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 fluorescently tagged RNA molecules that has, on average, less than a single molecule in the detection region. They collect the resulting weak fluorescence, separate it from the much stronger incident laser light, sort it by both polarization and color, and image it onto 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, and riboswitches, which regulate gene expression.

The Nesbitt group recently began probing the folding and unfolding of lysine riboswitches, which regulate the expression of the genes implicated in lysine metabolism. When lysine binds to these riboswitches, they fold into new three-dimensional structures that prevent gene expression. In the future, Nesbitt plans to extend the studies of the lysine riboswitch to more general investigations of RNA-protein interactions.

Nesbitt and his colleagues have recently undertaken studies of the temperature-dependence of RNA folding. Their goal is to identify the fundamental forces that dictate the RNA folding patterns they observe in the laboratory. Preliminary results support the notion that for RNA, it is a major task to overcome its natural propensity to exist in disordered states and assume a more ordered (folded) state. For instance, at physiological temperatures, RNA in the laboratory is very dynamic and does not remain stably folded. Eventually, Nesbitt plans to develop a model for how temperature affects RNA folding and function.

Undocked RNA molecules (a) emit green light under laser illumination. Docked molecules (b) emit mostly red light.

Credit: The Nesbitt group

Nesbitt's experiments require tethering small 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 plans to combine optical tweezers with single molecule microscopy.

In experiments with both immobilized and freely diffusing small RNA structures, Nesbitt's group has shown that light bonding, or docking, of an RNA 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 small 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 much larger RNA molecules.

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