Mechanisms of HCV NS3 Helicase Monitored by Optical Tweezers
As one of the essential enzymes for viral genome replication, the hepatitis C virus NS3 helicase is one of the best characterized RNA helicases to date in understanding the mechanistic cycles in a helicase-catalyzed strand separation reaction. Recently, single-molecule studies on NS3, in particular the use of optical tweezers with sub-base pair spatial resolution, have allowed people to examine the potential elementary steps of NS3 in unwinding the double-stranded RNA fueled by ATP binding and hydrolysis. In this chapter, I detail the essential technical elements involved in conducting a high-resolution optical tweezers study of NS3 helicase, starting from the purification of the recombinant helicase protein from E. coli to setting up a high-resolution single-molecule experiment using optical tweezers.
Key wordsRNA helicase Optical tweezers Hepatitis C virus NS3 Single-molecule
This work was supported by NSF CAREER Award CHE1149670 to WC, NIH Director’s New Innovator Award 1DP2OD008693 to WC, and also in part by Research Grant No. 5-FY10-490 to WC from the March of Dimes Foundation.
- 6.Raney KD, Sharma SD, Moustafa IM, Cameron CE (2010) Hepatitis C virus non-structural protein 3 (HCV NS3): a multifunctional antiviral target. J Biol Chem 22725–22731Google Scholar
- 21.Bustamante C, Chemla YR, Moffitt JR (2009) High-resolution dual-trap optical tweezers with differential detection: instrument design. CSH Protoc, pdb ip73Google Scholar