A single-molecule study of RNA catalysis and folding

Using fluorescence microscopy, we studied the catalysis by and folding of i ndividual Tetrahymena thermophila ribozyme molecules. The dye-labeled and s urface-immobilized ribozymes used were shown to be functionally indistingui shable from the unmodified free ribozyme in solution. A reversible Local fo lding step in which a duplex docks and undocks from the ribozyme core was o bserved 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 ob served. In the overall folding process, intermediate folding states and mul tiple folding pathways were observed. In addition to observing previously e stablished folding pathways, a pathway with an observed folding rate consta nt of 1 per second was discovered. These results establish single-molecule fluorescence as a powerful tool for examining RNA folding.