The SensoLyte® 520 SARS-CoV-2 3CL Protease Activity Assay Kit employs a fluorescence FRET peptide substrate for the detection of enzyme activity. The sequence of this substrate is based on the site-specific cleavage sequence for coronavirus 3CLpro enzymes. When active 3CL Protease cleaves the FRET substrate, it results in an increase of HiLyte Fluor™ 488 green fluorescence monitored at excitation/emission = 490 nm/520 nm. With superior fluorescence quantum yield and longer emission wavelength, the HiLyte™ Fluor 488/QXL® 520 based FRET peptide has less interference from the autofluorescence of test compounds and cellular components and provides better assay sensitivity. This assay can detect as low as 15.6 ng/mL active 3CL protease in the sample. This kit can be used to detect enzyme activity in purified enzyme preparations, biological samples and can also be applied for the compound screening.
SARS-CoV-2 3-chymotrypsin-like protease (3CL protease, also called the main protease) is essential for processing the polyproteins that are translated from the viral RNA. It is involved in the transcription and replication during the viral infection. 3CL protease cleaves the replicase 1ab at at least 11 cleavage sites producing proteins that are assembled into the virion. The advantage of choosing 3CL protease as the SARS-CoV-2 virus drug target is that the protein structure is well conserved across the Coronaviridae family.