Enzymatic RNA Capping Method
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.
1 - 28 . (canceled)
29 . The method of claim 38 , wherein the uncapped target RNA is at least 200 nt in length.
30 . 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.
31 . 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.
32 . The method of claim 38 , further comprising synthesizing the uncapped target RNA using solid-phase oligonucleotide synthesis chemistry.
33 . 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.
34 . 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.
35 . The method of claim 38 , wherein the uncapped target RNA comprises one or more pseudouridines.
36 . The method of claim 38 , wherein contacting further comprises contacting (i), (ii), (iii), (iv) and optionally (v) in a single location.
37 . (canceled)
38 . 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.
39 . The method of claim 38 , wherein the single-chain RNA capping enzyme comprises amino acid sequence at least 90% identical to SEQ ID NO:5 and/or SEQ ID NO:6.
40 . The method of claim 38 , wherein the single-chain RNA capping enzyme comprises an amino acid sequence at least 90% identical to SEQ ID NO:11 and/or SEQ ID NO:12.
41 . The method according to claim 38 , further comprising monitoring the appearance of capped target RNA.
42 . 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.
43 . The composition of claim 42 , wherein the composition has a temperature in the range of 23° C.-60° C.
44 . The composition of claim 42 , wherein the composition is RNase-free and optionally comprises (v) one or more RNase inhibitors.
45 . The composition of claim 42 , wherein the single-chain RNA capping enzyme comprises an amino acid sequence at least 90% identical to SEQ ID NO:5 and/or SEQ ID NO:6.
46 . The composition of claim 42 , wherein the single-chain RNA capping enzyme comprises an amino acid sequence at least 90% identical to SEQ ID NO:11 and/or SEQ ID NO:12.
47 . The composition of claim 42 , wherein the polynucleotide comprises a DNA template and the composition further comprises a bacteriophage polymerase and ribonucleotide triphosphates for transcribing the DNA template to form an uncapped target RNA.
48 . The composition of claim 42 , wherein the composition optionally comprises (vi) S-adenosyl methionine (SAM) and (vii) a cap 2′O methyltransferase enzyme.
49 . The composition of claim 42 , wherein the uncapped target RNA comprises one or more pseudouridines.
50 . The composition of claim 42 further comprising one or more detergents, dyes, solvents and/or preservatives.
51 . The composition of claim 42 , wherein the single-chain RNA capping enzyme comprises an amino acid sequence at least 95% identical to SEQ ID NO:11 and/or SEQ ID NO:12.
52 . 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.
53 . The kit of claim 52 , wherein the kit further comprises a bacteriophage polymerase and ribonucleotides, for transcribing a template polynucleotide encoding a target RNA.
54 . The kit of claim 52 , wherein the kit further comprises S-adenosyl methionine (SAM), cap 2′O methyltransferase enzyme (2′OMTase), or both SAM and 2′OMTase.
55 . The kit of claim 52 , wherein the single-chain RNA capping enzyme comprises an amino acid sequence at least 90% identical to SEQ ID NO:5 and/or SEQ ID NO:6.
56 . The kit of claim 52 , wherein the single-chain RNA capping enzyme comprises an amino acid sequence that is at least 90% identical to SEQ ID NO:11 or SEQ ID NO:12.
57 . The kit of claim 52 , wherein the kit further comprises one or more detergents, dyes, solvents and/or preservatives.
58 . The kit of claim 52 , wherein the single-chain RNA capping enzyme comprises an amino acid sequence that is at least 95% identical to SEQ ID NO:11 or SEQ ID NO:12.
59 - 73 . (canceled)