A BRET-based M^pro biosensor containing a nanobody and tandem cleavage sites shows an increased cleavage rate

Here, we report the engineering of a Bioluminescence Resonance Energy Transfer (BRET)-based SARS-CoV-2 main protease (Mpro) biosensor containing the Mpro N-terminal autocleavage sequence in tandem and a nanobody that shows an enhanced rate of Mpro-mediated proteolytic cleavage. Specifically, we designed Mpro biosensors containing 2x, 4x and 8x repeats of Mpro N-terminal autocleavage sequences and a combination of Mpro cleavage sequences containing a total of 12 cleavage sites sandwiched between mNeonGreen (mNG) and NanoLuc (NLuc). Gaussian accelerated molecular dynamics (GaMD) simulations of the predicted alpha-helical synthetic Mpro cleavage sequences revealed a dynamic nature of the cleavage sequences, which is critical for their efficient cleavage, and a relatively short end-to-end distances, which is required for high BRET. Live cell assays revealed a cleavage sequence length-dependent resonance energy transfer, except for the 12x -syn cleavage site, and an increased rate of cleavage and a decreased pharmacological inhibitor efficacy for the Mpro biosensor containing 2x cleavage sequences. Further, mutational analysis revealed a requirement for both cleavage sites to be intact for increased cleavage rate. Importantly, the inclusion of an Mpro-binding, but noninhibiting, NB2E3 nanobody at the N-terminal further increased the cleavage rate of the 2x cleavage sequence-containing Mpro biosensor. We envisage that the NB2E3 nanobody-2x Mpro biosensor engineered here will be useful in drug discovery and functional characterization of Mpro mutants in newly emerging SARS-CoV-2 variants as well as in detecting SARS-CoV-2 infection in a point-of-care testing (POCT) format.