Session: Current Topics in Biological Chemistry:

Adenosine Deaminases Acting on RNA (ADARs) convert adenosine to inosine in double stranded RNA. Inosine is recognized by cellular machinery as guanosine, affording an A-to-G transition. Therefore, endogenous human ADARs can be used to correct disease-causing point mutations at the RNA level, by directing them to target sites in the transcriptome via complementary guide strands. The use of endogenous editing enzymes can minimize barriers associated with off-target editing and enzyme delivery, and presents a potentially safer alternative to genome editing for therapeutic applications. This strategy requires the use of guide oligonucleotides that can stimulate efficient directed editing, leading to efforts aimed at optimizing guide RNAs. Here we use structural information for ADAR2-RNA complexes to guide the design of nucleoside analogs for the position in the guide strand that contacts a conserved glutamic acid residue in ADARs (E488 in ADAR2), where mutation of this residue to glutamate (E488Q) results in enzyme hyperactivity. We designed and evaluated chemical modifications that mimic this activating mutation, and found that Benner’s Z nucleotide (a cytidine analog) increased the observed rate when compared to the preferred canonical base. We obtained a high-resolution X-ray crystal structure of ADAR2 complexed with a duplex containing the Z nucleotide, which revealed its effect on the contact with E488. Finally, we report that this single nucleotide modification increased directed editing yields in human cells and mouse primary liver fibroblasts. Our results show that modification of the guide RNA can mimic the effect of hyperactive mutants of ADARs, and lays the groundwork for additional rational design of ADAR guide strands.

Dean Tantillo

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