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US 12,479,880 B2Granted
Enzymatic RNA capping method
AssigneeNew England Biolabs, Inc.
InventorsG. B. 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.
Claims23
A composition comprising:
- (i) a polynucleotide;
- (ii) 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:2, (b) SEQ ID NO:3, and (c) SEQ ID NO:4;
- (iii) guanosine triphosphate (GTP);
- (iv) a buffering agent;
- and
- (v) a methyl group donor,
- wherein the amino acid sequence further has at least 85% identity to (A) SEQ ID NO:7, (B) SEQ ID NO:
- 8, (C) SEQ ID NO:9, and/or (D) SEQ ID NO:10.
5 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:2, (b) SEQ ID NO:3, and (c) SEQ ID NO:4, wherein the enzyme is in a storage buffering agent;
- and a reaction buffering agent,
- wherein the amino acid sequence further has at least 85% identity to (A) SEQ ID NO:7, (B) SEQ ID NO:
- 8, (C) SEQ ID NO:9, and/or (D) SEQ ID NO:10.
1 more element
A method for efficiently capping RNA in vitro, comprising:
- contacting:
- (i) an RNA sample comprising an uncapped target RNA;
- (ii) a single-chain RNA capping enzyme that has RNA triphosphatase (TPase), guanylyltransferase (GTase) and guanine-N7 methyltransferase (N7 MTase) activities;
- (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, wherein the single-chain RNA capping enzyme comprises an amino acid sequence at least 90% identical to (a) SEQ ID NO:2, (b) SEQ ID NO:3, and (c) SEQ ID NO:4 and at least 85% identical to (A) SEQ ID NO:7, (B) SEQ ID NO:8, (C) SEQ ID NO:9, and/or (D) SEQ ID NO:10.
5 more elements
Dependencies are inferred from the claim text.