Selective 5′ ligation tagging of RNA
First Claim
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1. A method for generating 5′
- -ligation-tagged RNA from RNA in a sample that is uncapped and has a 5′
polyphosphate group, comprising;
(A) providing;
(i) a sample that contains uncapped RNA that has a 5′
polyphosphate group;
(ii) a RNA 5′
polyphosphatase comprising the sequence of amino acids 35 to 200 of SEQ ID NO;
2, wherein the polyphosphatase does not comprise the sequence of amino acids 1 to 34 of SEQ ID;
2;
(iii) an acceptor oligonucleotide that exhibits a tag; and
(iv) a RNA ligase;
(B) contacting the sample with the RNA 5′
polyphosphatase under conditions and for sufficient time wherein the uncapped RNA that has a 5′
polyphosphate group is converted to RNA that has a 5′
monophosphate group; and
(C) contacting the sample from step (B) with the acceptor oligonucleotide and the RNA ligase under conditions and for sufficient time wherein 5′
-ligation-tagged RNA is generated.
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Abstract
The present invention provides novel compositions, kits and methods employing RNA 5′ polyphosphatases, RNA 5′ monophosphatases, capping enzymes, decapping enzymes, nucleic acid pyrophosphatases and RNA ligases, as well as other enzymes, for selective 5′ ligation tagging of desired classes of RNA molecules that differ with respect to particular chemical moieties on their 5′ ends. The 5′ tagged RNA molecules can be used for synthesis of tagged first-stand cDNA, double-stranded cDNA, and sense or antisense RNA for a variety of uses.
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14 Claims
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1. A method for generating 5′
- -ligation-tagged RNA from RNA in a sample that is uncapped and has a 5′
polyphosphate group, comprising;(A) providing; (i) a sample that contains uncapped RNA that has a 5′
polyphosphate group;(ii) a RNA 5′
polyphosphatase comprising the sequence of amino acids 35 to 200 of SEQ ID NO;
2, wherein the polyphosphatase does not comprise the sequence of amino acids 1 to 34 of SEQ ID;
2;(iii) an acceptor oligonucleotide that exhibits a tag; and (iv) a RNA ligase; (B) contacting the sample with the RNA 5′
polyphosphatase under conditions and for sufficient time wherein the uncapped RNA that has a 5′
polyphosphate group is converted to RNA that has a 5′
monophosphate group; and(C) contacting the sample from step (B) with the acceptor oligonucleotide and the RNA ligase under conditions and for sufficient time wherein 5′
-ligation-tagged RNA is generated. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14)
- -ligation-tagged RNA from RNA in a sample that is uncapped and has a 5′
Specification