Phosphorothioate substitutions in RNA structure studied by molecular dynamics simulations, QM/MM calculations and NMR experiments

Phosphorothioates (PTs) are important chemical modifications of the RNA backbone where a single non-bridging oxygen of the phosphate is replaced with a sulphur atom. PT can stabilize RNAs by protecting them from hydrolysis and is commonly used as tool to explore their function. It is, however, unclear what basic physical effects PT has on RNA stability and electronic structure. Here, we present Molecular Dynamics (MD) simulations, quantum mechanical (QM) calculations, and NMR spectroscopy measurements, exploring the effects of PT modifications in the structural context of the Neomycin-sensing riboswitch (NSR). The NSR is the smallest biologically functional riboswitch with a well-defined structure stabilized by a U-turn motif. Three of the signature interactions of the U-turn; an H-bond, an anion-{pi} interaction and a potassium binding site; are formed by RNA phosphates, making the NSR an ideal model for studying how PT affects RNA dynamics, stability, and interaction with solvent. By comparing with high-level QM calculations, we reveal the distinct physical properties of the individual interactions facilitated by the PT. The sulphur force-field parameters commonly employed in the literature do not reflect these distinctions, leading to unsatisfactory description of PT in simulations of the NSR. We show that it is not possible to accurately describe the PT interactions using one universal set of van der Waals sulphur parameters and provide suggestions for improving the force-field performance.