clampFISH Probe Exponential Signal Amplification
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Solution Overview
Problem
Existing single molecule RNA FISH methods face challenges with weak signal intensities, especially in high-background tissue sections, and require long imaging times, while methods that amplify signals often compromise on accuracy, multiplexing capacity, or cost.
Innovation Solution
The development of primary click-amplifying FISH (clampFISH) probes, which consist of a target-specific oligonucleotide flanked by universal oligonucleotides, and secondary and tertiary amplifier probes, allowing for exponential signal amplification through click chemistry reactions.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Ease of manufacture
If direct fluorescent labeling of DNA probes is used, then the method is simple and cost-effective, but the signal intensity is weak and imaging time is long
Solution Approach 1:
The patent implements nested probe structures where secondary probes bind to primary probes, and tertiary probes bind to secondary probes, creating a hierarchical amplification system. This nesting approach allows multiple fluorescent labels to be accumulated around a single target RNA, significantly enhancing signal intensity while maintaining a relatively simple overall methodology.
Solution Approach 2:
The patent introduces intermediate amplifier probes that mediate between the directly labeled primary probe and the final fluorescent signal. These intermediary probes carry additional fluorescent labels and enable signal amplification without requiring complex direct labeling of the target RNA, thus maintaining ease of manufacture while improving signal intensity.
2Illumination intensity
If signal amplification methods are used, then the signal intensity is improved, but accuracy, multiplexing capacity, or cost is compromised
Solution Approach 1:
The patent divides the amplification process into distinct functional segments: primary probes for target recognition and binding, secondary probes for signal amplification, and tertiary probes for further enhancement. This segmentation allows each component to be optimized for its specific function, maintaining measurement precision while achieving signal amplification. The modular design also enables flexible multiplexing by assigning different fluorescent labels to different probe segments.
3Illumination intensity
If multiple amplifier probes are used for signal amplification, then the signal intensity is enhanced, but the device complexity increases
Solution Approach 1:
The patent employs universal adapter sequences and standardized binding regions that allow the same secondary and tertiary probe designs to be used across different target sequences. This universality reduces the complexity of probe design and implementation, as the amplification probes do not need to be custom-designed for each target, thereby enhancing signal intensity without proportionally increasing device complexity.
4Measurement precision
If long imaging times are used, then weak signals can be detected, but productivity is reduced
Solution Approach 1:
The patent performs preliminary signal amplification through the binding of multiple amplifier probes to the target RNA before imaging begins. The primary probe binds to the target and recruits secondary and tertiary amplifier probes that carry fluorescent labels, pre-amplifying the signal so that subsequent imaging requires shorter exposure times and can be performed at higher throughput without sacrificing detection 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
This approach enables accurate and flexible multiplexing with high sensitivity and specificity, reducing imaging time and improving signal intensity, while maintaining cost-effectiveness and high-throughput capabilities.
Implementation Method 1
the 3' end of the first oligonucleotide is covalently locked to the 5' end of the second oligonucleotide using click chemistry to circularize the primary clampFISH probe
Implementation Method 2
hybridizing the primary clampFISH probe to a target nucleic acid
Implementation Method 3
hybridizing a fluorescent dye-coupled DNA readout probe to the secondary or tertiary amplifier probe of the scaffold, wherein the signal from the readout probes is detected by a fluorescence microscopy
Data Source
AI summary
The present invention provides novel methods for exponential amplification of nucleic acid's fluorescence in situ hybridization (FISH) signal with high sensitivity and specificity. The present method thereby allows for FISH to be used in high-throughput screening methods and diagnostics. In one aspect, the invention comprises designing a primary click-amplifying FISH (clampFISH) probe for binding to a target sequence.