# US 2021/0172011 A1 · Enzyme method

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- Number: US 2021/0172011 A1
- Document ID: US20210172011A1
- Status: Published application
- Kind: A1
- Application number: 16/902,301
- Filed: 2020-06-16
- Published: 2021-06-10
- Earliest claimed: 2011-10-21
- Assignees: Oxford Nanopore Technologies PLC
- Applicants: Oxford Nanopore Technologies Ltd.
- Inventors: Ruth Moysey; Andrew John Heron
- Web page: https://patentgrep.com/patent/US20210172011A1

## CPC

- C12Q 1/6869: Methods for sequencing
- C12Q 2521/513: Winding/unwinding enzyme, e.g. helicase
- C12Q 2565/631: being a biochannel or pore
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES

## Abstract

The invention relates to a new method of characterizing a target polynucleotide. The method uses a pore and a Hel308 helicase or a molecular 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.

## Claims

### Claim at position 1

-43. (canceled)

### Claim 44

A method of characterising a target polynucleotide, comprising:
(a) contacting the target polynucleotide with a transmembrane pore such that a portion of the target polynucleotide is captured by a transmembrane pore, wherein the pore is present in a membrane;
(b) contacting the target polynucleotide with a Hel308 helicase such that the helicase binds to the target polynucleotide, thereby controlling the movement of the target polynucleotide through the transmembrane pore; and
(c) measuring one or more characteristics of the target polynucleotide as the target polynucleotide moves through the transmembrane pore, thereby characterising the target polynucleotide.

### Claim 45 (depends on 44)

The method according to claim 44, 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.

### Claim 46 (depends on 45)

The method according to claim 45, 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.

### Claim 47 (depends on 44)

The method according to claim 44, wherein at least a portion of the target polynucleotide is double stranded.

### Claim 48 (depends on 44)

The method according to claim 44, wherein the transmembrane pore is a protein pore or a solid state pore.

### Claim 49 (depends on 48)

The method according to claim 48, wherein the transmembrane protein pore is selected from the group consisting of: .alpha.-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.

### Claim 50 (depends on 44)

The method according to claim 44, 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.

### Claim 51 (depends on 50)

The method according to claim 50, wherein X2 is A, F, M, C, V, L, I, S, T or P.

### Claim 52 (depends on 44)

The method according to claim 44, wherein the Hel308 helicase binds to the target polynucleotide at an internal nucleotide.

### Claim 53 (depends on 44)

The method according to claim 44, wherein the one or more characteristics of the target polynucleotide are measured by electrical measurement and/or optical measurement.

### Claim 54 (depends on 53)

The method according to claim 53, wherein the electrical measurement is a current measurement, an impedance measurement, a tunnelling measurement, or a field effect transistor (FET) measurement.

### Claim 55 (depends on 44)

The method according to claim 44, wherein the method further comprises the step of applying a voltage across the pore to form a complex between the pore and the first helicase and wherein at least a portion of the polynucleotide is double stranded.

### Claim 56 (depends on 44)

The method according to claim 44, wherein the target polynucleotide moves from the cis side of the membrane to the trans side of the membrane.

### Claim 57 (depends on 56)

The method according to claim 56, wherein the portion of the target polynucleotide that is captured by a transmembrane pore is the 3′ end of the target polynucleotide.

### Claim 58 (depends on 44)

The method according to claim 44, wherein the target polynucleotide moves from the trans side of the membrane to the cis side of the membrane.

### Claim 59 (depends on 58)

The method according to claim 58, wherein the portion of the target polynucleotide that is captured by a transmembrane pore is the 5′ end of the target polynucleotide.

### Claim 60 (depends on 44)

The method according to claim 44, wherein the target polynucleotide is partially translocated through the pore in (a).

### Claim 61 (depends on 44)

The method according to claim 44, wherein method is carried out using a salt concentration of at least 0.3 M or at least 1.0 M and the salt is optionally KCl.

### Claim 62 (depends on 44)

The method according to claim 44, wherein an initial helicase controls the movement of the target polynucleotide into the pore in (a).

### Claim 63 (depends on 62)

The method according to claim 62, wherein the Hel308 helicase of (a) binds to an internal polynucleotide of the target polynucleotide at a location on the polynucleotide different than the initial helicase.