Modified T7-related RNA polymerases and methods of use thereof
The invention relates to modified T7-related RNA polymerases and methods of use thereof. In some embodiments, the invention relates to modified T7-related RNA polymerases that transcribe RNA with reduced abortive cycling and increased efficiency compared with native T7-related RNA polymerases.
1. A modified T7-related RNA polymerase having an amino acid insertion of up to five amino acids within its C-linker motif compared with a corresponding native form of the modified T7-related RNA polymerase, wherein the amino acid sequence of the corresponding native form of the T7-related RNA polymerase is as set forth in SEQ ID NO: 1, and wherein the amino acid insertion is immediately before the amino acid at position 252 or 259 of the corresponding native form of the T7-related RNA polymerase.
2. The modified T7-related RNA polymerase of claim 1 , wherein the insertion results in a reduced level of abortive product release compared with the corresponding native form of the T7-related RNA polymerase.
3. The modified T7-related RNA polymerase of claim 1 , wherein the insertion results in a reduced energetic barrier to rotation of its N-terminal platform compared with the corresponding native form of the T7-related RNA polymerase.
4. The modified T7-related RNA polymerase of claim 1 , wherein the insertion comprises a glycine, alanine, serine, threonine, cysteine, glutamine, asparagine, glutamate, aspartate, lysine, or arginine.
5. A modified T7-related RNA polymerase having the amino acid sequence of SEQ ID NO: 5 or 6.
6. A method for producing an RNA, the method comprising:
combining the modified T7-related RNA polymerase of claim 1 with a nucleic acid template that encodes the RNA and is operably linked with a promoter recognized by the modified T7-related RNA polymerase, under conditions suitable for RNA transcription, and maintaining the combination for a period of time sufficient to transcribe the RNA, thereby producing the RNA.
7. A method for producing a protein, the method comprising:
(i.) combining the modified T7-related RNA polymerase of claim 1 with a nucleic acid template that encodes an mRNA and is operably linked with a promoter recognized by the modified T7-related RNA polymerase, under conditions suitable for RNA transcription, and maintaining the combination for a period of time sufficient to transcribe the mRNA; and (ii.) subjecting the transcribed mRNA to a translation reaction, thereby producing the protein.
8. The method of claim 6 , wherein compared with the corresponding native form of the T7-RNA polymerase, the modified T7-related RNA polymerase produces the RNA with less release of abortive RNA fragments.
9. A kit comprising a container housing the modified T7-related RNA polymerase of claim 1 .
10. The kit of claim 9 , further comprising container housing a reaction buffer.
11. The kit of claim 10 , further comprising at least one container housing a reagent for preparing a labeled RNA probe or for performing an in vitro translation reaction.
12. The modified T7-related RNA polymerase of claim 1 , wherein the amino acid insertion comprises glycine and/or alanine residues.
13. The modified T7-related RNA polymerase of claim 1 wherein the amino acid insertion is up to four amino acids.
14. The modified T7-related RNA polymerase of claim 1 wherein the amino acid insertion is up to three amino acids.
15. The modified T7-related RNA polymerase of claim 1 wherein the amino acid insertion is up to two amino acids.
16. The modified T7-related RNA polymerase of claim 1 wherein the amino acid insertion is one amino acid.
17. The modified T7-related RNA polymerase of claim 5 having the amino acid sequence of SEQ ID NO: 5.
18. A method for producing an RNA, the method comprising:
combining the modified T7-related RNA polymerase of claim 17 with a nucleic acid template that encodes the RNA and is operably linked with a promoter recognized by the modified T7-related RNA polymerase, under conditions suitable for RNA transcription, and maintaining the combination for a period of time sufficient to transcribe the RNA, thereby producing the RNA.
19. A method for producing a protein, the method comprising:
(i.) combining the modified T7-related RNA polymerase of claim 17 with a nucleic acid template that encodes an mRNA and is operably linked with a promoter recognized by the modified T7-related RNA polymerase, under conditions suitable for RNA transcription, and maintaining the combination for a period of time sufficient to transcribe the mRNA; and (ii.) subjecting the transcribed mRNA to a translation reaction, thereby producing the protein.
20. A kit comprising a container housing the modified T7-related RNA polymerase of claim 17 .
21. The kit of claim 20 , further comprising container housing a reaction buffer.
22. The kit of claim 20 , further comprising at least one container housing a reagent for preparing a labeled RNA probe or for performing an in vitro translation reaction.
23. The modified T7-related RNA polymerase of claim 5 having the amino acid sequence of SEQ ID NO: 6.
24. A method for producing an RNA, the method comprising:
combining the modified T7-related RNA polymerase of claim 23 with a nucleic acid template that encodes the RNA and is operably linked with a promoter recognized by the modified T7-related RNA polymerase, under conditions suitable for RNA transcription, and maintaining the combination for a period of time sufficient to transcribe the RNA, thereby producing the RNA.
25. A method for producing a protein, the method comprising:
(i.) combining the modified T7-related RNA polymerase of claim 23 with a nucleic acid template that encodes an mRNA and is operably linked with a promoter recognized by the modified T7-related RNA polymerase, under conditions suitable for RNA transcription, and maintaining the combination for a period of time sufficient to transcribe the mRNA; and (ii.) subjecting the transcribed mRNA to a translation reaction, thereby producing the protein.
26. A kit comprising a container housing the modified T7-related RNA polymerase of claim 23 .
27. The kit of claim 26 , further comprising container housing a reaction buffer.
28. The kit of claim 27 , further comprising at least one container housing a reagent for preparing a labeled RNA probe or for performing an in vitro translation reaction.
29. The modified T7-related RNA polymerase of claim 5 further comprising an artificial tag, label, or chelating moiety.