Structural heterogeneity and dynamics in the apical stem loop of s2m from SARS-CoV-2 Delta by an integrative NMR spectroscopy and MD simulation approach

In structured RNAs, helical elements are often capped by apical loops that are integral structural elements, ranging from 3 to >20 nts of size on average, and display a highly heterogeneous energy landscape profile. This renders their structural characterization particularly challenging. We here provide an example for comprehensive characterization of the SARS-CoV-2 s2m element containing a highly dynamic nonaloop using an integrative approach of nuclear magnetic resonance spectroscopy (NMR), small angle X-ray scattering (SAXS) and molecular dynamics simulations (MD). As representation of the structure by a single conformation is unsuitable, we here present ensemble of NMR structures in agreement with SAXS data. We further explored the conformational space in the s2m nonaloop and its transient closing 5’-G-U-3’ base pair by MD simulations weighted by experimental NMR observables, leading to a comprehensive representation of the s2m nonaloop motif. Our deconvolution of the ensemble into conformations and dynamics provides a basis for future ensemble-functional characterisation of functional RNA structures featuring dynamic motifs.