Molecular Sensor Biased Hinge for Analyte Tracking
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Solution Overview
Problem
Existing molecular sensors face challenges in accurately tracking fine-scale changes in analyte concentrations due to over-expression, which interferes with endogenous systems and maintains the ligand binding domain and sensor domain in an inappropriate ON state, leading to difficulties in immunoassays and other applications.
Innovation Solution
A molecular sensor with a biased hinge mechanism, where the ligand binding domain and sensor domain are designed to frequently transition between bound and unbound conformations, allowing for dynamic switching between ON and OFF states, enabling sensitive detection of analyte concentrations without requiring tight binding.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If the ligand binding domain and sensor domain are designed to bind tightly to maintain a strong RET signal, then the signal-to-noise ratio is improved, but the sensor remains in the ON state too long and interferes with endogenous systems
Solution Approach 1:
The patent applies the dynamics principle by designing a hinge mechanism that enables continuous conformational transitions between bound and unbound states. The hinge allows the sensor to dynamically switch between ON and OFF states rather than remaining statically bound, which reduces interference with endogenous systems while maintaining detectable signal levels through transient binding events
Solution Approach 2:
The patent implements periodic action through the oscillatory binding-unbinding cycles of the hinge mechanism. The ligand binding domain and sensor domain repeatedly associate and dissociate, creating periodic RET signal pulses that maintain measurement capability while limiting the duration of endogenous system interference through transient rather than sustained binding
2Measurement precision
If the ligand binding domain and sensor domain are kept tightly bound to enhance RET signal, then detection sensitivity is improved, but the ability to track fine-scale changes in analyte concentration is reduced
Solution Approach 1:
The hinge mechanism provides dynamic conformational flexibility that allows the sensor to respond rapidly to changing analyte concentrations. By enabling fast association and dissociation rates, the hinge allows the sensor to track fine-scale temporal changes in analyte levels while maintaining sufficient binding affinity for sensitive detection
Solution Approach 2:
The patent applies parameter changes by modifying the binding affinity and kon/koff rates of the hinge mechanism. The hinge is designed with specific kinetic parameters that allow rapid equilibrium responses to analyte concentration changes, enabling the sensor to accurately track both the magnitude and temporal dynamics of analyte fluctuations
3Illumination intensity
If over-expression of the biosensor is increased to strengthen the RET signal, then signal intensity is improved, but interference with endogenous CaM molecules and proper cellular function increases
Solution Approach 1:
The hinge mechanism enables the biosensor to dynamically bind and release from endogenous CaM molecules rather than remaining permanently bound. This transient interaction mode allows higher expression levels to be tolerated because the dynamic on-off cycling prevents sustained interference with CaM's native functions while maintaining sufficient signal intensity through increased binding event frequency
Solution Approach 2:
The hinge mechanism provides self-regulation by allowing the biosensor to naturally cycle between bound and unbound states based on local analyte concentrations. This self-service behavior reduces the need for high expression levels to achieve adequate signal, as the transient binding events are sufficient to generate detectable RET signals without overwhelming endogenous systems
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
This approach enhances the signal-to-noise ratio and allows for real-time tracking of analyte concentrations, reducing interference with endogenous systems and improving the accuracy of immunoassays and other applications.
Implementation Method 1
The fluorophores or bioluminescent molecules may be photo-activatable (such as PA-mRFP l or PA-mCherryl), photo-convertible convertible (such as Kaede or Dendra2), photo-switchable (such as Dronpa or Pardon), fluorescent protein timers (such as DsRed-E5 or Fast-FT), or phosphorescent.
Implementation Method 2
In one embodiment, the detectable ON state signal is provided by resonance energy transfer (RET) between signal molecule B and signal molecule B'.
Implementation Method 3
The resonance energy transfer (RET) may Forster resonance energy transfer (FRET) or bioluminescent resonance energy transfer (BRET).
Implementation Method 4
The signal molecule B and/or B' may comprise a chromophore, fluorophore or bioluminescent molecule.
Data Source
AI summary
The invention relates to a sensor molecule for detecting a target molecule comprising: (a) a rod-like molecule L and a rod-like molecule R connected to each other by a joint molecule C to form a hinge; (b) a target binding molecule A bonded to the end of rod- like molecule L opposite to the joint molecule C; (c) a binding molecule A' bonded to the end of rod-like molecule R opposite the joint molecule C; wherein the target binding molecule A is arranged to bind to the target molecule to be detected, and binding molecule A' is arranged to bind to: i) the same target molecule as target binding molecule A; or ii) a complex of the target binding molecule A and the target; and wherein the hinge is biased into an open position, such that target binding molecule Band binding molecule A' are biased apart by the hinge. The invention further relates to nucleic acid encoding such sensors, host cells comprising such sensor, and uses and methods of such sensors.