SMFS raw data (Roquin B-site)

Local RNA structure is decisive for specific engagement with gene-regulatory proteins and, as a consequence, correct cellular function. However, its structure is often dynamic and, thus, challenging to study. Here, we show NMR and single-molecule force spectroscopy efficiently complement each other to provide high-resolution, time-resolved data on RNA folding intermediates during dynamic complex formation with the immune-regulating protein Roquin, which exploits multiple RNA-binding sites. Our data reveal a dual-mode binding of Roquin to RNA by firmly attaching to the stem-loop and, at the same time, destabilizing other regions, making them accessible to downstream interaction partners.