Molecular Beacon Locking Oligonucleotide for Cell Force Imaging
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Solution Overview
Problem
Current methods lack effective tools to measure forces applied to specific receptors on cell surfaces, which is crucial for understanding mechanotransduction and cellular processes such as adhesion, migration, and immune response.
Innovation Solution
The development of devices and methods using nucleic acid complexes with hairpin motifs and locking oligonucleotides that unravel and hybridize upon mechanical forces, allowing for the detection and imaging of receptor-ligand interactions through fluorescence or redox active agents.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If conventional fluorescent sensors are used to measure forces on membrane receptors, then force detection capability is provided, but the sensors cannot capture transient and rare mechanical events due to limited temporal resolution and signal stability
Solution Approach 1:
The molecular beacon is pre-configured in a closed hairpin structure with the fluorophore and quencher in close proximity, ready to undergo a conformational change upon force application. This preliminary setup allows the sensor to immediately respond to transient forces without requiring signal processing or activation steps, capturing rare mechanical events that occur on millisecond timescales
Solution Approach 2:
The molecular beacon undergoes a phase transition from a closed hairpin structure to an extended unfolded structure when mechanical force is applied. This phase transition separates the fluorophore from the quencher, generating a detectable fluorescence signal. The binary nature of this transition (closed/open) provides high temporal resolution for detecting transient forces
2Measurement precision
If molecular beacons are used to detect mechanical forces, then real-time force measurement is enabled, but the signal cannot be distinguished from background noise for rare events
Solution Approach 1:
A locking oligonucleotide acts as an intermediary that selectively binds to and stabilizes the force-unfolded molecular beacon structure. This locking step converts a transient conformational change into a stable, detectable signal that can be distinguished from background noise, enabling detection of rare mechanical events
Solution Approach 2:
The molecular beacon utilizes fluorescence emission (color change) as the readout mechanism. When the beacon is in the closed state, the fluorophore is quenched and no fluorescence is emitted. Upon force-induced unfolding, the fluorophore separates from the quencher and emits fluorescence, providing a clear visual signal for force detection
3Measurement precision
If high-force thresholds are used in tension sensors, then specific mechanical events can be detected, but forces below the threshold cannot be measured
Solution Approach 1:
The mechanical properties of the molecular beacon are tuned by modifying the hairpin structure (stem length, GC content, loop sequence) to achieve different force thresholds. This allows customization of the sensor for detecting forces across a wide range, from weak adhesive interactions to strong mechanotransduction events, while maintaining high specificity at each threshold level
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 the imaging of transient and rare mechanical events at the cellular level, including forces as low as 1 pN, with high mechanical selectivity and the ability to record and erase mechanical signals, enhancing the understanding of receptor dynamics and cellular interactions.
Implementation Method 1
a locking oligonucleotide that hybridizes to a portion of the hairpin turn and stem of the molecular beacon when the molecular beacon unravels or melts due to pulling forces on the ligand
Implementation Method 2
when the molecular beacon unravels or melts due to pulling forces on the ligand
Implementation Method 3
In certain embodiments, the label is horseradish peroxidase. In certain embodiments, the label is a fluorescent molecule and detecting is observing the fluorescence of the label
Data Source
AI summary
In certain embodiments, this disclosure relates to devices and methods for imaging transient mechanical events in cells. In certain embodiments, this disclosure contemplates devices comprising receptors, cells or cell membranes comprising receptors, a molecular beacon as a linker between a solid surface and a ligand, and a locking oligonucleotide that selectively binds a portion of the hairpin turn and stem of the molecular beacon when the beacon is mechanically melted with piconewton forces. In certain embodiments, this disclosure relates to methods of locking, unlocking, and imaging cellular events using labeled locking and unlocking oligonucleotides disclosed herein.