Compositions, kits, and methods for in vitro transcription
The present disclosure relates, according to some embodiments, to cold-active RNA polymerases, variants thereof, compositions and kits comprising cold-active RNA polymerases, and methods of using cold-active RNA polymerases. Cold-active polymerases may have, for example, an amino acid sequence ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% identical (e.g., ≥95% or ≥98% identical) to any of SEQ ID NOS: 1-19 and optionally may have at least one substitution relative to SEQ ID NO:1.
1 . A composition comprising:
a cold-active RNA polymerase having an amino acid sequence at least 95% identical to any of SEQ ID NOS: 1, 5, 6, 8-10, 12, 13, 18, and 19; and
a capping enzyme,
wherein the cold-active RNA polymerase is a non-naturally occurring cold-active RNA polymerase or a cold-active RNA polymerase of Pseudomonas phage Njord, Pseudomonas phage uligo, Vibrio phage φA318, Vibrio phage Vp670, Vibrio phage Vc1 , Vibrio phage VEN, and the capping enzyme is a non-naturally occurring capping enzyme or a capping enzyme of Faustovirus, mimivirus, or moumouvirus.
2 . A composition according to claim 1 , wherein the polymerase comprises at least one conservative substitution relative to SEQ ID NO:1.
3 . A composition according to claim 1 , wherein the amino acid sequence of the polymerase is less than 100% identical to SEQ ID NOS: 1, 5, 6, 8-10, 12, and 13 and/or wherein the amino acid sequence of the polymerase is 100% identical to SEQ ID NO:17.
4 . A composition according to claim 1 , wherein the polymerase is immobilized to a support or the capping enzyme is immobilized to a support or the polymerase and the capping enzyme are each immobilized to a separate support or the polymerase and the capping enzyme are each immobilized to a common support.
5 . A composition according to claim 1 , wherein the polymerase and the capping enzyme are included in a fusion comprising, in an N-terminal to C-terminal direction, (a) the polymerase and the capping enzyme or (b) the capping enzyme and the polymerase.
6 . A composition according to claim 5 , wherein the fusion further comprises a linker between the polymerase and the capping enzyme.
7 . A composition according to claim 1 further comprising one or more of:
guanosine triphosphate (GTP) or modified GTP;
a methyl group donor;
a 2′ O-methyltransferase; and
a buffering agent.
8 . A composition according to claim 1 further comprising a polynucleotide template comprising, in a 5′ to 3′ direction, a promoter corresponding to the polymerase and a sequence of interest.
9 . A composition according to claim 8 , wherein the promoter has a nucleotide sequence according to one of SEQ ID NOS: 31-34, 35-37, and 39-41 and/or wherein the sequence of interest comprises a coding sequence.
10 . A composition according to claim 1 further comprising a polyribonucleotide product of the polymerase, wherein the product has fewer double-stranded RNA molecules than a polyribonucleotide product of T7 RNA polymerase having the same nucleotide sequence.
11 . A composition according to claim 1 further comprising at least one of a buffering agent and/or a polyamine.
12 . A composition according to claim 11 , wherein the buffering agent comprises HEPES, MES, MOPS, TAPS, tricine, Tris, ACES, ADA, BES, Bicine, CAPS, carbonic acid/bicarbonic acid, CHES, citric acid, DIPSO, EPPS, histidine, MOPSO, phosphoric acid, PIPES, POPSO, TAPS, TAPSO, or triethanolamine.
13 . A composition according to claim 11 , wherein the polyamine comprises spermidine, spermine, putrescine, polyethylenimine, 1,4,7-triazacyclononane, cyclen, ethylenediamine, or 1, 3, 5,-triazinane.
14 . A method comprising:
(a) contacting:
(i) a composition according to claim 1 ;
(ii) a polynucleotide template comprising an expression control sequence of the RNA polymerase and a coding sequence encoding an artificial transcript, the coding sequence operably linked to the expression control sequence; and
(iii) ribonucleotide triphosphates,
to produce the artificial transcript.
15 . A method according to claim 14 further comprising:
(b) contacting the artificial transcript with the capping enzyme and one or more of
(i) guanosine triphosphate (GTP) or modified GTP, (ii) a methyl group donor, (iii) a 2′ O-methyltransferase, and (iv) a buffering agent,
to produce a capped artificial transcript.
16 . A method comprising contacting:
(a) a composition according to claim 1 ;
(b) a polynucleotide template comprising an expression control sequence of the RNA polymerase and a coding sequence encoding an artificial transcript, the coding sequence operably linked to the expression control sequence;
(c) ribonucleotide triphosphates;
(d) guanosine triphosphate (GTP) or modified GTP;
(e) a methyl group donor;
(f) a 2′ O-methyltransferase; and
(g) a buffering agent,
to produce the artificial transcript, wherein the artificial transcript is capped.