# US 8,785,211 B2 · Methods using pores

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- Number: US 8,785,211 B2
- Document ID: US8785211B2
- Status: Granted
- Kind: B2
- Application number: 13/338,794
- Filed: 2011-12-28
- Published: 2012-08-30
- Granted: 2014-07-22
- Earliest claimed: 2005-11-15
- Assignees: Isis Innovation Limited
- Applicants: Hagan Bayley; Yann Astier; Orit Braha
- Inventors: Hagan Bayley; Yann Astier; Orit Braha
- Web page: https://patentgrep.com/patent/US8785211B2

## CPC

- G01N 33/48721: {Investigating individual macromolecules, e.g. by translocation through nanopores (Coulter counters in general G01N15/12; fabrication methods for nanoscale apertures B81B1/00; sequencing of nucleic acids C12Q1/68)}
- C12Q 1/6869: Methods for sequencing
- G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q)
- 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 method of identifying an individual nucleotide, comprising (a) contacting the nucleotide with a transmembrane protein pore so that the nucleotide interacts with the pore and (b) measuring the current passing through the pore during the interaction and thereby determining the identity of the nucleotide. The invention also relates to a method of sequencing nucleic acid sequences and kits related thereto.

## Claims

### Claim 1

A method of distinguishing an individual monophosphate nucleotide, comprising:
(a) contacting the monophosphate nucleotide with a transmembrane protein pore so that the monophosphate nucleotide interacts with the pore, wherein the pore comprises a molecular adaptor that facilitates the interaction between the monophosphate nucleotide and the pore, wherein the molecular adaptor is a cyclodextrin; and
(b) measuring a current passing through the pore during the interaction and thereby distinguishing the monophosphate nucleotide from other monophosphate nucleotides.

### Claim 2 (depends on 1)

A method according to claim 1, wherein the interaction involves the monophosphate nucleotide reversibly binding to the channel of the pore.

### Claim 3 (depends on 1)

A method according to claim 1, wherein the pore is α-hemolysin as shown in SEQ ID NO: 2 or a variant thereof.

### Claim 4 (depends on 3)

A method according to claim 3, wherein the variant is (M113R)7.

### Claim 5 (depends on 1)

A method according to claim 1, wherein the molecular adaptor is a heptakis-6-amino-β-cyclodextrin (am7-β-CD).

### Claim 6 (depends on 1)

A method according to claim 1, wherein the individual monophosphate nucleotide is a monophosphate ribonucleotide.

### Claim 7 (depends on 6)

A method according to claim 6, further comprising before step (a) digesting a ribonucleic acid (RNA) sequence to provide the individual monophosphate nucleotide.

### Claim 8 (depends on 1)

A method according to claim 1, wherein the individual monophosphate nucleotide is a monophosphate deoxyribonucleotide.

### Claim 9 (depends on 8)

A method according to claim 8, further comprising before step (a) digesting a deoxyribonucleic acid (DNA) sequence to provide the individual monophosphate nucleotide.

### Claim 10 (depends on 7)

A method according to claim 7, wherein more than one of the individual monophosphate nucleotides of the RNA sequence are contacted with the pore in a sequential manner such that the identity of the whole or part of the sequence may be determined.

### Claim 11

A method of sequencing a target nucleic acid sequence, comprising:
(a) digesting an individual monophosphate nucleotide from one end of the target sequence using a processive exonuclease;
(b) contacting the monophosphate nucleotide with a transmembrane protein pore so that the monophosphate nucleotide interacts with the pore, wherein the pore comprises a molecular adaptor that facilitates the interaction between the monophosphate nucleotide and the pore, wherein the molecular adaptor is a cyclodextrin;
(c) measuring a current passing through the pore during the interaction and thereby determining the identity of the individual monophosphate nucleotide; and
(d) repeating steps (a) to (c) at the same end of the target sequence and thereby determining the sequence of the nucleic acid.

### Claim 12 (depends on 9)

A method according to claim 9, wherein more than one of the individual monophosphate nucleotides of the DNA sequence are contacted with the pore in a sequential manner such that the identity of the whole or part of the sequence may be determined.