IP Library › Granted Patent US 12,385,923
Granted Patent B2
US 12,385,923 · App. 17/294,281 · Granted Aug 12, 2025

Cleavable fluorescent tyramide for sensitive and multiplexed analysis of biological samples

Inventors: Jia Guo (Tempe, AZ); Manas Mondal (Tempe, AZ); Renjie Liao (Tempe, AZ); Lu Xiao (Tempe, AZ)
Assignee: Arizona Board of Regents on behalf of Arizona State University
G01N33/582G01N21/64G01N2021/6439
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Quick Facts
Patent No.
US 12,385,923
App. No.
17/294,281
Granted
Aug 12, 2025
Kind
B2
Abstract

Provided herein are methods for multiplexed in situ analysis of biomolecules in a tissue. In particular, provided herein are methods for multiplexed single-cell in situ protein and nucleic acid profiling in fixed or fresh tissues, and also allows the investigation of the different cell compositions and their spatial organizations in intact tissues through consecutive cycles of probe hybridization, fluorescence imaging, and signal removal.

Claims (17)

1. A method of multiplexed in situ analysis of biomolecules in a tissue, wherein the tissue comprises a plurality of biomolecules, the method comprising the following steps:

(a) performing a first contacting step comprising contacting the tissue with a plurality of horseradish peroxidase (HRP)-conjugated targeting agents that are configured to specifically bind or hybridize to a target biomolecule in the contacted tissue, wherein the first contacting step occurs under conditions that promote binding or hybridization of the targeting agents to the target biomolecule;

(b) performing a second contacting step comprising contacting the tissue with a cleavable detectably-labeled tyramide, wherein the second contacting step occurs under conditions that promote conjugation of the cleavable detectably-labeled tyramide to the target biomolecule;

wherein the cleavable detectably-labeled tyramide consists of, in order, a tyramide, a cleavable linker, and a detectable label;

(c) imaging the tissue after the second contacting step whereby a detectable signal generated from an interaction of HRP-conjugated targeting agents with the cleavable detectably-labeled tyramide is detected;

(d) only after the imaging step of (c), cleaving, at the cleavable linker, the detectable label from the detectably-labeled tyramide; and

(e) optionally consecutively repeating a cycle comprising: the first contacting step, second contacting step, imaging step, and cleaving step,

wherein the first contacting step of each consecutive cycle utilizes a new plurality of HRP-conjugate targeting agents that bind or hybridize to a different target biomolecule.

2. The method of claim 1 , wherein the plurality of biomolecules comprises proteins, RNA, or DNA, or a combination thereof.

3. The method of claim 1 , wherein the HRP-conjugated targeting agents are HRP-conjugated antibodies or HRP-conjugated oligonucleotides, or a combination thereof.

4. The method of claim 1 , wherein the cleavable detectably-labeled tyramide comprises a fluorophore.

5. The method of claim 4 , wherein the fluorophore is selected from the group consisting of Cy5, TAMRA, (13-[2-carboxy-(4 or 5)-(2,5-dioxopyrrolidin-1-yl)oxycarbonylphenyl]-6,7,7,19,19,20-hexamethyl-17-(sulfomethyl)-2-oxa-20-aza-6-azoniapentacyclo[12.8.0.03,12.05,10.016,21]docosa-1 (14),3,5,8,10,12,15,17,21-nonaen-9-yl) methanesulfonate (ALEXA FLUOR™ 594), ATTO 647N, and ATTO 700.

6. The method of claim 1 , wherein the cleavable detectably-labeled tyramide is a compound having formula II:

7. The method of claim 1 , wherein cleaving the detectable label from the cleavable detectably-labeled tyramide comprises chemically cleaving the detectable label.

8. The method of claim 1 , further comprising washing to remove unhybridized targeting agents and non-specifically hybridized targeting agents following the second contacting step.

9. The method of claim 1 , wherein the plurality of targeting agents comprises HRP-conjugated synthetic DNA oligonucleotide probes.

10. The method of claim 1 , wherein the plurality of targeting agents comprises HRP-conjugated polyclonal antibodies, HRP-conjugated monoclonal antibodies, or HRP-conjugated antigen-binding fragments thereof.

Assignments (2)
CONFIRMATORY LICENSE Recorded Dec 5, 2023
From: ARIZONA STATE UNIVERSITY, TEMPE CAMPUS
To: NATIONAL INSTITUTES OF HEALTH (NIH), U.S. DEPT. OF HEALTH AND HUMAN SERVICES (DHHS), U.S. GOVERNMENT
Reel/Frame 065774/0432 →
ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded Aug 15, 2023
From: GUO, JIA; MONDAL, MANAS; LIAO, RENJIE; XIAO, LU
To: ARIZONA BOARD OF REGENTS ON BEHALF OF ARIZONA STATE UNIVERSITY
Reel/Frame 064588/0817 →
Continuity (2)
Provisional Application 62767630 · Nov 15, 2018
Related Publication 20220026433A1 · Jan 27, 2022
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