In vitro selection of thermostable ribozymes

与蛋白质酶相比，核酶催化的结构和功能之间的关系仍然知之甚少。虽然对嗜热生物核糖体RNA的研究确定了它们热稳定性的决定因素，但关于高温下RNA催化的信息很少。体外筛选已经选择了许多新的催化活性，我们的实验室已经成功地实现了与拴系反应物的直接选择，以分离具有Diels-Alderase活性的核酶。在这个项目中，我们计划进化出具有更高热稳定性的Diels-Alderase核酶。将调查不同的选择策略，选择压力逐渐增加。分离的变异体将被测序，它们的动力学研究，并确定它们的温度-活性分布。然后将对选定的变异体进行核酶稳定的结构原理的比较。对核酶热稳定机制的预期洞察应该提供具有普遍意义的知识，并允许与蛋白质酶中各自的策略进行比较。

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.