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.
Claims32
1-28. (canceled)
The method of claim 38, wherein the uncapped target RNA is at least 200 nt in length.
The method of claim 38, wherein contacting further comprises increasing or decreasing the temperature to a second temperature of 37° C.-60° C., wherein the second temperature differs from the first temperature.
The method of claim 38, wherein (i), (ii), (iii), and (iv) are RNase-free and contacting optionally further comprises contacting (v) one or more RNase inhibitors.
The method of claim 38, further comprising synthesizing the uncapped target RNA using solid-phase oligonucleotide synthesis chemistry.
The method of claim 38, further comprising synthesizing the uncapped target RNA by contacting a DNA template encoding the uncapped RNA and a polymerase to produce the uncapped RNA.
The method of claim 38, wherein the methyl group donor is S-adenosyl methionine and contacting further comprises contacting (vi) a cap 2′O methyltransferase enzyme.
The method of claim 38, wherein the uncapped target RNA comprises one or more pseudouridines.
The method of claim 38, wherein contacting further comprises contacting (i), (ii), (iii), (iv) and optionally (v) in a single location.
(canceled)
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.
59-73. (canceled)
Dependencies are inferred from the claim text.