Enzyme-pore constructs

• US 10,077,471 B2
• Filed: 09/18/2015
• Issued: 09/18/2018
• Est. Priority Date: 07/07/2008
• Status: Active Grant

• Current Assignee: Oxford Nanopore Technologies Limited
• Original Assignee: Oxford Nanopore Technologies Limited
• Inventors: Jayasinghe, Lakmal, Bayley, John Hagan Pryce, Cheley, Stephen, McKeown, Brian, White, James, Clarke, James Anthony
• Primary Examiner(s): Woolwine, Samuel C
• Application Number:
  US14/858,138
• Publication Number:
  US 20160076092A1
• Time in Patent Office: 1,096 Days
• Field of Search: None
• CPC Class Codes: C07K 14/31 from Staphylococcus (G)
• CPC Class Codes: C12N 9/1247 DNA-directed RNA polymerase (2.7.7.6)
• CPC Class Codes: C12N 9/1252 DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase
• CPC Class Codes: C12N 9/127 RNA-directed RNA polymerase (2.7.7.48), i.e. RNA replicase
• CPC Class Codes: C12N 9/1276 RNA-directed DNA polymerase (2.7.7.49), i.e. reverse transcriptase or telomerase
• CPC Class Codes: C12N 9/16 acting on ester bonds (3.1)
• CPC Class Codes: C12N 9/22 Ribonucleases RNAses, DNAses catalytic nucleic acids C12N15/113
• CPC Class Codes: C12N 9/52 derived from bacteria or Archaea
• CPC Class Codes: C12N 9/90 Isomerases (5.)
• CPC Class Codes: C12N 9/96 Stabilising an enzyme by forming an adduct or a composition; Forming enzyme conjugates
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2565/631 being a biochannel or pore

Abstract

The invention relates to constructs comprising a transmembrane protein pore subunit and a nucleic acid handling enzyme. The pore subunit is covalently attached to the enzyme such that both the subunit and enzyme retain their activity. The constructs can be used to generate transmembrane protein pores having a nucleic acid handling enzyme attached thereto. Such pores are particularly useful for sequencing nucleic acids. The enzyme handles the nucleic acid in such a way that the pore can detect its component nucleotides by stochastic sensing.

Claims

1. A method of sequencing a target nucleic acid sequence, comprising:

(a) contacting the nucleic acid strand with (i) a polymerase and (ii) labelled nucleotides in the presence of a pore such that phosphate labelled species are sequentially released when nucleotides are added to the nucleic acid strand, wherein the phosphate species contain a label specific for each nucleotide, and wherein the phosphate labelled species interacts with the barrel or channel of the pore;

(b) detecting the phosphate labeled species using the pore and thereby detecting the addition of the nucleotide to the nucleic acid strand, wherein the detecting comprises measuring the current passing through the pore during each interaction; and

(c) using the order of the phosphate labeled species to determine the sequence of the nucleic acid strand.

2. A method according to claim 1, wherein the pore is a transmembrane protein pore.

3. A method according to claim 2, wherein the pore is covalently attached to the polymerase.

4. A method according to claim 2, wherein the pore is attached to the polymerase at a site in close proximity to the opening of the barrel or channel of the pore, or wherein the polymerase is attached to the pore such that its active site is orientated towards the opening of the pore.

5. A method according to claim 2, wherein the pore is attached to the polymerase at a site in close proximity to the opening of the barrel or channel of the pore and wherein the polymerase is attached to the pore such that its active site is orientated towards the opening of the pore.

6. A method according to claim 2, wherein the transmembrane protein pore is derived from α

-hemolysin.

7. A method according to claim 6, wherein the pore comprises seven subunits each having at least 55% homology based on amino acid identity to SEQ ID NO:

2 over its entire length.

8. A method according to claim 7, wherein at least one subunit has a glutamine at position 139 of SEQ ID NO:

2, an arginine at position 113 of SEQ ID NO;

2 or a cysteine at position 119, 121 or 135 of SEQ ID NO;

2.

9. A method according to claim 8, wherein (a) all seven subunits have a glutamine at position 139 of SEQ ID NO:

2 and one of the subunits has a cysteine at position 135 and/or (b) all seven subunits have an arginine at position 113 of SEQ ID NO;

2.

10. A method according to claim 1, wherein the pore is attached to the polymerase.

11. A method according to claim 1, wherein the polymerase is (a) a member of any of the Enzyme Classification (EC) groups 2.7.7.6, 2.7.7.7, 2.7.7.19, 2.7.7.48 and 2.7.7.49 or (b) a DNA-dependent DNA polymerase, an RNA-dependent DNA polymerase, a DNA-dependent RNA polymerase or an RNA-dependent RNA polymerase.