RNA sequences that direct selective ADAR editing from a SELEX library bearing 8-azanebularine

Abstract

Adenosine Deaminases Acting on RNA (ADARs) catalyze the deamination of adenosine to inosine in double-stranded RNA (dsRNA). ADARs’ ability to recognize and edit dsRNA is dependent on local sequence context surrounding the edited adenosine and the length of the duplex. A deeper understanding of how editing efficiency is affected by mismatches, loops, and bulges around the editing site would aid in the development of therapeutic gRNAs for ADAR-mediated site-directed RNA editing (SDRE). Here, a SELEX (systematic evolution of ligands by exponential enrichment) approach was employed to identify dsRNA substrates that bind to the deaminase domain of human ADAR2 (hADAR2d) with high affinity. A library of single-stranded RNAs was hybridized with a fixed-sequence target strand containing the nucleoside analog 8-azanebularine that mimics the adenosine deamination transition state. The presence of this nucleoside analog in the library biased the screen to identify hit sequences compatible with adenosine deamination at the site of 8-azanebularine modification. SELEX also identified non-duplex structural elements that supported editing at the target site while inhibiting editing at bystander sites.