Library Screening Reveals Sequence Motifs That Enable ADAR2 Editing at Recalcitrant Sites

Abstract

Adenosine deaminases acting on RNA (ADARs) catalyze the hydrolytic deamination of adenosine to inosine in duplex RNA. The inosine product preferentially base pairs with cytidine resulting in an effective A-to-G edit in RNA. ADAR editing can result in a recoding event alongside other alterations to RNA function. A consequence of ADARs' selective activity on duplex RNA is that guide RNAs (gRNAs) can be designed to target an adenosine of interest and promote a desired recoding event. One of ADAR's main limitations is its preference to edit adenosines with specific 5′ and 3′ nearest neighbor nucleotides (e.g., 5′ U, 3′ G). Current rational design approaches are wellsuited for this ideal sequence context, but limited when applied to difficult-to-edit sites. Here we describe a strategy for the in vitro evaluation of very large libraries of ADAR substrates (En Masse Evaluation of RNA Guides, EMERGe). EMERGe allows for a comprehensive screening of ADAR substrate RNAs that complements current design approaches. We used this approach to identify sequence motifs for gRNAs that enable editing in otherwise difficult-to-edit target sites. A guide RNA bearing one of these sequence motifs enabled the cellular repair of a premature termination codon arising from mutation of the MECP2 gene associated with Rett Syndrome. EMERGe provides an advancement in screening that not only allows for novel gRNA design, but also furthers our understanding of ADARs' specific RNA−protein interactions.

Site-specific regulation of RNA editing with ribose-modified nucleoside analogs in ADAR guide strands

“…ADAR2 wild-type and E488Q mutant overexpression and purification were performed as previously described ( 13 )…”

Structural and functional effects of inosine modification in mRNA

“…SDRE uses antisense oligonucleotides complementary to therapeutically relevant target sites to form the duplex structure required for ADAR recruitment and editing. Apart from reversing disease-causing G-to-A mutations in the transcript, SDRE also has the potential to suppress nonsense mutations by targeted inosine modification at premature termination codons (PTCs), as recently applied in PTCs causing the neurological disorder Rett syndrome ( Doherty et al 2022 ; Brinkman et al 2023 ; Jacobsen et al 2023 ).…”

RNA editing enzymes: structure, biological functions and applications

“…The two bases next to the edited adenosine in the RNA sequence play an important role in the efficiency of RNA editing [ 49 ]. RNA editing is more likely to occur when the edited adenosine is preceded by a pyrimidine and followed by a G (e.g., 5’-UAG-3’) [ 17 , 50 ]. In addition to adjacent nucleotides, mismatches at processing sites can also increase the processing efficiency.…”