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bioRxiv · 10.1101/2025.08.29.672938

A small cationic probe for accurate, punctate discovery of RNA tertiary structure

Ehrhardt, J. E.·Qiu, D. Y.·Jin, S.·Boerneke, M. A.·Aufgebauer, C. J.·Horner, S. M.·Weeks, K. M.
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Abstract

RNA molecules fold into intricate three-dimensional tertiary structures that are central to their biological functions. Yet reliably discovering new motifs that form true tertiary interactions remains a major challenge. Here we show that RNA tertiary folding occasionally generates electronegative motifs that react selectively with the small, positively-charged probe trimethyloxonium (TMO). Sites with enhanced reactivity to TMO, compared with the neutral reagent dimethyl sulfate (DMS), are indicative of tertiary structure and define T-sites. These positions share a structural signature in which a reactive nucleobase is adjacent to non-bridging phosphate oxygens, creating localized regions of negative charge. T-sites consistently map to the cores of higher-order structural interactions and functional centers across diverse RNAs, including distinct states in conformational ensembles. In the 10,723-nt dengue virus genome, three strong T-sites were detected, each within a complex structure required for viral replication. Cation-based covalent chemistry enables high-confidence discovery and analysis of functional RNA tertiary motifs across long and complex RNAs, opening new opportunities for transcriptome-wide structural analysis.

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Ehrhardt, J. E., Qiu, D. Y., Jin, S., Boerneke, M. A., Aufgebauer, C. J., Horner, S. M., Weeks, K. M.. 2025-08-30. A small cationic probe for accurate, punctate discovery of RNA tertiary structure. https://doi.org/10.1101/2025.08.29.672938

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