OmpG variants

Patent metadata

Field	Value
Publication number	US-10752658-B2
Application number	US-201615762092-A
Country	US
Kind code	B2
Filing date	Sep 20, 2016
Priority date	Sep 22, 2015
Publication date	Aug 25, 2020
Grant date	Aug 25, 2020

Abstract

The present disclosure provides variant OmpG polypeptides, compositions comprising the OmpG variant polypeptides, and methods for using the variant OmpG polypeptides as nanopores for determining the sequence of single stranded nucleic acids. The variant OmpG nanopores reduce the ionic current noise versus the parental OmpG polypeptide from which they are derived and thereby enable sequencing of polynucleotides with single nucleotide resolution. The reduced ionic current noise also provides for the use of these OmpG nanopore variants in other single molecule sensing applications, e.g., protein sequencing.

First claim

The invention claimed is:

1. An isolated OmpG variant comprising an amino acid sequence having at least 95% sequence identity to residues 1-268 of the amino acid sequence set forth as SEQ ID NO: 5, the variant comprising a deletion of one or more loop-6 amino acids, a deletion of amino acid D215 of SEQ ID NO: 5, and an A217 amino acid residue of SEQ ID NO: 5, and wherein the variant retains the ability to form a nanopore.

2. The isolated OmpG variant of claim 1 , further comprising a mutation of one or more of amino acids R211, E15, R68, Y50, E152, E174, E17, D215, K114, E174, F66, or E31 of SEQ ID NO: 5.

3. The isolated OmpG variant of claim 2 , wherein the mutation comprises one or more of a R211N, R68N, Y50K, Y50N, or E17K mutation of SEQ ID NO: 5.

4. The isolated OmpG variant of claim 3 , wherein the mutation of SEQ ID NO: 5 is a Y50K amino acid substitution.

5. The isolated OmpG variant of claim 1 , wherein the OmpG variant is operably linked to a SpyTag construct.

6. The isolated OmpG variant of claim 5 , wherein the OmpG variant and the operably linked SpyTag construct comprise an amino acid sequence having at least 95% sequence identity to the amino acid sequence set forth as SEQ ID NO:10 or SEQ ID NO:11.

7. The isolated OmpG variant of claim 5 , further comprising a polymerase, wherein the polymerase is operably linked to the OmpG variant via the SpyTag construct.

8. The isolated OmpG variant of claim 1 , wherein the variant comprises a deletion corresponding to each of amino acids 216-227 of SEQ ID NO:2.

9. An isolated OmpG variant comprising an amino acid sequence having at least 95% sequence identity to residues 1-268 of the amino acid sequence set forth as SEQ ID NO:5, the variant comprising one or more loop-6 amino acid deletions, a Y50K amino acid substitution of SEQ ID NO: 5, and an A217 amino acid residue of SEQ ID NO: 5, and wherein the variant retains the ability to form a nanopore.

10. The isolated OmpG variant of claim 9 , further comprising a mutation of one or more of amino acids R211, E15, R68, E152, E174, E17, D215, K114, E174, F66, or E31 of SEQ ID NO:2.

11. The isolated OmpG variant of claim 10 , wherein the mutation is a D215 deletion of SEQ ID NO: 5.

12. The isolated OmpG variant of claim 10 , wherein the mutation comprises one or more of a R211N or R68N mutations of SEQ ID NO: 5.

13. The isolated OmpG variant of claim 9 , wherein the variant is operably linked to a SpyTag construct.

14. The isolated OmpG variant of claim 13 , wherein a polymerase is operably linked to the OmpG variant via the SpyTag construct.

15. The isolated OmpG variant of claim 13 , wherein the OmpG variant and the operably linked SpyTag construct comprise an amino acid sequence having at least 95% sequence identity to the amino acid sequence set forth as SEQ ID NO:6.

16. The isolated OmpG variant of claim 9 , wherein the variant comprises a deletion of each amino acid corresponding to amino acid residues 216-227 of SEQ ID NO:2.

17. The isolated OmpG variant of claim 1 , wherein the variant comprises an amino acid sequence having at least 98% sequence identity to residues 1-268 of the amino acid sequence set forth as SEQ ID NO: 5.

18. The isolated OmpG variant of claim 9 , wherein the variant comprises an amino acid sequence having at least 98% sequence identity to residues 1-268 of the amino acid sequence set forth as SEQ ID NO: 6.

Assignees

• Roche Sequencing Solutions Inc

Inventors

• Cech Cynthia
• Craig Tim
• Tzitzilonis Christos
• Yang Alexander
• Jensen Liv
• Yang Charlotte
• Harris Corissa
• Dipietro Matthew
• Dalal Dhruti

Classifications

• C12Q1/6874

  involving nucleic acid arrays, e.g. sequencing by hybridisation · CPC title

• C12N9/1252

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

• C12Q1/68

  involving nucleic acids · CPC title

• C12Y207/07007

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

• G01N33/6803

  General methods of protein analysis not limited to specific proteins or families of proteins · CPC title