RNA polymerase variants

Patent metadata

Field	Value
Publication number	US-11066686-B2
Application number	US-201916657122-A
Country	US
Kind code	B2
Filing date	Oct 18, 2019
Priority date	Aug 18, 2017
Publication date	Jul 20, 2021
Grant date	Jul 20, 2021

Abstract

The present disclosure provides, in some aspects, variant RNA polymerases, the use of which increases transcription efficiency while reducing the number of double-stranded RNA contaminates and run-on transcripts produced during an in vitro transcription reaction.

First claim

What is claimed is:

1. A method of performing an in vitro transcription (IVT) reaction, comprising combining a deoxyribonucleic acid (DNA) with a T7 ribonucleic acid (RNA) polymerase, nucleoside triphosphates and buffer, wherein the T7 RNA polymerase variant comprises an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 1 modified to comprise an amino acid substitution at position 47, and the T7 RNA polymerase variant has RNA polymerase activity.

2. The method of claim 1 , wherein the amino acid substitution is selected from the group consisting of alanine, isoleucine, leucine, methionine, lysine, glutamine, and glutamate.

3. The method of claim 2 , wherein the amino acid substitution is alanine.

4. The method of claim 1 , wherein the T7 RNA polymerase variant comprises an amino acid sequence having at least 95% identity to SEQ ID NO:1.

5. The method of claim 1 , wherein the T7 RNA polymerase comprises an additional C-terminal amino acid.

6. The method of claim 5 , wherein the additional C-terminal amino acid comprises glycine (G).

7. The method of claim 1 , wherein the T7 RNA polymerase variant comprises the amino acid sequence of SEQ ID NO: 3.

8. The method of claim 5 , wherein the T7 RNA polymerase comprises two additional C-terminal amino acids.

9. The method of claim 8 , wherein the two additional C-terminal amino acids comprise the same type of amino acid.

10. The method of claim 8 , wherein the two additional C-terminal amino acids comprise two different types of amino acids.

11. A method of performing an in vitro transcription (IVT) reaction, comprising combining a deoxyribonucleic acid (DNA) with a T7 ribonucleic acid (RNA) polymerase, nucleoside triphosphates and buffer, wherein the T7 RNA polymerase variant comprises an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO: 1 modified to comprise an alanine at position 47, and the T7 RNA polymerase variant has RNA polymerase activity.

12. The method of claim 11 , wherein the T7 RNA polymerase variant further comprises a C-terminal glycine.

13. The method of claim 12 , wherein the T7 RNA polymerase variant comprises the amino acid sequence of SEQ ID NO: 110.

Assignees

• Modernatx Inc

Inventors

• Rabideau Amy E
• Dousis Athanasios
• Ravichandran Kanchana
• Hobert Elissa

Classifications

• C12P19/34 Primary

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

• C12N2310/317

  with an inverted bond, e.g. a cap structure · CPC title

• C12Y207/07006

  DNA-directed RNA polymerase (2.7.7.6) · CPC title

• C12Y207/11023

  [RNA-polymerase-subunit] kinase (2.7.11.23) · CPC title

• C12N2310/3521

  Methyl · CPC title