UV-Cross-Linked DNA Nanomaterials Enable Robust Nanopatterning of Folate Ligands for Enhanced Cellular Uptake

The arrangement of ligands on a nanomaterial scaffold is a powerful approach to enhance targeted cellular delivery. However, nanomaterial-mediated delivery often employs imprecise ligand conjugation, limiting the exploration of optimal ligand density and spatial organization. To address these challenges, we developed DNA nanomaterials with precisely spaced folate ligands and rigidified them via a postassembly UV-based thymine cross-linking. These materials exhibit exceptional nuclease stability and maintain structural integrity both under biologically relevant conditions and during internalization into live HeLa cells. We used these nanomaterials as scaffolds for folate patterning and identified optimal modes of folate presentation for cellular uptake. Each step of the uptake process was probed, revealing the synergistic effects of structural stabilization and precise ligand patterning on the uptake mechanism, intracellular retention, and export dynamics. We then used our nanopatterned nanomaterials as delivery vectors for a gene-silencing nucleic acid payload. By integrating optimized ligand presentation and structural immobilization, we successfully achieved targeted gene silencing in folate receptor alpha-expressing cancer cells. This work showcases the effect that DNA nanostructure fidelity and rigidity have on the presentation of ligand moieties. It introduces UV cross-linking as a critical tool for structural stabilization of DNA nanomaterials, enabling applications in therapeutic delivery, diagnostics, and nanoscale cell surface engineering. In addition, this study reveals spatial principles of folate nanopatterning to enhance future targeted delivery systems with precision, stability, and biocompatibility.