Long lifetime alpha-hemolysin nanopores

Patent metadata

Field	Value
Publication number	US-11613778-B2
Application number	US-202117157576-A
Country	US
Kind code	B2
Filing date	Jan 25, 2021
Priority date	Jun 30, 2016
Publication date	Mar 28, 2023
Grant date	Mar 28, 2023

Abstract

Described herein are variants of alpha-hemolysin having at least one amino acid substitution at H35G, E111N, M113A, and/or K147N in the mature, wild-type alpha-hemolysin amino acid sequence. In certain examples, the variant may have a substitution at E111S, M113S, T145S, K147S, or L135I in the mature alpha-hemolysin amino acid sequence. The α-hemolysin variants may also include a substitution at H144A and/or a series of glycine residues spanning residues 127 to 131 of the mature, wild-type alpha hemolysin. Also provided are nanopore assemblies including the alpha-hemolysin variants, the assembly having an increased nanopore lifetime. Further, provided are variants that, in addition to providing increased lifetime, provide a decreased time-to-thread. Hence, the variants provided herein both increase nanopore lifetime and improve efficiency and accuracy of DNA sequencing reactions using nanopores comprising the variants.

First claim

What is claimed is:

1. An isolated polypeptide comprising an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 20, wherein the amino acid sequence comprises (a) an asparagine at a position corresponding to position 111 of SEQ ID NO: 20, (b) an alanine at a position corresponding to position 113 of SEQ ID NO: 20, (c) a glycine at positions corresponding to each of positions 35 and 126-131 of SEQ ID NO: 20, (d) an asparagine residue at a position corresponding to position 147 of SEQ ID NO: 20, and (e) an alanine at a position corresponding to position 144 of SEQ ID NO: 20.

2. The isolated peptide of claim 1 , wherein the peptide further comprises a lysine at a position corresponding to position 149 of SEQ ID NO: 20.

3. The isolated peptide of claim 1 , wherein the isolated peptide further comprises an attachment linker.

4. The isolated peptide of claim 3 , wherein the attachment linker comprises an amino acid sequences corresponding to amino acid residues 1-10 of SEQ ID NO: 21.

5. The isolated peptide of claim 3 , wherein the attachment linker is joined to a SpyTag or SpyCatcher amino acid sequence.

6. The isolated peptide of claim 5 , wherein the attachment linker is joined to a SpyTag amino acid sequence, the joined attachment linker and SpyTag sequence comprising an amino acid sequence as set forth as SEQ ID NO: 21.

7. The isolated peptide of claim 5 , wherein the SpyTag or SpyCatcher amino acid sequence is bound to a polymerase.

8. A heptameric nanopore assembly, the assembly comprising seven monomer units, wherein at least six of the monomer units are polypeptides according to claim 1 .

9. The heptameric nanopore assembly of claim 8 , wherein each of the at least six monomer units further comprises a lysine at a position corresponding to position 149 of SEQ ID NO: 20.

10. The heptameric nanopore assembly of claim 8 , wherein at least 1 of the monomer units comprises an amino acid sequence that is bound to a DNA polymerase or that is configured to bind a DNA polymerase.

11. The heptameric nanopore assembly of claim 10 , wherein the at least 1 monomer unit comprises an attachment linker.

12. The heptameric alpha-hemolysin nanopore assembly of claim 11 , wherein the attachment linker comprises an amino acid sequences corresponding to residues 1-10 of SEQ ID NO: 21.

13. The heptameric alpha-hemolysin nanopore assembly of claim 11 , wherein the attachment linker is joined to a SpyTag or SpyCatcher amino acid sequence.

14. The heptameric alpha-hemolysin nanopore assembly of claim 13 , wherein the attachment linker is joined to s SpyTag amino acid sequence, the joined attachment linker and SpyTag sequence comprising an amino acid sequence as set forth as SEQ ID NO: 21.

15. The heptameric alpha-hemolysin nanopore assembly of claim 13 , wherein the SpyTag or SpyCatcher amino acid sequence is bound to a polymerase.

16. The heptameric alpha-hemolysin nanopore assembly of claim 8 , wherein the heptameric alpha-hemolysin nanopore assembly is disposed within a membrane of a sequencing chip.

17. The heptameric alpha-hemolysin nanopore assembly of claim 16 , wherein membrane comprising the heptameric alpha-hemolysin nanopore assembly is disposed in proximity to a sensing electrode.

18. The heptameric alpha-hemolysin nanopore assembly of claim 16 , wherein each of the at least six monomer units further comprises a lysine at a position corresponding to position 149 of SEQ ID NO: 20.

Assignees

• Roche Sequencing Solutions Inc

Inventors

• Ambroso Mark
• Craig Timothy
• Dipietro Matthew
• Harris Corissa
• Porter Marshall

Classifications

• C07K14/00

  Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof · CPC title

• C12N15/01

  Preparation of mutants without inserting foreign genetic material therein; Screening processes therefor · CPC title

• A61K38/02

  Peptides of undefined number of amino acids; Derivatives thereof · CPC title

• C12Q1/6869 Primary

  Methods for sequencing · CPC title

• C07K14/31 Primary

  from Staphylococcus (G) · CPC title