Enzymatic RNA Capping Method
AssigneeNew England Biolabs, Inc.
InventorsG. Brett Robb, Siu-hong Chan, Bijoyita Roy
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Abstract
Provided herein is a method for efficiently capping RNA in vitro. In some embodiments the capping reaction may be done at high temperature using Vaccinia capping enzyme or a variant thereof. In other embodiments, the capping reactions may comprise a capping enzyme from a large virus of amoeba, e.g., Faustovirus, mimivirus or moumouvirus, or a variant thereof. Compositions and kits for practicing the method are also provided.
Claims31
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An RNA capping enzyme comprising an amino acid sequence that is (a) at least 90% identical to SEQ ID NO:20 and/or (b)(i) at least 90% identical to SEQ ID NO:7 and (ii) at least 90% identical to positions 1419 to 1587 of SEQ ID NO:20.
A method for capping an uncapped target RNA in vitro, comprising:
- contacting:
- (a) an RNA sample comprising the uncapped target RNA;
- (b) an RNA capping enzyme comprising an amino acid sequence that is (i) at least 90% identical to SEQ ID NO:20 and/or (ii) at least 90% identical to SEQ ID NO:7 and (b) an amino acid sequence that is at least 90% identical to positions 1419 to 1587 of SEQ ID NO:20;
- (c) guanosine triphosphate (GTP) or modified GTP
- (d) a buffering agent;
- and
- (e) a methyl group donor,
- at a temperature of 23° C.-60° C. to form a capped target RNA.
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Dependencies are inferred from the claim text.