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.
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