Polymerase Variants and Uses Thereof

Patent metadata

Field	Value
Publication number	US-2016333327-A1
Application number	US-201615151364-A
Country	US
Kind code	A1
Filing date	May 10, 2016
Priority date	May 14, 2015
Publication date	Nov 17, 2016
Grant date	—

Abstract

Described herein is a variant pol6 polymerase having at least one mutation selected from H223, N224, Y225, H227, 1295, Y342, T343, I357, S360, L361, I363, S365, S366, Y367, P368, D417, E475, Y476, F478, K518, H527, T529, M531, N535, G539, P542, N545, Q546, A547, L549, I550, N552, G553, F558, A596, G603, A610, V615, Y622, C623, D624, I628, Y629, R632, N635, M641, A643, I644, T647, I648, T651, I652, K655, W656, D657, V658, H660, F662, L690 and combinations thereof.

First claim

What is claimed is:

1 . A modified DNA polymerase having a DNA polymerase activity comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence as set forth in SEQ ID NO: 1 or 2, which amino acid sequence includes one or more amino acid substitutions selected from the group consisting of H223, N224, Y225, H227, I295, Y342, T343, I357, S360, L361, I363, S365, S366, Y367, P368, D417, E475, Y476, F478, K518, H527, T529, M531, N535, G539, P542, N545, Q546, A547, L549, I550, N552, G553, F558, A596, G603, A610, V615, Y622, C623, D624, I628, Y629, R632, N635, M641, A643, I644, T647, I648, T651, I652, K655, W656, D657, V658, H660, F662, L690, and combinations thereof.

2 . The modified DNA polymerase of claim 1 , wherein the substitutions are selected from the group consisting of H223A, N224Y/L/Q/M/R/K, Y225L/T/I/F/A/M, H227P/E/F/Y, I295 W/F/M/E, Y342L/F, T343N/F, I357G/L/Q/H/W/M/A/E/Y/P, S360G/E/Y, L361M/W/V/F, I363V/A/R/M/W, S365Q/W/M/A/G, S366A/L, Y367L/E/M/P/N/F, P368G, D417P, E475D, Y476V, F478L, K518Q, H527 W/R/L/Y/T/M, T529M/F, M531H/Y/A/K/R/W/T/L/V/G, N535L/Y/M/K/I/R/W/Q, G539Y/F, P542E/S/G, N545K/D/S/L/R/Q/W, Q546 W/F, A547M/Y/W/F/V/S, L549Q/Y/H/G/R/K, I550A/W/T/G/F/S, N552L/M/S/T, G553S/T/E/Q/K/R/M, F558P/T, A596S, G603T/A/L, A610T/E, V615A/T, Y622A/M, C623G/S/Y/F, D624F/K, I628Y/V/F/L/M, Y629 W/H/M/K, R632L/C, N635D, M641L/Y, A643L, I644H/M/Y, T647G/A/E/K/S/Y, I648K/R/V/N/T/L, T651Y/F/M, I652Q/G/S/N/F/T/A/L/E/K/M, K655G/F/E/N/Q/M/A, W656E, D657R/P/A/E, V658L, H660A/Y, F662I/L, L690M and combinations thereof.

3 . The modified DNA polymerase of claim 1 , wherein said modified polymerase has an altered characteristic as compared to the parental polymerase.

4 . The modified DNA polymerase of claim 3 , wherein the altered characteristic is selected from fidelity, processivity, elongation rate, stability, solubility and the ability to bind and/or incorporate polyphosphate nucleotides such as a tetraphosphate, pentaphosphate, hexaphosphate, heptaphosphate or octophosphate nucleotide.

5 . The modified DNA polymerase of claim 3 , wherein the altered characteristic is the ability to incorporate a nucleotide polyphosphate having 4, 5, 6, 7 or 8 phosphates into a growing DNA strand.

6 . The modified DNA polymerase of claim 5 , wherein the nucleotide polyphosphate is tagged.

7 . The modified DNA polymerase of claim 1 , wherein said modified polymerase has a substitution corresponding to S366A/L.

8 . The modified DNA polymerase of claim 1 , wherein said modified polymerase has a substitution corresponding to E475D.

9 . The modified DNA polymerase of claim 1 , wherein said modified polymerase has a substitution corresponding to F478L.

10 . The modified DNA polymerase of claim 1 , wherein said modified polymerase has a substitution corresponding to K518Q.

11 . The modified DNA polymerase of claim 1 , wherein said modified polymerase has a substitution corresponding to T529M.

12 . The modified DNA polymerase of claim 1 , wherein said modified polymerase has a substitution corresponding to N535L.

13 . The modified DNA polymerase of claim 1 , wherein said modified polymerase has a substitution corresponding to N545K/L.

14 . The modified DNA polymerase of claim 1 , wherein said modified polymerase has a substitution corresponding to A547F.

15 . The modified DNA polymerase of claim 1 , wherein said modified polymerase has a substitution corresponding to G553S.

16 . The modified DNA polymerase of claim 1 , wherein said modified polymerase has a substitution corresponding to T647G/Y.

17 . The modified DNA polymerase of claim 1 , wherein said modified polymerase has a substitution corresponding to T651Y.

18 . The modified DNA polymerase of claim 1 , wherein said modified polymerase has a substitution corresponding to I652Q/A/L/E/K/M.

19 . The modified DNA polymerase of claim 1 selected from
H223A;
N224Y/L/Q/M/R/K;
Y225L/1/T/F/A/M;
H227P/E/F/Y;
I295F/E/M/W;
Y342L/F;
T343N/F;
I357G/L/Q/H/W/M/A/E/Y/P;
S360G/E/Y;
L361M/W/V/F;
I363V/A/R/M/W;
S365Q/W/M/A/G;
S366A/L;
Y367L/E/M/P/N/F;
P368G;
D417P;
E475D;
Y476V;
F478L;
K518Q;
H527W/R/L/Y/T/M;
T529M/F;
M531H/Y/A/K/R/W/T/L/V/G;
N535L/Y/M/K/I/R/W/Q;
P542E/S/G;
N545D/K/S/L/R/Q/W;
Q546 W/F;
A547F/M/W/Y/V/S;
L549H/Y/Q/G/R/K;
I550A/W;
I550T/G/F/S;
N552L/M/T;
G553S/T/E/Q/K/R/M;
F558P/T;
A596S;
G603T/A/L;
A610T/E;
V615A/T;
Y622A/M;
C623G/S/Y/A/F;
D624F/K;
I628Y/V/F/L/M;
Y629 W/H/M/K;
R632L/C;
N635D;
M641 L/Y;
A643L;
I644H/M/Y;
T647G/A/E/K/S/Y;
I648K/R/V/N/T/L;
T651Y/F/M;
I652Q/G/S/N/F/T/A/L/E/K/M;
K655G/F/E/N/Q/M/A;
W656E;
D657R/P/A/E;
V658L;
H660A/Y;
F662I/L;
L690M;
S366A+N535L;
T651Y+N535L;
Y342L+E475D+F478L;
T343N+D417P+K518Q;
N535L+N545K+T651Y;
I363V+E475D+Y476V;
S366L+G553S+F558P;
S366A+N535L+A547M;
S366A+P542E+N545K;
S366A+P542E+I652Q;
S366A+N535L+T529M;
S366A+N535L+I652Q;
S366A+N535L+N545K;
T651 Y+P542E+N545K;
T651Y+P542E+Q546 W;
T651Y+P542E+S366A;
T651 Y+N535L+N545K;
S366A+N535I+I652Q;
T651Y+S366A+A547F;
T647G+A547F+Y225T;
A547F+A610T+S366A;
A547F+A610T+Y225I;
S366A+T647G+A547F;
T529M+S366A+A547F;
T647E+S366A+A547F;
T529M+T647G+A547F;
N545K+S366A+A547F;
T647G+A547F+T529M;
T529M+A610T+A547F;
M641 Y+T529M+A547F;
T647G+C623G+A547F;
A610T+I295 W+T651Y;
V615A+M531Y+T647G;
S366 L+F478L+A596S+L690M;
H223A+G553S+A643 L+F662I;
N535L+N545K+T651Y+T529M;
N535L+N545K+T651Y+N635D;
N535L+N545K+T651 Y+I652Q;
S366A+N535L+I652Q+T529M;
S366A+S365A+P368G+G603T;
N535L+N545K+T651Y+T647G;
S366A+N535L+I652Q+A547Y;
S366A+N535L+A547M+T647G;
T529M+S366A+A547F+N545K;
T529M+S366A+A547F+N545R;
T529M+S366A+A547F+N552L;
T529M+S366A+A547F+Y629 W;
N535I+N545K+T651Y+T529M;
N535I+N545K+T651 Y+N635D;
N535I+N545K+T651 Y+I652Q;
N535L+N545K+T651Y+T647G+C623G;
N535L+N545K+T651 Y+T647G+I628Y;
S366A+N535L+A547M+T647G+S360G;
N535I+N545K+T651 Y+I652Q+Y225I;
N535L+N545K+T651Y+T647G+K655G;
N535L+N545K+T651Y+T647G+L549Q;
S366A+N535L+I652Q+A547Y+K655G;
T529M+S366A+A547F+N545L+Y629 W;
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;
T529M+S366A+A547F+N545L+Y225L+N552M
T529M+S366A+A547F+N545L+Y225L+D657R+N224R;
T529M+S366A+A547F+N545L+Y225L+D657R+I628M;
T529M+S366A+A547F+N545L+Y225L+D657R+K655A; and
T529M+S366A+A547F+N545L+Y225L+D657R+Y629 W.

20 . The modified DNA polymerase of claim 1 comprising S366A+T529M+N545L+A547F.

21 . The modified DNA polymerase of claim 20 further comprising at least one mutation selected from
a. Y225L/F/A/M;
b. M531A/G;
c. G539Y;
d. N552L/T;
e. Y629 W/K;
f. K655F/Q/M/A; and
g. D657R/E/P/A.

22 . The modified DNA polymerase of claim 1 comprising S365A+S366A+P368G+G603T.

23 . The modified DNA polymerase of claim 1 , wherein said polymerase comprises less than 10 mutations.

Assignees

• Genia Tech Inc

Inventors

• AYER Aruna
• Arnold Cleoma
• Schwab Charles Wayan
• Thai Eileen
• Lederman Ilya
• Mcgaw Colin Alexander
• SHULTZ Tyler O'Brien

Classifications

• C12Y207/07007

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

• C12N9/1252 Primary

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

• C12N9/1241 Primary

  Nucleotidyltransferases (2.7.7) · CPC title

• C12Y207/07

  Nucleotidyltransferases (2.7.7) · CPC title