Polynucleotide Detection Using Flap Cleavage and Type V CRISPR
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Solution Overview
Problem
Existing nucleic acid detection methods, such as invader assays and CRISPR-based techniques, face challenges including slow detection speed, complexity, and limitations in target sequence recognition, making them costly and time-consuming.
Innovation Solution
A method involving a first and second nucleic acid probe forming a cleavage structure with a structure-specific nucleic acid cleaving agent, followed by ligation to an adaptor, and interaction with a Type V CRISPR/Cas effector protein to detect polynucleotides using a detectable signal.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If invader assay is used for nucleic acid detection, then detection specificity is improved, but detection speed deteriorates and device complexity increases
Solution Approach 1:
The detection system is segmented into distinct functional modules: CRISPR-Cas12a complex for specific target recognition, FEN1 enzyme for flap cleavage, Taq polymerase for extension, and adaptor ligation system. Each module performs a specific function, allowing parallel optimization of speed and specificity without mutual interference.
Solution Approach 2:
The probe is pre-configured with a 5' flap structure containing the adaptor binding sequence before the reaction begins. When the target is detected, the flap is immediately cleaved by FEN1 and the adaptor is ligated in the same reaction vessel, eliminating the need for separate purification or preparation steps that would slow down detection.
2Measurement precision
If invader assay is used for nucleic acid detection, then detection specificity is improved, but device complexity increases
Solution Approach 1:
Multiple functions are merged into a single reaction vessel: target detection, flap cleavage, adaptor ligation, and signal amplification all occur simultaneously in one tube. This consolidation reduces the number of manual操作步骤 and minimizes contamination risks between steps.
Solution Approach 2:
The probe design is universal and can detect different target sequences by simply changing the probe sequence while maintaining the same 5' flap structure and adaptor binding region. The same enzymatic system (FEN1, Taq, adaptor ligase) works for all targets, reducing the need for multiple specialized reagents.
3Measurement precision
If CRISPR-based detection methods are used, then detection sensitivity is improved, but adaptability deteriorates due to PAM sequence requirements
Solution Approach 1:
The 5' flap structure acts as an intermediary between the probe and the CRISPR-Cas12a system. Instead of requiring the target sequence itself to contain a PAM motif, the flap provides a standardized PAM-containing adaptor binding site. This mediator allows CRISPR to detect any target sequence as long as the probe can hybridize to it.
Solution Approach 2:
The probe is segmented into a target-binding region and a separate 5' flap region. The flap region contains the adaptor binding sequence with PAM motif, while the target-binding region can be customized to match any target sequence. This segmentation decouples the PAM requirement from the target sequence specificity.
4Reliability
If qPCR is used for nucleic acid detection, then detection reliability is improved, but cost and time consumption increase
Solution Approach 1:
The reaction proceeds continuously in one direction without requiring thermal cycling. The isothermal conditions allow all enzymatic reactions (hybridization, cleavage, extension, ligation) to proceed simultaneously and continuously, eliminating the time lost during repeated heating and cooling cycles required by qPCR.
Solution Approach 2:
The time-consuming thermal cycling step is extracted and removed from the protocol. The detection is performed under isothermal conditions at a single temperature (e.g., 37°C or 50°C), eliminating the need for expensive thermal cyclers and reducing total detection time from hours to minutes.
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 rapid, sensitive, and specific detection of polynucleotides, including single nucleotide polymorphisms, with potential for visual or fluorescent readouts, suitable for complex samples.
Implementation Method 1
hybridization of the first nucleic acid probe to the first portion of the polynucleotide analyte and hybridization of the second nucleic acid probe to the second portion of the polynucleotide analyte forms a cleavage structure
Implementation Method 2
formation of the cleavage structure allows cleavage of the first nucleic acid probe by the cleaving agent to release a 5′ flap
Implementation Method 3
ligating the 5′ flap to a nucleic acid adaptor to form an adaptor-ligated product
Implementation Method 4
measuring a detectable signal produced by cleavage of the single stranded detector DNA by the type V CRISPR/Cas effector protein
Data Source
AI summary
The invention relates generally to the field of nucleic acid detection. In particular, the specification teaches a method of detecting a polynucleotide analyte in a sample. In one aspect, the method comprises the use of a cleaving agent with flap endonuclease activity and a type V CRISPR/Cas effector protein. In another aspect, the type V CRISPR/Cas effector protein is a Cast 2 protein.