T7 RNA polymerase variants

Patent metadata

Field	Value
Publication number	US-12129494-B2
Application number	US-202117558454-A
Country	US
Kind code	B2
Filing date	Dec 21, 2021
Priority date	Jun 30, 2017
Publication date	Oct 29, 2024
Grant date	Oct 29, 2024

Abstract

The present invention provides engineered RNA polymerase variants and compositions comprising these variants. The present invention further provides engineered T7 RNA polymerase variants and compositions comprising these variants. These variants have been evolved for selective incorporation of the m7G(5′)ppp(5′)m7G cap analog over GTP at the initiation of in vitro transcription. The present invention also provides methods for selective capping of RNA transcripts.

First claim

What is claimed is:

1. An engineered RNA polymerase having a polypeptide sequence comprising at least 95% sequence identity to SEQ ID NO: 4 or 15, wherein the polypeptide sequence comprises at least a substitution at position 514, wherein the position is relative to SEQ ID NO: 4 or 15.

2. The engineered RNA polymerase of claim 1 , wherein the substitution at position 514 is 514I, 514F, 514Y, or 514L.

3. The engineered RNA polymerase of claim 1 , wherein the polypeptide sequence comprises at least a substitution set at positions 167/514, 136/357/404/514, 136/357/514, 136/404/514, 136/514, 357/394/401/404/514, 357/394/446/514, 357/514, 394/446/514, or 401/404/514.

4. The engineered RNA polymerase of claim 1 , wherein the polypeptide sequence comprises at least a substitution set 167N/514L, 136E/357I/404Y/514I, 136I/357I/514F, 136I/357K/514F, 136E/404Y/514F, 136E/514F, 136I/514I, 357N/394R/446W/514I, 357R/394R/401V/404Y/514L, 357R/514F, 394R/446W/514I, or 401V/404Y/514L.

5. The engineered RNA polymerase of claim 1 , wherein the polypeptide sequence comprises at least 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 4 or 15.

6. The engineered RNA polymerase of claim 1 , wherein the engineered polymerase exhibits at least one improved property compared to wild-type T7 RNA polymerase.

7. The engineered RNA polymerase of claim 1 , wherein the engineered RNA polymerase exhibits improved selectivity for a cap analog relative to GTP during transcription initiation.

8. The engineered RNA polymerase of claim 7 , wherein the engineered RNA polymerase exhibits at least one additional improved property selected from improved protein expression, improved stability in storage buffer, and improved stability under reaction conditions.

9. The engineered RNA polymerase of claim 1 , wherein the RNA polymerase does not utilize a cap or cap analog during transcription elongation.

10. The engineered RNA polymerase of claim 1 , wherein the RNA polymerase generates greater than about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more capped RNA transcripts relative to uncapped transcripts.

11. The engineered RNA polymerase of claim 10 , wherein the RNA polymerase generates greater than 90% capped RNA transcripts relative to uncapped transcripts.

12. The engineered RNA polymerase of claim 1 , wherein the engineered polymerase is purified.

13. A composition comprising an engineered RNA polymerase of claim 1 .

14. A method for producing capped RNA transcripts, comprising providing a composition comprising: i) at least one engineered RNA polymerase of claim 1 , a dinucleotide cap analog, and ii) a DNA template; exposing the DNA template to the composition under conditions such that the engineered RNA polymerase produces a capped RNA transcript.

15. The method of claim 14 , where the dinucleotide cap analog is alpha, gamma-bis(N7-methylguanosine) triphosphate (m7G(5′)ppp(5′)m7G) or an anti-reverse cap analog 3′-O-Mem7G(5′)ppp(5′)G.

16. The method of claim 14 , wherein the dinucleotide cap analog is alpha, gamma bis(N7-methylguanosine) triphosphate.

17. The method of claim 14 , wherein the method comprises an in vitro transcription reaction.

Assignees

• Codexis Inc

Inventors

• Miller Mathew G
• Chng Chinping
• Alvizo Oscar
• Mayo Melissa Ann
• Riggins James Nicholas
• Yi Xiang
• Penfield Jonathan S
• Huisman Gjalt W
• Davis Jared
• Saotome Yasushi

Classifications

• C12P19/34

  Polynucleotides, e.g. nucleic acids, oligoribonucleotides · CPC title

• C12N9/1252 Primary

  DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase · CPC title

• C12Y207/07006

  DNA-directed RNA polymerase (2.7.7.6) · CPC title

• C12N15/00

  Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor (mutants or genetically engineered microorganisms, per se C12N1/00, C12N5/00, C12N7/00; new plants per se A01H; plant reproduction by tissue culture techniques A01H4/00; new animals per se A01K67/00; use of medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases, gene therapy A61K48/00) · CPC title

• C12N9/1247 Primary

  DNA-directed RNA polymerase (2.7.7.6) · CPC title