Nucleotide-specific RNA conformations and dynamics within ribonucleoprotein condensates

Ribonucleoprotein (RNP) condensates have distinct physiological and pathological significance, but the structure of RNA within them is not well understood. Using contrast-variation solution X-ray scattering, which discerns only the RNA structures within protein-RNA complexes, alongside ensemble-based structural modeling we characterize the conformational changes of flexible poly-A, poly-U and poly-C single stranded RNA as it interacts with polybasic peptides, eventually forming condensed coacervate mixtures. At high salt concentrations, where macromolecular association is weak, we probe association events that precede the formation of liquid-like droplets. Structural changes occur in RNA that reflect charge screening by the peptides as well as π − π interactions of the bases with basic residues. At lower salt concentrations, where association is enhanced, poly-A RNA within phase separated RNP mixtures exhibit a broad scattering peak, suggesting subtle ordering. Coarse-grained molecular dynamics simulations are used to elucidate the nucleotide-specific dynamics within RNP condensates. While adenine-rich condensates behave like stable semidilute solutions, uracil-rich RNA condensates appear to be compositionally fluctuating. This approach helps understand how RNA sequence contributes to the molecular grammar of RNA-protein phase separation.