3D confinement reshapes RNA folding and enhances circularization in the Zika virus

Many RNA molecules function within confined environments, but the effect of confinement on RNA folding remains poorly understood. Proximity ligation experiments reveal altered long-range contacts in confined vs. unconfined states, yet they do not explain how spatial constraints give rise to these differences. Here, we develop a physical modeling approach that incorporates proximity ligation data into coarse-grained molecular dynamics simulations to reconstruct RNA 3D structures under confinement. We test our model on the ∼ 11 kb genome of the Zika virus, comparing the folding in virions (confined) and in cells (unconfined). We observe that the probability of contact between two regions of the genome vs. linear distance follows different scaling laws in the confined and unconfined cases, in agreement with proximity ligation experiments. We find that genome circularization—an interaction that regulates replication in Zika—occurs more frequently under confinement in both experiments and simulations. Our model reveals that the formation of long double-stranded stems through stacking confers local nematic liquid crystalline order to the RNA, and predicts pseudoknot topologies consistent with those seen in crystallographic structures of shorter RNAs. These results provide insight into the mechanism through which confinement alters the ensemble of 3D structures accessible to a long RNA molecule.