Analysis of duplex unwinding by RNA helicases using stopped-flow fluorescence spectroscopy.
The characterization of unwinding reactions by RNA helicases often requires the determination of rate constants that are too fast to be measured by traditional, manual gel-based methods. Stopped-flow fluorescence measurements allow access to fast unwinding rate constants. In this chapter, we outline strategies and experimental considerations for the design of stopped-flow fluorescence experiments to monitor duplex unwinding by RNA helicases, with focus on DEAD-box helicases. We discuss advantages, disadvantages, and technical considerations for stopped-flow approaches, as well as substrate design. In addition, we list protocols and explain functional information obtained with these experiments.
Department of Biochemistry, Center for RNA Molecular Biology, School of Medicine, Case Western Reserve University, Cleveland, Ohio, USA.
Andrea
A
Putnam
Eckhard
E
Jankowsky
D012313:RNA / Q000737:chemistry / Q000378:metabolism*; D020365:RNA Helicases / Q000378:metabolism*; D012330:RNA, Double-Stranded / Q000737:chemistry / Q000378:metabolism; D013050:Spectrometry, Fluorescence / Q000379:methods*
D012330:RNA, Double-Stranded; D012313:RNA; D020365:RNA Helicases