The Effects of Chain Length and NaCl Concentration on Phase Separation and Morphology of Polylysine Complexes with...

Electrostatic complexation between cationic polymers and nucleic acids underlies both fundamental biomolecular assemblies and emerging therapeutic technologies. Among these systems, polylysine/nucleic acid complexes provide a simple yet powerful model for probing how the molecular architecture and chain length dictate phase separation and salt-induced interactions. Despite extensive but separate studies on nucleic acid complexes and the effects of polymer length, the influence of polylysine chain length on phase behavior and morphology across different nucleic acids remains limited. Here, we systematically investigate complexes formed between poly-l-lysine (PLK) of defined lengths (30-800 residues) and three nucleic acid systems: baker's yeast tRNA (75-80 bp) and two length ranges of salmon sperm dsDNA (200-500 and ≤2000 bp). Using turbidity assays and optical microscopy, we examined phase separation and morphological transitions across a broad NaCl concentration range (0-1500 mM) and compared them to previous work. Our results show that the presence of single- and double-stranded nucleic acids strongly influences polymer complex stability and morphology in the presence of salt. The salt resistance was strongly influenced by the shortest polymer in the complex, though the length of the longer partner showed only subtle shifts in this critical salt value. The nucleic acid type further modulated outcomes: in the absence of added salt, dsDNA at all length ranges exclusively formed precipitates, whereas tRNA consistently formed coacervate droplets with all PLK variants. Interestingly, nucleic acid chain lengths below 100 showed phase transitions in the presence of salt before transitioning to a single-phase solution, whereas the longer dsDNA chains did not show a transition. However, dsDNA consistently transitioned from precipitates to coacervates, while tRNA transitioned from coacervates to precipitates. We note that even with systems of two short polymers, complexes remained phase-separated well beyond physiological ionic strength. Together, these findings further establish that the chain length and nucleic acid architecture are key determinants of polyelectrolyte complex morphology and stability across a range of ionic strengths. By linking molecular features to macroscopic behavior, this work provides design principles for engineering nucleic acid-polymer assemblies with tunable properties for biomaterials and therapeutic applications.