Single molecular fluorescence and force spectroscopy

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The long-term goal of this work is to develop an improved understanding of the mechanics of folding/unfolding in biological macromolecules. We will use a variety of state-of-the-art biophysical techniques to study nucleic acids as a model system. Like proteins, RNA enzymes can carry out catalysis, and exhibit an array of primary, secondary, and tertiary structural elements in their active, folded configurations. However, in contrast to polypeptides, RNA is based on a polymer repertoire of 4 bases instead of 20 amino acids. They also display a more hierarchical relation between secondary and tertiary structural motifs. RNA enzymes are also more easily synthesized, modified, manipulated, and modeled, all of which make them attractive candidates for biophysical studies. It is anticipated that some principles of RNA- and DNA-folding will generalize into the protein world, in addition to providing further insights into nucleic acid biochemistry. This proposal will concentrate on one of the best-characterized ribonucleic acid enzymes, the Group I intron ribozyme from T. thermophila and its derivatives. Our recent work has demonstrated the feasibility of studying folding, unfolding, and catalysis in the Tetrahymena ribozyme at the single molecule level. Single molecule methods such as single-molecule fluorescence energy transfer (FRET) and single-molecule optical force spectroscopy can reveal details of folding intermediates, enzyme stochasticity, kinetic rates and paths, that are not readily accessible through bulk methods. By placing fluorescent dye molecules in a variety of locations on the ribozyme, we propose to study the first stages of rapid collapse and the role of fluctuations in folding, as well as search for new intermediate states and further map the folding landscape of this enzyme. We also plan to use high resolution optical tweezers to measure how the ribozyme denatures under specific force loads. High-resolution measurements of the end-to-end distance of the molecule as a function of load should allow to assign specific features of this spectra to specific structural states. Finally, these physically-based studies of the how the well characterized Tetrahymena ribozyme folds and unfolds will no doubt add to our understanding of more complicated ribozymes and medically relevant RNA enzymes, such as the ribosome.