Charge Inversion of Single-Stranded DNA under High-Concentration Monovalent Salts

Single-stranded DNA (ssDNA) plays important roles in biology and is widely used in nanotechnology, biosensors, and drug delivery, where its functions depend critically on its physical properties. Here, we unexpectedly found that at high monovalent salt concentrations, ssDNA undergoes charge inversion, effectively switching from negatively to positively charged. This effect was consistently observed across three independent assays. Nanopore electrophoresis and dynamic light scattering experiments showed reversed electrophoretic mobility, shifting from anode- to cathode-directed migration at high salt concentrations. Single-molecule magnetic tweezers further revealed that ssDNA unexpectedly elongates and stiffens under high-salt conditions, as indicated by increased extension and persistence length. Complementary molecular dynamics simulations showed that excess cations accumulate near the phosphate backbone, reversing the net charge to approximately +0.3e per nucleotide and increasing intrachain electrostatic repulsion by ∼0.2 k_BT per nucleotide. These findings uncover a previously unrecognized electrostatic regime for ssDNA, provide new insights into ion-nucleic acid interactions, and enable programmable control of DNA behavior in high-salt environments, with implications for nanodevice engineering and biomolecular regulation. © 2025 American Chemical Society.