UC Davis

Adenosine deaminases acting on RNA (ADAR) are a family of enzymes responsible for the hydrolytic deamination of adenosine (A) to inosine (I), specifically in double-stranded RNA. This editing event can have coding as well as non-coding effects in the transcriptome and proteome, considering that (I) may induce secondary RNA structures and because (I) is translated as guanosine (G) by cellular machinery. The family of ADARs (ADAR1, ADAR2, and ADAR3) are endogenous to humans, but only ADAR1 and ADAR2 are catalytically active. ADARs have multiple crucial roles in RNA processing, thus, dysregulation of ADAR activity has been linked to many diseases, including cancer. While there is abundant work in characterizing the mechanism, structure, and function of these enzymes, there is substantial work in harnessing ADARs to correct mutagenic transcripts; a technique often called Site-Directed RNA Editing (SDRE). In brief, a therapeutic guide oligonucleotide (complementary to the mutated RNA target of interest) is inserted to create a double-stranded RNA substrate and site-specifically recruit ADAR for RNA editing. Knowledge of this enzyme’s mechanism, crystal structures, and substrate selectivity will allow us to understand ADAR’s role in disease biology and speed up the development of ADAR-based therapeutics. This dissertation describes the understanding of known high-resolution crystal structures of ADAR2 to rationally design guide oligonucleotides to direct ADAR editing and probe unknown structural features of ADAR1. More specifically, this dissertation details the use of nucleoside analogs and chemical biology to test most of the hypotheses presented herein. Chapter 1 provides a general introduction tailored to the content of the following chapters. It describes the detailed function of ADAR enzymes, their association to diseases, the mechanism, and its use for SDRE. Chapter 2 and Chapter 3 focus on designing guide oligonucleotides to direct therapeutic and highly selective editing in 5’-UA and 5’-AA sequence contexts (A is target adenosine). Chapter 4 provides a structure rationale to design ADAR1 inhibitors and explores methods to covalently cross-link RNA duplexes with modified nucleosides. Chapter 5 details X-ray crystallography studies to design purine analogs that enhance the ADAR reaction at disfavored sites, like 5’-GA. Lastly, Chapter 6 presents efforts to synthesize a 128 nucleotide (nt) long pegRNA with a guanosine analog for tethering to the reverse transcriptase of a prime editor for complex stabilization and cryo-electron microscopy studies.