# US 11,028,115 B2 · Labelled nucleotides

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- Number: US 11,028,115 B2
- Document ID: US11028115B2
- Status: Granted
- Kind: B2
- Application number: 17/034,725
- Filed: 2020-09-28
- Published: 2021-01-14
- Granted: 2021-06-08
- Earliest claimed: 2008-07-24
- Assignees: Illumina Cambridge Limited
- Applicants: Illumina Cambridge Limited
- Inventors: Xiaohai Liu; John Milton; Silke Ruediger; Xiaolin Wu
- Web page: https://patentgrep.com/patent/US11028115B2

## CPC

- C07H 19/04: Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
- C07H 19/06: Pyrimidine radicals
- C07H 19/10: with the saccharide radical esterified by phosphoric or polyphosphoric acids
- C07H 19/14: Pyrrolo-pyrimidine radicals
- C07H 19/16: Purine radicals
- C07H 19/20: with the saccharide radical esterified by phosphoric or polyphosphoric acids
- 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
- C07H 21/04: with deoxyribosyl as saccharide radical
- C12Q 1/6869: Methods for sequencing
- C12Q 1/6874: involving nucleic acid arrays, e.g. sequencing by hybridisation
- Y10T 436/143333: Saccharide [e.g., DNA, etc.]
- 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)
- 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
- Y10T: TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION

## Abstract

The invention provides a nucleotide or nucleoside having a base attached to a detectable label via a cleavable linker, characterised in that the cleavable linker contains a moiety selected from the group comprising: Formula (I) (wherein X is selected from the group comprising O, S, NH and NQ wherein Q is a C1-10 substituted or unsubstituted alkyl group, Y is selected from the group comprising O, S, NH and N(allyl), T is hydrogen or a C1-10 substituted or unsubstituted alkyl group and * indicates where the moiety is connected to the remainder of the nucleotide or nucleoside).

## Claims

### Claim 1

An oligonucleotide comprising a nucleotide residue, wherein said nucleotide residue comprises the structure:
wherein Z is —CH2N3;
B is a nucleobase selected from the group consisting of a purine, a pyrimidine and a deazapurine; and
Linker-Label comprises
wherein R is an optionally present electron-withdrawing group; Fluor is a fluorophore; and the dotted line connecting the Fluor and the benzene ring indicates that substitution may be at any free position on the benzene ring and further atoms not shown may be present.

### Claim 2 (depends on 1)

The oligonucleotide of claim 1, wherein R is not present.

### Claim 3 (depends on 2)

The oligonucleotide of claim 2, wherein Linker-Label comprises

### Claim 4 (depends on 3)

The oligonucleotide of claim 3, wherein the dotted line connecting the Fluor and the benzene ring comprises one or more spacer units such that the Fluor is held a sufficient distance from the nucleobase so as not to interfere with any interaction between the nucleotide and an enzyme.

### Claim 5 (depends on 4)

The oligonucleotide of claim 4, wherein the spacer unit comprises —O—, —NH—, or —N(alkyl)-.

### Claim 6 (depends on 3)

The oligonucleotide of claim 3, wherein the Linker-Label comprises:
wherein the double asterisks ** indicates the attachment point to the nucleobase.

### Claim 7 (depends on 6)

The oligonucleotide of claim 6, wherein the Fluor comprises a cyanine, a rhodamine, or a coumarin dye.

### Claim 8 (depends on 7)

The oligonucleotide of claim 7, wherein the Fluor is a rhodamine dye.

### Claim 9 (depends on 7)

The oligonucleotide of claim 7, wherein the Fluor is a Cy5 dye.

### Claim 10 (depends on 7)

The oligonucleotide of claim 7, wherein the Fluor is attached to the Linker by reacting an N-hydroxysuccinimide ester of the Fluor with an amino moiety of the Linker.

### Claim 11 (depends on 6)

The oligonucleotide of claim 6, wherein the nucleobase is
wherein the asterisk * indicates the attachment point to the sugar moiety of the nucleotide residue.

### Claim 12 (depends on 11)

The oligonucleotide of claim 11, wherein the nucleobase is
and the Fluor is a Cy5 dye.

### Claim 13 (depends on 11)

The oligonucleotide of claim 11, wherein the nucleobase is
and the Fluor is a rhodamine dye.

### Claim 14 (depends on 6)

The oligonucleotide of claim 6, wherein the nucleotide residue comprises the structure:

### Claim 15 (depends on 6)

The oligonucleotide of claim 6, wherein the oligonucleotide is in contact with an aqueous solution comprising ethylenediaminetetraacetic acid (EDTA).

### Claim 16 (depends on 6)

The oligonucleotide of claim 6, wherein the oligonucleotide is in contact with a mutant polymerase.

### Claim 17 (depends on 6)

The oligonucleotide of claim 6, wherein the oligonucleotide is in contact with a water soluble phosphine.

### Claim 18 (depends on 6)

The oligonucleotide of claim 6, wherein the oligonucleotide is hybridized to a target polynucleotide, and wherein the target polynucleotide is immobilized to a solid support.

### Claim 19 (depends on 18)

The oligonucleotide of claim 18, wherein the solid support comprises a plurality of immobilized target polynucleotides, wherein at least one immobilized target polynucleotide is hybridized to a second oligonucleotide, and wherein the second oligonucleotide comprises a different fluorophore than the Fluor.

### Claim 20 (depends on 19)

The oligonucleotide of claim 19, the solid support comprises a planar surface.