Optogenetic Phase Separation Screening System
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Solution Overview
Problem
Current methods lack the capability to efficiently and effectively map or screen intracellular interactions, particularly those involving phase separation in cells, which is crucial for understanding biomolecular modifications and identifying agents that modulate these interactions, due to limitations in detecting weak interactions and requiring direct molecular detection.
Innovation Solution
A high-throughput method involving cells expressing phase separation or aggregation systems controlled by light, where cells are irradiated with specific wavelengths to form condensates, and fluorescence is used to quantify phase separation, allowing for the identification of agents impacting these interactions.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If direct molecular detection methods are used to detect weak interactions, then measurement precision may be improved, but device complexity and difficulty of detecting and measuring increase significantly
Solution Approach 1:
The patent uses light as an intermediary to indirectly detect phase separation events. Instead of directly detecting weak molecular interactions, the system uses optically controllable proteins (e.g., CRY2-CIB1) that undergo phase separation in response to light irradiation. The light serves as a controllable trigger and readout mechanism, converting difficult-to-detect molecular interactions into easily measurable optical signals (fluorescence intensity, condensate formation), thereby reducing detection complexity while maintaining precision.
Solution Approach 2:
The patent changes the detection parameter from direct molecular interaction detection to optical property detection. By using fluorescently tagged proteins and measuring fluorescence intensity or condensate formation under different light conditions, the system transforms the measurement of weak interactions into measurements of optical parameters (fluorescence intensity, light absorption, condensate morphology), which are easier to detect and quantify with high precision.
2Productivity
If traditional low-throughput screening methods are used, then measurement precision for individual compounds may be maintained, but productivity decreases
Solution Approach 1:
The patent segments the screening process into multiple parallel independent experiments using microtiter plates with 96 or 384 wells. Each well contains cells expressing optically controllable phase separation systems with different test compounds. This segmentation allows simultaneous screening of numerous compounds while maintaining precise optical detection in each well, thereby increasing productivity without sacrificing measurement precision.
Solution Approach 2:
The patent changes the detection approach to use optical parameters (fluorescence intensity, light-induced condensate formation) that can be rapidly and automatically measured across many samples. This enables high-throughput automated screening while maintaining precision through consistent optical measurement protocols, unlike traditional methods that require manual or low-throughput analysis.
3Ease of operation
If optically controllable phase separation systems are used, then ease of operation and productivity improve, but device complexity increases due to light irradiation equipment
Solution Approach 1:
The patent employs multi-functional optical components that serve multiple purposes: the same light source is used to both trigger phase separation (via CRY2-CIB1 optogenetic system) and to detect it (via fluorescence excitation). This universality reduces the need for separate triggering and detection equipment, thereby reducing overall device complexity while maintaining ease of operation through integrated optical control and measurement.
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 sensitive detection of weak interactions and high-throughput screening of compounds, providing insights into phase behavior and potential therapeutic strategies for diseases related to protein aggregation, with the ability to observe drug effects on aggregate dynamics and structure.
Implementation Method 1
Liquid-liquid phase separation (LLPS) is a fundamental mechanism for organizing the contents of cells. LLPS is now recognized as important for driving assembly of a wide range of membrane-less condensates
Implementation Method 2
irradiating the at least one of the plurality of cells with an additional wavelength of light to cause the fluorescent protein or an attached fluorophore to fluoresce
Data Source
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AI summary
Disclosed is a high-throughput method and system for mapping or screening biomolecular interactions, where the method includes providing a plurality of cells, or in some examples purified proteins themselves, expressing a phase separation or aggregation system, including those capabl e of being controlled by at least one wavelength of light, with the phase separation or aggregation system comprising a target protein possibly fused to a fluorescent protein. The ceils are placed in a well, and chemical and/or biological agent are then introduced to the well. The well may then be the irradiated with the wavelength that controls the phase separation or aggregation system, after which the well is irradiated so as to cause the fluorescent proteins or fluorophores to fluoresce, after which the phase separation or aggregation can be quantified based on an amount of fluorescence within a first region and a second region of the sample, the first region containing a condensate and the second region not containing a condensate. Also disclosed is a method utilizing a non-optically controlled phase separation or aggregation system.