Dual Acceptor BRET Biosensor for Simultaneous Protease Detection
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Solution Overview
Problem
Current Bioluminescence Resonance Energy Transfer (BRET) technologies are limited to detecting a single molecular event due to the use of a single acceptor moiety, which restricts their application in monitoring multiple biological processes simultaneously.
Innovation Solution
The development of a BRET sensor that utilizes two acceptor moieties, including a monomeric mNeonGreen (mNG) fluorescent protein and a red fluorescent protein (RFP), allowing for the simultaneous monitoring of the activity of two proteins in a living cell.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If a single acceptor moiety is used in BRET sensor, then the device complexity is low, but the adaptability or versatility is limited to detecting only one molecular event
Solution Approach 1:
The patent combines two acceptor moieties (mNeonGreen fluorescent protein and red fluorescent protein) into a single BRET sensor construct, allowing simultaneous detection of two different molecular events (proteolytic activities) while maintaining a unified sensor structure that reduces overall system complexity
Solution Approach 2:
The BRET sensor is designed with universal functionality to detect multiple types of molecular events simultaneously through two different acceptor moieties, enabling the same sensor platform to monitor diverse biological processes (e.g., different protease activities) without requiring separate sensors for each target
2Adaptability or versatility
If two acceptor moieties are used in BRET sensor, then the adaptability or versatility improves for simultaneous monitoring of two molecular events, but the device complexity increases
Solution Approach 1:
The sensor is segmented into distinct functional modules: a bioluminescent donor protein, a first acceptor moiety (mNeonGreen) for detecting one molecular event, and a second acceptor moiety (red fluorescent protein) for detecting another molecular event. This modular segmentation allows independent optimization of each detection channel while maintaining overall sensor functionality
Solution Approach 2:
The sensor employs composite fluorescent protein structures combining mNeonGreen and red fluorescent protein in a single construct, creating a multi-functional detection system that leverages the spectral properties of different fluorescent proteins to achieve simultaneous multi-parameter detection
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 dual protease sensor effectively detects the activity of two proteases, such as SARS-CoV-2 Mpro and PLpro, by reducing resonance energy transfer between the bioluminescent protein and the acceptor proteins upon proteolytic cleavage, enabling robust and simultaneous monitoring of proteolytic activities.
Implementation Method 1
Bioluminescence Resonance Energy Transfer (BRET) is a phenomenon of proximity-dependent non-radiative, Forster resonance energy transfer (FRET) between a bioluminescent luciferase donor protein and a fluorescent acceptor protein
Implementation Method 2
a first BRET acceptor comprising a monomeric mNeonGreen (mNG) fluorescent protein; and a second BRET acceptor comprising a red fluorescent protein (RFP)
Data Source
AI summary
A biosensor for detecting proteolytic cleavage activity of two proteases, coronavirus main protease (Mpro) and papain-like protease (PLpro) is provided. The biosensor utilizes Bioluminescence Resonance Energy Transfer (BRET) between a donor and two acceptor moieties.