Polymerase variants

Patent metadata

Field	Value
Publication number	US-10829747-B2
Application number	US-201715710674-A
Country	US
Kind code	B2
Filing date	Sep 20, 2017
Priority date	Sep 22, 2016
Publication date	Nov 10, 2020
Grant date	Nov 10, 2020

Abstract

The present disclosure provides variant Pol6 polymerase polypeptides, compositions comprising the Pol6 variant polypeptides, and methods for using the variant Pol6 polypeptides for determining the sequencing of nucleic acids, for example, by nanopore sequencing. The variant Pol6 polymerases possess decreased rates of dissociation of template from the polymerase-template complex, which result in increased processivity relative to the parental Pol6 polypeptides from which they are derived.

First claim

What is claimed is:

1. An isolated polypeptide comprising an amino acid sequence having at least 90% sequence identity to a reference amino acid sequence consisting of SEQ ID NO: 1, wherein the amino acid sequence of the isolated polypeptide comprises one or more amino acid substitutions relative to SEQ ID NO: 2, wherein one of said amino acid substitutions relative to SEQ ID NO: 2 is a K561A/Q/M/G/Y substitution, and wherein said isolated polypeptide is capable of catalyzing template-directed synthesis of a DNA polynucleotide.

2. The isolated polypeptide of claim 1 , wherein said isolated polypeptide further has a substitution corresponding to Y242A.

3. The isolated polypeptide of claim 1 , wherein said isolated polypeptide further has a substitution corresponding to E585K.

4. The isolated polypeptide of claim 1 , wherein said isolated polypeptide further has substitutions corresponding to Y242A and E585K.

5. A method for sequencing a nucleic acid sample with the aid of a nanopore in a membrane adjacent to a sensing electrode, the method comprising:
a. providing tagged nucleotides into a reaction chamber comprising said nanopore, wherein an individual tagged nucleotide of said tagged nucleotides contains a tag coupled to a nucleotide, which tag is detectable with the aid of said nanopore;
b. carrying out a polymerization reaction with the aid of a single isolated polypeptide coupled to the nanopore, thereby incorporating an individual tagged nucleotide of said tagged nucleotides into a growing strand complementary to a single stranded nucleic acid molecule from said nucleic acid sample, wherein said isolated polypeptide is the isolated polypeptide according to claim 1 ; and
c. detecting, with the aid of said nanopore, a tag associated with said individual tagged nucleotide during incorporation of said individual tagged nucleotide, wherein said tag is detected with the aid of said nanopore while said nucleotide is associated with said isolated polypeptide.

6. The method of claim 5 , wherein the nanopore is alpha-hemolysin.

7. A polypeptide comprising an alpha-hemolysin monomer, wherein said alpha-hemolysin monomer is attached to the isolated polypeptide of claim 1 .

8. A nanopore heptamer comprising seven alpha-hemolysin monomers, wherein one of the seven alpha-hemolysin monomers is attached to the isolated polypeptide of claim 1 .

9. The isolated polypeptide of claim 1 , wherein the amino acid sequence of the isolated polypeptide has at least 95% sequence identity to the reference amino acid sequence when aligned using CLUSTAL-W program in MacVector version 13.0.7, operated with default parameters, including an open gap penalty of 10.0, an extended gap penalty of 0.1, and a BLOSUM 30 similarity matrix.

10. The isolated polypeptide of claim 1 , wherein the amino acid sequence of the isolated polypeptide has at least 98% sequence identity to the reference amino acid sequence when aligned using a CLUSTAL-W program in MacVector version 13.0.7, operated with default parameters, including an open gap penalty of 10.0, an extended gap penalty of 0.1, and a BLOSUM 30 similarity matrix.

11. The isolated polypeptide of claim 1 , wherein the amino acid sequence of the isolated polypeptide comprises a set of substitutions relative to SEQ ID NO: 2 selected from the group consisting of:
(a) T529M+S366A+A547F+N545L+Y225L+D657R+K561G;
(b) T529M+S366A+A547F+N545L+Y225L+D657R+K561G+K541R+T544A+S692Y;
(c) T529M+S366A+A547F+N545L+Y225L+D657R+K561G+K541R+T544A+S692Y+I652Q;
(d) T529M+S366A+A547F+N545L+Y225L+D657R+K561G+K541R+T544A+S692Y+D693P;
(e) T529M+S366A+A547F+N545L+Y225L+D657R+K561G+K541R+T544A+S692Y+D693P+V299F; and
(f) T529M+S366A+A547F+N545L+Y225L+D657R+K561G+K541R+T544A+S692Y+I652Q+D684M.

12. The isolated polypeptide of claim 1 , wherein the amino acid sequence of the isolated polypeptide comprises a set of substitutions relative to SEQ ID NO: 2 selected from the group consisting of:
(A) T529M+S366A+A547F+N545L+Y225L+D657R+K561G+K541R+T544A+S692Y, and further comprising one or more of:
a. N224T;
b. Y242A;
c. S293Q;
d. S294R/T;
e. I295A;
f. V299A/R;
g. L361M;
h. S369F;
i. Y373A/T;
j. F528Y;
k. K530G/E/F;
l. L549T/R;
m. G553A/K/T;
n. A554Q;
o. G556R;
p. K557R;
q. F558M;
r. T560F/Y;
s. E585K;
t. Y629 W;
u. C630A;
v. I638A/E;
w. N642E/K/F/Y/W;
x. T647G/L/Q/M;
y. K650Q;
z. T651M;
aa. I652G/A/E/Q/M;
bb. L653M;
cc. K655G/W;
dd. V658Y;
ee. D693R/M/P;
ff. A694L;

(B) T529M+S366A+A547F+N545L+Y225L+D657R+K561G+K541R+T544A+S692Y+I652Q, and further comprising one or more of:
a. Q221L;
b. F222L;
c. Y242A;
d. Y338L;
e. L361F;
f. Q370A;
g. K537E;
h. L543K/R;
i. G553A/K/Y/R/T/Q;
j. A554T;
k. E585K;
l. I628M;
m. Y629K;
n. N631T;
o. R632K;
p. N642Q/F/Y;
q. A643L;
r. D684G/F/W/M/Q;
s. D693A/K/M/Y;

(C) T529M+S366A+A547F+N545L+Y225L+D657R+K561G+K541R+T544A+S692Y+D693P, and further comprising one or more of:
a. N224K;
b. H227G/A/Q/T;
c. Y242A;
d. S293A/Q/T;
e. V299F;
f. C359T;
g. L361M;
h. F406Y;
i. F521M;
j. P523L/E/Q/K/R/T/M/A;
k. H527F/L/M/R;
l. K530G/E;
m. N535Y;
n. G553A;
o. Y555A;
p. T560Y;
q. E585K;
r. G603A;
s. C623G;
t. Y629K;
u. N642M;
v. T647K/F/Y;
w. I652G/L/Q/K/R/W/M;
x. K655A;
y. D684F/Y/W/M;
z. K686R;

(D) T529M+S366A+A547F+N545L+Y225L+D657R+K561G+K541R+T544A+S692Y+D693P+V299F, and further comprising one or more of:
a. Q221K/M;
b. H223L;
c. S294E/T;
d. F297L/M;
e. S293G;
f. N298G;
g. Y338L;
h. C359T;
i. S360A;
j. L361F;
Y367F;
l. S369A;
m. E396F;
k. 522Q;
o. D526E;
p. K530R;
q. M531A;
r. L538A;
s. L543T;
t. G553K/R/M;
u. K557R/E/Q;
v. T560L;
w. L621 W;
x. C623T;
y. C630Q;
z. N631A;
aa. I638K/F/E;
bb. D640E/Q/T;
cc. V658T/M;
dd. E680A/Q/K/F/Y/W/M;
ee. T683F/W;
ff. D684E/T/K;
gg. L685A;
hh. K686M;
ii. C687A;
jj. A694M/T/Y;
kk. R695K;
ll. 1697A; and

(E) T529M+S366A+A547F+N545L+Y225L+D657R+K561G+K541R+T544A+S692Y+I652Q+D684M, and further comprising one or more of:
a. Q221R;
b. N224K;
c. Y242K;
d. S293Q/G;
e. N298L/K/W;
f. V299R;
g. S360A/F;
h. L361F/M/A;
i. K522R;
j. H527M;
k. K530F;
l. K537E;
m. L543K;
n. G553Y/R/T/M;
o. A554Q/T;
p. E585K;
q. G603A;
r. Y629 W/K;
s. N631 W;
t. I638F;
u. N642M;
v. K650A;
w. K655F;
x. K672T;
y. K686T;
z. D693R/W;
aa. A694F.

13. The isolated polypeptide of claim 1 , wherein the amino acid sequence of the isolated polypeptide comprises T529M+S366A+A547F+N545L+Y225L+D657R+K561G+K541R+T544A+S692Y+Y242A+E585K substitutions relative to SEQ ID NO: 2.

14. The isolated polypeptide of claim 1 , wherein the amino acid sequence of the isolated polypeptide has at least 95% sequence identity to SEQ ID NO: 1.

Assignees

• Roche Sequencing Solutions Inc
• Roche Molecular Systems Inc

Inventors

• AYER Aruna
• Arnold Cleoma
• Schwab Charles
• Sarvabhowman Preethi
• Thai Eileen
• Lederman Ilya
• Mcgaw Colin
• Amato Evan
• ECKERT Barbara
• Suko Shawn
• Boenitz-Dulat Mara
• Woersdoerfer Bigna
• Wunderlich David

Classifications

• B82Y5/00

  Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery · CPC title

• C12Y207/07007

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

• C12Q1/6869

  Methods for sequencing · CPC title

• C12N9/1252 Primary

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

• C12Q2565/631

  being a biochannel or pore · CPC title