Cas Protein Reagent Stabilization via Particle Binding
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Solution Overview
Problem
The enzyme activity of Cas12a protein complexes in aqueous solutions tends to decrease rapidly, making them unsuitable for long-term use in nucleic acid detection applications.
Innovation Solution
A reagent containing complex particles with Cas protein and guide RNA bound to particles, which reduces hydration effects and maintains enzyme activity by hindering water access, allowing the complex to remain active even at low concentrations without the need for dilution.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If the complex of Cas protein and guide RNA is stored in aqueous liquid, then the reagent can be used for detection, but the enzyme activity decreases rapidly
Solution Approach 1:
The patent introduces particles as an intermediary carrier to bind the Cas protein-guide RNA complex. This particle-mediated approach isolates the complex from direct contact with aqueous environment, reducing hydration-induced enzyme activity loss while maintaining detectability through the particle-bound complex
Solution Approach 2:
The particle surface acts as a protective interface between the Cas protein complex and the aqueous storage environment. This boundary layer reduces harmful water access to the enzyme while allowing necessary molecular interactions for detection function
2Reliability
If the complex is bound to particles, then enzyme activity is maintained, but the device complexity increases
Solution Approach 1:
The detection system is segmented into distinct functional components: particles serve as stable carriers, while the Cas protein-guide RNA complex remains as a separate functional unit bound to the particle surface. This segmentation allows independent optimization of storage stability (particle) and detection function (complex)
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 reagent effectively maintains enzyme activity over time, enabling sensitive detection of target nucleic acids without significant loss of sensitivity, even in aqueous environments.
Implementation Method 1
A complex made up of Cas12a, a type of Cas protein (CRISPR-associated protein), and crRNA (CRISPR RNA, which can be reworded as guide RNA) recognizes and bind to the target DNA sequence
Implementation Method 2
Cas12a cleaves the target DNA bound to the complex. In this cleavage, when a reporter molecule defined by a fluorescent material and a quencher that are linked with a single-stranded DNA has been added to the reaction system in advance, Cas12a cleaves the single-stranded DNA acting as the linkage, and the fluorescent substance emits fluorescence
Data Source
AI summary
A reagent contains complex particles, each including a particle and a complex bound to the particle, and an aqueous liquid in which the complex particles are dispersed. The complex includes a Cas protein and a guide RNA bound to the Cas protein.