Method for characterising a polynucleotide by using a XPD helicase

Patent metadata

Field	Value
Publication number	US-9617591-B2
Application number	US-201214369024-A
Country	US
Kind code	B2
Filing date	Dec 28, 2012
Priority date	Dec 29, 2011
Publication date	Apr 11, 2017
Grant date	Apr 11, 2017

Abstract

The invention relates to a new method of characterizing a target polynucleotide. The method uses a pore and an XPD helicase. The helicase 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 a transmembrane nanopore and a membrane in an aqueous solution, wherein the transmembrane nanopore is present in the membrane, and wherein the aqueous solution comprises a salt at a concentration in a range of 0.3 M to 1 M;
(b) combining in the aqueous solution of step (a) a target polynucleotide and a XPD helicase under conditions in which the helicase binds to the target polynucleotide and controls the movement of the target polynucleotide through the nanopore; and
(c) measuring, during application of a potential across the nanopore, ion flow through the transmembrane nanopore as the polynucleotide moves through the nanopore wherein the ion flow measurements are indicative of one or more characteristics of the target polynucleotide, 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, and thereby characterising the target polynucleotide.

2. The method according to claim 1 , 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.

3. The method according to claim 1 , wherein the ion flow measurements comprise a current measurement, an impedance measurement, a tunnelling measurement or a field effect transistor (FET) measurement.

4. The method according to claim 1 , wherein the method further comprises the step of applying a voltage across the nanopore to form a complex between the nanopore and the helicase.

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

6. The method according to claim 1 , wherein the nanopore is a transmembrane protein nanopore or a solid state nanopore, optionally wherein the transmembrane protein nanopore is selected from 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. The method according to claim 6 , wherein the transmembrane protein nanopore is (a) formed of eight identical subunits as shown in SEQ ID NO: 2 or a variant thereof in which one or more of the seven subunits has at least 50% homology to SEQ ID NO: 2 based on amino acid identity over the entire sequence and retains pore activity, or (b) α-hemolysin formed of seven identical subunits as shown in SEQ ID NO: 4 or a variant thereof in which one or more of the seven subunits has at least 50% homology to SEQ ID NO: 4 based on amino acid identity over the entire sequence and retains pore activity.

8. The method according to claim 1 , wherein the XPD helicase comprises:
the amino acid motif X1-X2-X3-G-X4-X5-X6-E-G (SEQ ID NO: 8), wherein X1, X2, X5 and X6 are independently selected from any amino acid except D, E, K and R and wherein X3 and X4 may be any amino acid residue: and/or
the amino acid motif Q-Xa-Xb-G-R-Xc-Xd-R-(Xe) 3 -Xf-(Xg) 7 -D-Xh-R (SEQ ID NO: 9), wherein Xa, Xe and Xg may be any amino acid residue and wherein Xb, Xc and Xd are independently selected from any amino acid except D, E, K and R,
optionally wherein X1, X2, X5 and X6 and/or Xb, Xc and Xd are independently selected from G, P, A, V, L, I, M, C, F, Y, W, H, Q, N, S and T.

9. The method according to claim 8 , wherein the helicase comprises the following motifs:

(a)

(SEQ ID NO: 11)

YLWGTLSEG

and/or

(SEQ ID NO: 12)

QAMGRVVRSPTDYGARILLDGR;

(b)

(SEQ ID NO: 14)

SLWGTLAEG

and/or

(SEQ ID NO: 15)

QAIGRVVRGPDDFGVRILADRR;

(c)

(SEQ ID NO: 11)

YLWGTLSEG

and/or

(SEQ ID NO: 17)

QAMGRVVRSPGDFGVRILLDAR;

(d)

(SEQ ID NO: 11)

YLWGTLSEG

and/or

(SEQ ID NO: 19)

QAMGRVVRSPSDYGARILLDGR

(e)

(SEQ ID NO: 14)

SLWGTLAEG

and/or

(SEQ ID NO: 21)

QALGRVVRSPTDFGVRVLVDER;

(f)

(SEQ ID NO: 23)

VTGGVFAEG

and/or

(SEQ ID NO: 24)

QAAGRVLRTPEDRGVIALLGRR;

(g)

(SEQ ID NO: 26)

LGTGAFWEG

and/or

(SEQ ID NO: 27)

QGVGRLIRDERDRGVLILCDNR;

(h)

(SEQ ID NO: 29)

Assignees

• Oxford Nanopore Tech Ltd
• Oxford Nanopore Tech Ltd

Inventors

• Moysey Ruth
• Heron Andrew John
• Soeroes Szabolcs

Classifications

• C12Q1/6869 Primary

  Methods for sequencing · CPC title

• C12Q2565/631

  being a biochannel or pore · CPC title

• C12Q2565/607

  being a sensor, e.g. electrode · CPC title

• C12Q2521/513

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

• C12Q2527/137

  Concentration of a component of medium · CPC title