# US 11,028,116 B2 · Labelled nucleotides

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- Number: US 11,028,116 B2
- Document ID: US11028116B2
- Status: Granted
- Kind: B2
- Application number: 17/035,053
- 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/US11028116B2

## 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

A method for determining the sequence of a single-stranded target polynucleotide, comprising:
(a) contacting a primer polynucleotide/target polynucleotide complex with one or more of nucleoside triphosphates A, G, C and T, wherein at least one of said nucleoside triphosphate has the structure:
wherein
B is a nucleobase selected from the group consisting of a purine, a pyrimidine and a deazapurine;
Z is —CH2N3;
X is a triphosphate group;
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 maybe present;
(b) incorporating one nucleoside triphosphate into the primer polynucleotide;
(c) performing one or more fluorescent measurements to determine the identity of the incorporated nucleoside triphosphate; and
(d) cleaving the Linker after performing the one or more fluorescent measurements.

### Claim 2 (depends on 1)

The method of claim 1, wherein R is not present and the Linker-Label comprises:

### Claim 3 (depends on 2)

The method of claim 2, 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 nucleoside triphosphate and a polymerase.

### Claim 4 (depends on 2)

The method of claim 2, wherein the Linker-Label comprises:
wherein the double asterisks ** indicates the attachment point to the nucleobase.

### Claim 5 (depends on 4)

The method of claim 4, wherein the Fluor comprises a cyanine, a rhodamine, or a coumarin dye.

### Claim 6 (depends on 5)

The method of claim 5, wherein the Fluor is a Cy3 or a Cy5 dye.

### Claim 7 (depends on 5)

The method of claim 5, 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 8 (depends on 6)

The method of claim 6, wherein said at least one nucleoside triphosphate is a G nucleoside triphosphate and the Fluor is a Cy5 dye.

### Claim 9 (depends on 4)

The method of claim 4, further comprising (e) removing the —CH2N3 to expose a 3′-OH.

### Claim 10 (depends on 9)

The method of claim 9, wherein the —CH2N3 removing step (e), and the Linker cleaving step (d) are performed in a single chemical treatment step.

### Claim 11 (depends on 10)

The method of claim 10, wherein the —CH2N3 is removed and the Linker is cleaved by a water soluble phosphine reagent.

### Claim 12 (depends on 11)

The method of claim 11, wherein the water soluble phosphine reagent is a trialkyl phosphine derivatized with one or more hydroxyl functionalities.

### Claim 13 (depends on 9)

The method of claim 9, wherein steps (a) through (e) are repeated multiple times to determine the sequence of the target polynucleotide.

### Claim 14 (depends on 1)

The method of claim 1, wherein the primer polynucleotide/target polynucleotide complex is formed by contacting the target polynucleotide with a single-stranded primer polynucleotide complementary to at least a portion of the target polynucleotide.

### Claim 15 (depends on 14)

The method of claim 14, wherein the primer polynucleotide/target polynucleotide complex is immobilized to a solid support.

### Claim 16 (depends on 1)

The method of claim 1, wherein said incorporating step is accomplished by a polymerase.

### Claim 17 (depends on 1)

The method of claim 1, wherein any unincorporated nucleoside triphosphates are removed prior to said determining step.

### Claim 18 (depends on 17)

The method of claim 17, wherein the unincorporated nucleoside triphosphates are removed by using a washing buffer comprising ethylenediaminetetraacetic acid (EDTA).

### Claim 19 (depends on 1)

The method of claim 1, wherein at least one of the A, G, C, or T nucleoside triphosphates has the structure:
respectively.

### Claim 20 (depends on 4)

The method of claim 4, wherein at least one nucleoside triphosphate has the structure:
or a salt thereof.