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A Single-Molecule Study of RNA Catalysis and Folding

Science  16 Jun 2000:
Vol. 288, Issue 5473, pp. 2048-2051
DOI: 10.1126/science.288.5473.2048

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Abstract

Using fluorescence 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 indistinguishable from the unmodified 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 determination 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 fluorescence as a powerful tool for examining RNA folding.

  • * These authors contributed equally to this work.

  • To whom correspondence should be addressed. E-mail: herschla{at}cmgm.stanford.edu (D.H.) and schu{at}stanford.edu (S.C.)

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