Granted Patent
B2
US 12,577,611 · App. 18/978,794 · Granted Mar 17, 2026
Methods for in situ transcriptomics and proteomics
Assignee:
Singular Genomics Systems, Inc.
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Abstract
Disclosed herein, inter alia, are compositions and methods of use thereof for interrogating a cell.
Claims (43)
1 . A method of detecting targets of a cell, said method comprising:
(i) binding a first polynucleotide probe to a first nucleic acid molecule in the cell;
forming a first amplification product comprising multiple complements the first polynucleotide probe; and binding a first fluorescently labeled nucleotide to the first amplification product;
(ii) binding a second polynucleotide probe to an oligonucleotide of an antibody-oligonucleotide conjugate, wherein the antibody-oligonucleotide conjugate is attached to a protein in or on said cell; forming a second amplification product comprising multiple complements of the second polynucleotide probe; and binding a second fluorescently labeled nucleotide to the second amplification product;
(iii) binding a fluorescent stain to an organelle of the cell; and
(iv) detecting the targets by detecting the first fluorescently labeled nucleotide, the second fluorescently labeled nucleotide, and the fluorescent stain.
2 . The method of claim 1 , wherein forming a first amplification product and forming a second amplification product comprises rolling circle amplification or exponential rolling circle amplification.
3 . The method of claim 1 , wherein (iv) comprises:
a) detecting the second fluorescently labeled nucleotide;
b) followed by detecting the first fluorescently labeled nucleotide;
c) followed by detecting the fluorescent stain.
4 . The method of claim 1 , wherein the fluorescent stain comprises an ER stain, Golgi stain, F-actin stain, lysosomal stain, mitochondrial stain, nucleolar stain, or plasma membrane stain.
5 . The method of claim 1 , wherein the fluorescent stain is eosin.
6 . The method of claim 1 , wherein binding the first fluorescently labeled nucleotide comprises hybridizing a primer to the first amplification product and incorporating the first fluorescently labeled nucleotide.
7 . The method of claim 6 , wherein the first fluorescently labeled nucleotide comprises a reversible terminator.
8 . The method of claim 7 , further comprising incorporating a plurality of fluorescently labeled nucleotides into the primer, wherein the fluorescent label is detected and the reversible terminator is removed prior to the incorporation of a next nucleotide.
9 . The method of claim 1 , wherein the fluorescently labeled nucleotide forms part of a detection oligonucleotide, and binding a fluorescently labeled nucleotide comprises hybridizing the detection oligonucleotide to the first amplification product.
10 . The method of claim 1 , wherein the cell forms part of a tissue, and said tissue comprises liver tissue, kidney tissue, bone tissue, lung tissue, thymus tissue, adrenal tissue, skin tissue, bladder tissue, colon tissue, spleen tissue, or brain tissue.
11 . The method of claim 10 , wherein said tissue comprises breast tissue, lung tissue, colon tissue, lymph tissue, kidney tissue, bone tissue, or brain tissue.
12 . The method of claim 1 , wherein the first nucleic acid molecule is an RNA molecule.
13 . The method of claim 1 , wherein the first nucleic acid molecule is a DNA molecule.
14 . The method of claim 1 , further comprising identifying the cell based on the detected targets.
15 . The method of claim 1 , wherein (iv) comprises:
a) detecting the first fluorescently labeled nucleotide;
b) followed by detecting the second fluorescently labeled nucleotide;
c) followed by detecting the fluorescent stain.
16 . The method of claim 1 , wherein (iv) comprises:
a) detecting the first fluorescently labeled nucleotide;
b) followed by detecting the fluorescent stain;
c) followed by detecting the second fluorescently labeled nucleotide.
17 . The method of claim 1 , wherein (iv) comprises:
a) detecting the second fluorescently labeled nucleotide;
b) followed by detecting the fluorescent stain;
c) followed by detecting the first fluorescently labeled nucleotide.
18 . The method of claim 1 , wherein (iv) comprises:
a) detecting the fluorescent stain;
b) followed by detecting the first fluorescently labeled nucleotide;
c) followed by detecting the second fluorescently labeled nucleotide.
19 . The method of claim 1 , wherein (iv) comprises:
a) detecting the fluorescent stain;
b) followed by detecting the second fluorescently labeled nucleotide;
c) followed by detecting the first fluorescently labeled nucleotide.
20 . The method of claim 1 , wherein the antibody-oligonucleotide conjugate is bound to CD3, CD4, CD8, FOXP3, TIM-3, PD-1, CTLA-4, Ki67, IFNG, IL-10, IL-17, LAG-3, TIGIT, CD40, GITR, ICOS, OX40, CD25, KLRG1, CD27, CCR7, CXCR5, CD127, or CD39.
Assignments (1)
ASSIGNMENT OF ASSIGNOR'S INTEREST
Recorded Feb 18, 2025
From: GLEZER, ELI N.; CANG, HU; HONG, ZHENMIN
To: SINGULAR GENOMICS SYSTEMS, INC.
Reel/Frame 070246/0636 →
Continuity (10)
Provisional Application
63209886
· Jun 11, 2021
Provisional Application
63140700
· Jan 22, 2021
Provisional Application
63062054
· Aug 6, 2020
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