BRET Mpro Biosensor with Repeated Cleavage Sites
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Solution Overview
Problem
Current assays for monitoring Mpro activity in living cells are limited by low sensitivity and high costs, particularly due to the need for double labeling of peptides and the inability to implement these assays in living cells.
Innovation Solution
Development of a BRET-based Mpro biosensor with increased Mpro-mediated proteolytic cleavage rate, achieved by incorporating synthetic Mpro cleavage sequences with repeated N-terminal autocleavage sites between mNeonGreen and NanoLuc reporter proteins, and further enhanced by inclusion of an Mpro-binding nanobody.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If a BRET-based Mpro biosensor is developed to monitor Mpro activity in living cells, then the ability to perform real-time monitoring in living cells is improved, but the cleavage rate and sensitivity remain insufficient
Solution Approach 1:
The biosensor is segmented into multiple functional domains: mNeonGreen reporter protein, repeated Mpro cleavage sites (2x, 4x, or 8x repeats), NanoLuc reporter protein, and Mpro-binding nanobody. This segmentation allows each component to perform its specific function optimally, with the repeated cleavage sites providing multiple targets for Mpro to increase cleavage rate while maintaining the BRET signaling capability for living cell monitoring
Solution Approach 2:
The Mpro-binding nanobody is nested within the biosensor construct, positioned to bind Mpro and recruit it to the cleavage sites. This nesting strategy increases the local concentration of Mpro at the cleavage sites without requiring overexpression of Mpro itself, thereby enhancing cleavage rate and sensitivity while maintaining compatibility with living cell monitoring
2Productivity
If synthetic Mpro cleavage sequences with repeated N-terminal autocleavage sites are incorporated to increase cleavage rate, then the cleavage rate is improved, but the biosensor design complexity increases
Solution Approach 1:
The cleavage rate is optimized by varying the number of repeated Mpro cleavage sites (2x, 4x, or 8x repeats) and by modifying the nanobody fusion position (N-terminal or C-terminal). These parameter changes allow systematic optimization of cleavage rate while providing a modular design framework that manages complexity through standardized repeating units
Solution Approach 2:
The biosensor design incorporates universal modular elements: the Mpro-binding nanobody serves multiple functions by both recruiting Mpro to enhance cleavage rate and by providing a standardized platform for fusion to different cleavage site repeats. This multi-functionality reduces overall design complexity by using a single versatile component for multiple purposes
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 biosensor exhibits a significantly enhanced cleavage rate and sensitivity, allowing for more effective monitoring of Mpro activity in living cells, which is useful for drug discovery and functional genomics applications, including characterization of Mpro mutations and point-of-care testing for SARS-COV-2 infection.
Implementation Method 1
NanoLuc (NLuc) reporter protein
Implementation Method 2
Bioluminescence Resonance Energy Transfer (BRET)-based Mpro biosensor
Implementation Method 3
Mpro-mediated proteolytic cleavage rate
Data Source
AI summary
Example systems, methods, and apparatus are disclosed herein for a BRET-based Miro biosensor including an mNeonGreen (mNG) reporter protein, a NanoLuc (NLuc) reporter protein, and x repeats of an N-terminal autocleavage peptide sequence of Mpro. The x repeats of an N-terminal autocleavage peptide sequence of Mpro are located between the mNG reporter protein and the NLuc reporter protein.