CoV Protease Substrate-1 TF5 allows for efficient detection of coronavirus (CoV) proteases. This fluorescence resonance energy transfer (FRET)-based peptide substrate is cleaved by 3C-like proteases (3CLᵖʳᵒ), the CoV main protease involved in regulating the coronavirus replication complex. 3CLᵖʳᵒ is also a potential target for therapeutic development.

CoV Protease Substrate-1 TF5 has an excitation wavelength of 649 nm, an emission wavelength of 664 nm, and an extinction coefficient of 250,000 cm⁻¹ M⁻¹. It consists of a 14 amino acid sequence (KTSAVLQSGFRKME) that also contains the Tide Quencher™ 5 (TQ5; quencher) and Tide Fluor™ 5 (TF5; donor) fragments at the N- and C- termini, respectively. When the peptide substrate is intact, favorable energetic overlap of the TF5 excited state and the TQ5 absorption leads to efficient intramolecular fluorescence quenching of TF5 by the TQ5 fragment. Cleavage of the substrate by CoV proteases, however, dissociates the TF5 fragment from TQ5, restoring the fluorescence of the TF5 moiety that is directly correlated to the CoV protease activity.