In vitro selection of thermostable ribozymes

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Compared to protein enzymes, the relationships between structure and function in ribozyme catalysis are still poorly understood. While the investigation of ribosomal RNAs from thermophilic organisms identified determinants of their thermal stability, there is very little information available on RNA catalysis at elevated temperatures. In vitro selection has allowed to select numerous new catalytic activities, and our lab has successfully implemented direct selection with tethered reactants to isolate ribozymes with Diels-Alderase activities. In this project, we plan to evolve Diels-Alderase ribozymes with increased thermal stability. Different selection strategies will be investigated, and selection pressure gradually increased. Isolated variants will be sequenced, their kinetics studied, and their temperature-activity profile determined. Selected variants will then be compared for structural principles of ribozyme stabilization. The anticipated insight into mechanisms of thermal stabilization of ribozymes should provide knowledge of general significance and should allow comparison with the respective strategies in protein enzymes.