Super-Resolution Molecular Patterning via Transient Probe Binding
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Solution Overview
Problem
Current methods struggle to label and manipulate target proteins with nanometer precision in small regions without inadvertently labeling nearby targets, limiting the ability to study or modify specific molecular locations in biological systems.
Innovation Solution
The development of super-resolution labeling methods using transiently binding nucleic acid probes with photocrosslinkers and photocleavable linkers, allowing for precise binding and irradiation only when a single binding event occurs within a diffraction-limited region, enabling nanometer-scale manipulation and patterning of molecules.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If conventional optical labeling methods are used, then labeling can be performed, but the resolution is limited by the diffraction limit of light and nearby targets cannot be distinguished
Solution Approach 1:
The method segments the labeling process into distinct temporal phases: first allowing probes to bind transiently to multiple targets, then using feedback-controlled irradiation to selectively crosslink only those probes bound to targets within the specified region of interest. This segmentation enables super-resolution labeling by separating the binding phase from the fixation phase.
Solution Approach 2:
The system employs real-time feedback by monitoring binding events through fluorescence detection and using this information to control the timing and location of irradiation. The feedback loop ensures that crosslinking occurs only when a single probe is detected in the ROI, enabling precise spatial control beyond the diffraction limit.
2Productivity
If irradiation is applied continuously to label targets, then labeling efficiency increases, but nearby targets within the diffraction limited region are also labeled indiscriminately
Solution Approach 1:
The method uses periodic, pulse-like irradiation rather than continuous illumination. Irradiation is delivered in brief pulses only when binding events are detected in the ROI, maintaining high labeling efficiency while preventing indiscriminate labeling of nearby targets through temporal gating.
Solution Approach 2:
The system performs preliminary detection of binding events before initiating irradiation. By first identifying when a probe is bound to a target in the ROI through fluorescence monitoring, the system prepares for selective crosslinking, ensuring that subsequent irradiation affects only the intended target.
3Measurement precision
If transiently binding probes are used to achieve super-resolution, then nanometer precision labeling is possible, but the binding stability decreases
Solution Approach 1:
The method changes the binding parameters by using transiently binding probes with optimized affinity that allow them to associate and dissociate dynamically. This transient binding regime, combined with feedback-controlled crosslinking, enables super-resolution precision while maintaining sufficient binding stability during the labeling window.
Solution Approach 2:
The system replaces stable mechanical binding with a combination of transient binding and photochemical crosslinking. The transient binding provides the necessary dynamics for super-resolution, while the photochemical crosslinking step locks in the desired configuration, substituting permanent chemical bonding for prolonged physical binding.
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 allows for precise optical manipulation and labeling of molecules at user-specified locations, enhancing applications in nanoscale single-cell spatial proteomics and optogenetics with improved resolution and accuracy.
Implementation Method 1
the probes comprise a photocrosslinker... irradiating the diffraction limited region of the substrate, wherein the probes comprise a photocrosslinker
Implementation Method 2
the probes have a hairpin secondary structure and have a photocleavable linker or spacer along their length (such that breakage of the linker or spacer will induce a covalent break in the chain)
Implementation Method 3
The fluorophore may have characteristics similar to those of standard Points Accumulation for Imaging in Nanoscale Topography (PAINT) probes
Data Source
AI summary
This disclosure provides methods for generating super-resolution patterns of molecules on substrates. In one aspect, disclosed herein is a method comprising contacting a plurality of transiently binding nucleic acid probes to their respective targets wherein the targets are immobilized on a substrate, detecting a binding event in a select region or set of select regions within a diffraction limited region of the substrate, and irradiating the diffraction limited region of the substrate, wherein the probes comprise a photocrosslinker. In one embodiment, the photocrosslinker is 3-cyanovinylcarbazole.