# US 12,180,542 B2 · Methods for in SITU transcriptomics and proteomics

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- Number: US 12,180,542 B2
- Document ID: US12180542B2
- Status: Granted
- Kind: B2
- Application number: 17/816,841
- Filed: 2022-08-02
- Published: 2022-12-22
- Granted: 2024-12-31
- Earliest claimed: 2020-08-06
- Assignees: Singular Genomics Systems, Inc.
- Applicants: Singular Genomics Systems, Inc.
- Inventors: Eli N. Glezer; Hu Cang; Zhenmin Hong
- Web page: https://patentgrep.com/patent/US12180542B2

## CPC

- C12Q 1/6841: In situ hybridisation
- C12Q 1/6804: Nucleic acid analysis using immunogens (immunoassay G01N33/53)
- G01N 33/5308: {for analytes not provided for elsewhere, e.g. nucleic acids, uric acid, worms, mites}
- G01N 33/6842: {Proteomic analysis of subsets of protein mixtures with reduced complexity, e.g. membrane proteins, phosphoproteins, organelle proteins}
- G01N 33/53: Immunoassay; Biospecific binding assay; Materials therefor
- G01N 2458/10: Oligonucleotides as tagging agents for labelling antibodies
- G01N 2570/00: Omics, e.g. proteomics, glycomics or lipidomics; Methods of analysis focusing on the entire complement of classes of biological molecules or subsets thereof, i.e. focusing on proteomes, glycomes or lipidomes
- 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
- G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q)

## Abstract

Disclosed herein, inter alia, are compositions and methods of use thereof for interrogating a cell.

## Claims

### Claim 1

A method of monitoring a disease state of a subject; said method comprising:
obtaining a first sample from the subject and obtaining a second sample from the subject at a later time, wherein the first sample and the second sample each comprise a tissue or cell comprising a plurality of proteins associated with the disease state; followed by
concurrently contacting both the first sample and the second sample with a plurality of specific binding reagents;
binding a specific binding reagent to a protein associated with the disease state in the first sample, and binding a specific binding reagent to a protein associated with the disease state in the second sample, wherein each specific binding reagent comprises an oligonucleotide barcode from a known set of oligonucleotide barcodes associated with each protein; and
sequencing each oligonucleotide barcode in said tissue or in or on said cell.

### Claim 2 (depends on 1)

The method of claim 1, further comprising:
i) contacting both the first sample and the second sample with a plurality of oligonucleotide probes and forming a circular probe on each oligonucleotide barcode, wherein forming a circular probe comprises hybridizing a first sequence of an oligonucleotide probe to a first sequence of the oligonucleotide barcode, hybridizing a second sequence of the oligonucleotide probe to a second sequence of the oligonucleotide barcode, and ligating the first sequence of the oligonucleotide probe to the second sequence of the oligonucleotide probe;
ii) amplifying each oligonucleotide barcode to generate amplification products comprising the oligonucleotide barcode, or a complement thereof; and
iii) sequencing the amplification products.

### Claim 3 (depends on 2)

The method of claim 2, wherein the first sample and the second sample further comprise one or more nucleic acid molecules associated with the disease state, the method further comprises:
contacting both the first sample and the second sample with a plurality of detection oligonucleotides and binding a detection oligonucleotide to a nucleic acid molecule associated with the disease state in the first sample, and binding a detection oligonucleotide to a nucleic acid molecule associated with the disease state in the second sample, and sequencing each detection oligonucleotide in said tissue or in or on said cell.

### Claim 4 (depends on 2)

The method of claim 2, wherein each oligonucleotide probe is a single-stranded polynucleotide having a 5′ and a 3′ end.

### Claim 5 (depends on 4)

The method of claim 4, wherein binding each oligonucleotide probe comprises hybridizing the 5′ and 3′ ends of each oligonucleotide probe to two adjacent nucleic acid sequences of each oligonucleotide barcode.

### Claim 6 (depends on 4)

The method of claim 4, wherein binding each oligonucleotide probe to each oligonucleotide barcode comprises hybridizing the 3′ end of the oligonucleotide probe to a first complementary region of the oligonucleotide barcode, and hybridizing the 5′ end of each oligonucleotide probe to a second complementary region of the oligonucleotide barcode, wherein the second complementary region is about 5 or more nucleotides in the 5′ direction with respect to the first complementary region.

### Claim 7 (depends on 6)

The method of claim 6, wherein the first complementary region and the second complementary region of each oligonucleotide barcode are separated by about 5 to about 75 nucleotides.

### Claim 8 (depends on 2)

The method of claim 2, wherein amplifying comprises extending an amplification primer hybridized to the circular probe with a strand-displacing polymerase, wherein the primer extension generates an extension product comprising multiple complements of the circular probe.

### Claim 9 (depends on 1)

The method of claim 1, wherein the specific binding reagent comprises an antibody, single-chain Fv fragment (scFv), antibody fragment-antigen binding (Fab), or an aptamer.

### Claim 10 (depends on 2)

The method of claim 2, wherein sequencing comprises hybridizing a sequencing primer to a nucleic acid molecule comprising the oligonucleotide barcode, or a complement thereof, and contacting the sequencing primer with a sequencing solution comprising one or more modified nucleotides comprising a reversible terminator, and monitoring sequential incorporation of complementary nucleotides to generate one or more sequencing reads, wherein the reversible terminator is removed prior to the introduction of the next complementary nucleotide.

### Claim 11 (depends on 1)

The method of claim 1, wherein each sample comprises a plurality of cells.

### Claim 12 (depends on 11)

The method of claim 11, wherein the plurality of cells are permeabilized and immobilized to a solid support surface.

### Claim 13 (depends on 12)

The method of claim 12, wherein the surface comprises a patterned surface suitable for immobilization of a plurality of cells in an ordered pattern.

### Claim 14 (depends on 1)

The method of claim 1, wherein the method does not comprise lysing the cell.

### Claim 15 (depends on 1)

The method of claim 1, wherein the second sample is obtained about 1 to about 10 days after obtaining the first sample.

### Claim 16 (depends on 1)

The method of claim 1, wherein the second sample is obtained about 2 to about 56 weeks after obtaining the first sample.

### Claim 17 (depends on 1)

The method of claim 1, wherein prior to obtaining the second sample, the method further comprises administering a therapeutically effective amount of a drug to the subject.

### Claim 18 (depends on 1)

The method of claim 1, wherein the proteins associated with the disease state comprise an MDC, NME-2, KGF, PlGF, Flt-3L, HGF, MCP1, SAT-1, MIP-1-b, GCLM, OPG, TNF RII, VEGF-D, ITAC, MMP-10, GPI, PPP2R4, AKRIB1, Amy1A, MIP-1b, P-Cadherin, or an EPO protein.

### Claim 19

A method of monitoring a disease state of a subject; said method comprising:
obtaining a first sample from the subject and obtaining a second sample from the subject at a later time, wherein the first sample and the second sample each comprise a tissue or cell comprising one or more nucleic acid molecules associated with the disease state and one or more proteins associated with the disease state; followed by
contacting both the first sample and the second sample with a plurality of circularizable probes and binding a circularizable probe to a nucleic acid molecule associated with the disease state in the first sample and binding a circularizable probe to a nucleic acid molecule associated with the disease state in the second sample, wherein each circularizable probe comprises a sequence complementary to the nucleic acid molecule associated with a disease state, a primer binding sequence, and a barcode sequence; amplifying each circularizable probe to generate amplification products comprising the barcode sequence, or a complement thereof; and
sequencing the amplification products in or on said tissue or cell; and
concurrently contacting both the first sample and the second sample with a plurality of specific binding reagents and binding a specific binding reagent to a protein associated with the disease state, wherein each specific binding reagent comprises an oligonucleotide barcode; and
sequencing each oligonucleotide barcode in or on said tissue or cell.

### Claim 20 (depends on 19)

The method of claim 19, wherein the one or more nucleic acids associated with the disease comprise a ABL1, AKT1, ALK, APC, ATM, BRAF, CDH1, CDKN2A, CSFIR, CTNNB1, EGFR, ERBB2, ERBB4, EZH2, FBXW7, FGFR1, FGFR2, FGFR3, FLT3, GNA11, GNAQ, GNAS, HNF1A, HRAS, IDH1, IDH2, JAK2, JAK3, KDR, KIT, KRAS, MET, MLH1, MPL, NOTCH1, NPM1, NRAS, PDGFRA, PIK3CA, PTEN, PTPN11, RB1, RET, SMAD4, SMARCB1, SMO, SRC, STK11, TP53, or VHL gene.

### Claim 21

A kit, comprising:
i) a solid support;
ii) a first antibody-oligonucleotide conjugate, comprising a first oligonucleotide barcode and a second antibody-oligonucleotide conjugate comprising a second oligonucleotide barcode, wherein the first and second oligonucleotide barcodes comprise different sequences;
iii) a first oligonucleotide probe complementary to the first oligonucleotide barcode comprising a first primer binding sequence and a second oligonucleotide probe complementary to the second oligonucleotide barcode comprising a second primer binding sequence; and
iv) a first primer comprising a sequence complementary to the first primer binding sequence and a second primer comprising a sequence complementary to the second primer binding sequence.

### Claim 22

A plurality of samples from a subject, wherein each sample comprises a tissue or cell and is obtained from the subject at a different time, each sample comprising:
a) one or more nucleic acid molecules each bound to a polynucleotide probe in said tissue or cell, wherein the polynucleotide probe is a circular polynucleotide comprising a first primer binding sequence and a first barcode sequence, wherein the first barcode sequence is from a known set of barcodes associated with the bound nucleic acid molecule; and
b) one or more proteins each bound to a specific binding reagent in said tissue or in or on said cell, wherein the specific binding reagent comprises an oligonucleotide comprising a second primer binding sequence and a second barcode sequence associated with the specific binding reagent, wherein the second barcode sequence is from a known set of barcodes associated with the bound protein.

### Claim 23 (depends on 1)

The method of claim 1, wherein said second sample is obtained 1 to 30 days later.

### Claim 24 (depends on 1)

The method of claim 1, wherein the cell comprising one or more proteins associated with the disease state is a non-adherent cell.

### Claim 25 (depends on 1)

The method of claim 1, further comprising contacting the cell with a cell-permeable dye and imaging the cell.

### Claim 26 (depends on 1)

The method of claim 1, wherein the proteins associated with the disease state comprise a 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 a CD39 protein.

### Claim 27 (depends on 1)

The method of claim 1, further comprising amplifying each oligonucleotide barcode to generate amplification products in or on a cell; and binding labeled probes to the amplification products and detecting the labeled probes in or on the cell.

### Claim 28 (depends on 1)

The method of claim 1, wherein each oligonucleotide barcode is 1 nucleotide.

### Claim 29 (depends on 1)

The method of claim 1, wherein sequencing comprises:
amplifying each oligonucleotide barcode to generate a nucleic acid molecule comprising the oligonucleotide barcode, or a complement thereof;
hybridizing a sequencing primer to the nucleic acid molecule;
incorporating a labeled nucleotide into the sequencing primer; and
detecting the labeled nucleotide.

### Claim 30

A method of monitoring samples, said method comprising:
depositing a first sample at a first location on a solid support and depositing a second sample at a second location on the solid support, wherein the first sample comprises a plurality of cells and the second sample comprises a plurality of cells; fixing the plurality of cells of the first sample; fixing the plurality of cells of the second sample at a later time;
contacting the first sample and the second sample with a plurality of specific binding reagents;
binding a specific binding reagent to a protein in the first sample, and binding a specific binding reagent to a protein in the second sample, wherein each specific binding reagent comprises an oligonucleotide barcode from a known set of oligonucleotide barcodes; and
sequencing each oligonucleotide barcode in each sample.

### Claim 31 (depends on 30)

The method of claim 30, wherein fixing comprises contacting the plurality of cells sample with ethanol, methanol, 2-propanol, acetone, glyoxal, formaldehyde, paraformaldehyde, glutaraldehyde, or a mixture thereof.

### Claim 32 (depends on 30)

The method of claim 30, prior to fixing the plurality of cells of the second sample, the method comprises contacting the second sample with a genetically modifying agent.

### Claim 33 (depends on 32)

The method of claim 32, wherein the genetically modifying agent is a virus.

### Claim 34 (depends on 32)

The method of claim 32, wherein the genetically modifying agent is a pharmaceutical agent.

### Claim 35 (depends on 30)

The method of claim 30, further comprising contacting the first sample and the second sample with a stain and detecting the stain, wherein the stain comprises an ER stain, Golgi stain, F-actin stain, lysosomal stain, mitochondrial stain, nucleolar stain, or plasma membrane stain.