Controllable Reconfiguration of DNA Nanostar Hydrogel Networks via Sticky-End Ligation

• Covalent ligation suppresses strand rearrangement within DNA hydrogels. • Topology freezing enhances stiffness and mechanical stability. • Ligation extends DNA hydrogel lifetime in serum by at least sevenfold. • Mechanical robustness of ligated DNA hydrogels facilitates their handling. • Ligated hydrogels support 3D cell culture. DNA nanostar hydrogels hold great promise as biomaterials, but their mechanical properties and nuclease susceptibility limit their use. Here, we propose ligation of nanostar sticky ends as a strategy to fundamentally transform the mechanical and dynamic properties of DNA nanostar hydrogels and enhance their nuclease resistance. Ligation eliminates free ends of DNA nanostars, conferring strong exonuclease resistance; suppresses strand rearrangement, converting liquid-like assemblies into gel-like materials; and prevents macroscopic reversibility despite preserving microscopic motif hybridization. Ligation also stiffens the material, enabling facile handling and 3D printing, and markedly slows digestion in serum-supplemented medium. In the presence of low concentrations of the DNase I inhibitor actin, ligated hydrogels remain stable for at least two weeks. As a proof-of-concept application, we captured and maintained cells within ligated hydrogels for one week in culture. By overcoming the limitations of DNA nanostar hydrogels, this work enables their use as programmable biomaterials that fully leverage the DNA nanotechnology toolbox.