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CoV Protease Substrate-2 EDANS allows for efficient detection of coronavirus (CoV) proteases. This fluorescence resonance energy transfer (FRET)-based peptide substrate can be cleaved by 3C-like protease (3CL???), the CoV main protease involved in regulating the coronavirus replication complex. 3CL??? is also a potential target for therapeutic applications. CoV Protease Substrate-2 EDANS has an excitation wavelength of 336 nm, an emission wavelength of 455 nm, and an extinction coefficient of 5900 cm?1 M?1. It consists of a 12 amino acid sequence (VNSTLQSGLRKM) that also contains the DABCYL (quencher) and EDANS (donor) fragments at the N- and C- termini, respectively. When the peptide substrate is intact, favorable energetic overlap of the EDANS excited state and the DABCYL absorption leads to efficient intramolecular fluorescence quenching of EDANS by the DABCYL fragment. Cleavage of the substrate by CoV proteases, however, dissociates the EDANS fragment from DABCYL, restoring the fluorescence of the EDANS moiety that is directly correlated to the CoV protease activity.