David Nesbitt and his group use ultrasensitive time, color, and polarization-resolved fluorescence to detect single RNA molecules in a confocal microscope. They focus the pulse train from a mode-locked laser into a sample of dilute RNA molecules that has, on average, less than single molecule in the detection region. They collimate the resulting weak fluorescence, separate it from the much stronger incident laser light, sort it by both polarization and color, and image it on single photon-counting avalanche photodiodes. Using FRET techniques, they can measure distances of 2-8 nm between specifically labeled sites on the RNA. This allows them to investigate the folding kinetics for RNA in real time at the single molecule level. Their efforts are focused on simplifying complex RNA structures to understand the mechanisms that stabilize specific structural folds. This information is crucial to understanding RNA-based enzymes, or ribozymes. In the future, their techniques should make it possible to probe the folding and unfolding of biomolecules in chemically active states.

Nesbitt's experiments require tethering RNA molecules to a glass cover slip. The researchers accomplish this with a conventional biotin-streptavidin biochemical technique. To eliminate the possible impact of surface effects on the folding dynamics, they have also developed methods for studying "free" RNA dynamics by exploiting "burst-mode" single-molecule microscopy. This technique allows them to watch species diffuse into and out of the confocal region. In the future, Nesbitt and his colleagues plan to combine optical tweezers with single molecule microscopy.

In experiments with both immobilized and freely diffusing small RNA structures, Nesbitt's group showed that light bonding, or docking, of a tetraloop with its receptor is strongly influenced by the presence of magnesium ions. For instance, the docking rate of immobilized structures increased 12-fold with increasing magnesium concentrations; at the same time, undocking rates fell slightly. Similar results were obtained for freely diffusing RNA structures. The group also found that eliminating both magnesium and sodium ions prevents docking, suggesting that cation enhancement of docking may not be specific. Such studies are important because structures like the tetraloop-receptor are what form and maintain the three-dimensional structure of large RNA molecules.