Interactome Analysis Kit Using Photo-Crosslinking for Living Cells
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Solution Overview
Problem
Existing interactome analysis methods suffer from low selectivity, sensitivity, and spatiotemporal resolution, limited applicability to specific proteins of interest, and inability to analyze interactomes in living cells, particularly in the nucleus.
Innovation Solution
A kit comprising vectors for expressing a protein of interest (POI) fused with a fluorescent protein, and a HaloTag system with a nuclear localization sequence, along with a photocatalyst for photo-crosslinking, allowing for precise analysis of interactomes within living cells and the nucleus.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If immunoprecipitation is used to isolate interactomes, then protein-protein interactions can be detected, but selectivity and spatiotemporal resolution are very low due to physical adsorption capturing surrounding proteins
Solution Approach 1:
The patent replaces the mechanical/physical adsorption mechanism of immunoprecipitation with a photochemical reaction mechanism. Photocrosslinking uses light-induced chemical bonds to specifically tag interactomes, eliminating the non-specific physical adsorption that causes low selectivity in traditional methods.
Solution Approach 2:
The patent introduces photoactivation as a controllable parameter to achieve spatiotemporal resolution. By using light-induced photocrosslinking, the interaction can be captured at specific moments and locations, transforming the static, non-resolvable interactome analysis into a dynamic, time-and space-resolved process.
2Measurement precision
If enzyme-based labeling with biotin-phenoxyl radical is used, then interactants can be effectively labeled, but spatiotemporal resolution is low due to long lifetime and wide diffusion distance of the radical
Solution Approach 1:
The patent employs a short-lived photocatalyst intermediate that rapidly forms and decomposes, similar to the disposable nature of photoproducts. This short lifetime prevents diffusion away from the target, ensuring the labeling reaction occurs only at the precise location and moment of protein interaction, thereby achieving high spatiotemporal resolution.
3Measurement precision
If chemical crosslinking method is used, then protein interactions can be captured, but indiscriminate reactions cause overall crosslinking of proteins without spatiotemporal resolution and cannot be applied to living cells
Solution Approach 1:
The patent replaces non-specific chemical crosslinking with light-induced photocrosslinking. The photoactivation step acts as a spatial and temporal gatekeeper, allowing crosslinking to occur only where and when light is applied, thereby achieving high resolution and enabling application in living cells without indiscriminate protein crosslinking.
Solution Approach 2:
The patent uses periodic light activation to control the crosslinking process. By applying light in controlled periods, the photocrosslinking reaction can be activated only when needed, providing temporal resolution and allowing the system to remain adaptable to living cell conditions throughout the cell cycle.
4Measurement precision
If μMap method with carbene intermediate is used, then labeling radius is improved to several nm, but target protein selection is limited to extracellular region due to antibody targeting requirements
Solution Approach 1:
The patent creates a universal photocrosslinking system that can target any protein with a fluorescent tag, regardless of location. The photocatalyst can be delivered to any cellular compartment and will photocrosslink interactomes of tagged proteins anywhere in the cell, making the system universally applicable to both extracellular and intracellular proteins without being limited by antibody accessibility.
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 method achieves high precision, sensitivity, and spatiotemporal resolution in interactome analysis by forming covalent bonds between proteins, enabling analysis of interactomes in living cells and the nucleus with controlled timing.
Implementation Method 1
irradiating a light source to induce a photo-crosslinking chemical reaction between the POI and the interactome
Data Source
AI summary
The present invention relates to a kit for analyzing interactomes, and a method for analyzing interactomes through photo-crosslinking by using the kit. According to the kit for analyzing interactomes and the method for analyzing interactomes by using same, in one aspect of the present invention, interactomes are linked through a covalent bond one by one so that analysis errors can be reduced. In addition, according to the present invention, interactomes in living cells can be analyzed and interactomes in the nucleus of cells can be analyzed. Furthermore, according to the present invention, the formation time point of interactomes can be regulated so that target proteins and interactomes can be analyzed in a specific situation or time point.