Long lifetime alpha-hemolysin nanapores

Patent metadata

Field	Value
Publication number	US-10934582-B2
Application number	US-201916519251-A
Country	US
Kind code	B2
Filing date	Jul 23, 2019
Priority date	Jun 30, 2016
Publication date	Mar 2, 2021
Grant date	Mar 2, 2021

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 L1351 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. A heptameric alpha-hemolysin nanopore assembly, the assembly comprising:
a plurality of alpha-hemolysin variant monomers, wherein each of the plurality of variant monomers comprises an amino acid sequence having at least 85% or more sequence identity to the amino acid sequence set forth as SEQ ID NO: 14 and an amino acid substitution corresponding to V149K of SEQ ID NO: 14; and,
an alpha-hemolysin monomer that is configured to bind a DNA polymerase to the alpha-hemolysin monomer.

2. The heptameric alpha-hemolysin nanopore assembly of claim 1 , wherein the plurality of alpha-hemolysin variant monomers comprises six alpha-hemolysin variant monomers.

3. The heptameric alpha-hemolysin nanopore assembly of claim 2 , wherein one or more of the alpha-hemolysin variants further comprise an amino acid substitution corresponding to H35G, E111N, M113A, 126-131G, K147N, or H144A of SEQ ID NO: 14.

4. The heptameric alpha-hemolysin nanopore assembly of claim 2 , wherein the alpha-hemolysin monomer that is configured to bind the DNA polymerase comprises an amino acid sequence having at least 85% sequence identity to the amino acid sequence set forth as SEQ ID NO: 14 and an amino acid substitution corresponding to V149K of SEQ ID NO: 14.

5. The heptameric alpha-hemolysin nanopore assembly of claim 2 , wherein the alpha-hemolysin monomer that is configured to bind the DNA polymerase comprises an amino acid sequence having at least 85% sequence identity to the amino acid sequence set forth as SEQ ID NO: 14 and wherein the amino at position 149 is an amino acid other than a lysine residue.

6. The heptameric alpha-hemolysin nanopore assembly of claim 4 , wherein the alpha-hemolysin monomer that is configured to bind the DNA polymerase comprises an amino acid sequence having at least 85% sequence identity to the amino acid sequence set forth as SEQ ID NO: 14 and wherein the monomer further comprises an amino acid substitution corresponding to E111N, M113A, 126-131G, or K147N of SEQ ID NO: 14.

7. The heptameric alpha-hemolysin nanopore assembly of claim 1 , wherein the plurality of alpha-hemolysin variant monomers comprises six alpha-hemolysin variant monomers, each variant monomer comprising an amino acid substitution corresponding to H35G, E111N, M113A, 126-131G, K147N, and H144A of SEQ ID NO: 14, and
wherein the alpha-hemolysin monomer that is configured to bind the DNA polymerase comprises an amino acid sequence having at least 85% sequence identity to the amino acid sequence set forth as SEQ ID NO: 14 and an amino acid substitution corresponding to E111N, M113A, 126-131G, and K147N of SEQ ID NO: 14.

8. The heptameric alpha-hemolysin nanopore assembly of claim 1 , wherein the alpha-hemolysin monomer that is configured to bind the DNA polymerase comprises an attachment linker.

9. The heptameric alpha-hemolysin nanopore assembly of claim 8 , wherein the attachment linker comprises a SpyTag or SpyCatcher amino acid sequence.

10. A method for sequencing a target nucleic acid sequence, comprising:
providing a chip, the chip comprising a plurality of sensing electrodes and a membrane that is disposed adjacent or in proximity to the sensing electrodes;
disposing, within the membrane, a heptameric nanopore assembly, the heptameric nanopore assembly comprising (i) an alpha-hemolysin DNA polymerase binding monomer and (ii) a plurality of alpha-hemolysin variant monomers, wherein each variant monomer comprises an amino acid sequence that is least 85% identical to the amino acid sequence set forth as SEQ ID NO: 14 and an amino acid substitution corresponding to V149K of SEQ ID NO: 14;
contacting the chip with a target nucleic acid sequence and a plurality of negatively charged tagged nucleotides;
applying a voltage across the membrane;
determining, by one or more of the sensing electrodes, one or more current changes associated with the heptameric nanopore assembly; and
determining, with the aid of a computer processor and based on the one or more of the determined current changes associated with the heptameric nanopore assembly, a sequence for the target nucleic acid sequence.

11. The method of claim 10 , wherein the plurality of alpha-hemolysin variant monomers comprises six alpha-hemolysin variant monomers.

12. The method of claim 11 , wherein one or more of the alpha-hemolysin variants further comprise an amino acid substitution corresponding to H35G, E111N, M113A, 126-131G, or H144A of SEQ ID NO: 14.

13. The method of claim 10 , wherein the alpha-hemolysin DNA polymerase binding monomer comprises an amino acid sequence at least 85% identical to the amino acid sequence set forth as SEQ ID NO: 14 and an amino acid substitution corresponding to an E111N, M113A, 126-131G, or K147N of SEQ ID NO: 14.

14. The method of claim 10 , wherein the plurality of alpha-hemolysin variant monomers comprises six alpha-hemolysin variant monomers, each variant monomer comprising an amino acid substitution corresponding to H35G, E111N, M113A, 126-131G, and H144A of SEQ ID NO: 14, and
wherein the alpha-hemolysin DNA polymerase binding monomer comprises an amino acid sequence that is at least 85% identical to the amino acid sequence set forth as SEQ ID NO: 14 and an amino acid substitution corresponding to E111N, M113A, 126-131G, and K147N substitution of SEQ ID NO: 14.

15. The method of claim 14 , wherein the chip comprises a well and wherein the nanopore assembly is disposed within the membrane over the well.

16. The method of claim 10 , wherein the heptameric nanopore assembly has an increased lifetime relative to a nanopore consisting of native alpha-hemolysin.

17. A system for sequencing a nucleic acid comprising:
a plurality of heptameric nanopore assemblies, wherein the plurality of heptameric nanopore assemblies comprise six alpha-hemolysin variant monomers and one DNA polymerase binding monomer, wherein each of the variant monomers comprises an amino acid sequence having at least 85% or more sequence identity to the amino acid sequence set forth as SEQ ID NO: 14 and an amino acid substitution corresponding to V149K of SEQ ID NO: 14;
a chip comprising a plurality of sensing electrodes and a membrane that is disposed adjacent or in proximity to one or more of the sensing electrodes, wherein the plurality of heptameric nanopore assemblies are disposed within the membrane and wherein each of the sensing electrodes is configured to detect an ionic current or change in resistance, conductance, charge, or voltage upon a nucleic acid incorporation or tag capture event associated with the plurality of heptameric nanopore assemblies; and,
a control system comprising a computer processor, wherein the computer processor is configured to determine, based on the detection of an ionic current or change in resistance, conductance, charge, or voltage, a sequence of the nucleic acid.

18. The system of claim 17 , wherein the chip comprises a well and wherein a heptameric nanopore assembly is disposed within the membrane over the well.

19. The system of claim 17 , wherein the system further comprises a plurality of tagged nucleotides.

20. The system of claim 19 , wherein the tagged nucleotides are negatively charged.

21. The system of claim 17 , wherein one or more of the six alpha-hemolysin variant monomers further comprise an amino acid substitution corresponding to H35G, E111N, M113A, 126-131G, K147N, or H144A of SEQ ID NO: 14.

22. The system of claim 17 , wherein the alpha-hemolysin DNA polymerase bindin

Assignees

• Roche Sequencing Solutions Inc

Inventors

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

Classifications

• C07K14/31 Primary

  from Staphylococcus (G) · CPC title

• C12N15/01

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

• C12Q1/6869 Primary

  Methods for sequencing · CPC title

• A61K38/02

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

• C07K14/00

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