Harnessing Nature's Molecular Recognition Capabilities to Map and Study RNA Modifications.

Area of Science:

• Molecular Biology
• Genetics
• Biochemistry

Background:

• RNA editing, or epitranscriptomic modification, alters RNA after transcription, impacting gene expression and function.
• Over 170 RNA modifications exist, with dysregulation linked to diseases, necessitating precise detection methods.
• Current methods like antibodies and chemical labeling have limitations in selectivity and ease of use.

Purpose of the Study:

• To develop novel, selective methods for identifying and mapping RNA editing sites.
• To overcome limitations of existing antibody-based and chemical labeling techniques for epitranscriptomic studies.
• To leverage protein-based recognition for enhanced RNA modification analysis.

Main Methods:

• Repurposing Endonuclease V (EndoV), an enzyme that recognizes inosine in RNA.
• Developing EndoVIPER-seq for preparative enrichment of inosine-edited RNAs for RNA sequencing.
• Creating EndoVLISA, a plate-based immunoassay for quantifying inosine in cellular RNA.

Main Results:

• EndoVIPER-seq improved RNA sequencing coverage and identified new A-to-I editing sites.
• EndoVLISA enabled cost-effective, high-throughput quantification of differential A-to-I editing levels.
• EndoV demonstrated efficacy as a selective 'anti-inosine antibody' for RNA analysis.

Conclusions:

• Repurposing enzymes like EndoV offers a promising new direction for epitranscriptomics.
• These novel methods provide sensitive and selective tools for studying RNA modifications.
• Exploiting natural molecular recognition capabilities enhances the study of RNA editing and its role in health and disease.