Cleavable Fluorescent Tyramide for Low-Copy Multiplexed Tissue Detection
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Solution Overview
Problem
Existing methods for in situ analysis of proteins and nucleic acids in tissues are limited in their ability to detect a small number of molecules and often miss low-copy transcripts, lacking sensitivity and multiplexing capabilities.
Innovation Solution
A method utilizing cleavable fluorescent tyramide (CFT) for multiplexed analysis, involving cycles of target staining, fluorescence imaging, and fluorophore cleavage with HRP-conjugated agents, allowing detection of multiple biomolecules with single-molecule sensitivity.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If conventional immunohistochemistry and immunofluorescence methods are used, then the analysis can be performed with simple procedures, but only a handful of proteins can be detected in one tissue sample
Solution Approach 1:
The patent implements periodic action through cyclic imaging rounds where fluorescent labels are sequentially added, imaged, and then removed. Each cycle detects a new target biomolecule, enabling multiplexed detection of many proteins and nucleic acids in the same tissue sample without permanent label accumulation, thus resolving the contradiction between detection capacity and procedural complexity
Solution Approach 2:
The patent introduces cleavable fluorescent labels as intermediaries that temporarily mark target biomolecules during imaging and can be selectively removed after each imaging round. This intermediary mechanism enables repeated detection cycles with different targets, significantly increasing the number of biomolecules detectable in a single tissue sample while maintaining manageable procedural complexity
2Measurement precision
If conventional imaging methods are used, then the procedure is simple, but transcripts present at low copy numbers are missed
Solution Approach 1:
The patent applies preliminary action by performing target-specific enrichment and amplification steps before the actual imaging detection. This includes hybridization of probes to target transcripts and incorporation of cleavable fluorescent labels, which enhances the signal from low-copy transcripts to detectable levels while preserving the ability to perform multiple detection cycles
Solution Approach 2:
The cleavable fluorescent label serves as an intermediary that amplifies the detection signal for low-copy transcripts. The label is incorporated during a pre-imaging preparation step, allowing sensitive detection of rare transcripts, and can be removed after imaging to enable subsequent detection of other targets, thus improving measurement precision without prohibitive complexity
3Adaptability or versatility
If multiple biomolecules are detected simultaneously, then comprehensive profiling is achieved, but the complexity of the method increases
Solution Approach 1:
The patent uses periodic action to achieve multiplexing by cycling through multiple imaging rounds, each detecting a different target biomolecule. Fluorescent labels are added, imaged, and removed in repeated cycles, allowing comprehensive detection of many proteins and nucleic acids sequentially rather than simultaneously, thus maintaining operational simplicity while achieving high multiplexing capability
Solution Approach 2:
The cleavable fluorescent label acts as a removable intermediary that enables multiplexed detection. Each label type is introduced as an intermediary for a specific target, imaged, and then selectively removed to allow introduction of the next label type. This intermediary approach achieves comprehensive biomolecule profiling while keeping each individual imaging step operationally simple
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 comprehensive and sensitive detection of over 50 different proteins and nucleic acids in individual cells of intact tissues, maintaining protein antigenicity and efficiently removing fluorescent signals.
Implementation Method 1
contacting a tissue comprising a plurality of biomolecules to cleavable detectably-labeled tyramide, wherein the first contacting step occurs under conditions that promote conjugation of the cleavable fluorophore-labeled tyramide to a target biomolecule
Implementation Method 2
contacting the tissue with a plurality of horseradish peroxidase (HRP)-conjugated targeting agents that are configured to specifically bind or hybridize to the target biomolecule
Implementation Method 3
imaging the cell after the second contacting step whereby a detectable signal generated from an interaction of HRP-conjugated targeting agents with the cleavable fluorophore-labeled tyramide is detected
Implementation Method 4
removing the detectable label from the detectably-labeled tyramide
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.