaptor;

c) amplifying said cDNA using primers that bind to said first adaptor and second adaptors.

12. The method of claim 11, wherein further comprising sequencing said cDNA.

13. The method of claim 5, wherein said first adaptor contains an affinity tag, and said method further comprises purifying said ligation products from other molecules in said ligated sample using said affinity tag.

14. The method of claim 1, wherein said initial RNA sample comprises adaptor-ligated RNA molecules that have a 3′

adaptor and a 5′

adaptor.

15. The method of claim 14, wherein said method comprises, after said contacting, making cDNA using a primer that hybridizes with said 3′

adaptor.

18. The method of claim 1, wherein said method comprises contacting an initial RNA sample containing a population of different RNA molecules with a divalent cation and a first DNAzyme and a second DNAzyme, wherein said first and second DNAzymes both the cleave the same RNA molecule to produce an RNA product that has a defined sequence of nucleotides at its ends.

19. The method of claim 18, further comprising ligating the ends of said RNA product to an adaptor to produce a circular product molecule.

16. The method of any of the prior embodiments including embodiment 14, amplifying cDNA copied from said uncleaved RNA molecules using primers that are complementary to the 3′

and 5′

adaptors.

17. The method of claim 16, wherein said method further comprises sequencing said cDNA.

20. A composition comprising a plurality of DNAzymes that are designed to cleave multiple target RNAs.