UC Davis

Adenosine Deaminases Acting on RNA (ADARs) are responsible for the deamination of adenosine (A) to inosine in double-stranded RNAs (dsRNA). Inosine (I) is read as guanosine (G) by molecular machinery so ADARs effectively catalyze an A to G point mutation. This RNA editing ability has designated the ADAR family as a target for therapeutic development in a method called ADAR-mediated site directed RNA editing (SDRE). This modality involves introducing guide oligonucleotides which specifically hybridize to a disease-causing mutation in the target RNA and recruiting ADAR to edit that specific mutation. Thus, ADAR-mediated RNA editing has strong potential to treat genetic disorders at the RNA level and mitigate or eliminate symptoms. However, ADARs do not edit every adenosine in dsRNA equally; details such as sequence context, RNA secondary structures, and chemical modifications factor into how well ADAR can edit a particular A. The broad goal of the works in this dissertation is to gain insight into the structures and modifications that can be included in the substrate RNA which can facilitate specific efficient editing at a desired site. Chapter 1 provides a general introduction of methods used to treat genetic disorders and how ADARs have been adopted into this field of therapeutics. A brief background of the work done to investigate the structure and function of ADARs is included to describe the potential and limitations associated with ADAR-mediated SDRE. In Chapter 2, a systematic evolution of ligands by exponential enrichment (SELEX) approach is used to filter a large library of RNA substrates for the highest affinity binding substrates to determine how secondary structures in the RNA duplex can impact binding to ADAR and the subsequent editing with the discovered structures. Chapters 3 and 4 investigate the effect of 2’-sugar modifications on ADAR editing activity. Chapter 3 details my contribution to a fellow lab member’s project investigating the effect of 2’-Fluoro (2’-F) modifications to RNA residues within a particular binding domain on the substrate RNA. My work here provides insight into the effects a 2’-F has on ADAR-RNA interactions that led to increased editing rates using a 2’-F. Chapter 4 establishes the baseline for the use of copper-click handles on the 2’-sugar to be used in substrate RNAs so we can investigate the effects of propargyl groups and a variety of triazole products on RNA editing.