Granted Patent B2

US 12,486,532 · App. 18/645,232 · Granted Dec 2, 2025

Inventor: Eli N. Glezer (Del Mar, CA)

Assignee: Singular Genomics Systems, Inc.

C12Q1/6841

Abstract

Claims (52)

1 . A method of detecting molecules in a tissue, said method comprising:

(i) binding a first polynucleotide probe to a first nucleic acid molecule in said tissue and incorporating a sequence of the first nucleic acid molecule into the first polynucleotide probe; forming a first amplification product comprising multiple complements of the first polynucleotide probe; and binding a first primer to the first amplification product and incorporating a first fluorescently labeled nucleotide into the first primer;

(ii) binding a second polynucleotide probe to an oligonucleotide of an antibody-oligonucleotide conjugate, wherein said antibody-oligonucleotide conjugate is attached to a protein in said tissue; forming a second amplification product comprising multiple complements of the second polynucleotide probe; and binding a second primer to the second amplification product and incorporating a second fluorescently labeled nucleotide into the second primer;

(iii) contacting the tissue with a stain; and

(iv) detecting the first fluorescently labeled nucleotide, the second fluorescently labeled nucleotide, and the stain.

2 . The method of claim 1 , wherein 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.

3 . The method of claim 1 , wherein said tissue comprises breast tissue, lung tissue, colon tissue, lymph tissue, kidney tissue, bone tissue, tonsil tissue, or brain tissue.

4 . The method of claim 1 , wherein the first fluorescently labeled nucleotide comprises a reversible terminator moiety.

5 . The method of claim 4 , further comprising incorporating a plurality of fluorescently labeled nucleotides into the first primer, wherein the label is detected and the reversible terminator is removed prior to the incorporation of a next nucleotide.

6 . The method of claim 1 , wherein the first nucleic acid molecule is an RNA molecule.

7 . The method of claim 6 , wherein the RNA molecule encodes for a chimeric antigen receptor.

8 . The method of claim 6 , wherein the RNA molecule comprises a T cell receptor alpha variable (TRAV) gene sequence, T cell receptor alpha joining (TRAJ) gene sequence, T cell receptor alpha constant (TRAC) gene sequence, T cell receptor beta variable (TRBV) gene sequence, T cell receptor beta diversity (TRBD) gene sequence, T cell receptor beta joining (TRBJ) gene sequence, T cell receptor beta constant (TRBC) gene sequence, T cell receptor gamma variable (TRGV) gene sequence, T cell receptor gamma joining (TRGJ) gene sequence, T cell receptor gamma constant (TRGC) gene sequence, T cell receptor delta variable (TRDV) gene sequence, T cell receptor delta diversity (TRDD) gene sequence, T cell receptor delta joining (TRDJ) gene sequence, or T cell receptor delta constant (TRDC) gene sequence.

9 . The method of claim 6 , wherein the RNA molecule comprises a T cell receptor alpha (TCRA) gene sequence or a T cell receptor beta (TCRB) gene sequence.

10 . The method of claim 1 , wherein the first nucleic acid molecule is a DNA molecule.

11 . The method of claim 1 , wherein the stain comprises an ER stain, Golgi stain, F-actin stain, lysosomal stain, mitochondrial stain, nucleolar stain, or plasma membrane stain.

12 . The method of claim 1 , wherein the stain is eosin.

13 . 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 stain.

14 . The method of claim 1 , wherein (iv) comprises:

a) detecting the first fluorescently labeled nucleotide;

b) followed by detecting the stain;

c) followed by detecting the second fluorescently labeled nucleotide.

15 . 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 stain.

16 . The method of claim 1 , wherein (iv) comprises:

a) detecting the second fluorescently labeled nucleotide;

b) followed by detecting the stain;

c) followed by detecting the first fluorescently labeled nucleotide.

17 . The method of claim 1 , wherein (iv) comprises:

a) detecting the stain;

b) followed by detecting the first fluorescently labeled nucleotide;

c) followed by detecting the second fluorescently labeled nucleotide.

18 . The method of claim 1 , wherein (iv) comprises:

a) detecting the stain;

b) followed by detecting the second fluorescently labeled nucleotide;

c) followed by detecting the first fluorescently labeled nucleotide.

19 . 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.

20 . The method of claim 1 , wherein binding a first polynucleotide probe to a first nucleic acid molecule in said tissue and incorporating a sequence of the first nucleic acid molecule into the first polynucleotide probe comprises:

hybridizing a first hybridization sequence of the first polynucleotide probe to a first sequence of the first nucleic acid molecule, and hybridizing a second hybridization sequence of the first polynucleotide probe to a second sequence of the first nucleic acid molecule, wherein said first nucleic acid molecule comprises a target sequence between the first sequence and the second sequence and extending the first polynucleotide probe along the target sequence to generate a complement of the target sequence, and ligating the complement of the target sequence to the first hybridization sequence thereby forming a circular oligonucleotide.

21 . 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 and incorporating a sequence of the first nucleic acid molecule into the first polynucleotide probe; forming a first amplification product comprising multiple complements the first polynucleotide probe; and binding a first primer to the first amplification product and incorporating a first fluorescently labeled nucleotide into the first primer;

(ii) binding a second polynucleotide probe to an oligonucleotide of an antibody-oligonucleotide conjugate, wherein the antibody-oligonucleotide conjugate is attached to a protein on said cell; forming a second amplification product comprising multiple complements of the second polynucleotide probe; and binding a second primer to the second amplification product and incorporating a second fluorescently labeled nucleotide into the second primer;

(iii) binding a fluorescent stain to an organelle of the cell; and

(iv) detecting the first fluorescently labeled nucleotide, the second fluorescently labeled nucleotide, and the fluorescent stain.

22 . The method of claim 21 , further comprising identifying the cell based on the detected targets.

23 . The method of claim 21 , wherein the sequence of the first nucleic acid molecule is introduced to the cell using a gene editing enzyme.

24 . The method of claim 1 , wherein the sequence of the first nucleic acid molecule is 1 nucleotide.

25 . The method of claim 1 , wherein the sequence of the first nucleic acid is 6 to 12 nucleotides.

Assignments (2)

Continuity (9)

References Cited (284)