Ribonucleic acid polymers (RNA) are similar to proteins in that they can fold into defined three-dimensional shapes that are important for their function. RNAs have many functions in the cell in addition to coding for protein synthesis, and some long non-coding RNAs are now known to fold into structures that possess protein-like functions. This project will develop novel methods that will allow researchers to understand the complex process of RNA folding in biology, and to control this process to produce designed RNA molecules with desired three-dimensional shapes for use in synthetic biology. The project has the potential to revolutionize the study of RNA folding, providing a better understanding of how RNAs fold in living cells. To bring an appreciation of such biomolecular structures to the general public, the project will expand the "Molecular Playground," a system that projects animated, architectural scale molecular images in public spaces, with which the public can directly interact through a simple wave of the hand (a passerby can, for example, "grab" and rotate the molecule to see it from all sides). Specifically, the project will facilitate installation of inexpensive exhibits in middle and high schools, including those serving groups underrepresented in science, with an aim towards exciting young people towards the exploration of STEM fields.
Sequential, cotranscriptional folding of long noncoding RNAs (ncRNA), often accompanied by programmed RNA polymerase pausing, is now appreciated to be critical to proper ncRNA function. Current in vitro RNA folding studies do not mimic the resulting directed topological constraints. Using toehold mediated strand displacement this proposal will develop a novel system that allows controlled, sequential release of RNA from an unstructured RNA-DNA duplex, modeling sequential, cotranscriptional folding. Breaking the exogenous displacing DNA strand into two segments or introducing mismatches in it allows the introduction of controlled pausing in folding. Initial characterization of the system will follow folding of fluorescent aptamers, and later studies will be directed at understanding folding of the coenzyme B12 riboswitch, a system known to require polymerase pausing. Critically, perturbations will not be introduced into the RNA itself, but will be engineered only into the displacing strand, providing a native fold in all cases. This novel approach has the potential to revolutionize the study of RNA folding, providing a better understanding of how ncRNAs fold in vivo, and providing new tools for synthetic biology.
