Granted Patent B2

US 12,595,508 · App. 18/598,995 · Granted Apr 7, 2026

Inventors: Ronald Graham (Carlsbad, CA); Olga Adelfinskaya (San Marcos, CA); Megha Cila (San Diego, CA); Rodrigo Rodriguez (San Diego, CA)

Assignee: Singular Genomics Systems, Inc.

C12Q1/6869C07F9/65515C07H19/06C07H19/10C07H19/14C07H19/16C07H19/20C12N9/1241C12Q1/6806C12Q2334/40C12Q2521/101C12Q2525/101C12Q2535/00

Abstract

Claims (27)

1 . A nucleotide comprising a nucleobase and a cleavable linker, wherein said cleavable linker covalently links a detectable moiety to the nucleobase, wherein said cleavable linker comprises a thio-trigger moiety having the formula

wherein

X is —O—, —NH—, or —S—;

R 100 is —SR 102 or —CN; and

R 102 and R 102a are independently hydrogen, halogen, —CCl 3 , —CBr 3 , —CF 3 , —CI 3 , —CHCl 2 , —CHBr 2 , —CHF 2 , —CHI 2 , —CH 2 Cl, —CH 2 Br, —CH 2 F, —CH 2 I, —CN, —OH, —NH 2 , —COOH, —CONH 2 , —NO 2 , —SH, —SO 3 H, —SO 4 H, —SO 2 NH 2 , —NHNH 2 , —ONH 2 , —NHC(O)NHNH 2 , —NHC(O)NH 2 , —NHSO 2 H, —NHC(O)H, —NHC(O)OH, —NHOH, —OCCl 3 , —OCF 3 , —OCBr 3 , —OCI 3 , —OCHCl 2 , —OCHBr 2 , —OCHI 2 , —OCHF 2 , —OCH 2 Cl, —OCH 2 Br, —OCH 2 I, —OCH 2 F, —N 3 , —SF 5 , substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl.

2 . The nucleotide of claim 1 , wherein R 100 is —CN.

3 . The nucleotide of claim 1 , wherein R 100 is —SR 102 .

4 . The nucleotide of claim 3 , wherein R 102 is halogen, —CCl 3 , —CBr 3 , —CF 3 , —CI 3 , —CHCl 2 , —CHBr 2 , —CHF 2 , —CHI 2 , —CH 2 Cl, —CH 2 Br, —CH 2 F, —CH 2 I, —CN, —OH, —NH 2 , —COOH, —CONH 2 , —NO 2 , —SH, —SO 3 H, —SO 4 H, —SO 2 NH 2 , —NHNH 2 , —ONH 2 , —NHC(O)NHNH 2 , —NHC(O)NH 2 , —NHSO 2 H, —NHC(O)H, —NHC(O)OH, —NHOH, —OCCl 3 , —OCF 3 , —OCBr 3 , —OCI 3 , —OCHCl 2 , —OCHBr 2 , —OCHI 2 , —OCHF 2 , —OCH 2 Cl, —OCH 2 Br, —OCH 2 I, —OCH 2 F, —N 3 , —SF 5 , substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl.

5 . The nucleotide of claim 3 , wherein R 102 is substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl.

6 . The nucleotide of claim 3 , wherein R 102 is unsubstituted alkyl, unsubstituted heteroalkyl, unsubstituted cycloalkyl, unsubstituted heterocycloalkyl, unsubstituted aryl, or unsubstituted heteroaryl.

7 . The nucleotide of claim 3 , wherein R 102 is unsubstituted alkyl.

8 . The nucleotide of claim 3 , wherein R 102 is substituted or unsubstituted C 1 -C 4 alkyl.

9 . The nucleotide of claim 1 , wherein X is —O—.

10 . The nucleotide of claim 1 , wherein X is —NH—.

11 . The nucleotide of claim 1 , wherein X is —S—.

12 . The nucleotide of claim 1 , further comprising a reversible terminator moiety.

13 . The nucleotide of claim 1 , wherein the thio-trigger moiety has the formula:

14 . The nucleotide of claim 1 , wherein the thio-trigger moiety has the formula:

15 . The nucleotide of claim 1 , wherein the detectable moiety is a fluorescent dye moiety.

16 . The nucleotide of claim 1 , wherein the detectable moiety comprises the formula:

17 . A method of detecting a nucleic acid molecule, said method comprising:

contacting a primer hybridized to the nucleic acid molecule with a nucleotide of claim 1 ;

incorporating the nucleotide into the primer with a polymerase; and

detecting the detectable label, thereby detecting the nucleic acid molecule.

18 . The method of claim 17 , further comprising cleaving the linker with a cleaving reagent.

19 . The method of claim 18 , wherein said cleaving agent is Tris-(2-carboxyethyl)phosphines trisodium salt (TCEP), tris(hydroxypropyl)phosphine (THPP), guanidine, urea, cysteine, 2-mercaptoethylamine, or dithiothreitol (DTT).

20 . The method of claim 18 , wherein said cleaving agent is Tris-(2-carboxyethyl)phosphines trisodium salt (TCEP) or tris(hydroxypropyl)phosphine.

Assignments (2)

Continuity (4)

References Cited (212)