Method for characterising a polynucleotide by using a XPD helicase

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)

YIWGTLSEG

and/or

(SEQ ID NO: 30)

QAMGRVVRSPTDYGARILIDGR;

(i)

(SEQ ID NO: 11)

YLWGTLSEG

and/or

(SEQ ID NO: 32)

QAMGRIVRSPDDYGVRILLDSR;

(j)

(SEQ ID NO: 14)

SLWGTLAEG 

and/or

(SEQ ID NO: 34)

QALGRVIRAPDDFGVRVLADKR;

(k)

(SEQ ID NO: 36)

VSGGRLSEG

and/or

(SEQ ID NO: 37)

QEIGRLIRSAEDTGACVILDKR;

(l)

(SEQ ID NO: 39)

VMGGRNSEG

and/or

(SEQ ID NO: 40)

QAAGRVHRSEEEKGAVVVLDYR;

(m)

(SEQ ID NO: 39)

VMGGRNSEG

and/or

(SEQ ID NO: 42)

QAAGRVHRSEEEKGSIVILDYR;

(n)

(SEQ ID NO: 14)

SLWGTLAEG

and/or

(SEQ ID NO: 45)

QAMGRVIRSPEDFGVRMLVDRR;

(o)

(SEQ ID NO: 47)

LATGRFAEG

and/or

(SEQ ID NO: 48)

QMIGRLIRTENDYGVVVIQDKR;

(p)

(SEQ ID NO: 50)

IARGKLAEG

and/or

(SEQ ID NO: 51)

QSIGRAIRGPTDNATIWLLDKR;

(q)

(SEQ ID NO: 53)

VGKGKLAEG

and/or

(SEQ ID NO: 54)

QAIGRAIRDVNDKCNVWLLDKR;

(r)

(SEQ ID NO: 39)

VMGGRNSEG

and/or

(SEQ ID NO: 56)

QAAGRVHRSAEEKGAIIILDYR;

(s)

(SEQ ID NO: 14)

SLWGTLAEG

and/or

(SEQ ID NO: 58)

QALGRVIRSPEDVGVRALLDRR;

(t)

(SEQ ID NO: 14)

SLWGTLAEG

and/or

(SEQ ID NO: 60)

QALGRVIRSPEDFGVRILLDKR;

or

(u)

(SEQ ID NO: 11)

YLWGTLSEG

and/or

(SEQ ID NO: 17)

QAMGRVVRSPGDFGVRILLDAR.

10. The method according to claim 1 , wherein the XPD helicase is one of the helicases shown in Table 4 or 5 or a variant thereof, optionally wherein the XPD helicase comprises (a) the sequence shown in any one of SEQ ID NOs: 10, 13, 16, 18, 20, 22, 25, 28, 31, 33, 35, 38, 41, 43, 44, 46, 49, 52, 55, 57, 59, 61 and 62 or (b) a variant thereof having at least 30% homology to the relevant sequence based on amino acid identity over the entire sequence and retains helicase activity.

11. The method according to claim 10 , wherein the XPD helicase comprises (a) the sequence shown in SEQ ID NO: 10 or (b) a variant thereof having at least 40% homology to the SEQ ID NO: 10 based on amino acid identity over the entire sequence and retains helicase activity.

12. The method according to claim 1 , wherein the salt is KCl.