A169 FLUORESCENT LABELING OF THE HCV HELICASE TO MONITOR NUCLEIC ACID UNWINDING BY FRET. (1st March 2018) - Ablenas, C
Abstract: Background: The hepatitis C virus (HCV) non-structural protein 3 (NS3) contains a helicase activity essential for viral replication. The helicase binds to single-stranded (ss) regions of nucleic acids and unwinds duplexes in an ATP-dependent manner. The mechanism by which the helicase disrupts RNA secondary structure in the viral genome to make way for the replication machinery remains elusive. Several mechanisms have been proposed, which include an active mechanism whereby the helicase actively engages the ss/double-stranded (ds) junction of the substrate to unwind the duplex, and a passive mechanism where the helicase binds and translocates along a ss nucleic acid overhang, taking advantage of transient melting at the ss/ds junction. Aims: To generate site-specific fluorescently labeled HCV helicase as a tool to track the movement of the enzyme during unwinding and monitor the dynamics of this process. Methods: The unnatural amino acid p-azido phenylalanine was incorporated in the recombinant HCV helicase during protein expression in E. coli . Using a strain-promoted azide-alkyne click reaction we developed a one-step process to screen for both protein expression and reactivity of the azido group from the incorporated unnatural amino acid. After successfully identifying a position in the helicase for incorporation of the unnatural amino acid and fluorescent labeling with a Cy5 fluorophore, we used the site-specific fluorescently labeled enzyme to monitor the location of binding by Förster Resonance Energy Transfer (FRET) to DNA substrates modified with an appropriate Cy3 donor fluorophore. Results: Using our approach to simultaneously screen for protein expression with the unnatural amino acid as well as reactivity of the incorporated unnatural amino acid, we identified a position in the HCV helicase suitable for incorporation of p-azido phenylalanine and fluorescent labeling with a Cy5 fluorescent dye. We then developed a plate-based FRET assay to confirm that we could detect the location of binding on a DNA substrate in a distance-dependent manner. Finally, using single molecule fluorescence microscopy we were able to detect binding by FRET for individual enzyme-substrate complexes. Conclusions: The FRET-based assay has the potential to monitor distinct steps of the unwinding process. Single molecule FRET experiments will provide a deeper understanding of the mechanism by which the helicase interacts with its substrate during unwinding and the dynamics involved in this process. Funding Agencies: CIHRthe Canadian Network on Hepatitis C (CanHepC), and the Fonds de recherche du Québec – Santé (FRQS)