Publication
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.