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;
(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.
28 . iously Presented) 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 .- 60 . (canceled)
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 His 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 His 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 .- 73 . (canceled)