Proximity-Dependent Enzyme Labeling for Weak Protein Interaction Detection

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Solution Overview

Problem

Current methods for evaluating protein-protein, peptide-protein, nucleic acid-protein, medium-molecular-weight compound-protein, and low-molecular-weight compound-protein interactions are insufficient, particularly for detecting weak interactions, and existing protein array technologies face challenges with protein denaturation and drying issues.

Innovation Solution

A method involving the use of a proximity-dependent modifying enzyme to label a substance to be analyzed, which is then added to an immobilized substance on a substrate, allowing for detection of interactions through a labeling substance, while maintaining the immobilized substance in a non-denatured state to prevent structural changes.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Productivity

If protein array technology is used to analyze interactions, then high throughput and comprehensive analysis are achieved, but protein denaturation and drying occur on the substrate

Engineering Contradiction:

Improvehigh throughput analysisVSAvoidprotein activity

Core Design Contradiction:

ProductivityVSReliability

Solution Approach 1:

The patent introduces a proximity-dependent modifying enzyme as an intermediary that labels the substance to be analyzed when it interacts with the immobilized substance. This mediator enables detection without requiring the immobilized substance to remain in a dry state, thus maintaining protein activity while achieving high throughput analysis through protein array technology.

Inventive Principle:

Principle #24Intermediary (Mediator)

2Reliability

If living cells are used for interaction analysis, then physiological conditions are maintained, but comprehensiveness is limited due to cell species and cell cycle variations

Engineering Contradiction:

Improvephysiological condition maintenanceVSAvoidcomprehensiveness

Core Design Contradiction:

ReliabilityVSAdaptability or versatility

Solution Approach 1:

The patent extracts the interaction analysis from the complex living cell system and transfers it to a controlled in vitro protein array system. By immobilizing purified proteins on the substrate and using proximity-dependent labeling, the method maintains reliability of interaction detection while achieving comprehensiveness through the ability to analyze multiple protein-substance interactions simultaneously without cell cycle or species variations.

Inventive Principle:

Principle #2Taking out (Extraction)

3Measurement precision

If surface plasmon resonance method is used, then highly accurate measurement is achieved, but comprehensive evaluation and high throughput are not possible

Engineering Contradiction:

Improvemeasurement accuracyVSAvoidhigh throughput capability

Core Design Contradiction:

Measurement precisionVSProductivity

Solution Approach 1:

The patent merges the high measurement precision capability with high throughput analysis by combining the proximity-dependent labeling method with protein array technology. The labeling substance provides accurate detection signals similar to SPR, while the array format enables simultaneous analysis of multiple interactions, achieving both precision and productivity.

Inventive Principle:

Principle #5Merging (Combining)

4Difficulty of detecting and measuring

If immobilized substance is analyzed to identify interaction, then interaction detection is achieved, but labor and time are required for identification

Engineering Contradiction:

Improveinteraction detectionVSAvoididentification time

Core Design Contradiction:

Difficulty of detecting and measuringVSLoss of time

Solution Approach 1:

The patent uses labeling substances that produce detectable signals (such as fluorescent or colorimetric changes) when bound to the immobilized substance through interaction. This allows direct visualization and identification of interacting substances on the array without additional labor-intensive identification steps, reducing both time and effort required.

Inventive Principle:

Principle #32Color changes

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 the detection of interactions with a binding dissociation constant of 1×10−8 M or more, overcoming the limitations of existing technologies by maintaining protein activity and preventing denaturation, thus facilitating comprehensive and high-throughput analysis of previously undetectable interactions.

Implementation Method 1

a substance to be analyzed that is labeled with a proximity-dependent modifying enzyme

Methodology Applied

Scientific EffectEnzymatic modification: Enzyme

Data Source

PatentUS20230251254A1Method for evaluating interaction between immobilized substance immobilized directly or indirectly on substrate and proximity-dependent modifying enzymelabeled substance to be analyzed

Publication Date: 2023.08.10 CELLFREE SCIENCE CO LTD

AI summary

A related-art interaction analysis method has been insufficient for detecting weak interactions. The inventors of the present invention have recognized that a method of evaluating an interaction between an immobilized substance immobilized directly or indirectly on a substrate and a substance to be analyzed that is labeled with a proximity-dependent modifying enzyme can solve the above-mentioned problem. Thus, the present invention has been completed.