Secondary Structureof i-Motif DNA ModulatesPhase Separation

Abstract Liquid–liquid phase separation is central to nucleic acid organization in cells, yet the role of DNA secondary structures in phase separation is largely underexplored. Using confocal fluorescence microscopy, we investigated phase separation of i-motif (iM)-forming sequences in the presence of poly-l-lysine under varying pHs. iMs are four-stranded DNA structures formed in cytosine-rich sequences, often enriched in oncogenes and telomeres. At acidic pH, fully folded iMs suppressed droplet formation, indicating that structural stiffness inhibits phase separation. Near the transition pH, partially folded iMs formed gel-like droplets with high contact angles. At basic pH, unfolded iMs exhibited fluid-like droplets with lower contact angles. Disrupting the iM secondary structure via cytosine deamination restored liquid-like droplets at transitional pH, confirming the role of structure. In contrast, stable G-quadruplex structures consistently formed high-contact-angle droplets. These findings highlight the DNA secondary structure as a key determinant of biomolecular condensate formation, morphology, and dynamics, with potential implications for gene expression.