25 RNA Folding, Unfolding, and Dynamics, One Molecule at a Time

Abstract

Single-molecule methods allow the measurement of the properties and reactions of one molecule at a time. In bulk, or ensemble methods, the effects of all the molecules in the sample are measured; even in 1 μl of a 1 μM solution, there are about 10¹² molecules. Each molecule in the solution has a different conformation and a different interaction with solvent and other solutes. The conformations and interactions change with time as the molecules collide, flex, and breathe—as they undergo the thermal motions consistent with the free energy landscape of all the molecules in the solution. A fluorescence signal from the solution is a consequence of the different absorbance and emission properties of all the molecules with their varied dynamics. If a chemical reaction is occurring, or if there is an equilibrium between different species, the fluorescence signal becomes even more complex. Multiple species may have very different absorbance and emission properties, and their concentrations change with time during a reaction. The effective averaging over all conformations and species obscures the effects of minor contributions to the signal; the major contributors dominate the spectrum. Note that a bulk measurement provides an average value, but by observing single molecules, the investigator can learn about the distributions of properties, not just average values.

The signal for a single molecule depends on the molecular conformations and interactions of the molecule averaged over the time resolution of the measurement. A trajectory of the behavior of the molecule as a function of time is...