Tongyin Zheng, Nicolas L. Fawzi
RNA G-quadruplexes (rG4s) are remarkably stable secondary structures with critical regulatory roles in gene expression, RNA metabolism, and telomere maintenance. However, their behavior within cells remains controversial, partly due to challenges in detecting rG4s in complex environments. Here, we use solution NMR spectroscopy to investigate how condensates formed by the low-complexity and RGG domains of the RNA-binding protein FUS affect the structure of TERRA, a highly stable model rG4. We show that FUS LC-RGG1 interacts with TERRA in dilute solution and that binding perturbs, but does not disrupt, the G-quadruplex structure. When co-phase separated with FUS LC-RGG1, however, NMR signatures of TERRA's folded state disappear, and the remaining observable resonances indicate an unfolded conformation, even in buffer containing potassium where TERRA rG4 is exceptionally stable when outside a condensate. Quantitative comparisons with a mutant form of TERRA, used as a baseline for fully unfolded RNA, suggest that at minimum a third of TERRA RNA becomes unfolded in the condensed phase. Thus, our results demonstrate that condensates can shift the structural ensemble of rG4 towards unfolded species, offering a potential mechanistic explanation for their apparent lack of stability in vivo and revealing how phase-separated environments may actively modulate RNA structure and function.