A single-molecule study of RNA catalysis and folding

Using ßuorescence microscopy, we studied the catalysis by and folding of individual Tetrahymena thermophila ribozyme molecules. The dye-labeled and surface-immobilized ribozymes used were shown to be functionally indistin-guishable from the unmodiÞed free ribozyme in solution. A reversible local folding step in which a duplex docks and undocks from the ribozyme core was observed directly in single-molecule time trajectories, allowing the determi-nation of the rate constants and characterization of the transition state. A rarely populated docked state, not measurable by ensemble methods, was observed. In the overall folding process, intermediate folding states and multiple folding pathways were observed. In addition to observing previously established folding pathways, a pathway with an observed folding rate constant of 1 per second was discovered. These results establish single-molecule ßuorescence as a pow-erful tool for examining RNA folding. Virtually all knowledge of chemical processes has been deduced from ensemble measure