Cleavable Fluorescent Streptavidin for Sensitive Multiplexed Tissue Analysis
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Solution Overview
Problem
Existing methods for multiplexed in situ molecular analysis, such as immunofluorescence and fluorescence in situ hybridization, are limited by low detection sensitivity, which impedes the analysis of low-expression proteins and short RNA, requiring long imaging exposure times and reducing sample throughput.
Innovation Solution
A method involving cleavable biotin-conjugated targeting agents and cleavable fluorescent streptavidin (CFS) for layer-by-layer signal amplification, followed by chemical cleavage and streptavidin blocking, allowing reiterative cycles for comprehensive biomolecule profiling.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If conventional immunofluorescence or FISH methods are used for multiplexed analysis, then spectral overlap of fluorophores limits the number of targets to a couple of proteins or nucleic acids, but the method is simpler to implement
Solution Approach 1:
The patent introduces biotin as an intermediary molecule that bridges the targeting agents (antibodies or oligonucleotides) and the detectable labels (fluorophores). Instead of directly conjugating fluorophores to targeting agents, biotin is conjugated to targeting agents, and then streptavidin (which binds biotin with high affinity) is used as a bridge to attach fluorophores. This intermediary system enables multiplexed detection without spectral overlap because different biotin-streptavidin pairs can be used in sequential cycles, resolving the contradiction between versatility and complexity.
Solution Approach 2:
The detection process is segmented into multiple cycles, where each cycle detects a subset of targets using biotin-conjugated targeting agents and streptavidin-conjugated fluorophores. After imaging, the fluorophores are cleaved and streptavidin is blocked, allowing the next cycle to begin. This segmentation enables the detection of many more targets than would be possible in a single step, resolving the contradiction between the number of detectable targets and method complexity.
2Measurement precision
If detection tags are directly conjugated to affinity probes, then the method is simpler, but detection sensitivity is limited and long imaging exposure time is required
Solution Approach 1:
The patent employs a nested structure where biotin is conjugated to the targeting agent, streptavidin binds to biotin, and fluorophores are conjugated to streptavidin. This nested arrangement (targeting agent-biotin-streptavidin-fluorophore) creates a signal amplification effect because multiple fluorophores can be attached to each targeting agent through the streptavidin-biotin complex. This nesting resolves the contradiction by enhancing detection sensitivity without requiring longer exposure times.
Solution Approach 2:
The patent changes the binding affinity parameter by using the extremely high affinity interaction between biotin and streptavidin (Kd in the range of 10^-15 M). This high affinity ensures stable complex formation and signal amplification, dramatically improving detection sensitivity compared to direct conjugation methods. The parameter change in binding affinity resolves the contradiction between detection sensitivity and imaging time.
3Measurement precision
If long imaging exposure time is used to compensate for low sensitivity, then detection sensitivity improves, but sample throughput decreases and assay time increases
Solution Approach 1:
The patent performs preliminary signal amplification by forming the biotin-streptavidin complex before imaging. This preliminary action ensures that sufficient signal is generated during the imaging step, eliminating the need for long exposure times. The signal amplification occurs during the incubation steps, allowing for rapid imaging and high sample throughput, thus resolving the contradiction between detection sensitivity and productivity.
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
The method significantly enhances detection sensitivity by at least one order of magnitude, enabling the analysis of low-expression proteins and short nucleic acids, reducing imaging and assay times, and allowing multiple targets to be assessed in a single sample without damage.
Implementation Method 1
contacting a tissue with a plurality of targeting agents conjugated to biotin via a cleavable linker, wherein the targeting agents are configured to specifically bind or hybridize to a target biomolecule
Implementation Method 2
the targeting agents are configured to specifically bind or hybridize to a target biomolecule in the contacted tissue
Implementation Method 3
contacting a tissue comprising a plurality of biomolecules to streptavidin comprising a detectable label via a cleavable linker, wherein the second contacting step occurs under conditions that promote conjugation of the detectably-labeled streptavidin to biotin of the targeting agents
Implementation Method 4
The cleavable detectably-labeled streptavidin can comprise a fluorophore
Implementation Method 5
The cleavable linker can be chemically cleavable, enzymatically cleavable, nucleophilically cleavable, electrophilically cleavable, photocleavable, metal cleavable, cleavable under reductive conditions, cleavable under oxidative conditions, cleavable using an acidic reagent, or cleavable using a basic reagent
Data Source
AI summary
Provided herein are methods and systems for sensitive and multiplexed in situ analysis of samples such as biological samples using cleavable fluorescent streptavidin. In particular, provided herein are methods for multiplexed single-cell in situ biomolecule profiling in samples, including 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.