A NanoLuc-Based Protease Biosensor for Highly Sensitive Detection of Intracellular Protease Activity: Applications to...

FlipNanoLuc is a highly sensitive protease biosensor based on the β-strand-flipping principle of NanoLuc luciferase, which is derived from Oplophorus gracilirostris. In the inactive configuration, one β-strand of NanoLuc is repositioned, thereby suppressing luciferase activity. Upon cleavage of the embedded protease recognition sequence by a target protease, the flipped β-strand is released, and luciferase activity is reconstituted. Incorporation of the LgBiT fragment (NanoBiT technology) yields strong luminescent output once the reporter is reconstituted, whereas the CL1-PEST1 degradation tag lowers background luminescence by promoting the degradation of the uncleaved, inactive form; together, these two modifications widen the dynamic range. A firefly luciferase normalization cassette connected via a P2A self-cleaving peptide is encoded in the same reporter plasmid, thereby eliminating the need for separate co-transfection. Because the readout directly reports intracellular protease activity in living cells, the system is suitable for detecting protease activation during apoptosis or viral infection and, in principle, for evaluating protease inhibitors and antiviral compounds. This protocol describes the following: (1) generation of HEK293T cells stably expressing FlipNanoLuc by retroviral transduction; (2) validation of reporter activity by protease overexpression; (3) detection of apoptosis using staurosporine; (4) detection of human coronavirus OC43 infection; and (5) detection of SARS-CoV-2 infection in BHK cells stably expressing hACE2. All luminescence assays employ dual-luciferase normalization and are compatible with standard 384-well plate readers. Key features • Highly sensitive detection of intracellular protease activity using a β-strand-flipping NanoLuc biosensor with dual-luciferase normalization encoded in a single plasmid. • Detects protease activity in living cells, including caspase-3 during apoptosis and 3CLpro during authentic OC43 and SARS-CoV-2 infection. • Quantitative luminescence readout in a 384-well plate format with a standard plate reader. • Adaptable to other proteases and to inhibitor or antiviral screening by exchanging the embedded recognition sequence.