MFB: Continuous evolution of RNAs with novel functions in mammalian cells

Functional RNA molecules hold tremendous potential for applications in biological research, bioengineering, and medicine. Importantly, RNAs can now be readily identified that bind virtually any other biomolecule of interest and display unique functions, including enabling the catalysis of chemical reactions, fluorescence, and more. But there is a major challenge obstructing progress – RNA structure is incredibly sensitive to the local environment. Current strategies to identify functional RNAs almost universally proceed in test tubes, mostly yielding functional RNAs that also work well only in a test tube and unfortunately fail when translated into biological environments. This project seeks to deliver and optimize a new platform that will enable rapid and robust identification of functional RNAs directly in the mammalian cellular environment. The approach involves application of next-generation strategies to attain laboratory-timescale RNA evolution in cells, enabling efficient selection for desired RNA functions. Because the novel RNAs are identified within cellular milieus to begin with, they will consistently function in biological settings – unlike functional RNAs discovered using current test tube-based methods. Alongside this progress, the project will provide opportunities for PhD student training as well as enable substantive expansion of an ongoing, successful high school internship program. The overarching hypothesis of this work is that in-cell based directed evolution can solve the environment-sensitive folding and structure challenge that has hamstrung the functional RNA field. The research aims to deliver a bespoke, custom-designed mammalian phage-assisted continuous evolution system to enable the robust and efficient identification of mammalian cell-functional RNA aptamers, allosteric activators, and glue RNAs. The selection couples developed will be highly modular and generally useful for anyone in the field to evolve functional RNAs of interests. The resulting RNA-mPACE platform is intended to deliver ease-of-use, strategies to support parallelization, scaling, and speed of directed evolution campaigns, and methods to specifically target mutagenesis to the gene encoding the RNA of interest to further accelerate functional RNA development. Successful execution of this research will enable the enormous potential of functional RNAs in biology to finally be unleashed. For research applications, it will become far more feasible to modulate the activity of any biomolecule of interest. For medical applications, RNAs could become a preferred modality for all types of targets. Key fundamental insights will also become accessible, especially related to how cellular environments shape RNA structure-function landscapes. This work is jointly supported by the Division of Molecular and Cellular Biosciences (MCB) in the Directorate for Biological Sciences, by the Division of Chemistry in the Directorate for Mathematical and Physical Sciences, and by the Systems and Synthetic Biology cluster in MCB. This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.