Enzyme method

Patent metadata

Field	Value
Publication number	US-9758823-B2
Application number	US-201214351038-A
Country	US
Kind code	B2
Filing date	Oct 18, 2012
Priority date	Oct 21, 2011
Publication date	Sep 12, 2017
Grant date	Sep 12, 2017

Abstract

The invention relates to a new method of characterizing a target polynucleotide. The method uses a pore and a Hel308 helicase or amolecular motor which is capable of binding to the target polynucleotide at an internal nucleotide. The helicase or molecular motor controls the movement of the target polynucleotide through the pore.

First claim

The invention claimed is:

1. A method of characterising a target polynucleotide, comprising:
(a) providing in an aqueous solution a transmembrane pore and a membrane, wherein the transmembrane pore is present in the membrane, and wherein the aqueous solution comprises a salt at a concentration in a range of 0.3 M to 3 M;
(b) combining, in the aqueous solution of step (a), the target polynucleotide and a Hel308 helicase, wherein the helicase binds to the target polynucleotide to form a polynucleotide helicase complex, thereby controlling the movement of the target polynucleotide through the transmembrane pore; and
(c) measuring, during an application of a potential across the transmembrane pore, an ionic current as the target polynucleotide moves through the transmembrane pore, to determine one or more characteristics of the target polynucleotide and thereby characterising the target polynucleotide.

2. A method according to claim 1 , wherein the one or more characteristics are selected from (i) the length of the target polynucleotide, (ii) the identity of the target polynucleotide, (iii) the sequence of the target polynucleotide, (iv) the secondary structure of the target polynucleotide and (v) whether or not the target polynucleotide is modified.

3. A method according to claim 2 , wherein the target polynucleotide is modified by methylation, by oxidation, by damage, with one or more proteins or with one or more labels, tags or spacers.

4. A method according to claim 1 , wherein at least a portion of the polynucleotide is double stranded.

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

6. A method according to claim 5 , wherein the transmembrane protein pore is selected from the group consisting of: α-hemolysin, leukocidin, Mycobacterium smegmatis porin A (MspA), outer membrane porin F (OmpF), outer membrane porin G (OmpG), outer membrane phospholipase A, Neisseria autotransporter lipoprotein (NalP) and WZA.

7. A method according to claim 1 , wherein the Hel308 helicase comprises the amino acid motif Q-X1-X2 G R A G R (SEQ ID NO: 8), wherein X1 is C, M or L and X2 is any amino acid residue.

8. A method according to claim 7 , wherein X2 is A, F, M, C, V, L, I, S, T or P.

9. A method according to claim 1 , wherein the Hel308 helicase is capable of binding to the target polynucleotide at an internal nucleotide.

10. A method according to claim 1 , wherein the salt is KCl.

11. A method according to claim 10 , wherein the salt concentration is at least 1.0 M.

Assignees

• Oxford Nanopore Tech Ltd

Inventors

• Moysey Ruth
• Heron Andrew John

Classifications

• C12Q2565/631

  being a biochannel or pore · CPC title

• C12Q1/6869 Primary

  Methods for sequencing · CPC title

• C12Q2521/513

  Winding/unwinding enzyme, e.g. helicase · CPC title

• C12Q1/6811 Primary

  Selection methods for production or design of target specific oligonucleotides or binding molecules · CPC title