The RNA editing ADAR enzymes convert adenosines to inosines in RNA. This phenomenon is widespread with thousands of A to I sites known in human transcripts. Since inosine is decoded as guanosine during translation, this modification can lead to changes in the meaning of codons (recoding). There are at least 50 different A to I sites in human mRNAs that cause recoding and recoding is common in the nervous system. Indeed, ADARs are necessary for a properly functioning nervous system and are known to regulate behavior in metazoans. Mutations in the human ADAR1 gene cause the skin disorder Dyschromatosis Symmetrica Hereditaria (DSH) and the autoimmune disease Aicardi-Goutieres Syndrome (AGS). Also, ADAR1 upregulation and hyper editing has been observed in several different cancers. Despite the significance of this form of regulation of RNA structure and function, our understanding of the mechanism of A to I editing is incomplete. For instance, the selectivity for specific adenosines by different ADARs remains difficult to fully explain. Furthermore, pharmacological methods for controlling RNA editing do not currently exist. In addition, given ADARs’ ability to change RNA sequence, there is growing interest in harnessing this property and redirecting it to correct disease-associated G-to-A mutations. However, current strategies for directed A to I editing are not sufficiently selective or efficient. In this competitive renewal of an R01 project, we will address these knowledge gaps through the application of nucleic acid chemistry coupled with techniques from structural biology and biochemistry. The results of these studies will extend our basic understanding of the process of RNA editing as well as lead to new methods for its control. We will stabilize ADAR-RNA complexes using RNA bearing nucleoside analogs and solve their structures by X-ray crystallography in collaboration with Prof. Andrew Fisher. The studies proposed for the next funding period extend this fruitful collaboration to include a fragment of human ADAR2 comprising the deaminase domain and a double stranded RNA-binding domain 2 (dsRBD2) as well as full length human ADAR2. In addition, advances in our ability to obtain high concentrations of highly pure human ADAR1 deaminase domain enable crystallography with this protein and its RNA complexes to be carried out by us in the next funding period. A novel screen is proposed for the discovery of complementary mutant ADAR/modified guide RNA combinations for efficient directed editing applications. In addition, selections will be carried out to discover new guide strand modifications that facilitate ADAR editing. Finally, experiments are proposed to discover low molecular weight inhibitors of ADAR activity. !