Proximity Probe Assays for Joint Protein Expression and Interaction Detection
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Solution Overview
Problem
Current multiplexed protein detection methods primarily focus on detecting expression levels and protein-protein interactions, neglecting the detection of individual protein expression levels and post-translational modifications, while proximity assays like in situ PLA only detect proximity events and not expression levels.
Innovation Solution
A method using proximity probes and nucleic acid reagents, such as padlock probes, to perform three separate detection reactions that generate distinct signals for individual target molecules and their interaction, allowing simultaneous detection of both target molecules and their interaction.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If proximity assays like in situ PLA are used to detect protein-protein interactions, then interaction detection is improved, but individual protein expression level detection is lost
Solution Approach 1:
The assay is segmented into three independent detection reactions: (1) a first reaction detecting the first target molecule using a first probe, (2) a second reaction detecting the second target molecule using a second probe, and (3) a third reaction detecting the interaction between the two target molecules using both probes. Each reaction generates a distinct signal, allowing simultaneous measurement of individual protein levels and their interaction.
2Measurement precision
If multiplexed protein detection methods are used to detect expression levels, then individual protein detection is improved, but functional aspects like protein-protein interactions are neglected
Solution Approach 1:
The detection system is designed with multi-functionality to perform both expression level detection and interaction detection within a single multiplexed assay. By using probes with nucleic acid domains that can participate in different reaction types (individual detection reactions and interaction detection reaction), the system universally handles multiple detection objectives simultaneously.
3Adaptability or versatility
If in situ PLA is applied in a multiplexing context, then multiple interactions can be detected, but the complexity of the assay increases and individual expression levels cannot be detected
Solution Approach 1:
Different regions or domains of the probes are assigned different functions: the binding domain recognizes the target protein while the nucleic acid domain participates in specific detection reactions. This local differentiation allows the same probe structure to serve multiple purposes in different reaction contexts, enabling multiplexing without proportionally increasing overall assay complexity.
Data Source
AI summary
The present invention provides a method for detecting two target molecules in a sample, both as individual molecules and in proximity, or interaction with one another. The method involves performing three separate assay reactions to detect the first and second target molecules, and their interaction, using proximity probes which are common between the three assay reactions, and nucleic acid reagents which interact with the probes. In particular the methods use unique nucleic acid substrate molecules, such as padlock probes, to detect probes bound to their target and to determine when probes for the two targets are in close proximity, indicating an interaction between the targets.