Analysis of duplex unwinding by RNA helicases using stopped-flow fluorescence spectroscopy

Methods Enzymol. 2012;511:1-27. doi: 10.1016/B978-0-12-396546-2.00001-2.

Abstract

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.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • RNA / chemistry
  • RNA / metabolism*
  • RNA Helicases / metabolism*
  • RNA, Double-Stranded / chemistry
  • RNA, Double-Stranded / metabolism
  • Spectrometry, Fluorescence / methods*

Substances

  • RNA, Double-Stranded
  • RNA
  • RNA Helicases