Discovery and Tuning of RNA Editing Guides via High-Throughput Screening and Chemical Modification

Area of Science:

• Molecular Biology
• RNA Therapeutics
• Genetic Engineering

Background:

• Adenosine deaminases acting on RNA (ADAR) enzymes perform A-to-G recoding by deaminating adenosine to inosine in double-stranded RNA.
• Inosine is recognized as guanosine during translation, enabling programmable RNA editing for therapeutic applications.
• Rett syndrome is a neurological disorder associated with mutations in the MeCP2 transcript, often involving premature termination codons.

Purpose of the Study:

• To discover novel guide RNA motifs for efficient ADAR-mediated RNA editing.
• To optimize these motifs for therapeutic applications, enhancing stability and cellular viability.
• To establish a platform for identifying and refining guide RNA features for site-directed RNA editing.

Main Methods:

• Utilized EMERGe, a high-throughput screening platform, to identify guide RNA motifs targeting the MeCP2 transcript.
• Conducted structure-activity relationship studies and chemical modifications (2'-O-methyl, 2'-fluoro, phosphorothioate) to optimize guide RNA.
• Performed reporter assays in HEK293T cells to assess editing efficiency and dose-dependency at MECP2 loci.

Main Results:

• Identified a novel guide RNA motif (5'-GUG-3') that supports efficient ADAR2-mediated RNA editing in vitro.
• Developed a chemically modified guide RNA that retains editing activity and exhibits enhanced nuclease resistance.
• Demonstrated dose-dependent editing at two MECP2 loci in HEK293T cells using the optimized guide RNA.

Conclusions:

• The EMERGe platform is effective for discovering novel editing-enabling guide RNA motifs.
• Targeted chemical modification can render discovered motifs therapeutically viable in cellular contexts.
• This study establishes a pipeline for optimizing guide RNAs beyond conventional design principles for RNA editing therapies.