Integrated NMR and MD structure and dynamics of the stem–loop-II motif (s2m) from the Omicron variant of SARS-CoV-2

Abstract

The stem–loop-II motif (s2m) is a conserved viral RNA element located in the 3′UTR of different viruses including SARS-CoV-2. High-resolution 3D structural data for s2m are only available for the fundamentally different SCoV-1 version and difficult to access for SARS-CoV-2 due to the highly dynamic nature of the s2m RNA element. With the Omicron variant, a large deletion occurred for s2m, resulting in a relatively short hairpin with an apical pentaloop. We determined the NMR solution structure of s2m_omicron using a variety of torsion-angle sensitive NMR parameters in addition to NOE distance restraints. Surprisingly, relatively high {¹H},¹³C heteronuclear NOE values, averaged ribose ³J_HH-coupling constants (H1′H2′; H3′H4′), and dipole(H1′-C1′),‐dipole(H6/8-C6/8)-CCRs hinted toward significant dynamics for the small pentaloop making structure calculations solely relying on NMR data insufficient. To address this problem, we performed ten 1 microsecond MD-simulations from the NMR structure bundle as a starting point and applied Bayesian maximum entropy (BME) reweighting to refine the ensemble with the ³J-coupling constant data. Our results from the combined methodology provide a detailed view of the conformational dynamics of the Omicron variant of s2m characterized by different stacking patterns, ribose repuckering, and overall heterogeneity of the torsion angles for the loop nucleotides. Strikingly, despite the deletion of the initial nonaloop, as present in the Wuhan and Delta variants of s2m, our combined methodology reveals substantial dynamics and reorganization of a conserved UAC triplet at the tip of the pentaloop, adding physical insight that may be leveraged for the ultimate determination of the still unknown function of the RNA element.