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.
Claims47
1-27. (canceled)
A method for capping an uncapped target RNA in vitro, comprising:
- contacting:
- (i) an RNA sample comprising the uncapped target RNA;
- (ii) an RNA capping enzyme comprising an amino acid sequence that is at least 90% identical to SEQ ID NO:1, 7, or 20;
- (iii) guanosine triphosphate (GTP) or modified GTP
- (iv) a buffering agent;
- and
- (v) a methyl group donor,
- at a temperature of 40° C.-60° C. to form a capped target RNA.
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.
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;
- (iii) guanosine triphosphate (GTP);
- (iv) a buffering agent;
- and
- (v) a methyl group donor.
A kit comprising:
- a single-chain RNA capping enzyme that has RNA triphosphatase (TPase), guanylyltransferase (GTase) and guanine-N7 methyltransferase (N7 MTase) activities, wherein the enzyme is in a storage buffering agent;
- and
- a reaction buffering agent.
An RNA capping enzyme comprising an amino acid sequence that is at least 90% identical to SEQ ID NO:20.
An RNA capping enzyme fusion comprising (a) an amino acid sequence that is 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.
A method comprising contacting:
- (a) a composition comprising one or more capped RNAs;
- (b) a targeting oligonucleotide, and
- (c) an RNase H,
- to form cleavage products of the capped RNAs,
- wherein the RNase H is guided by the targeting oligonucleotide.
An Escherichia coli cell comprising a single-chain RNA capping enzyme that has RNA triphosphatase (TPase), guanylyltransferase (GTase) and guanine-N7 methyltransferase (N7 MTase) activities.
An Escherichia coli cell lysate comprising a single-chain RNA capping enzyme that has RNA triphosphatase (TPase), guanylyltransferase (GTase) and guanine-N7 methyltransferase (N7 MTase) activities.
Dependencies are inferred from the claim text.