Split Cas Enzyme Dimerization Screening Assay
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Solution Overview
Problem
Current methods for detecting molecular dimerization events in biochemistry lack high-throughput capabilities, robustness, and clear readouts, often resulting in false positives due to non-specific signal generation.
Innovation Solution
The development of dimerization screening assays using a split Cas enzyme that forms a functional ribonucleoprotein complex with both cis- and trans-cleavage activity, employing blocked nucleic acid molecules or guide RNA to prevent non-specific activation, allowing for robust and reprogrammable detection of dimerization events at attomolar limits.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If conventional dimerization detection methods are used, then detection capability is provided, but high-throughput capability and robustness are lacking
Solution Approach 1:
The Cas enzyme is divided into two separate fragments (N-terminal and C-terminal portions) that cannot function independently. Each fragment is fused to a different binding partner, and only when the binding partners dimerize do the fragments come together to form a functional enzyme. This segmentation enables high-throughput screening while maintaining robustness through specific dimerization-dependent activation.
Solution Approach 2:
The binding partners are pre-fused to the Cas enzyme fragments before the dimerization event occurs. This preliminary configuration ensures that when dimerization happens, the functional complex is immediately assembled and activated, enabling rapid detection suitable for high-throughput applications without requiring additional assembly steps.
2Measurement precision
If detection sensitivity is increased, then detection limit is improved, but false positives due to non-specific signal generation increase
Solution Approach 1:
Blocked nucleic acid molecules are introduced that prevent non-specific activation of the Cas enzyme. These blocked molecules remain inactive unless specifically unblocked by the correct target sequence, thereby preventing false positive signals while maintaining high detection sensitivity for specific targets.
Solution Approach 2:
Blocked nucleic acid molecules act as intermediaries between the Cas enzyme and the target. They serve as a controlled activation mechanism that requires specific recognition to become unblocked and activate the enzyme, thereby filtering out non-specific signals while allowing specific target detection.
3Difficulty of detecting and measuring
If clear readout is achieved, then detection clarity is improved, but assay complexity increases
Solution Approach 1:
The assay utilizes fluorescent reporter molecules that emit light when activated by the Cas enzyme. This color/fluorescence change provides a clear, easily detectable readout signal that can be measured in high-throughput formats without requiring complex detection instrumentation, thus improving readout clarity while managing assay complexity.
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 assays provide accurate and sensitive detection of putative binding pairs, minimizing false positives by ensuring the ribonucleoprotein complex is only activated upon specific dimerization events, enabling high-throughput screening with minimal non-specific signal generation.
Implementation Method 1
a first ribonucleoprotein complex (RNP1) that, when activated, exhibits cis- and trans-cleavage activity
Implementation Method 2
employing blocked nucleic acid molecules or guide RNA to prevent non-specific activation
Implementation Method 3
dimerization screening assays that detect dimerization events
Data Source
AI summary
The present disclosure relates to compositions of matter and assay methods used to screen for molecular dimerization events using a two-ribonucleoprotein complex signal boost assay. The compositions and methods provide a readout upon detection of dimerization of molecules and may be implemented in a high throughput manner using libraries of tens to hundreds to thousands of putative binding partners.