# US 2019/0376134 A1 · Long lifetime alpha-hemolysin nanapores

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- Number: US 2019/0376134 A1
- Document ID: US20190376134A1
- Status: Published application
- Kind: A1
- Application number: 16/519,251
- Filed: 2019-07-23
- Published: 2019-12-12
- Earliest claimed: 2016-06-30
- Assignees: Roche Sequencing Solutions, Inc.
- Applicants: Roche Sequencing Solutions, Inc.
- Inventors: Mark Ambroso; Timothy Craig; Matthew DePietro; Corissa Harris; Marshall Porter
- Web page: https://patentgrep.com/patent/US20190376134A1

## CPC

- C12Q 1/6869: Methods for sequencing
- A61K 38/02: Peptides of undefined number of amino acids; Derivatives thereof
- C07K 14/31: from Staphylococcus (G)
- C12N 15/01: Preparation of mutants without inserting foreign genetic material therein; Screening processes therefor
- C07K 14/00: Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D)
- C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00)
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## 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 a-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.

## Claims

### Claim 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, thereby attaching the DNA polymerase to the nanopore assembly.

### Claim 2 (depends on 1)

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

### Claim 3 (depends on 2)

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, E111 N, M113A, 126-131G, K147N, or H144A of SEQ ID NO: 14.

### Claim 4 (depends on 2)

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.

### Claim 5 (depends on 2)

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.

### Claim 6 (depends on 4)

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 E111 N, M113A, 126-131G, or K147N of SEQ ID NO: 14.

### Claim 7 (depends on 1)

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, E111 N, 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 E111 N, M113A, 126-131G, and K147N of SEQ ID NO: 14.

### Claim 8 (depends on 1)

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.

### Claim 9 (depends on 8)

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

### Claim 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.

### Claim 11 (depends on 10)

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

### Claim 12 (depends on 11)

The method of claim 11, wherein one or more of the alpha-hemolysin variants further comprise an amino acid substitution corresponding to H35G, E111 N, M113A, 126-131G, or H144A of SEQ ID NO: 14.

### Claim 13 (depends on 10)

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.

### Claim 14 (depends on 10)

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, E111 N, 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.

### Claim 15 (depends on 14)

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

### Claim 16 (depends on 10)

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

### Claim 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.

### Claim 18 (depends on 17)

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.

### Claim 19 (depends on 17)

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

### Claim 20 (depends on 19)

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

### Claim 21 (depends on 17)

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.

### Claim 22 (depends on 17)

The system of claim 17, 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 an E111 N, M113A, 126-131G, or K147N substitution of SEQ ID NO: 14.

### Claim 23 (depends on 17)

The system of claim 17, wherein the six alpha-hemolysin variant monomers further comprise a substitution at H35G, E111N, M113A, 126-131G, K147N, 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 E111 N, M113A, 126-131G, and K147N of SEQ ID NO: 14.