A Small Cationic Probe for Accurate, Punctate Discovery of RNA Tertiary Structure

Area of Science:

• Biochemistry
• Molecular Biology
• Structural Biology

Background:

• RNA molecules fold into complex 3D structures essential for their biological functions.
• Identifying novel RNA tertiary structural motifs remains a significant challenge in molecular biology.

Purpose of the Study:

• To develop a reliable method for discovering RNA tertiary structural motifs.
• To characterize the structural signature of these motifs and their functional relevance.

Main Methods:

• Utilized trimethyloxonium (TMO), a positively charged probe, to selectively react with electronegative sites on RNA.
• Compared TMO reactivity with dimethyl sulfate (DMS) to identify sites indicative of tertiary structure (T-sites).
• Mapped T-sites to identify higher-order structural interactions and functional centers in diverse RNA molecules, including the dengue virus genome.

Main Results:

• Identified T-sites, characterized by a nucleobase adjacent to nonbridging phosphate oxygens, indicating localized negative charge and tertiary structure.
• Demonstrated that T-sites consistently map to core regions of RNA higher-order structures and functional centers.
• Detected three strong T-sites within the dengue virus genome, associated with essential viral replication structures.

Conclusions:

• Cation-based covalent chemistry provides a high-confidence approach for discovering and analyzing functional RNA tertiary motifs.
• This method enables structural analysis across long and complex RNA molecules, including transcriptome-wide applications.
• Opens new avenues for understanding RNA structure-function relationships and developing novel RNA-targeted therapeutics.