Adenosine deaminases that act on RNA (ADARs) can be directed to predetermined sites in transcriptomes by forming duplex structures with exogenously delivered guide RNAs (gRNAs). They can then catalyze the hydrolytic deamination of adenosine to inosine in double stranded RNA, which is read as guanosine during translation. High resolution structures of ADAR2-RNA complexes revealed a unique conformation for the nucleotide in the guide strand base paired to the editing site's 5' nearest neighbor (-1 position). Here we describe the effect of 16 different nucleoside analogs at this position in a gRNA that targets a 5'-UA̲-3' site. We found that several analogs increase editing efficiency for both catalytically active human ADARs. In particular, 2'-deoxynebularine (dN) increased the ADAR1 and ADAR2 in vitro deamination rates when at the -1 position of gRNAs targeting the human MECP2 W104X site, the mouse IDUA W392X site, and a site in the 3'-UTR of human ACTB. Furthermore, a locked nucleic acid (LNA) modification at the -1 position was found to eliminate editing. When placed -1 to a bystander editing site in the MECP2 W104X sequence, bystander editing was eliminated while maintaining on-target editing. In vitro trends for four -1 nucleoside analogs were validated by directed editing of the MECP2 W104X site expressed on a reporter transcript in human cells. This work demonstrates the importance of the -1 position of the gRNA to ADAR editing and discloses nucleoside analogs for this site that modulate ADAR editing efficiency.

Adenosine deaminases acting on RNA (ADARs) are RNA editing enzymes that catalyze the hydrolytic deamination of adenosine (A) to inosine (I) in dsRNA. In humans, two catalytically active ADARs, ADAR1 and ADAR2, perform this A-to-I editing event. The growing field of nucleotide base editing has highlighted ADARs as promising therapeutic agents while multiple studies have also identified ADAR1's role in cancer progression. However, the potential for site-directed RNA editing as well as the rational design of inhibitors is being hindered by the lack of detailed molecular understanding of RNA recognition by ADAR1. Here, we designed short RNA duplexes containing the nucleoside analog, 8-azanebularine (8-azaN), to gain insight into molecular recognition by the human ADAR1 catalytic domain. From gel shift and in vitro deamination experiments, we validate ADAR1 catalytic domain's duplex secondary structure requirement and present a minimum duplex length for binding (14 bp, with 5 bp 5' and 8 bp 3' to editing site). These findings concur with predicted RNA-binding contacts from a previous structural model of the ADAR1 catalytic domain. Finally, we establish that neither 8-azaN as a free nucleoside nor a ssRNA bearing 8-azaN inhibits ADAR1 and demonstrate that the 8-azaN-modified RNA duplexes selectively inhibit ADAR1 and not the closely related ADAR2 enzyme.

ADARs (adenosine deaminases acting on RNA) can be directed to sites in the transcriptome by complementary guide strands allowing for the correction of disease-causing mutations at the RNA level. However, ADARs show bias against editing adenosines with a guanosine 5' nearest neighbor (5'-GA sites), limiting the scope of this approach. Earlier studies suggested this effect arises from a clash in the RNA minor groove involving the 2-amino group of the guanosine adjacent to an editing site. Here we show that nucleosides capable of pairing with guanosine in a syn conformation enhance editing for 5'-GA sites. We describe the crystal structure of a fragment of human ADAR2 bound to RNA bearing a G:G pair adjacent to an editing site. The two guanosines form a Gsyn:Ganti pair solving the steric problem by flipping the 2-amino group of the guanosine adjacent to the editing site into the major groove. Also, duplexes with 2'-deoxyadenosine and 3-deaza-2'-deoxyadenosine displayed increased editing efficiency, suggesting the formation of a Gsyn:AH+anti pair. This was supported by X-ray crystallography of an ADAR complex with RNA bearing a G:3-deaza dA pair. This study shows how non-Watson-Crick pairing in duplex RNA can facilitate ADAR editing enabling the design of next generation guide strands for therapeutic RNA editing.