Probing RNA structural dynamics and function by fluorescence resonance energy transfer (FRET).
Biological function of RNA is often mediated by cyclic switching between several (meta-)stable arrangements of tertiary structure. Fluorophore labeling of RNA offers a unique view into these folding and conformational switching events, since a fluorescence signal is sensitive to its molecular environment and can be continuously monitored in real time to produce kinetic rate information. This unit focuses on the practical implications of using fluorescence resonance energy transfer (FRET) to probe RNA structural dynamics and function. FRET is a particularly powerful fluorescence technique since, in addition to kinetic data, it provides insights into the structural basis of a conformational rearrangement. Protocols describe how to postsynthetically label RNA for FRET and how to acquire and analyze FRET data. Support protocols describe methods for deprotecting synthetic RNA and for purifying RNA by gel electrophoresis and HPLC. Considerations for selecting appropriate RNA, fluorophores, and labeling strategies are discussed in detail in the commentary.
Walter
Nils G
NG
University of Michigan, Ann Arbor, Michigan.
D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D005456:Fluorescent Dyes; D007700:Kinetics; D015336:Molecular Probe Techniques; D009690:Nucleic Acid Conformation; D012313:RNA / Q000737:chemistry*
D005456:Fluorescent Dyes; D012313:RNA