RNA Selectively Modulates Activity of Virulent Amyloid PSMα3 and Host Defense LL-37 via Phase Separation and Aggregation Dynamics

Abstract Amyloid-forming peptides are increasingly recognized as dynamic regulators at the host–pathogen interface, yet how environmental factors control their assembly and activity remains poorly understood. Here we show that RNA acts as a concentration-dependent regulator of two sequence-related α-helical peptides with fundamentally different assembly behaviors: the cross-α amyloid-forming Staphylococcus aureus virulence factor PSMα3 and the non-amyloidogenic human host defense peptide LL-37. RNA drives PSMα3 through distinct assembly states, from liquid-like condensates to fibrillar polymorphs, while preserving cytotoxic and antimicrobial activity over time. In contrast, RNA attenuates LL-37 cytotoxicity toward host cells while maintaining antibacterial activity, consistent with a host-protective immunomodulatory effect. Together with the opposing effects of EGCG, which redirects both peptides into amorphous, inactive assemblies, these findings support a mechanistic model in which biological activity is governed not by monomer abundance or mature fibrils alone, but by supramolecular architecture, assembly trajectory, and dynamics. The contrasting RNA-dependent outcomes further suggest that the amyloid forming capacity of PSMα3 enables RNA to sustain membrane-active states associated with bacterial virulence, whereas LL-37 is shifted toward less cytotoxic states that preserve antimicrobial defense while limiting host damage. More broadly, our findings identify RNA as an environmental regulator of α-helical peptide assemblies and establish assembly-state control as a tunable determinant of biological function. Competing Interest Statement The authors have declared no competing interest. Footnotes We substantially expanded and refined the Discussion and Introduction to more clearly distinguish between mature fibrils, transient assembly intermediates, and broader assembly-state-dependent mechanisms. We also incorporated additional literature representing different perspectives from the field. We revised the section -PSMα3 cytotoxicity arises from dynamic assembly intermediates-. We additionally revised the manuscript to clarify the significance of the EGCG comparison. Importantly, we also broadened the manuscript substantially by incorporating independent studies from multiple unrelated systems supporting the principle that supramolecular organization influences biological function. We expanded the -Biological and therapeutic implications- section to discuss biologically plausible extracellular environments in which PSMα3 may encounter nucleic acids. Overall, the revised manuscript reflects a substantially expanded discussion of PSMα3 assembly- state-dependent activity, the role of RNA in modulating assembly trajectories, and the broader conceptual implications for membrane-active peptide assemblies.