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 - 27 . (canceled)
28 . 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.
29 . The method of claim 28 , wherein the uncapped target RNA is at least 200 nt in length.
30 . The method of claim 28 , 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 28 , 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 28 , further comprising synthesizing the uncapped target RNA using solid-phase oligonucleotide synthesis chemistry.
33 . The method of claim 28 , 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 28 , 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 28 , wherein the uncapped target RNA comprises one or more pseudouridines.
36 . The method of claim 28 , wherein contacting further comprises contacting (i), (ii), (iii), (iv) and optionally (v) in a single location.
37 . The method according to claim 28 , further comprising monitoring the appearance of capped target RNA.
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 (a) SEQ ID NO:2, (b) SEQ ID NO:3, (c) SEQ ID NO:4, (d) SEQ ID NO:5 and/or (e) 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 (a) SEQ ID NO:7, (b) SEQ ID NO:8, (c) SEQ ID NO:9, (d) SEQ ID NO:10), (e) SEQ ID NO:11 and/or (f) 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 (a) SEQ ID NO:2, (b) SEQ ID NO:3, (c) SEQ ID NO:4, (d) SEQ ID NO:5 and/or (e) 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 (a) SEQ ID NO:7, (b) SEQ ID NO:8, (c) SEQ ID NO:9, (d) SEQ ID NO:10), (e) SEQ ID NO:11 and/or (f) 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 (a) SEQ ID NO:7, (b) SEQ ID NO:8, (c) SEQ ID NO:9, (d) SEQ ID NO:10), (e) SEQ ID NO:11 and/or (f) 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 (a) SEQ ID NO:2, (b) SEQ ID NO:3, (c) SEQ ID NO:4, (d) SEQ ID NO:5 and/or (e) 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 (a) SEQ ID NO:7, (b) SEQ ID NO:8, (c) SEQ ID NO:9, (d) SEQ ID NO:10), (e) SEQ ID NO:11 or (f) 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 (a) SEQ ID NO:7, (b) SEQ ID NO:8, (c) SEQ ID NO:9, (d) SEQ ID NO:10), (e) SEQ ID NO:11 or (f) SEQ ID NO:12.
59 . An RNA capping enzyme comprising an amino acid sequence that is at least 90% identical to SEQ ID NO:20.
60 . 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.
61 . 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.
62 . The method according to claim 61 , wherein the cleavage products comprise capped fragments having a length of 24 nucleotides.
63 . The method according to claim 61 , wherein the RNase H is E. coli RNase H or Thermus thermophilus RNase H.
64 . The method according to claim 61 , wherein the targeting oligonucleotide comprises 5′ deoxynucleotides and 3′ ribonucleotides.
65 . The method according to claim 61 further comprising adding a FAM-labeled nucleotide to the 3′ end of the cleavage products for form FAM-labeled cleavage products.
66 . The method according to claim 65 further comprising analyzing the FAM-labeled cleavage products by capillary electrophoresis.
67 . The method according to claim 66 further comprising analyzing the ratio of relative quantity of m7GpppGm(Cap-1), m7G- (Cap-0), unmethyl-G-capped and uncapped RNA in the FAM-labeled cleavage products by mass spectrometry.
68 . 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.
69 . The Escherichia coli cell of claim 68 , wherein the single-chain RNA capping enzyme has an amino acid sequence at least 90% identical to a sequence selected from the group consisting of (a) SEQ ID NO:2, (b) SEQ ID NO:3, (c) SEQ ID NO:4, (d) SEQ ID NO:5, and (e) SEQ ID NO:6.
70 . The Escherichia coli cell of claim 68 , wherein the single-chain RNA capping enzyme has an amino acid sequence at least 90% identical to a sequence selected from the group consisting of (a) SEQ ID NO:7, (b) SEQ ID NO:8, (c) SEQ ID NO:9, (d) SEQ ID NO:10, (e) SEQ ID NO:11, and (f) SEQ ID NO:12, and (g) SEQ ID NO:20.
71 . 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.
72 . The Escherichia coli cell lysate of claim 71 , wherein the single-chain RNA capping enzyme has an amino acid sequence at least 90% identical to a sequence selected from the group consisting of (a) SEQ ID NO:2, (b) SEQ ID NO:3, (c) SEQ ID NO:4, (d) SEQ ID NO:5, and (e) SEQ ID NO:6.
73 . The Escherichia coli cell lysate of claim 71 , wherein the single-chain RNA capping enzyme has an amino acid sequence at least 90% identical to a sequence selected from the group consisting of (a) SEQ ID NO:7, (b) SEQ ID NO:8, (c) SEQ ID NO:9, (d) SEQ ID NO:10, (e) SEQ ID NO:11, and (f) SEQ ID NO:12, and (g) SEQ ID NO:20.