RNA folding dynamics by single-molecule fluorescence resonance energy transfer

Single-molecule fluorescence resonance energy transfer (smFRET) microscopy has become an increasingly popular tool to study the structural dynamics of RNA molecules. It reveals, in real time, the structural dynamics of these molecules that would be otherwise hidden in ensemble-averaged measurements. Here we present a detailed protocol for performing smFRET using total internal reflection fluorescence microscopy, including RNA preparation, optical setup, separation of the dual color channels, sample immobilization and data acquisition and analysis.