Protein crowders remodel RNA electrostatics, hydration, and dynamics: a challenge to steric crowding models.

The intracellular environment is densely populated with macromolecules, creating crowded conditions. Whether in vitro environments or synthetic crowders like polyethylene glycol (PEG) accurately capture the complexity of RNA interactions in vivo remains unclear. Using all-atom molecular dynamics simulations, we investigated the HIV-1 TAR RNA hairpin in dilute, PEG-crowded, and realistic protein-crowded solutions. We found that PEG primarily exerts excluded-volume effects, maintaining RNA hydration and Na$^{+}$ ion condensation similar to dilute conditions. In contrast, protein crowders significantly altered RNA electrostatics, reducing Na$^{+}$ condensation by nearly 60%, displacing surface hydration water, and forming chemically specific contacts dominated by positively charged residues, notably arginine, and lysine. These interactions led to local RNA expansion and reshaped its conformational landscape without disrupting the global fold. Moreover, protein crowding dramatically slowed RNA translational and rotational dynamics, as well as local water and ion mobility, whereas these effects were minimal with PEG. Our findings emphasize that crowder identity critically determines RNA behavior and challenge the use of PEG as a universal model for intracellular conditions, providing mechanistic predictions for RNA studies in biologically relevant environments.