Control of ADAR2 Dimerization and RNA Editing Efficiency by Site-Specific 2'-Fluoro Modification of Guide RNAs

Area of Science:

• Molecular Biology
• RNA Therapeutics
• Enzymology

Background:

• Adenosine Deaminases Acting on RNA (ADARs) are enzymes that convert adenosine to inosine in double-stranded RNA (dsRNA).
• Site-directed RNA editing (SDRE) employs guide RNAs (gRNAs) to direct ADARs for correcting disease-causing mutations.
• Modifications in gRNAs can enhance ADAR-mediated RNA editing efficiency, but modifications at the dsRNA binding domain (dsRBD)-RNA interface are less understood.

Purpose of the Study:

• To investigate the impact of 2'-modifications in the gRNA dsRBD binding site on ADAR-catalyzed RNA editing rates.
• To identify specific gRNA modifications that enhance the efficiency of therapeutic RNA editing.

Main Methods:

• Synthesis of gRNAs with 2'-modifications (2'-F, 2'-OMe) at specific positions within the dsRBD binding site.
• In vitro and in cellulo assays to measure ADAR1 and ADAR2 catalyzed adenosine deamination rates.
• Structural analysis (crystal structure) and biochemical assays (EMSA, mass photometry) to elucidate the mechanism of enhanced editing.

Main Results:

• Replacing a 2'-OH with a 2'-F at specific gRNA positions significantly increased in vitro ADAR2 editing rates for two sequences.
• 2'-OMe modifications at these positions were inhibitory, while 2'-F modifications were validated in cellulo.
• ADAR1-catalyzed deamination was not stimulated by these 2'-F modifications; structural and biochemical data suggest 2'-F at +13 promotes ADAR2 dimerization.

Conclusions:

• Specific 2'-F modifications on gRNAs can substantially enhance ADAR2-mediated RNA editing efficiency.
• These findings provide insights into RNA features that optimize ADAR substrates and guide the design of improved gRNAs for therapeutic applications.
• The observed enhancement is specific to ADAR2 and may involve facilitated enzyme dimerization.