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.