Method for analyzing activation state of signaling pathway and method for selecting personalized medicine using same
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Solution Overview
Problem
Current methods for analyzing protein-protein interactions in cells are limited in accurately measuring interactions at a single-molecule level in a real-time, complex cellular environment, leading to inaccurate predictions of disease progression and therapeutic responses.
Innovation Solution
A method involving immobilizing a target protein on a substrate, forming a complex with a fluorescence-labeled protein, and analyzing their interaction using a total internal reflection fluorescence microscope to determine the activation state of signaling pathways, allowing for real-time analysis of protein-protein interactions in cells or tissues.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If conventional methods (chromatography, yeast two-hybrid, FRET) are used to analyze protein-protein interactions, then the analysis can be performed under controlled conditions, but the accuracy of measuring single-molecule interactions in real-time is insufficient
Solution Approach 1:
The patent replaces conventional mechanical and chemical analysis methods with optical detection. Specifically, it uses total internal reflection fluorescence microscopy to detect fluorescence signals from fluorescently labeled proteins, enabling real-time observation of protein-protein interactions at the single-molecule level without the limitations of conventional methods
Solution Approach 2:
The patent employs fluorescent labeling of proteins and detects protein-protein interactions through fluorescence signal changes. When the fluorescently labeled protein binds to the target protein, the fluorescence signal is detected, providing real-time information about interaction events at the single-molecule level
2Measurement precision
If FRET method is used to detect protein binding, then fluorescent transition can indicate binding events, but the success rate is low because weak or short-duration bindings are not reliably detected
Solution Approach 1:
The patent replaces the FRET mechanism with total internal reflection fluorescence microscopy. This optical detection method can capture fluorescence signals from bound proteins with high sensitivity and can detect weak or transient interactions that FRET misses, thereby improving both detection sensitivity and success rate
Solution Approach 2:
The patent changes the detection parameter from FRET efficiency (which requires close proximity and specific orientation) to total internal reflection fluorescence signal intensity. This parameter change allows detection of a broader range of binding events including weak and transient interactions
3Difficulty of detecting and measuring
If chromatography is used to purify and analyze proteins, then protein interactions can be studied in isolation, but it is difficult to identify interactions at single-molecule level in a complex cellular environment
Solution Approach 1:
The patent replaces chromatographic separation with direct optical detection in the cellular environment. By using total internal reflection fluorescence microscopy, it can detect single-molecule interactions directly in cells or tissue extracts without purification, maintaining both ease of analysis and single-molecule detection capability
Solution Approach 2:
The patent extracts only the fluorescently labeled protein of interest from the complex cellular environment and introduces it to the substrate where target proteins are immobilized. This selective extraction allows specific interaction detection without the need to purify all proteins
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 method enables comprehensive analysis of signaling pathways in cells or tissues, facilitating personalized medicine selection and predicting therapeutic efficacy by accurately assessing protein interactions, thereby improving disease prognosis and treatment outcomes.
Implementation Method 1
analyzing their interaction using a total internal reflection fluorescence microscope
Implementation Method 2
forming a complex with a fluorescence-labeled protein
Data Source
AI summary
The present invention relates to a method for analyzing the activation state of a signaling pathway in a cell or tissue separated from a subject through real time single molecule protein-protein interaction analysis, and a method for selecting a personalized medicine or predicting a therapeutic efficacy to a medicine using the same.