# US 12,006,540 B2 · Synthesis of novel disulfide linker based nucleotides as reversible terminators for DNA sequencing by synthesis

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- Number: US 12,006,540 B2
- Document ID: US12006540B2
- Status: Granted
- Kind: B2
- Application number: 17/816,555
- Filed: 2022-08-01
- Published: 2023-01-26
- Granted: 2024-06-11
- Earliest claimed: 2015-09-28
- Assignees: The Trustees of Columbia University in the City of New York
- Applicants: The Trustees of Columbia University in the City of New York
- Inventors: Jingyue Ju; Xiaoxu Li; Xin Chen; Zengmin Li; Shiv Kumar; Shundi Shi; Cheng Guo; Jianyi Ren; Min-Kang Hsieh; Minchen Chien; Chuanjuan Tao; Ece Erturk; Sergey Kalachikov; James J. Russo
- Web page: https://patentgrep.com/patent/US12006540B2

## CPC

- C12Q 1/6869: Methods for sequencing
- C07H 19/10: with the saccharide radical esterified by phosphoric or polyphosphoric acids
- C07H 19/14: Pyrrolo-pyrimidine radicals
- C07H 19/20: with the saccharide radical esterified by phosphoric or polyphosphoric acids
- 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
- 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)

## Abstract

Disclosed herein, inter alia, are compounds, compositions, and methods of use thereof in the sequencing of a nucleic acid.

## Claims

### Claim 1

A nucleotide analogue comprising (i) a base, (ii) a deoxyribose or ribose, and (iii) a detectable label bound to the base via a linker, wherein said linker has the formula:

### Claim 2 (depends on 1)

The nucleotide analogue of claim 1, wherein the nucleotide analogue has the formula:
B is a base;
R3 is —OH, monophosphate, polyphosphate or a nucleic acid;
R5 is a detectable label;
R6 is hydrogen or a polymerase-compatible cleavable moiety;
R7 is hydrogen or —OH.

### Claim 3 (depends on 2)

The nucleotide analogue of claim 2, wherein B is a divalent cytosine or a derivative thereof, a divalent guanine or a derivative thereof, a divalent adenine or a derivative thereof, a divalent thymine or a derivative thereof, a divalent uracil or a derivative thereof, a divalent hypoxanthine or a derivative thereof, a divalent xanthine or a derivative thereof, a deaza-adenine or a derivative thereof, a deaza-guanine or a derivative thereof, a deaza-hypoxanthine or a derivative thereof, a divalent 7-methylguanine or a derivative thereof, a divalent 5,6-dihydrouracil or a derivative thereof, a divalent 5-methylcytosine or a derivative thereof, or a divalent 5-hydroxymethylcytosine or a derivative thereof.

### Claim 4 (depends on 2)

The nucleotide analogue of claim 2, wherein B is

### Claim 5 (depends on 2)

The nucleotide analogue of claim 2, wherein R3 is —OH.

### Claim 6 (depends on 2)

The nucleotide analogue of claim 2, wherein R3 is triphosphate.

### Claim 7 (depends on 2)

The nucleotide analogue of claim 2, wherein R5 is a fluorescent dye.

### Claim 8 (depends on 2)

The nucleotide analogue of claim 2, wherein R5 is

### Claim 9 (depends on 2)

The nucleotide analogue of claim 2, wherein R6 is a polymerase-compatible cleavable moiety comprising an azido moiety.

### Claim 10 (depends on 2)

The nucleotide analogue of claim 2, wherein R6 is a polymerase-compatible cleavable moiety comprising a dithio moiety.

### Claim 11 (depends on 2)

The nucleotide analogue of claim 2, wherein R6 is a polymerase-compatible cleavable moiety comprising C2-C6 allyl.

### Claim 12 (depends on 2)

The nucleotide analogue of claim 2, wherein R6 is a polymerase-compatible cleavable moiety comprising methoxyalkyl.

### Claim 13 (depends on 2)

The nucleotide analogue of claim 2, wherein R6 is —NH2, —CN, —CH3, or —CH2N3.

### Claim 14 (depends on 2)

A kit comprising the nucleotide analogue of claim 2.

### Claim 15 (depends on 10)

A method for sequencing a nucleic acid, comprising:
(i) incorporating in series with a nucleic acid polymerase, within a reaction vessel, one of four different labeled nucleotide analogues into a primer to create an extension strand, wherein said primer is hybridized to said nucleic acid and wherein each of the four different labeled nucleotide analogues comprise a unique detectable label;
(ii) detecting said unique detectable label of each incorporated nucleotide analogue, so as to thereby identify each incorporated nucleotide analogue in said extension strand, thereby sequencing the nucleic acid; and
wherein each of said four different labeled nucleotide analogues are of the structure of claim 10, wherein
in the first of said four different labeled nucleotide analogues, B is a thymidine or uridine hybridizing base;
in the second of said four different labeled nucleotide analogues, B is an adenosine hybridizing base;
in the third of said four different labeled nucleotide analogues, B is a guanosine hybridizing base; and
in the fourth of said four different labeled nucleotide analogues, B is a cytosine hybridizing base.

### Claim 16 (depends on 15)

The method of claim 15, wherein R3 of at least one of the four different labeled nucleotide analogues is triphosphate.

### Claim 17 (depends on 15)

The method of claim 15, wherein R5 of at least one of the four different labeled nucleotide analogues is a fluorescent dye.

### Claim 18 (depends on 15)

The method of claim 15, wherein R5 of at least one of the four different labeled nucleotide analogues is selected from

### Claim 19 (depends on 15)

The method of claim 15, wherein R6 of at least one of the four different labeled nucleotide analogues is a polymerase-compatible cleavable moiety.

### Claim 20 (depends on 1)

A kit comprising the nucleotide analogue of claim 1.