Polymerase variants

Patent metadata

Field	Value
Publication number	US-10590480-B2
Application number	US-201715443964-A
Country	US
Kind code	B2
Filing date	Feb 27, 2017
Priority date	Feb 29, 2016
Publication date	Mar 17, 2020
Grant date	Mar 17, 2020

Abstract

The present of disclosure provides variant Pol6 polymerase polypeptide, 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. A variant-polypeptide capable of catalyzing polymerization of deoxynucleotides said polypeptide comprising an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 2, wherein the amino sequence comprises an E585K/R/H substitution relative to SEQ ID NO: 2.

2. The polypeptide of claim 1 , further comprising a member of an attachment selected from the group (a) a Spycatcher/SpyTag peptide system, (b) a native chemical ligation system, (c) a sortase system, (d) a transglutaminase systems, (e) a formylglycine linkage system, (f) a system comprising reaction between 6-hydrazino-nicotinic acid and 4-formylbenzoate, and (g) a zinc finger system attaching the polypeptide to a biological nanopore.

3. The polypeptide of claim 1 , wherein said amino acid sequence further comprises an M738K/R/H substitution relative to SEQ ID NO: 2.

4. The polypeptide of claim 1 , wherein said amino acid sequence further comprises a D44A, S366A, T529M, and A547F relative to SEQ ID NO: 2.

5. The polypeptide of claim 2 , wherein said amino acid sequence further comprises a D44A, S366A, T529M, and A547F relative to SEQ ID NO: 2.

6. The polypeptide of claim 3 , wherein said amino acid sequence further comprises a D44A, S366A, T529M, and A547F relative to SEQ ID NO: 2.

7. The polypeptide of claim 4 , wherein said polypeptide has a reduced rate of dissociation in the presence of 500 mM potassium glutamate relative to a polypeptide consisting of SEQ ID NO: 4.

8. The polypeptide of claim 5 , wherein said polypeptide has a reduced rate of dissociation in the presence of 500 mM potassium glutamate relative to a polypeptide consisting of SEQ ID NO: 4.

9. The polypeptide of claim 6 , wherein said polypeptide has a reduced rate of dissociation in the presence of 500 mM potassium glutamate relative to a polypeptide consisting of SEQ ID NO: 4.

10. The polypeptide of claim 1 , wherein the amino acid sequence comprises residues 141-869 of SEQ ID NO: 6, residues 141-869of SEQ ID NO: 7, or residues 141-869 of SEQ ID NO: 8.

11. The polypeptide of claim 1 , further comprising an affinity tag.

12. The polypeptide of claim 1 , further comprising means for attaching the polypeptide to a biological nanopore.

13. A composition comprising the polypeptide of claim 1 attached to a biological nanopore.

14. The composition of claim 13 , wherein the attachment between the polypeptide and the biological nanopore results from an attachment system selected from the group consisting of: (a) a SpyCatcher/SpyTag peptide system, (b) a native chemical ligation system, (c) a sortase system, (d) a transglutaminase systems, (e) a formylglycine linkage system, (f) a system comprising reaction between 6-hydrazino-nicotinic acid and 4-formylbenzoate, and (g) a zinc finger system.

15. The composition of claim 13 , further comprising a polynucleotide complexed with the polypeptide.

16. A biochip comprising a plurality of nanopore sequencing complexes, wherein each nanopore sequencing complex comprises:
a nanopore inserted in a membrane adjacent to an electrode of a sensing circuit,
the polypeptide of claim 1 attached to the nanopore, and
a polynucleotide complexed with the polypeptide.

17. The biochip of claim 16 , wherein the nanopore is a biological nanopore.

18. The biochip of claim 16 , wherein the biochip comprises at least 500 nanopore sequencing complexes per 1 mm 2 .

Assignees

• Roche Sequencing Solutions Inc

Inventors

• AYER Aruna
• Sarvabhowman Preethi
• Dalal Dhruti Vasudev

Classifications

• C12Q1/6869 Primary

  Methods for sequencing · CPC title

• C12Y207/07007

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

• C12N9/1252

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

• C12Q2521/101

  DNA polymerase · CPC title

• C12Q2565/631

  being a biochannel or pore · CPC title