Influence of polycation modification on droplet size and internal structure in RNA/poly-l-lysine coacervates

Abstract

Complex coacervation between RNA and poly(L-lysine) (PLL) provides a minimal and tunable model system to study biomolecular condensates and nucleic acid complexation for gene delivery. Here, we compare coacervates formed with linear PLL, hydrophobically modified PLL, and compact PLL nanoparticles to study the effect of polycation architecture and charge density. Droplet formation and size distributions are characterized by dynamic light scattering and confocal microscopy. Fluorescence confocal microscopy, using independently labelled fluorescein-RNA and rhodamine-PLL, demonstrates co-localization of both components with enhanced fluorescence quenching upon PLL modification, consistent with a reduced average separation between the two species. Fluorescence lifetime analysis shows a systematic decrease in the fluorescein lifetime from linear PLL to modified PLL and PLL nanoparticles, supporting increasingly close RNA–PLL proximity. Consistently, small-angle X-ray scattering (SAXS) indicates that all coacervates behave as strongly screened semidilute polyelectrolyte complexes, with nanometre-scale correlation lengths that decrease systematically upon PLL modification, reflecting a tighter internal organization. Together, these results demonstrate that increasing polycation compactness and lower charge density lead to tighter internal organization of RNA–PLL coacervates and highlight the complementarity of SAXS and fluorescence lifetime measurements for resolving nanoscale structure in complex coacervates.