Structural analysis of human ADAR2-RNA complexes by X-ray crystallography

Adenosine deaminases acting on RNAs (ADARs) are a class of RNA editing enzymes found in metazoa that catalyze the hydrolytic deamination of adenosine to inosine in duplexed RNA. Inosine is a nucleotide that can base pair with cytidine, therefore, inosine is interpreted by cellular processes as guanosine. ADARs are functionally important in RNA recoding events, RNA structure modulation, innate immunity, and can be harnessed for therapeutically-driven base editing to treat genetic disorders. Guide RNAs (gRNAs) bearing various modifications can be used to recruit ADARs to edit sites of interest in a process called site-directed RNA editing (SDRE). To help advance the rational design of gRNAs for therapeutics, characterizing the structure-to-activity relationship of ADARs' recognition and binding of substrate duplex RNA at atomic resolution is critical. In this chapter, we describe the process of determining the structure of human ADAR2 bound to duplex RNA using X-ray crystallography. Solid phase synthesis of 8-azanebularine-modified RNAs and purification for binding and crystallographic studies are described. The overexpression and purification of ADARs and assembly of the protein-RNA complex are detailed. Lastly, methods for crystallizing ADAR-RNA complexes and X-ray structure determination and data refinement strategies are outlined.