A BRET-based Mpro 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 (M pro ) biosensor containing the M pro N-terminal autocleavage sequence in tandem and a nanobody that shows an enhanced rate of M pro -mediated proteolytic cleavage. Specifically, we designed M pro biosensors containing 2×, 4× and 8× repeats of M pro N-terminal autocleavage sequences and a combination of M pro 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 M pro 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 12× -syn cleavage site, and an increased rate of cleavage and a decreased pharmacological inhibitor efficacy for the M pro biosensor containing 2× cleavage sequences. Further, mutational analysis revealed a requirement for both cleavage sites to be intact for increased cleavage rate. Importantly, the inclusion of an M pro -binding, but non-inhibiting, NB2E3 nanobody at the N-terminal further increased the cleavage rate of the 2× cleavage sequence-containing M pro biosensor. We envisage that the NB2E3 nanobody-2× M pro biosensor engineered here will be useful in drug discovery and functional characterization of M pro 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.