RNA (ribonucleic acid) is a central molecule in all cells, and plays a vital role in helping living beings adapt to stress, or grow in new environments. Certain RNAs act by turning other genes on and off or by ensuring the faithful synthesis of proteins. Many RNA molecules fold into specific three-dimensional shapes that allow them to perform these different functions in the cell. The goal of this research project is to learn the physical rules that govern how RNA molecules fold into specific three-dimensional shapes. This is important for understanding how RNAs evolve over time and in different organisms. It is also important for learning how to engineer RNA for applications such as toxin sensing or biofuel production. Sensitive microscopes will be used to observe how single RNA molecules fold over time and show whether the structure of a particular RNA is flexible or rigid. New photo-reactive chemical groups will be synthesized and used to turn on the function of the RNA with light. The ability to control RNA with light will produce new research tools that can be used by other scientists. In addition, this research project will contribute to science education and increase STEM education among underrepresented groups by providing research experience to undergraduate students and by engaging undergraduate teams to develop simplified protocols for laboratory modules on RNA structure and evolution. This research project will contribute to US competitiveness and is expected to produce new chemical tools for controlling RNA with light that can be used by other scientists and biotechnology companies.
Non-coding RNAs self-assemble into complex structures to catalyze biochemical reactions, regulate gene activity by diverse mechanisms, and scaffold cellular compartments in the nucleus and cytoplasm. They are important drivers of molecular evolution and essential for the normal growth of all life forms. This research aims to identify fundamental principles of RNA self-assembly, using catalytic RNAs (ribozymes) as model systems. Previous studies of a bacterial group I ribozyme demonstrated that cooperative networks of tertiary interactions in different regions of the RNA drive assembly toward a unique native conformation. It is not known whether this is a general property of all naturally evolved RNAs, nor how tertiary interactions alter the conformational dynamics of RNA. Single-molecule FRET (smFRET) will be used to monitor millisecond to second motions of RNA helices under different conditions and distinguish native and non-native conformations. The folding dynamics of the stable Azoarcus ribozyme will be contrasted with that of Twister ribozyme, which requires less Mg(II) for self-cleavage than for folding. New photo-reactive nucleotides will be synthesized to control folding and self-cleavage of the small Twister ribozyme with light. New photocaged nucleotides will be synthesized and combined with smFRET to directly observe the dynamical motions in the RNA before and after RNA self-cleavage. This project is jointly funded by the Molecular Biophysics Cluster in the Division of Molecular and Cellular Biosciences of the Biological Sciences Directorate and the Chemistry of Life Processes Program in the Division of Chemistry of the Mathematical and Physical Sciences Directorate.
