UC Davis

An important class of enzymes involved in RNA editing is the ADAR family (adenosine deaminases acting on RNA), which facilitate the deamination of adenosine (A) to inosine (I) in double-stranded RNA (dsRNA). Inosines are decoded as guanosines (G) in most cellular processes; hence, A-to-I editing can be considered an A-to-G substitution. Among the RNA editing enzymes, ADARs are of particular interest because a large portion of RNA editing events are due to A-to-I editing by the two catalytically active human ADARs (ADAR1 and ADAR2). ADARs have diverse roles in RNA processing, gene expression regulation, and innate immunity; and mutations in the ADAR genes and dysregulated ADAR activity have been associated with cancer, autoimmune diseases, and neurological disorders. A-to-I editing is also currently being explored for correcting disease-causing mutations in the RNA, where therapeutic guide oligonucleotides complementary to the target transcript are used to form a dsRNA substrate and site-specifically direct ADAR editing. Knowledge of the mechanism of ADAR-catalyzed reaction and the origin of its substrate selectivity will allow understanding of ADAR’s role in disease biology and expedite the process of developing ADAR-targeted therapeutics.

This dissertation describes some biochemical and structural studies that were performed to gain more detailed insights into substrate recognition by both ADAR1 and ADAR2 leading to the informed design of ADAR-selective inhibitors and editing-enabling guide oligonucleotides for directed editing applications with ADARs. Chapter 1 gives a general introduction to human ADAR1 and ADAR2, their association with human diseases, and the search for ADAR inhibitors. It also describes the potential of ADARs for the emerging field of therapeutic site-directed RNA editing and expounds on what is currently known about ADAR mechanism, substrate specificity and selectivity. Chapter 2 details some biochemical experiments aimed to characterize the ADAR2 dimerization interface and illuminate the function of ADAR dimerization on substrate recognition and editing. The information derived from these studies were then utilized to design and test protein and peptide blockers of ADAR dimerization for editing inhibition in Chapter 3. Given the lag in the molecular understanding of ADAR1 compared to ADAR2, Chapter 4 presents efforts to probe substrate recognition, obtain high resolution structure, and develop selective inhibitors of ADAR1 using 8-azanebularine-modified RNA duplexes. Finally, Chapter 5 details X-ray crystallography studies conducted to characterize a chemical modification and unique sequence motifs in guide oligonucleotides that enabled editing at non-sequence-preferred therapeutic target sites for directed editing with ADARs.