Conditionally Stable Ligand-Binding Domains for In Vivo Small Molecule Detection
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Solution Overview
Problem
Current methods for detecting small molecules in living cells are limited, as existing techniques are inapplicable or impractical for in vivo detection due to requirements for genetic encoding, membrane impermeability, or limited applicability to only well-characterized cases with dramatic conformational changes.
Innovation Solution
Development of biosensors with conditionally stable ligand-binding domains (LBDs) engineered to respond to specific small molecules, fused to reporter proteins or transcription factors, allowing for modular and generalizable detection of small molecule localization and abundance in vivo through fluorescence, catalysis, signaling, or gene transcription.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If common detection methods (FRET reporters, peptide ligation, protein complementation) are used, then detection capability is achieved, but applicability to small molecules is limited or inapplicable
Solution Approach 1:
The invention changes the stability parameter of the ligand-binding domain by introducing destabilizing mutations, creating a conditional stability state that is rescued by small molecule binding. This allows the biosensor to respond to small molecules through stability changes rather than relying on conformational rearrangements or genetic encoding requirements
Solution Approach 2:
The invention introduces a destabilizing mutation as an intermediary element that mediates between the ligand-binding domain and the reporter protein. This intermediary creates a conditional stability mechanism where the destabilizing mutation serves as a switch that is turned off by ligand binding, enabling small molecule detection
2Measurement precision
If metabolite binding proteins with dramatic conformational rearrangements are used, then detection sensitivity is improved, but applicability is limited to well-characterized cases
Solution Approach 1:
Instead of relying on dramatic conformational rearrangements, the invention changes the stability parameter of the ligand-binding domain through destabilizing mutations. This creates a more generalizable approach that can be applied to any ligand-binding protein, not just those with well-characterized conformational changes
Solution Approach 2:
The invention inverts the traditional approach by instead of using stable proteins that change conformation upon binding, it uses destabilized proteins that are stabilized upon binding. This inversion allows the biosensor to report ligand presence through stability rescue rather than conformational change
3Ease of manufacture
If SNAP or HaloTag methods are used, then modular detection approach is achieved, but in vivo use is prohibited due to membrane impermeability
Solution Approach 1:
The invention extracts the detection capability from the ligand-binding domain and separates it from the reporter protein through genetic fusion. The destabilizing mutation is introduced into the ligand-binding domain itself, eliminating the need for separate chemical labeling or membrane-permeable compounds
Solution Approach 2:
The biosensor system is self-sufficient as the destabilizing mutation is genetically encoded within the ligand-binding domain. The system does not require external chemical probes or membrane-permeable compounds, making it fully functional in vivo through endogenous protein expression and stability regulation
4Speed
If LBD is directly fused to reporter protein, then rapid response time is achieved, but sensitivity and dynamic range are reduced
Solution Approach 1:
The invention segments the transcription factor into separate functional domains (DNA-binding domain, ligand-binding domain, activation domain) that can independently function. This segmentation allows the destabilizing mutation in the LBD to be rescued by ligand binding, enabling sensitive detection while maintaining rapid transcriptional response
Solution Approach 2:
The invention introduces transcriptional activation as an intermediary step between ligand binding and reporter expression. The destabilizing mutation creates a pool of unstable TF that is selectively stabilized by ligand binding, allowing sensitive detection through accumulation of active TF while maintaining rapid response through transcriptional activation
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 and dynamic range-enhanced detection of small molecules, applicable to various biological processes, including environmental contaminants, gene regulation, and metabolic pathway optimization, with potential for in vivo monitoring and bioproduction enhancements.
Implementation Method 1
addition of the cognate ligand stabilizes the ligand-binding domain and increases in vivo levels of the TF
Data Source
AI summary
Disclosed is a biosensor engineered to conditionally respond to the presence of specific small molecules, the biosensors including conditionally stable ligand-binding domains (LBDs) which respond to the presence of specific small molecules, wherein readout of binding is provided by reporter genes or transcription factors (TFs) fused to the LBDs.