# US 12,215,122 B2 · Nucleotide analogues

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- Number: US 12,215,122 B2
- Document ID: US12215122B2
- Status: Granted
- Kind: B2
- Application number: 18/522,886
- Filed: 2023-11-29
- Published: 2024-05-30
- Granted: 2025-02-04
- Earliest claimed: 2018-10-25
- Assignees: Singular Genomics Systems, Inc.
- Applicants: Singular Genomics Systems, Inc.
- Inventors: Ronald Graham; Olga Adelfinskaya; Megha Cila; Rodrigo Rodriguez; Abrehet Abdu; Eli N. Glezer
- Web page: https://patentgrep.com/patent/US12215122B2

## CPC

- C07H 19/14: Pyrrolo-pyrimidine radicals
- C07H 19/10: with the saccharide radical esterified by phosphoric or polyphosphoric acids
- C07H 19/20: with the saccharide radical esterified by phosphoric or polyphosphoric acids
- C07H 21/04: with deoxyribosyl as saccharide radical
- C12Q 1/6869: Methods for sequencing
- C12Q 1/6876: Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- 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

## Abstract

Disclosed herein, inter alia, are compounds, compositions, and methods of using the same for the sequencing of a nucleic acid.

## Claims

### Claim 1

A method of sequencing a nucleic acid molecule comprising:
(a) incorporating in series a plurality of nucleotides into a primer hybridized to the nucleic acid molecule to create an extension strand, wherein each nucleotide comprises:
(i) a cleavable moiety covalently bound to the 3′ oxygen of the nucleotide, said cleavable moiety having the formula -L1P-S—S—R6P (I); wherein, L1P is a substituted methylene, wherein L1P is substituted with a substituted or unsubstituted alkyl, or a substituted or unsubstituted heteroalkyl; R6P is unsubstituted C1-C4 alkyl; and
(ii) a detectable moiety attached to said nucleotide via a covalent linker, wherein said covalent linker comprises
wherein, R102 is unsubstituted C1-C4 alkyl; and
(b) detecting the detectable label of the incorporated nucleotide to identify the incorporated nucleotide in the extension strand, thereby sequencing the nucleic acid molecule.

### Claim 2 (depends on 1)

The method of claim 1, wherein the plurality of nucleotides comprises four different labeled nucleotides.

### Claim 3 (depends on 1)

The method of claim 1, following incorporation of a nucleotide, the method comprises cleaving the cleavable moiety by contacting the incorporated nucleotide with a reducing agent at about 55° C. to about 65° C.

### Claim 4 (depends on 1)

The method of claim 1, wherein the nucleotide comprises a cytosine or a derivative thereof, guanine or a derivative thereof, adenine or a derivative thereof, thymine or a derivative thereof, uracil or a derivative thereof, hypoxanthine or a derivative thereof, xanthine or a derivative thereof, 7-methylguanine or a derivative thereof, 5,6-dihydrouracil or a derivative thereof, 5-methylcytosine or a derivative thereof, or 5-hydroxymethylcytosine or a derivative thereof.

### Claim 5 (depends on 1)

The method of claim 1, wherein L1P is substituted with unsubstituted alkyl or unsubstituted heteroalkyl.

### Claim 6 (depends on 1)

The method of claim 1, wherein L1P is

### Claim 7 (depends on 1)

The method of claim 1, wherein L1P is

### Claim 8 (depends on 1)

The method of claim 1, wherein-L1P-S—S—R6P is:

### Claim 9 (depends on 1)

The method of claim 1, wherein -L1P-S—S—R6P is:

### Claim 10 (depends on 1)

The method of claim 1, wherein said detectable moiety is a xanthene derivative, cyanine derivative, naphthalene derivative, coumarin derivative, oxadiazole derivative, anthracene derivative, pyrene derivative, oxazine derivative, acridine derivative, arylmethine derivative, or tetrapyrrole derivative.

### Claim 11 (depends on 1)

The method of claim 1, wherein said nucleic acid molecule is in a cell.

### Claim 12 (depends on 1)

The method of claim 1, prior to step (b), the extension strand comprises an incorporated nucleotide having the formula:
wherein
L100 is said covalent linker, “
” is the attachment point to the remainder of the extension strand, and R4 is said detectable moiety.

### Claim 13 (depends on 1)

The method of claim 1, wherein the covalent linker comprises

### Claim 14 (depends on 1)

The method of claim 1, wherein the covalent linker comprises

### Claim 15 (depends on 1)

The method of claim 1, wherein R102 is unsubstituted methyl, unsubstituted ethyl, or unsubstituted propyl.

### Claim 16 (depends on 1)

The method of claim 1, wherein covalent linker is:

### Claim 17 (depends on 1)

The method of claim 1, wherein the covalent linker is

### Claim 18 (depends on 3)

The method of claim 3, wherein the reducing agent is tris(hydroxypropyl)phosphine (THPP), tris-(2-carboxyethyl)phosphine (TCEP), or dithiothreitol (DTT).

### Claim 19 (depends on 1)

The method of claim 1, wherein said detectable moiety comprises a cyanine moiety.