RNA polymerase, methods of purification and methods of use
Cyanophage Syn5 RNA polymerase (RNAP) and methods of purifying it are provided. Cyanophage Syn5 RNAP having one or more promoter mutations are provided. Methods of expressing cyanophage Syn5 RNAP are provided. Novel promoter sequences and in vitro transcription systems utilizing Syn5 RNAP are provided. Methods of making high range ssRNA ladders are also provided. Methods of incorporating 2′-modified dNTPs using Syn5 RNA polymerase are provided.
1. A method of performing in vitro transcription comprising the steps of:
providing a cDNA comprising a cyanophage Syn5 RNA polymerase (RNAP) promoter sequence and a nucleic acid template sequence, nucleotides and a Y564F mutant cyanophage Syn5 RNAP, wherein said Syn5 RNA Polymerase (RNAP) has at least 90% sequence identity to the amino acid sequence of SEQ ID NO:23; and
incubating the cDNA comprising the cyanophage Syn5 RNA polymerase (RNAP) promoter sequence and the nucleic acid template sequence, the nucleotides and the Y564F mutant cyanophage Syn5 RNAP together for a sufficient time to produce transcripts.
2. The method of claim 1 , wherein the incubating step is performed in the presence of salt at a concentration of at least about 100 mM.
3. The method of claim 2 , wherein the salt is KCl or NaCl.
4. The method of claim 1 , wherein the incubating step is performed at between about 4° C. and about 37° C.
5. The method of claim 1 , wherein the nucleotides are modified nucleotides.
6. The method of claim 1 , wherein the Y564F mutant cyanophage Syn5 RNAP includes a protein tag.
7. The method of claim 1 , wherein the transcripts are greater than about 10,000 nucleotides in length.
8. The method of claim 1 , wherein the nucleic acid template sequence comprises one or more T7 terminator-like sequences and the transcripts produced are full-length.
9. The method of claim 1 , wherein the in vitro transcription is performed in the presence of one or any combination of Mg 2+ , Mn 2+ and Fe 2+ .
10. The method of claim 1 , wherein greater than about 90% of the transcripts lack a nucleotide overhang at the 3′ ends.
11. A method of performing in vitro transcription comprising the steps of:
providing a reaction mixture of a cDNA comprising a cyanophage Syn5 RNA polymerase (RNAP) promoter sequence and a nucleic acid template sequence, nucleotides, one or more 2′-modified nucleotides, Mg 2+ and Mn 2+ r and a Y564F mutant cyanophage Syn5 RNAP, wherein said Syn5 RNA Polymerase (RNAP) has at least 90% sequence identity to the amino acid sequence of SEQ ID NO:23 and
incubating the reaction mixture for a sufficient time to produce a transcript including the one or more 2′-modified nucleotides.
12. The method of claim 11 wherein the Mg 2+ and Mn 2+ are present in a ratio of between about 2 to 1 to about 1 to 2.
13. The method of claim 11 wherein the Mg 2+ and Mn 2+ are each present in the reaction mixture at a concentration of about 5 mM or higher.
14. The method of claim 11 wherein the one or more 2′-modified nucleotides is 2′-F-dATP, 2′-F-dGTP, 2′-F-dCTP, 2′-F-dUTP, 2′-NH 2 -dATP, 2′-NH 2 -dGTP, 2′-NH 2 -dCTP, 2′-NH 2 -dUTP, 2′-OMe-dATP, 2′-OMe-dGTP, 2′-OMe-dCTP, or 2′-OMe-dUTP.
15. The method of claim 11 wherein the nucleic acid template sequence is a DNA template sequence and the transcript includes fewer than three 2′-modified nucleotides within the first 12 nucleotides of the transcript and wherein the three 2′-modified nucleotides within the first 12 nucleotides are non-consecutive.
16. The method of claim 11 wherein the nucleic acid template sequence includes a sequence which when transcribed by the cyanophage Syn5 RNAP produces a transcript including fewer than three 2′-modified nucleotides within the first 12 nucleotides of the transcript.
17. The method of claim 11 wherein discrimination between the modified nucleotides is reduced.