Glutaraldehyde Cross-Linking of Oligolysines Coating DNA Origami Greatly Reduces Susceptibility to Nuclease Degradation

DNA nanostructures (DNs) have garnered a large amount of interest as a potential therapeutic modality. However, DNs are prone to nuclease-mediated degradation and are unstable in low Mg²⁺ conditions; this greatly limits their utility in physiological settings. Previously, PEGylated oligolysines were found to protect DNs against low-salt denaturation and to increase nuclease resistance by up to ∼400-fold. Here we demonstrate that glutaraldehyde cross-linking of PEGylated oligolysine-coated DNs extends survival by up to another ∼250-fold to >48 h during incubation with 2600 times the physiological concentration of DNase I. DNA origami with cross-linked oligolysine coats are non-toxic and are internalized into cells more readily than non-cross-linked origami. Our strategy provides an off-the-shelf and generalizable method for protecting DNs in vivo.