In the usual bulk, or ensemble, measurements, the properties of all the molecules in the sample, contribute to the signal. For example, a fluorescence signal from the solution is a consequence of the different absorbance and emission properties of all the molecules with their varied dynamics. In ensemble measurements, the averaging over all conformations and species obscures the effects of minor contributions to the signal; the major contributors dominate the spectrum. However, single-molecule methods allow the measurement of the properties and reactions of one molecule at a time. By observing the conformational changes of each single molecule over time, one can learn about the distributions of properties and their dynamics, not just averages.
Because of the stochastic nature of kinetics, single-molecule methods are especially advantageous for studying reactions. Each molecule has a probability of reacting, but when it will actually react is not predictable. Thus, in bulk, reactions do not remain synchronized, and the resulting population average hides many kinetic details; intermediates may be difficult to detect. However, following the progression of a single molecule from reactant to product can reveal each intermediate; a detailed mechanism can be obtained. The kinetics and thermodynamics of the reaction can be obtained from the lifetimes of each conformation.
Any property that can be measured for one molecule at a time can be used to characterize molecules and their reactions. The two most widely used single-molecule methods applied to RNA structures and functions are fluorescence resonance energy transfer (FRET), and force versus extension measurements. A unique capability of studying one molecule at a time is to apply force to the molecule while not perturbing the rest of the solution. In such experiments, the molecule is attached to two beads controlled by optical tweezers, or it is attached to a surface and an atomic force microscope cantilever; the force on the molecule and the distance between the attached points are measured. Force becomes a thermodynamic variable, like temperature or pressure, that can influence a reaction. Force affects the equilibrium if there is a change in the length of the molecule during the reaction. Similarly, force affects rates of reactions depending on the distances to the transition states. Thus, force can be used to study thermodynamics and kinetics of reactions—such as unfolding of RNA—that would otherwise only occur at high temperatures, or in the presence of a denaturant. Unfolding and refolding of an RNA can be studied in the presence of proteins, other RNAs, ligands, and even mixtures approximating the contents of biological cells. Although the process is not the same as what occurs for RNA molecules in cells, it should be a better approximation than the conventional unfolding and folding studies in high concentrations of urea, or by thermal melting curves. The reversible mechanical work (force times distance) for unfolding an RNA is equal to the Gibbs free energy of unfolding. The temperature dependence of this work gives the enthalpy, and thus the entropy of the process.
In this article, we briefly describe methods used in studying single molecules, and review the applications of these methods to RNA reactions, including mechanical unfolding and folding of RNA secondary and tertiary structures, interactions with HIV reverse transcriptase, unfolding by helicases, and translation of messenger RNAs.