# US 9,040,678 B2 · Functionalization and purification of molecules by reversible group exchange

> Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt

- Number: US 9,040,678 B2
- Document ID: US9040678B2
- Status: Granted
- Kind: B2
- Application number: 13/565,400
- Filed: 2012-08-02
- Published: 2013-02-07
- Granted: 2015-05-26
- Earliest claimed: 2011-08-05
- Assignees: Illumina, Inc.
- Applicants: Frank J. Steemers; Kevin L. Gunderson; Kerri York; Ryan Christopher Smith
- Inventors: Frank J. Steemers; Kevin L. Gunderson; Kerri York; Ryan Christopher Smith
- Web page: https://patentgrep.com/patent/US9040678B2

## CPC

- C07H 1/06: Separation; Purification
- C07D 495/04: Ortho-condensed systems
- C07H 21/00: Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
- C12N 15/1003: {Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor}
- C12Q 1/6811: Selection methods for production or design of target specific oligonucleotides or binding molecules
- C07H 1/00: Processes for the preparation of sugar derivatives
- C07H: SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS (derivatives of aldonic or saccharic acids C07C, C07D; aldonic acids, saccharic acids C07C59/105, C07C59/285; cyanohydrins C07C255/16; glycals C07D; compounds of unknown constitution C07G; polysaccharides, derivatives thereof C08B; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification C12N15/00; sugar industry C13)
- C07D: HETEROCYCLIC COMPOUNDS (macromolecular compounds C08)
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- 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

Embodiments of the present disclosure include methods and compositions for functionalizing molecules, such as oligonucleotides, with functional groups, including polyhistidine tags useful in affinity methods. Some embodiments include methods for modifying and purifying complex mixtures of molecules by exchange of functional tags.

## Claims

### Claim 1

A method for modifying a nucleic acid, said method comprising:
(a) performing a first exchange reaction comprising contacting:
a nucleic acid comprising an aldehyde group, and
a bifunctional linking reagent comprising a hydrazine or amine moiety under conditions to form an imine bond coupling the nucleic acid to the bifunctional linking reagent; wherein the bifunctional linking reagent further comprises a functional tag;
(b) contacting the sample with a first binding partner to the first functional tag; and
(c) performing a first purification step comprising separating the first binding partner from the sample, thereby isolating a nucleic acid from a sample;
(d) performing a second exchange reaction comprising contacting the nucleic acid isolated in step (c) with an exchange reagent under conditions sufficient for the imine bond to be broken between the nucleic acid and the bifunctional linking reagent and for a new imine bond to form between the exchange reagent and the nucleic acid;
(f) contacting the nucleic acid with a second binding partner to the second functional tag; and
(g) separating the second binding partner from the nucleic acid that is not associated with the second affinity tag, thereby isolating the nucleic acid.

### Claim 2 (depends on 1)

The method of claim 1, wherein the bifunctional linking reagent comprises a 6-hydrazinonicotinate acetone hydrazone moiety or derivative thereof.

### Claim 3 (depends on 1)

The method of claim 1, wherein the first functional tag is selected from the group consisting of a histidine, biotin, and glutathione-S-transferase.

### Claim 4 (depends on 1)

The method of claim 1, wherein the first binding partner is selected from the group consisting of nickel, avidin, streptavidin and glutathione.

### Claim 5 (depends on 1)

The method of claim 1, wherein said bifunctional linking reagent is reversibly or irreversibly associated with a substrate.

### Claim 6 (depends on 1)

The method of claim 1, wherein the second binding partner is selected from the group consisting of nickel, avidin, streptavidin and glutathione.

### Claim 7 (depends on 1)

The method of claim 1, wherein the nucleic acid comprises DNA.

### Claim 8 (depends on 1)

The method of claim 1, wherein the 5′ terminal nucleotide of the nucleic acid comprises the aldehyde group.

### Claim 9 (depends on 1)

The method of claim 1, wherein the sample comprises a plurality of synthesized nucleic acids comprising full-length synthesized nucleic acid molecules and partially synthesized nucleic acid molecules.

### Claim 10 (depends on 1)

The method of claim 1, wherein the second exchange reaction comprises a nonreversible exchange.

### Claim 11 (depends on 1)

The method of claim 1, wherein the second exchange reaction comprises a reversible exchange.

### Claim 12 (depends on 1)

The method of claim 1, wherein any one of steps (a) or (b) is performed in a buffer comprising urea.

### Claim 13 (depends on 1)

The method of claim 1, wherein said exchange reagent comprises at least a second functional tag comprising a reversible or irreversible association between said exchange reagent and said second functional tag.

### Claim 14 (depends on 13)

The method of claim 13, wherein the second functional tag is different from the first functional tag.

### Claim 15 (depends on 13)

The method of claim 13, wherein the second functional tag is selected from the group consisting of histidine, biotin, and glutathione-S-transferase.

### Claim 16 (depends on 13)

The method of claim 13, wherein said second affinity tag is reversibly or irreversibly bound to a substrate.

### Claim 17 (depends on 1)

The method of claim 1, wherein one or more of the nucleic acids and the bifunctional linking reagent comprises an additional functional tag that is a label tag.

### Claim 18 (depends on 17)

The method of claim 17, wherein the label tag is selected from the group consisting of a fluorophore, radioisotope, chromogen, enzyme, and epitope.

### Claim 19 (depends on 1)

The method of claim 1, wherein said bifunctional linking reagent comprises a second functional tag.

### Claim 20 (depends on 19)

The method of claim 19, wherein the first functional tag and second functional tag are different.