Visible-Light Azide Crosslinkers for Precise Protein Interactome Mapping
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Solution Overview
Problem
Current protein-protein interaction (PPI) mapping methods face limitations in accurately identifying interactomes due to diffusive labeling and toxicity from ultraviolet light irradiation, particularly with unstable visible light active moieties in physiological systems.
Innovation Solution
Development of novel compounds represented by Chemical Formula 1, which can be photo-crosslinked by visible light, allowing precise protein or peptide interaction analysis with a radius of 0.01 to 1 nm, overcoming diffusive labeling and toxicity issues.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If UV light activation is used for photo-crosslinking, then crosslinking ability is improved, but toxicity to living cells increases
Solution Approach 1:
The patent changes the wavelength parameter of light activation from UV to visible light range. The azide group absorbs visible light (405 nm) to generate reactive nitrene species, achieving photo-crosslinking without the cytotoxicity associated with UV irradiation. This parameter change resolves the contradiction by maintaining crosslinking effectiveness while eliminating harmful UV effects on living cells.
2Object-affected harmful factors
If visible light active moieties are used for photo-crosslinking, then toxicity is reduced, but stability in physiological systems deteriorates
Solution Approach 1:
The patent creates a composite structure by conjugating the azide photo-crosslinking moiety to a HaloTag ligand framework. This composite molecule combines the photoreactivity of the azide group with the structural stability and cell permeability of the HaloTag ligand, achieving both visible light responsiveness and physiological stability simultaneously.
3Area of stationary object
If conventional proximity labeling is used, then labeling radius is sufficient for sub-compartmental mapping, but precision for interactome analysis deteriorates
Solution Approach 1:
The patent introduces a photo-crosslinking intermediary mechanism between the HaloTag and proximal proteins. The azide-containing ligand acts as a mediator that, upon visible light activation, forms covalent bonds with interacting proteins within a very small radius (0.01-1 nm). This intermediary approach enables precise capture of direct protein-protein interactions while maintaining the beneficial labeling radius of proximity labeling.
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
The novel compounds enable accurate protein interactome identification in living cells by visible light irradiation, enhancing the precision of PPI mapping and reducing toxicity, thus improving the accuracy of protein interaction analysis.
Implementation Method 1
photo-crosslinking reactions have been utilized to capture the physical interactome, and this method utilizes aryl azide, diazo or diazitine moieties that can be converted into reactive nitrene or carbene species by ultraviolet (UV) light activation
Implementation Method 2
converted into reactive nitrene or carbene species by ultraviolet (UV) light activation
Data Source
AI summary
The present invention relates to novel compounds that are photo-crosslinked by visible light and spatiotemporal proximity photo-crosslinking by visible light activation (spotlight) using the same. When a target nucleic acid or target protein is bound to the novel compounds by the property of being photo-crosslinked to the proximal protein or peptide by irradiation with visible light and it is irradiated with visible light, the novel compounds according to the present invention can be photo-crosslinked with a protein or peptide that physically interacts with the target protein or target nucleic acid, and thus, there are advantages in that it is possible to overcome diffusive labeling, which is a limitation of the conventional proximity labeling technology, by photo-crosslinking by visible light irradiation, which is a safe method for a living body, and to improve the accuracy of protein interactome identification.