Modification of Nucleic Acid Analogs with Protein Domains and Photoswitches in Order to Regulate Gene Function

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Recently, we have been demonstrating the feasibility of applying adenosine to inosine (A-to-I) RNA editing to repair point mutations on the RNA-level. For this the catalytic deaminase domain of hADAR1 was engineered into an artificial guideRNA-dependent enzyme. This allowed for the efficient introduction of single point mutations at user-defined sites into mRNAs in a highly rational and simple way. Since A-to-I editing changes the sense of codons and the meaning of RNA processing signals, directed RNA-editing has immense potential as a tool in basic biology research and for application in medicine.In this proposal, we aim to further develop our directed editing strategy into a practical tool for the manipulation of protein and RNA function. Specifically, we want to establish our successful in-vitro-approach in cell culture and aim to apply it in the repair of disease-related point mutations on the RNA-level. To overcome a potential limitation of our initial approach, we aim to further develop a complementary approach which benefits from the possibility that all components are genetically encodable, and that endogenous hADAR can be harnessed for directed editing. Specifically, we want to further elaborate and understand this tool in vitro, but we also aim to establish its functioning inside the cell. If successful, the proposed work will pave the way for the application of directed RNA editing in basic biology and life sciences.