Photo‐Cross‐linked DNA Structures Greatly Improves Their Serum Nuclease Resistances and Gene Knock‐In Efficiencies

The stabilization and structural integrity of DNA architectures remain significant challenges in their biomedical applications, particularly when inserting functional units into the genome using long single-stranded DNA (lssDNA). To address these challenges, a site-specific photo-cross-linking method is employed. Single-stranded oligonucleotides, containing one or two photosensitive cyanovinylcarbazole nucleoside (^CNVK) molecules, are precisely incorporated and cross-linked at the specific sites of ssDNA through base-pairing, followed by rapid UV irradiation at 365 nm. This interstrand photo-cross-linking improves the thermal stability of DNA duplexes and allows this study to afford a tetrahedral DNA nanostructure in a yield of >94%. Most importantly, the photo-cross-linked DNA architectures exhibit high resistances against serum degradation, especially prevent digestion of exonuclease III (exo III), which is common in conventional lambda-processing method. Meanwhile, this photo-cross-linking treatment can significantly improve the knock-in (KI) efficiencies of lssDNA in different cells including 293T, K562, and HepG2, approximately three to eightfold those of the uncross-linked lssDNA, and remain a low cytotoxicity. Given the significantly enhanced nuclease resistance in serum and improved KI efficiencies, this study anticipates that this photo-cross-linking method will become a valuable tool in technologically advanced biomedical applications, such as nanotechnology and nucleic acid therapy.