Boerneke, M. A. | Search bioRxiv

bioRxiv2025

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

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.

biochemistry↗

bioRxiv2022

Structure-first identification of conserved RNA elements that regulate dengue virus genome architecture and replication.

The genomes of RNA viruses encode the information required for replication in host cells in both their linear sequence and in complex higher-order structures. A subset of these complex functional RNA genome structures show clear sequence conservation. However, the extent to which viral RNA genomes contain conserved structural elements - that cannot be detected by sequence alone - that nonetheless are critical to viral fitness is largely unknown. Here, we take a structure-first approach to identify motifs conserved across the coding sequences of the RNA genomes for the four dengue virus (DENV) serotypes. We used SHAPE-MaP to identify 22 candidate motifs with conserved RNA structures, but no prior association with viral replication. At least ten of these motifs are important for viral fitness, revealing a significant unnoticed extent of RNA structure-mediated regulation within viral coding sequences. These conserved viral RNA structures promote a compact global genome architecture, interact with proteins, and regulate the viral replication cycle. These motifs are constrained at the levels of both RNA structure and protein sequence and are potential resistance-refractory targets for antivirals and live-attenuated vaccines. Structure-first identification of conserved RNA structure is poised to guide efficient discovery of RNA-mediated regulation in viral genomes and other cellular RNAs.

biochemistry↗