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.
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74 . An RNA capping enzyme comprising an amino acid sequence that is (a) at least 90% identical to SEQ ID NO:20 and/or (b)(i) at least 90% identical to SEQ ID NO:7 and (ii) at least 90% identical to positions 1419 to 1587 of SEQ ID NO:20.
75 . An RNA capping enzyme according to claim 74 , wherein the enzyme is an RNA capping enzyme fusion.
76 . An RNA capping enzyme according to claim 74 , wherein the enzyme has RNA polymerase activity and/or RNA capping activity.
77 . An RNA capping enzyme according to claim 74 , wherein the enzyme has (a) RNA polymerase activity and/or (b)(i) RNA triphosphatase (TPase) activity, (ii) guanylyltransferase (GTase) activity, and (iii) guanine-N7 methyltransferase (N7 MTase) activity.
78 . An RNA capping enzyme according to claim 74 , wherein the amino acid comprises, in an N-terminal to C-terminal direction, the sequence (i) that is at least 90% identical to SEQ ID NO:7 and the sequence (ii) that is at least 90% identical to positions 1419 to 1587 of SEQ ID NO:20.
79 . A composition comprising:
(a) a polynucleotide;
(b) an RNA capping enzyme according to claim 74 ;
(c) guanosine triphosphate (GTP);
(d) a buffering agent; and
(e) a methyl group donor.
80 . The composition of claim 79 , wherein the composition is RNase-free and optionally comprises (e) one or more RNase inhibitors.
81 . The composition of claim 79 , wherein the RNA capping enzyme amino acid sequence is at least 90% identical to (a) SEQ ID NO:2, (b) SEQ ID NO:3, and/or (c) SEQ ID NO:4.
82 . The composition of claim 79 , wherein the RNA capping enzyme amino acid sequence is at least 95% identical to SEQ ID NO:7.
83 . The composition of claim 79 , wherein the polynucleotide comprises a DNA template and the composition further comprises ribonucleotide triphosphates for transcribing the DNA template to form an uncapped target RNA.
84 . The composition of claim 83 , wherein the uncapped target RNA comprises one or more pseudouridines.
85 . The composition of claim 79 , wherein the composition comprises (f) S-adenosyl methionine (SAM) and (g) a cap 2′O methyltransferase enzyme.
86 . The composition of claim 79 further comprising one or more detergents, dyes, solvents and/or preservatives.
87 . A kit comprising:
an RNA capping enzyme according to claim 74 ; and
a reaction buffering agent.
88 . The kit of claim 87 , wherein the kit further comprises ribonucleotides, for transcribing a template polynucleotide encoding a target RNA.
89 . The kit of claim 87 , wherein the kit further comprises S-adenosyl methionine (SAM), cap 2′O methyltransferase enzyme (2′OMTase), or both SAM and 2′OMTase.
90 . The kit of claim 87 , wherein the RNA capping enzyme amino acid sequence is at least 90% identical to (a) SEQ ID NO:2, (b) SEQ ID NO:3, and/or (c) SEQ ID NO:4.
91 . The kit of claim 87 , wherein the RNA capping enzyme amino acid sequence is at least 95% identical to SEQ ID NO:7.
92 . The kit of claim 87 , wherein the kit further comprises one or more detergents, dyes, solvents and/or preservatives.
93 . An Escherichia coli cell and/or an Escherichia coli cell lysate comprising an RNA capping enzyme according to claim 74 .
94 . A method for capping an uncapped target RNA in vitro, comprising:
contacting:
(a) an RNA sample comprising the uncapped target RNA;
(b) an RNA capping enzyme comprising an amino acid sequence that is (i) at least 90% identical to SEQ ID NO:20 and/or (ii) 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;
(c) guanosine triphosphate (GTP) or modified GTP
(d) a buffering agent; and
(e) a methyl group donor,
at a temperature of 23° C.-60° C. to form a capped target RNA.
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