Alpha-Hemolysin Variants and Uses Thereof

Patent metadata

Field	Value
Publication number	US-2019367975-A1
Application number	US-201916425743-A
Country	US
Kind code	A1
Filing date	May 29, 2019
Priority date	Apr 21, 2016
Publication date	Dec 5, 2019
Grant date	—

Abstract

Described herein are variants of alpha-hemolysin having at least one mutation, such as a mutation to a positive charge. In certain examples, the mutation is selected from V149K, E287R, H35G, T109K, P151K, K147N, E111N, M113A, or combinations thereof in the mature, wild-type 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 a decreased time-to-thread. The decreased time-to-thread, for example, increases DNA sequencing efficiency and accuracy.

First claim

What is claimed is:

1 . An isolated polypeptide comprising an amino acid sequence having at least 90% sequence identity to one or more SEQ ID NO: 5-14, wherein said amino acid sequence has a set of substitutions relative to SEQ ID NO: 14 comprising a combination selected from the group consisting of:
(a) H35G+V149K; (b) V149K+E287R+H35G; (c) V149K+E287R; (d) T109K+H35G; (e) P151K+H35G; (f) V149K+P151K+H35G; (g) T109K+V149K+H35G; (h) V149K+K147N+E111N+D127G+D128G+T129G+K131G+M113A+H35G; (i) V149K+K147N+E111N+D127G+D128G+T129G+K131G+M113A; and (j) T109K+V149K+P151K+H35G.

2 . The isolated polypeptide of claim 1 , wherein said set of substitutions consists of substitutions corresponding to positions of SEQ ID NO: 14 selected from the group consisting of 1-6, 8-11, 13-22, 24-33, 35-37, 40, 44-51, 56, 62, 64-75, 79, 82, 83, 85, 87, 89-95, 97, 102-118, 120-132, 134-156, 158-164, 168, 170-191, 193, 194, 197-205, 207, 208, 210-216, 218, 221, 222, 224-229, 235-241, 244, 246, 250, 252, 253, 255, 257, 259-264, 266, 268-278, 280-283, 285-289, and 291-293.

3 . The isolated polypeptide of claim 1 , wherein said set of substitutions further comprises an H144A substitution.

4 . The isolated polypeptide of claim 1 , wherein said set of substitutions further comprises an alanine, asparagine, or lysine substitution at one or more of residues 1-17 of SEQ ID NO: 14.

5 . The isolated polypeptide of claim 1 , wherein said amino acid sequence comprises a series of glycine residues at positions corresponding to each of residues 127-131 of SEQ ID NO: 14.

6 . The isolated polypeptide of claim 1 , wherein the amino acid sequence comprises SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13.

7 . The isolated polypeptide of claim 1 , wherein the amino acid sequence consists of SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13.

8 . The isolated polypeptide of claim 1 , wherein the at least 90% sequence identity to SEQ ID NO: 4 is a sequence identity according to CLUSTAL-W program in MacVector version 13.0.7, operated with an open gap penalty of 10.0, an extended gap penalty of 0.1, and a BLOSUM 30
similarity matrix.

9 . The isolated polypeptide of claim 1 , further comprising a purification tag.

10 . The isolated polypeptide of claim 1 , further comprising a polymerase attached thereto.

11 . The isolated polypeptide of claim 10 , wherein the polymerase is a DNA polymerase.

12 . The isolated polypeptide of claim 10 , wherein the DNA polymerase is covalently attached to the isolated polypeptide.

13 . A heptameric nanopore assembly comprising 7 alpha-hemolysin subunits arranged in a nanopore, wherein at least one of the seven alpha-hemolysin subunits comprises the isolated polypeptide of claim 1 and a DNA polymerase covalently attached to one of the seven alpha-hemolysin subunits.

14 . The heptameric nanopore assembly of claim 13 , wherein the nanopore is a heteromeric pore.

15 . The heptameric nanopore assembly of claim 13 , wherein the nanopore is a homomeric pore.

16 . The heptameric nanopore assembly of claim 13 , wherein the nanopore has a decreased time to thread (TTT) relative to a heptameric nanopore assembly consisting of native alpha-hemolysins.

17 . The heptameric nanopore assembly of claim 13 , wherein the nanopore has at least a 10% decreased TTT relative to the heptameric nanopore assembly including native alpha-hemolysins.

18 . The heptameric nanopore assembly of claim 13 , wherein the nanopore has at least a 50% decreased TTT relative to the heptameric nanopore assembly including native alpha-hemolysins.

19 . The heptameric nanopore assembly of claim 13 , wherein the nanopore has at least a 80% decreased TTT relative to the heptameric nanopore assembly including native alpha-hemolysins.

20 . A system for sequencing a nucleic acid comprising:
a heptameric nanopore assembly according to claim 13 ; 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; and a set of negatively charged tagged nucleotides.

21 . 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 of claim 13 ; 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.

22 . The method of claim 21 , wherein the chip comprises a well and wherein the nanopore assembly is disposed within the membrane over the well.

23 . The method of claim 21 , wherein the nucleic acid comprises a tag.

24 . The method of claim 23 , wherein the tag is a negatively charged tag.

Assignees

• Roche Sequencing Solutions Inc

Inventors

• Craig Timothy K
• Ambroso Mark R
• Harris Corissa J
• Dipietro Matthew M
• Zou Yaozhong
• Porter Marshall W

Classifications

• C07K14/31

  from Staphylococcus (G) · CPC title

• C12Q2600/156

  Polymorphic or mutational markers · CPC title

• C12Q1/6869 Primary

  Methods for sequencing · CPC title

• G01N33/48721

  Investigating individual macromolecules, e.g. by translocation through nanopores (Coulter counters in general G01N15/12; fabrication methods for nanoscale apertures B81B1/00; sequencing of nucleic acids C12Q1/68) · CPC title

• C12Q1/689

  for bacteria · CPC title