Freezing-induced solid-liquid microphase separation for self-assembly of functional nucleic acid nanoparticles

In nature, nuclear DNA complexed with histone form highly condensed chromatin via loop extrusion and microphase separation, yielding physically constrained solid-like structures. Inspired by this natural compaction, here we demonstrate that freezing-induced crystallization pressure and freeze-concentration effects trigger solid-liquid microphase separation for efficient DNA compression. Freezing compress various nucleic acids (single stranded DNA, molecular beacons, siRNA, plasmid DNA) into high-density states that self-assemble into nanoparticles (DNA-NPs, MB-NPs, siBcl-2-NPs, siPLK1-NPs, pEGFP-NPs). Molecular dynamics simulations reveal that ice crystal growth increases solute concentration, enhancing DNA intermolecular interactions to form supramolecular chains. Stress at advancing ice-liquid interfaces further orders and compacts these supramolecular chains into nanoparticles. In breast cancer cells, MB-NPs detect miRNA-21; siBcl-2-NPs downregulate Bcl-2 protein; siPLK1-NPs exhibited antitumor effects; and pEGFP-NPs mediate green fluorescent protein (GFP) expression. This freeze-induced self-assembly strategy replaces chemical synthesis with physical constraints, establishing a versatile platform for facile production of nucleic acid nanoparticles. Efficent delivery of various nucleic acids in vitro and in vivo remains challenging. Here, the authors use freezing-induced condensation and microphase separation to compress nucleic acids into functional nanoparticles, without excipients, validated the bioactivity in vitro and in vivo.