# US 12,760,352 B2 · High-throughput and highly multiplexed imaging with programmable nucleic acid probes

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- Number: US 12,760,352 B2
- Document ID: US12760352B2
- Status: Granted
- Kind: B2
- Application number: 17/811,486
- Filed: 2022-07-08
- Published: 2023-01-26
- Granted: 2026-10-13
- Earliest claimed: 2014-03-11
- Assignees: President and Fellows of Harvard College
- Applicants: President and Fellows of Harvard College
- Inventors: Peng Yin; Sarit Agasti; Xi Chen; Ralf Jungmann
- Web page: https://patentgrep.com/patent/US12760352B2

## CPC

- C12Q 1/6804: Nucleic acid analysis using immunogens (immunoassay G01N33/53)
- C12Q 1/6818: involving interaction of two or more labels, e.g. resonant energy transfer
- C12Q 2537/143: Multiplexing, i.e. use of multiple primers or probes in a single reaction, usually for simultaneously analyse of multiple analysis
- C12Q 2563/179: the label being a nucleic acid
- 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

## Abstract

The present invention provides, inter alia, methods and compositions for imaging, at high spatial resolution, targets of interest.

## Claims

### Claim 1

A method for spatial imaging a plurality of protein targets in situ within a sample comprising cells and/or tissue comprising:
(1) contacting the sample comprising a plurality of targets with a plurality of target-specific binding partners, wherein each of the target-specific binding partners is linked to a docking strand, and wherein target-specific binding partners of different specificity are linked to different docking strands,
(2) contacting the sample with labeled imager strands, wherein the labeled imager strands comprise a sequence that hybridizes to the docking strand a moiety cleavable by a nucleic acid-DNA glycosylase and a fluorescent label,
(3) imaging the sample to detect a fluorescent signal from the hybridized imager strands,
(4) adding a nucleic acid-DNA glycosylase to the sample and,
removing the fluorescent signal from the hybridized imager strands, and
(5) repeating steps (2) to (4), each time with a labeled imager strand having a unique nucleotide sequence relative to all other labeled imager strands, whereby a plurality of targets are imaged in situ.

### Claim 2 (depends on 1)

The method of claim 1, wherein the nucleic acid-DNA glycosylase is a uracil-DNA glycosylase.

### Claim 3 (depends on 1)

The method of claim 1, wherein the labeled imager strands nucleic acids comprise at least one deoxyuridine (dU).

### Claim 4 (depends on 1)

The method of claim 1, wherein the labeled imager strands are labeled identically.

### Claim 5 (depends on 1)

The method of claim 1, wherein the labeled imager strands comprise a distinct label.

### Claim 6 (depends on 1)

The method of claim 1, wherein one or more targets of the plurality are proteins and/or the sample is a cell or tissue sample, a cell lysate, a tissue lysate, or a bodily fluid.

### Claim 7 (depends on 1)

The method of claim 1, wherein in step (5) the sample is imaged using confocal or epi-fluorescence microscopy.

### Claim 8 (depends on 1)

The method of claim 1, wherein the target-specific binding partner is an antibody or an antibody fragment.

### Claim 9 (depends on 1)

The method of claim 1, wherein the target-specific binding partner is a natural or engineered ligand, a small molecule, an aptamer, a peptide or an oligonucleotide.