Single Molecules in Attoliter Droplets: A Comparison of FRET from Free and Confined RNA

Single molecular-pair fluorescence resonance energy transfer (FRET) has become a cross-disciplinary tool for understanding molecular folding and interactions. To the extent that much of this work is in vitro, many methods have been devised to hold molecules in a detection volume. Among others, these include surface functionalization and attachment, liposomal confinement, and gel encapsulation. Here we demonstrate the use of free, but slowly diffusing, attoliter volume aqueous droplets in perfluorinated oil as nanoenvironments for single-molecule sensitive measurement. FRET from dye-labeled RNA confined to droplets is compared with FRET from freely-diffusing RNA. We show that droplet-confinement results in dramatically higher signal-to-noise and longer observation times than are possible for freely-diffusing molecules. The effects of confinement on the molecular environment will be discussed, and a method for determining the pH in attoliter droplets will be described.