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Phosphorothioates (PTs) are important chemical modifications of the RNA backbone where a single nonbridging oxygen of the phosphate is replaced with a sulfur atom. PT can stabilize RNAs by protecting them from hydrolysis and is commonly used as a 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-π interaction, and a potassium binding site; are formed by RNA phosphates, making the NSR an ideal model for studying how PT affects RNA structure and dynamics. By comparing with high-level QM calculations, we reveal the distinct physical properties of the individual interactions facilitated by the PT. The sulfur substitution, besides weakening the direct H-bond interaction, reduces the directionality of H-bonding while increasing its dispersion and induction components. It also reduces the induction and increases the dispersion component of the anion-π stacking. The sulfur force-field parameters commonly employed in the literature do not reflect these distinctions, leading to the 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 sulfur parameters and provide suggestions for improving the force-field performance.