# US 10,308,918 B2 · Polymerase variants

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- Number: US 10,308,918 B2
- Document ID: US10308918B2
- Status: Granted
- Kind: B2
- Application number: 15/012,317
- Filed: 2016-02-01
- Published: 2016-08-04
- Granted: 2019-06-04
- Earliest claimed: 2015-02-02
- Assignees: Roche Molecular Systems, Inc.; Genia Technologies, Inc.
- Applicants: Genia Technologies, Inc.; Roche Molecular Systems, Inc.
- Inventors: Aruna Ayer; Arkadiusz Bibillo; Cleoma Renetta Arnold; Charles Wayan Schwab; Eileen Thai; Ilya Lederman; Colin Alexander McGaw; Tyler O'Brien Shultz; Barbara Eckert; Shawn Suko; Mara Boenitz-Dulat; Bigna Woersdoerfer; David Daniel Wunderlich
- Web page: https://patentgrep.com/patent/US10308918B2

## CPC

- C12N 9/1252: {DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase}
- C07K 2319/21: containing a His-tag
- C12Y 207/07007: DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00)
- C12Y: ENZYMES

## Abstract

Described herein are variant pol6 polymerase having at least having at least two substitutions, wherein at least one of the substitutions is at position S366 of SEQ ID NO: 2. The disclosed polymerases may be coupled to alpha-hemolysin nanopores, and may be used in nanopore-based sequencing apparatuses.

## Claims

### Claim 1

An isolated polypeptide having a DNA polymerase activity comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 1, wherein the amino acid sequence having at least 95% sequence identity to SEQ ID NO: 1 comprises at least two amino acid substitutions relative to SEQ ID NO: 2, wherein one of said at least two amino acid substitutions corresponds to S366 of SEQ ID NO: 2 and wherein said amino acid corresponding to position S366 is substituted with A or L.

### Claim 2 (depends on 1)

The isolated polypeptide of claim 1, wherein said at least two amino acid substitutions comprises a set of amino acid substitutions selected from the group consisting of:
S366A+N535L;
S366L+G553S+F558P;
S366A+N535L+A547M;
S366A+P542E+N545K;
S366A+P542E+I652Q;
S366A+N535L+T529M;
S366A+N535L+I652Q;
S366A+N535L+N545K;
S366A+N535I+I652Q;
T651Y+S366A+A547F;
A547F+A610T+S366A;
S366A+T647G+A547F;
T529M+S366A+A547F;
T647E+S366A+A547F;
N545K+S366A+A547F;
S366L+F478L+A596S+L690M;
S366A+N535L+I652Q+T529M;
S366A+S365A+P368G+G603T;
S366A+N535L+I652Q+A547Y;
S366A+N535L+A547M+T647G;
T529M+S366A+A547F+N545K;
T529M+S366A+A547F+N545R;
T529M+S366A+A547F+N552L;
T529M+S366A+A547F+Y629W;
S366A+N535L+A547M+T647G+S360G;
S366A+N535L+I652Q+A547Y+K655G;
T529M+S366A+A547F+N545L+Y629W;
T529M+S366A+A547F+N545L+Y225L;
T529M+S366A+A547F+N545L+Y225F;
T529M+S366A+A547F+N545L+K655F;
T529M+S366A+A547F+N545L+N552L;
T529M+S366A+A547F+N545R+M531A;
T529M+S366A+A547F+N545R+G539Y;
T529M+S366A+A547F+N545R+V658L;
T529M+S366A+A547F+N545L+Y225L+D657R;
T529M+S366A+A547F+N545L+Y225L+N552L;
T529M+S366A+A547F+N545L+Y225L+I652G;
T529M+S366A+A547F+N545L+Y225L+I652Q; and
T529M+S366A+A547F+N545L+Y225L+N552M.

### Claim 3 (depends on 1)

The isolated polypeptide of claim 1 comprising S366A+T529M+N545L+A547F.

### Claim 4 (depends on 3)

The isolated polypeptide of claim 3 further comprising at least one substitution selected from the group consisting of:
a. Y225L/F/A;
b. M531A;
c. G539Y;
d. N552L;
e. Y629W; and
f. K655F.

### Claim 5 (depends on 1)

The isolated polypeptide of claim 1 comprising S365A+S366A+P368G+G603T.

### Claim 6 (depends on 1)

The isolated polypeptide of claim 1, comprising T529M+S366A+A547F.

### Claim 7 (depends on 1)

The isolated polypeptide of claim 1, comprising T529M+S366A+A547F+N545L+Y225L.

### Claim 8 (depends on 1)

The isolated polypeptide of claim 1, comprising T529M+S366A+A547F+N545L+Y225L+D657R.

### Claim 9 (depends on 1)

The isolated polypeptide of claim 1, wherein said at least two amino acid substitutions further comprises one or more substitutions selected from the group consisting of: F478L, T529M, N535L, N545K/L, A547F, G553S, T647G, and I652Q.