Cleavable Tetrazine Click Chemistry for Multiplexed Protein Imaging
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Solution Overview
Problem
Current multiplexed protein imaging technologies suffer from low detection sensitivity and multiplexing capacity, making it difficult to perform highly sensitive and multiplexed in situ protein profiling using off-the-shelf antibodies.
Innovation Solution
A system and method utilizing a tyramide conjugated to trans-cylooctene (TCO-tyramide) and a cleavable detectably-labeled tetrazine, such as tetrazine-(N3)n-Cy5, for multiplexed in situ protein analysis through click chemistry and reiterative cycles of target staining, fluorescence imaging, and fluorophore cleavage.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If current multiplexed protein imaging technologies are used, then protein detection is possible, but detection sensitivity is low
Solution Approach 1:
The imaging process is divided into sequential cycles, each dedicated to detecting a specific protein target. In each cycle, HRP-conjugated antibodies specific to one protein bind to the target, followed by addition of TCO-tyramide and cleavable fluorescent tetrazine. After imaging, the fluorescent signal is cleaved and HRP is deactivated, allowing the next protein to be detected in a new cycle. This segmentation enables high sensitivity for each protein while maintaining multiplexing capacity across many different proteins.
Solution Approach 2:
The method employs periodic cycles of protein detection, signal cleavage, and HRP deactivation. Each cycle repeats the same sequence of operations for different proteins: antibody binding, tyramide conjugation, fluorescent labeling, imaging, signal removal, and enzyme inactivation. This periodic action allows systematic detection of multiple proteins with high sensitivity in each cycle while preventing signal carryover to subsequent cycles.
2Adaptability or versatility
If current multiplexed protein imaging technologies are used, then protein profiling is possible, but multiplexing capacity is limited
Solution Approach 1:
The system uses universal components that can detect multiple different proteins: HRP-conjugated antibodies can be raised against any protein of interest, TCO-tyramide serves as a universal substrate for HRP catalysis, and the cleavable fluorescent tetrazine acts as a universal fluorescent reporter. This universality allows the same basic protocol to be applied to detect dozens of different proteins by simply changing the antibody, without requiring different detection chemistry for each protein.
Solution Approach 2:
After imaging, the fluorescent signal from the tetrazine is chemically cleaved and removed from the tissue section, and HRP is simultaneously deactivated. This discarding of the fluorescent signal and enzyme activity allows the tissue to be prepared for detection of the next protein in the sequence without carryover signal or enzyme activity interfering with subsequent detection cycles.
3Measurement precision
If conventional imaging methods are used, then protein detection is achieved, but detection sensitivity is insufficient for single-molecule level
Solution Approach 1:
The method introduces HRP-conjugated antibodies as intermediaries that bind to the protein target and catalyze the conversion of TCO-tyramide into reactive species that covalently bind to nearby tyrosine residues. This intermediary mechanism amplifies the signal from each protein molecule, transforming single-molecule protein detection into detectable fluorescent signals while maintaining specificity through the antibody-target interaction.
Solution Approach 2:
The detection system combines multiple functional components into a composite labeling architecture: HRP enzyme, TCO-tyramide substrate, cleavable fluorescent tetrazine reporter, and protein target. This composite material approach integrates signal amplification, specific binding, fluorescent signaling, and signal removal functions into a single coordinated system that achieves single-molecule sensitivity.
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 highly sensitive and multiplexed quantification of numerous proteins and nucleic acids in individual cells of intact tissues with single-molecule sensitivity, allowing for detailed protein expression profiling and cell clustering.
Implementation Method 1
contacting a tissue sample with a first plurality of horseradish peroxidase (HRP)-conjugated targeting agents that are configured to specifically bind to or hybridize to a first target biomolecule in the tissue sample
Implementation Method 2
contacting the tissue sample with the compound of Formula I under conditions that promote conjugation of the compound to the target biomolecule
Implementation Method 3
A system and method utilizing a tyramide conjugated to trans-cylooctene (TCO-tyramide) and a cleavable detectably-labeled tetrazine, such as tetrazine-(N3)n-Cy5, for multiplexed in situ protein analysis through click chemistry
Implementation Method 4
a detectable marker, a cleavable linker, and a tetrazine residue
Data Source
AI summary
Disclosed herein are compositions, systems, platforms, and kits comprising a tyramide-TCO and a tetrazine molecule linked to a detectable marker. Also disclosed are methods of using the compositions, systems, platforms, and kits to detect one or more target molecules, such as proteins or nucleic acids, in a sample.