Pol6 polymerase variants

Patent metadata

Field	Value
Publication number	US-10851353-B2
Application number	US-201916358580-A
Country	US
Kind code	B2
Filing date	Mar 19, 2019
Priority date	Sep 22, 2016
Publication date	Dec 1, 2020
Grant date	Dec 1, 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 having a DNA polymerase activity comprising an amino acid sequence having at least 95% sequence identity SEQ ID NO: 1, wherein the amino acid sequence having at least 95% sequence identity to SEQ ID NO: 1 comprises a V299Y/R/E/A/F/K substitution relative to SEQ ID NO: 2.

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. The isolated polypeptide of claim 1 , comprising a set of substitutions selected from the group consisting of: T529M+S366A+A547F+N545L+Y225L+D657R+S692F+V299Y/R/E/A, T529M+8366A+A547F+N545L+Y225L+D657R+K561G+K541R+T544A+S692Y+V299A/R, T529M+S366A+A547F+N545L+Y225L+D657R+K561G+K541R+T544A+S692Y+D693P+V299F, and T529M+S366A+A547F+N545L+Y225L+D657R+K561G+K541R+T544A+S692Y+I652Q+D684M+V299R.

6. A method for sequencing a nucleic acid sample with the aid of an alpha-hemolysin 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 according to claim 1 , said isolated polypeptide being 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; 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 variant polymerase.

7. A composition comprising the isolated polypeptide of claim 1 covalently linked to an alpha-hemolysin monomer.

8. The composition of claim 7 , wherein the alpha-hemolysin monomer to which the isolated polypeptide is covalently linked is a constituent of a heptamer nanopore.

9. The composition of claim 8 , wherein the heptamer nanopore comprises 6 alpha-hemolysin monomers that are not covalently attached to the isolated polypeptide and one alpha-hemolysin monomer that is attached to the isolated polypeptide.

10. 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: 2 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.

11. The isolated polypeptide of claim 1 , wherein the amino acid sequence of the isolated polypeptide has at least 98% sequence identity to SEQ ID NO: 2 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.

12. The isolated polypeptide of claim 1 , wherein said isolated polypeptide is capable of catalyzing template-directed synthesis of a DNA polynucleotide.

Assignees

• Roche Sequencing Solutions Inc
• Roche Molecular Systems Inc

Inventors

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

Classifications

• C12Q1/6869

  Methods for sequencing · CPC title

• 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

• B82Y5/00

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

• C12Q2565/631

  being a biochannel or pore · CPC title