Chemical Modifications in RNA: Elucidating the Chemistry of dsRNA-Specific Adenosine Deaminases (ADARs)

ConspectusThe term RNA editing refers to any structural change in an RNA molecule (e.g., insertion, deletion, or base modification) that changes its coding properties and is not a result of splicing. An important class of enzymes involved in RNA editing is the ADAR family (adenosine deaminases acting on RNA), which facilitates 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 to be 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 to site-specifically direct ADAR editing. Knowledge of the mechanism of the ADAR-catalyzed reaction and the origin of its substrate selectivity will lead to a broader understanding of ADAR's role in disease biology and expedite the process of developing ADAR-targeted therapeutics.Chemically modified oligonucleotides provide a versatile platform for modulating the activity and interrogating the structure, function, and selectivity of nucleic acid binding and modifying proteins. In this Account, we provide an overview of oligonucleotide modifications that have allowed us to gain a deeper understanding of ADAR's molecular mechanisms, which we utilize in the rational design and optimization of ADAR activity modulators. First, we describe the use of the nucleoside analog 8-azanebularine (8-azaN) to generate high-affinity ADAR-RNA complexes for biochemical and biophysical studies with ADARs, with a particular emphasis on X-ray crystallography. We then discuss key observations derived from the crystal structures of ADAR2 bound to 8-azaN-modified RNA duplexes and describe how these findings provided insight into ADAR editing optimization by introducing nucleoside modifications at various positions in the synthetic guide strands. We also present the informed design of 8-azaN-modified RNA duplexes that selectively bind and inhibit ADAR1 but not the closely related ADAR2 enzyme. Finally, we conclude with some open questions on ADAR structure and substrate recognition and share our current endeavors in the development of ADAR guide oligonucleotides and inhibitors.