# US 2021/0172013 A1 · Compositions and methods for improving nanopore sequencing

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- Number: US 2021/0172013 A1
- Document ID: US20210172013A1
- Status: Published application
- Kind: A1
- Application number: 17/111,167
- Filed: 2020-12-03
- Published: 2021-06-10
- Earliest claimed: 2012-08-03
- Assignees: University of Washington through its Center for Commercialization
- Applicants: University of Washington through its Center for Commercialization
- Inventors: Jens H. Gundlach; Andrew Laszlo
- Web page: https://patentgrep.com/patent/US20210172013A1

## CPC

- C12Q 1/6869: Methods for sequencing
- 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 present disclosure provides methods and reagents for improving nanopore-based analyses of polymers. Specifically, the disclosure provides a method of analyzing a polymer that includes a polymer analyte that contains an end domain that has at least one charged moiety. The disclosure also provides a method of increasing the interaction rate between a polymer analyte and a nanopore, wherein the polymer analyte contains an end domain that has at least one charged moiety. The disclosure also provide compositions for use with the described methods, including adapter compositions that contain charged moieties, such as phosphate or sulfate groups, and that are configured to being linked to an polymer analyte domain.

## Claims

### Claim at position 1

-27. (canceled)

### Claim 28

A polymer adapter composition, comprising:
a polynucleic acid with between 1 and 20 nucleotides,
a charged moiety linked to at least one of the nucleotides of the polynucleic acid,
wherein the charged moiety comprises one of: at least two phosphate groups arranged in linear and/or branched configuration, a sulfate group, a charged amino acid, a modified charged nucleotide, and an acidic residue forming an anion.

### Claim 29 (depends on 28)

The composition of claim 28, comprising at least two sulfate groups arranged in linear and/or branched configuration.

### Claim 30 (depends on 29)

The composition of claim 29, wherein the sulfate groups are disposed in a branched configuration with two or more charged groups in each branch.

### Claim at position 5

-33. (canceled)

### Claim 34 (depends on 28)

The composition of claim 28, wherein the phosphate groups are disposed in a branched configuration with two or more charged groups in each branch.

### Claim 35 (depends on 28)

The composition of claim 28, wherein the charged moiety comprises at least one phosphate and at least one sulfate.

### Claim 36 (depends on 35)

The composition of claim 35, wherein the at least one phosphate and at least one sulfate are arranged in linear and/or branched configuration.

### Claim 37 (depends on 28)

The composition of claim 28, wherein the charged moiety is linked to a nucleotide that is within three residue positions of a terminal nucleotide of the polynucleic acid.

### Claim 38 (depends on 28)

The composition of claim 28, wherein the charged moiety is linked to a terminal nucleotide of the polynucleic acid.

### Claim 39 (depends on 38)

The composition of claim 38, wherein the terminal nucleotide of the polynucleic acid is the 5′ terminal nucleotide.

### Claim 40 (depends on 38)

The composition of claim 38, wherein the terminal nucleotide of the polynucleic acid is the 3′ terminal nucleotide.

### Claim 41 (depends on 28)

The composition of claim 28, wherein the charged moiety increases the energetic favorability for the polynucleic acid to interact with a nanopore comprising an opening or vestibule with a net positive charge.

### Claim 42 (depends on 28)

The composition of claim 28, wherein the polynucleic acid is configured to be ligated to a terminal end of a nucleic acid polymer analyte.

### Claim 43 (depends on 28)

A kit for nanopore-based polymer sequencing, comprising the adapter composition of claim 28.

### Claim 44 (depends on 43)

The kit of claim 43, further comprising reagents for ligating the adapter to a polymer analyte domain.

### Claim 45 (depends on 43)

The kit of claim 43, further comprising a nanopore system.

### Claim 46 (depends on 45)

The kit of claim 45, wherein the nanopore system comprises a nanopore, a multi-chamber assay container, apparatus for applying and measuring electric fields, a molecular motor that translocates a polymer through the nanopore enzymatically, or any combination thereof.

### Claim 47 (depends on 46)

The kit of claim 46, wherein the nanopore is a protein nanopore with a tunnel structure comprising a vestibule and a constriction zone, and wherein the vestibule and/or constriction zone has a net positive charge.