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US 12,398,173 B2Granted
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.
Claims18
A composition comprising:
- (i) a polynucleotide;
- a single-chain RNA capping enzyme that has RNA triphosphatase (TPase), guanylyltransferase (GTase) and guanine-N7 methyltransferase (N7 MTase) activities and comprises an amino acid sequence at least 90% identical to (a) SEQ ID NO:7, (b) SEQ ID NO:8, (c) SEQ ID NO:9, and/or (d) SEQ ID NO:10;
- (iii) guanosine triphosphate (GTP);
- (iv) a buffering agent;
- and
- (v) a methyl group donor.
3 more elements
A kit comprising:
- a single-chain RNA capping enzyme that has RNA triphosphatase (TPase), guanylyltransferase (GTase) and guanine-N7 methyltransferase (N7 MTase) activities and comprises an amino acid sequence at least 90% identical to (a) SEQ ID NO:7, (b) SEQ ID NO:8, (c) SEQ ID NO:9, and/or (d) SEQ ID NO:10, wherein the enzyme is in a storage buffering agent;
- and a reaction buffering agent.
A method for efficiently capping RNA in vitro, comprising:
- contacting:
- (i) an RNA sample comprising an uncapped target RNA;
- a single-chain RNA capping enzyme that has RNA triphosphatase (TPase), guanylyltransferase (GTase) and guanine-N7 methyltransferase (N7 MTase) activities and comprises an amino acid sequence at least 90% identical to (a) SEQ ID NO:7, (b) SEQ ID NO:8, (c) SEQ ID NO:9, and/or (d) SEQ ID NO:
- 10;
- (iii) guanosine triphosphate (GTP) or modified GTP
- (iv) a buffering agent;
- and
- (v) a methyl group donor,
- at a temperature of 23° C.-60° C., to form a capped target RNA.
6 more elements
Dependencies are inferred from the claim text.