Phase Separation Sensors for Biomolecular Condensate Detection
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Solution Overview
Problem
Current methods for studying cellular phase separation are limited by reliance on truncated protein mutants, reconstituted systems in non-physiological buffers, and overexpression/knockin of tagged fusions, which can alter phase separation behavior, making it challenging to effectively evaluate, detect, monitor, and manipulate biomolecular condensates, especially in living cells and in vivo.
Innovation Solution
Development of novel phase separation sensors comprising at least two domains, with one domain including an accessory protein and another domain featuring an artificial client protein or intrinsically disordered sequence, capable of targeting and associating with biomolecular condensates without disrupting them, allowing for detection, monitoring, and modulation of these condensates in vitro or in vivo.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If truncated protein mutants or overexpression/knockin of tagged fusions are used to study phase separation, then phase separation behavior can be observed, but the phase separation behavior is altered and cannot accurately reflect native conditions
Solution Approach 1:
The sensor is divided into two separate domains: an accessory protein domain and an artificial client protein domain. This segmentation allows each domain to perform its specific function independently while working together to achieve accurate detection of native phase separation behavior without the need for truncated mutants or tagged fusions.
Solution Approach 2:
The accessory protein acts as an intermediary that bridges the artificial client protein to the biomolecular condensate. This intermediary enables the sensor to detect phase separation behavior accurately by mediating the interaction between the artificial client and the condensate components, reflecting native conditions without disruption.
2Measurement precision
If conventional sensors are used to detect biomolecular condensates, then detection is possible, but the sensors disrupt the condensates and alter their natural behavior
Solution Approach 1:
The accessory protein is designed with specific local properties that enable it to interact with condensate components in a physiologically relevant manner. This local quality ensures the sensor detects condensates accurately without disrupting their natural structure or behavior, as the interaction occurs at specific localized sites rather than globally affecting the condensate.
3Adaptability or versatility
If existing methods are used to study phase separation, then some detection is achieved, but evaluation and monitoring in living cells and in vivo remains challenging
Solution Approach 1:
The sensor design is universal and can be applied across different cellular contexts and experimental conditions, including in living cells and in vivo. The accessory protein can be configured to target various condensate types, making the sensor versatile for evaluating and monitoring phase separation in diverse biological settings without requiring method development for each specific case.
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 precise targeting and assessment of biomolecular condensates, including nascent or preassembled ones, without independent phase separation behavior, allowing for the modulation of these condensates in various cellular contexts, thereby overcoming the limitations of existing methods.
Implementation Method 1
Many condensates behave as dynamic liquids and appear to form through liquid-liquid phase separation (LLPS) driven by weak, multivalent interactions between macromolecules
Implementation Method 2
condensate formation has emerged as a fundamental mechanism for the organization of biomolecules within the nucleus and cytosol and at membranes... driven by weak, multivalent interactions between macromolecules
Data Source
AI summary
The present invention provides phase separation sensors capable of targeting or associating with one or more biomolecular condensate or membraneless compartment in cells. The phase separation sensors comprise at least two domains wherein a first domain comprises one or more accessory protein or molecule and a second domain comprises an artificial client protein or intrinsically disordered sequence. The artificial client protein possesses intrinsic disorder and is capable of engaging in ultra-weak phase separation-specific interactions with one or more component protein or molecule in a biomolecular condensate. Methods and applications utilizing the sensors are provided including targeting, detecting, visualizing, manipulating, monitoring a biomolecular condensate and delivering one or more functional protein, label, drug or agent to a biomolecular condensate.