T7 RNA polymerase variants

Patent metadata

Field	Value
Publication number	US-10793841-B2
Application number	US-201816012478-A
Country	US
Kind code	B2
Filing date	Jun 19, 2018
Priority date	Jun 30, 2017
Publication date	Oct 6, 2020
Grant date	Oct 6, 2020

Abstract

The present invention provides engineered T7 RNA polymerase variants and compositions thereof. These variants have been evolved for selective incorporation of the symmetrical capped GTP analog over GTP at the initiation of in vitro transcription. The present invention also provides methods for using the variants provided herein. The present invention further provides for the use of the compositions provided herein.

First claim

What is claimed is:

1. An engineered RNA polymerase comprising a polypeptide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence of SEQ ID NO:4, wherein said engineered RNA polymerase comprises a substitution at position 404 in said polypeptide sequence, and wherein the amino acid positions of said polypeptide sequence are numbered with reference to SEQ ID NO:4.

2. The engineered RNA polymerase of claim 1 , wherein said polypeptide further comprises a substitution set selected from 397/513/635, 397/513/635/660, 513/660/664, 513/635/660, 513/635/664, 513/660/664, and 660/664, and/or any combinations thereof, wherein the amino acid positions are numbered with reference to SEQ ID NO:4.

3. The engineered RNA polymerase of claim 1 , wherein said polypeptide further comprises at least one substitution or substitution set selected from 397, 397/513, 397/513/635, 397/513/635, 397/513/635/656, 397/513/635/656/660, 397/513/635/656/660/664, 397/513/635/656/664, 397/513/635/660, 397/513/635/660/664, 397/513/635/664, 397/513/656/660, 397/513/660, 397/513/660/664, 397/513/664, 397/513, 397/635, 397/635/656/660/664, 397/635/656/664, 397/635/660, 397/635/664, 397/635/664/850, 397/660, 397/664, and/or any combinations thereof, wherein the amino acid positions are numbered with reference to SEQ ID NO:4.

4. The engineered RNA polymerase of claim 1 , wherein at least one substitution or substitution set is selected from 113/137/513, 136/357/514, 136/357/514, 136/394/446, 136/401, 136/401, 136/446, 136/514, 136/446, 136/514, 137, 137/401, 137/401/513, 137/401/513, 137/513, 137/513/621, 137/635, 137/656, 357/394/401/514, 357/394/446/514, 357/514, 394/446/514, 401, 401/514, 401/513/635, 401/635, 513/635, 513/635/656, 513/660, 635/656, 635/660, and 660, and/or any combinations thereof, wherein the amino acid positions are numbered with reference to SEQ ID NO:15.

5. The engineered RNA polymerase of claim 1 , wherein said engineered RNA polymerase comprises a polypeptide sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the sequence of at least one engineered RNA polymerase variant set forth in SEQ ID NO: 35, or 37.

6. The engineered RNA polymerase of claim 1 , wherein said engineered RNA polymerase comprises a variant engineered polymerase set forth in SEQ ID NO: 29, 35, or 37.

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

8. The engineered RNA polymerase of claim 7 , wherein said at least one improved property is selected from improved selectivity for cap analog relative to GTP during transcription initiation, improved protein expression, improved stability in storage buffer, and improved stability under reaction conditions.

9. The engineered RNA polymerase of claim 1 , wherein the polymerase maintains RNA yield, transcription fidelity, thermostability, protein expression, stability at −20° C., or stability in reaction conditions equivalent to the wild-type T7 RNA polymerase.

10. The engineered RNA polymerase of claim 1 , wherein said engineered polymerase is purified.

11. A polynucleotide sequence encoding at least one engineered RNA polymerase of claim 1 .

12. A polynucleotide sequence encoding at least one engineered RNA polymerase comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence of SEQ ID NO: 4, wherein said engineered RNA polymerase comprises a substitution at position 404.

13. A polynucleotide sequence encoding at least one engineered RNA polymerase comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to SEQ ID NO: 29, 35, 37.

14. A polynucleotide sequence encoding at least one engineered RNA polymerase, wherein said polynucleotide sequences comprises at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity SEQ ID NO: 28, 34, or 36.

15. The polynucleotide sequence of claim 11 , wherein said polynucleotide sequence is operably linked to a control sequence.

16. The polynucleotide sequence of claim 11 , wherein said polynucleotide sequence is codon optimized.

17. An expression vector comprising at least one polynucleotide sequence of claim 11 .

18. An isolated host cell comprising at least one expression vector of claim 17 .

19. A method of producing an engineered RNA polymerase in a host cell, comprising culturing the host cell of claim 18 , under suitable cultures conditions, such that at least one engineered RNA polymerase is produced.

20. The method of claim 19 , further comprising recovering at least one engineered RNA polymerase from the culture and/or host cell.

21. The method of claim 19 , further comprising the step of purifying said at least one engineered RNA polymerase.

22. A composition comprising at least one engineered RNA polymerase of claim 1 .

23. A method for producing capped RNA transcripts in vitro, 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 said DNA template to said composition under conditions such that said engineered RNA polymerase produces a capped RNA transcript.

24. The method of claim 23 , 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-Me-m 7 G(5′)ppp(5′)G.

25. The method of claim 23 , wherein the dinucleotide cap analog is alpha, gamma-bis(N7-methylguanosine) triphosphate.

26. The method of claim 23 , further comprising the addition of inorganic pyrophosphatase.

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

Classifications

• C12Y207/07006

  DNA-directed RNA polymerase (2.7.7.6) · CPC title

• C12P19/34

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

• C12R2001/19

  Escherichia coli · CPC title

• C12N15/70

  Vectors or expression systems specially adapted for E. coli · CPC title

• C12N9/1247 Primary

  DNA-directed RNA polymerase (2.7.7.6) · CPC title