Granted Patent
B2
US 12,215,122 · App. 18/522,886 · Granted Feb 4, 2025
Nucleotide analogues
Inventors:
Ronald Graham (Carlsbad, CA); Olga Adelfinskaya (San Marcos, CA); Megha Cila (San Diego, CA); Rodrigo Rodriguez (San Diego, CA); Abrehet Abdu (San Diego, CA); Eli N. Glezer (Del Mar, CA)
Assignee:
Singular Genomics Systems, Inc.
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Abstract
Disclosed herein, inter alia, are compounds, compositions, and methods of using the same for the sequencing of a nucleic acid.
Claims (27)
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 -L 1P -S—S—R 6P (I); wherein, L 1P is a substituted methylene, wherein L 1P is substituted with a substituted or unsubstituted alkyl, or a substituted or unsubstituted heteroalkyl; R 6P is unsubstituted C 1 -C 4 alkyl; and
(ii) a detectable moiety attached to said nucleotide via a covalent linker, wherein said covalent linker comprises
wherein, R 102 is unsubstituted C 1 -C 4 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.
2. The method of claim 1 , wherein the plurality of nucleotides comprises four different labeled nucleotides.
3. 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.
4. 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.
5. The method of claim 1 , wherein L 1P is substituted with unsubstituted alkyl or unsubstituted heteroalkyl.
6. The method of claim 1 , wherein L 1P is
7. The method of claim 1 , wherein L 1P is
8. The method of claim 1 , wherein-L 1P -S—S—R 6P is:
9. The method of claim 1 , wherein -L 1P -S—S—R 6P is:
10. 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.
11. The method of claim 1 , wherein said nucleic acid molecule is in a cell.
12. The method of claim 1 , prior to step (b), the extension strand comprises an incorporated nucleotide having the formula:
wherein
L 100 is said covalent linker, “
” is the attachment point to the remainder of the extension strand, and R 4 is said detectable moiety.
13. The method of claim 1 , wherein the covalent linker comprises
14. The method of claim 1 , wherein the covalent linker comprises
15. The method of claim 1 , wherein R 102 is unsubstituted methyl, unsubstituted ethyl, or unsubstituted propyl.
16. The method of claim 1 , wherein covalent linker is:
17. The method of claim 1 , wherein the covalent linker is
18. The method of claim 3 , wherein the reducing agent is tris(hydroxypropyl)phosphine (THPP), tris-(2-carboxyethyl)phosphine (TCEP), or dithiothreitol (DTT).
19. The method of claim 1 , wherein said detectable moiety comprises a cyanine moiety.
Assignments (2)
SECURITY INTEREST
Recorded Mar 7, 2025
From: SINGULAR GENOMICS SYSTEMS, INC.
To: FIRST-CITIZENS BANK & TRUST COMPANY
Reel/Frame 070440/0465 →
ASSIGNMENT OF ASSIGNOR'S INTEREST
Recorded May 20, 2024
From: GRAHAM, RONALD; ADELFINSKAYA, OLGA; CILA, MEGHA; RODRIGUEZ, RODRIGO; ABDU, ABREHET; GLEZER, ELI N.
To: SINGULAR GENOMICS SYSTEMS, INC.
Reel/Frame 067468/0850 →
Continuity (7)
Continuation
17287255
Provisional Application
62841146
· Apr 30, 2019
Provisional Application
62789877
· Jan 8, 2019
Provisional Application
62750552
· Oct 25, 2018
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