Cleavable Fluorescent Tyramide for Multiplexed Tissue Profiling
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Solution Overview
Problem
Current methods for in situ analysis of proteins and nucleic acids in tissues are limited by their inability to comprehensively profile multiple biomolecules simultaneously with high sensitivity and specificity, often missing low-copy number transcripts and requiring cumbersome antibody-tag conjugation processes.
Innovation Solution
A cleavable detectably-labeled tyramide (CLT) system using horseradish peroxidase (HRP)-conjugated targeting agents and a combination of 1,3,5-Triaza-7-phosphaadamantane (PTA) and tris(2-carboxyethyl)phosphine (TCEP) for multiplexed imaging, allowing for repeated cycles of staining, imaging, and signal removal to quantify numerous biomolecules in intact tissues with single-molecule sensitivity.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If conventional immunohistochemistry and immunofluorescence methods are used, then protein detection is achieved, but only a handful of proteins can be detected simultaneously and sensitivity is limited
Solution Approach 1:
The patent segments the detection process into multiple cycles, where in each cycle a specific protein target is detected using HRP-conjugated antibodies and cleavable fluorescent tyramide. After imaging, the fluorescent signal is removed and the process is repeated for the next protein target. This segmentation enables sequential detection of multiple proteins in the same tissue sample, increasing the number of detectable proteins while maintaining high sensitivity through signal amplification in each cycle.
Solution Approach 2:
The patent employs parameter changes by using different detectable markers (fluorophores) for different protein targets and by dynamically adjusting the detection cycle parameters. The cleavable fluorescent tyramide allows the fluorescent signal to be removed after imaging, resetting the system for the next detection cycle. This dynamic parameter adjustment enables multiplexed detection of numerous proteins while maintaining single-molecule sensitivity through controlled signal amplification and removal.
2Adaptability or versatility
If multiple proteins are detected using conventional methods, then comprehensive profiling is achieved, but the process is time-consuming and complex
Solution Approach 1:
The patent implements continuity of useful action by establishing a rapid cyclic process where protein detection, imaging, fluorescent signal removal, and HRP deactivation occur in continuous cycles. The cleavable fluorescent tyramide enables quick signal removal without disrupting the tissue structure, allowing the next detection cycle to begin immediately. This continuous cycling through multiple protein detections significantly reduces total imaging time compared to sequential conventional methods while maintaining comprehensive profiling capability.
Solution Approach 2:
The patent applies discarding and recovering by removing the fluorescent signal from the tyramide after imaging using PTA and TCEP, which cleave the fluorophore-tyramide bond. This signal removal (discarding) allows the same tissue sample to be reused for detecting the next protein target without carryover fluorescence. The HRP enzyme is also deactivated during this process, recovering the system for the next detection cycle. This reuse of the same tissue sample across multiple cycles dramatically reduces time loss.
3Measurement precision
If high sensitivity detection is achieved, then single-molecule detection is possible, but the ability to detect low-copy number transcripts is limited
Solution Approach 1:
The patent applies preliminary action by using HRP-conjugated antibodies that first bind to the target protein, followed by the addition of cleavable fluorescent tyramide. The HRP enzyme catalyzes the formation of a fluorescent signal at the exact location of the target molecule before imaging occurs. This preliminary signal generation amplifies the detection capability, allowing single-molecule sensitivity even for low-copy number transcripts that would be undetectable by conventional direct fluorescence methods.
Solution Approach 2:
The patent uses an intermediary approach by introducing HRP-conjugated antibodies and cleavable fluorescent tyramide as intermediaries between the target protein and the detectable fluorescent signal. The HRP enzyme acts as a catalyst that converts the target protein binding event into an amplified fluorescent signal. This intermediary signal amplification mechanism enables detection of single molecules and low-copy number transcripts while maintaining the spatial information of the original target in the tissue context.
4Adaptability or versatility
If repeated detection cycles are performed, then multiple proteins are profiled, but protein antigenicity may be compromised
Solution Approach 1:
The patent applies taking out by selectively removing only the fluorescent signal component (fluorophore) from the tyramide using PTA and TCEP, while leaving the tissue structure and protein antigens intact. The cleavage reaction specifically targets the fluorophore-tyramide bond, extracting the fluorescent signal for removal while preserving the underlying protein epitopes and tissue architecture. This selective extraction enables repeated detection cycles without compromising protein antigenicity, as the actual protein targets remain undamaged after each cycle.
Solution Approach 2:
The patent uses a disposable approach by employing cleavable fluorescent tyramide that serves its detection function in one cycle and is then discarded (cleaved) to remove the fluorescent signal. The tyramide itself remains in the tissue but its fluorescent functionality is eliminated through cleavage. This disposable fluorescent signal approach allows multiple detection cycles without requiring replacement of the tissue sample or concern for signal carryover, maintaining protein antigenicity while enabling comprehensive multiplexed profiling.
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
Enables high-throughput, sensitive, and accurate multiplexed protein and nucleic acid profiling in situ, maintaining protein antigenicity and reducing imaging time, with potential for profiling hundreds of proteins in a single specimen, enhancing understanding of complex biological systems and disease diagnosis.
Implementation Method 1
contacting the tissue with the cleavable detectably-labeled tyramide (CLT) compound of Formula I, under conditions that promote conjugation of the cleavable labeled tyramide to the target biomolecule
Implementation Method 2
contacting the tissue sample with a composition comprising 1,3,5-Triaza-7-phosphaadamantane (PTA) and tris(2-carboxyethyl)phosphine (TCEP)
Implementation Method 3
Exemplary detectable makers include, without limitation, fluorophores, luminescent agents (e.g., chemiluminescent agents), fluorescent proteins, and radioisotopes
Data Source
AI summary
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.