Dissecting the basis for differential substrate specificity of ADAR1 and ADAR2

To understand how secondary structure governs ADAR2 vs ADAR1 substrate selectivity, we employed two oligo libraries of structure and sequence variants previously reported in Uzonyi, A. et al, 2021. Each oligo pool was transfected into ADAR1-knockout HEK293T cells, alongside a plasmid expressing different Adenosine deaminases acting on RNA (ADAR) proteins or neither of them (No-ADAR), as a negative control. Based on the drawn conclusion of ADAR-mediated editing, novel ADAR recruiting RNAs(arRNA) were designed not only to improve the target editing efficiency but also to tune editing selectivity in ADAR-specific expressing context. Each arRNA-expressing plasmid was independently transfected into ADAR1-knockout HEK293T cells, alongside a plasmid expressing ADAR1 or ADAR2.