Photolyases are flavoenzymes responsible for the repair of carcinogenic DNA damage caused by ultraviolet radiation. They non-covalently harbor a catalytic cofactor flavin adenine dinucleotide (FAD). The blue light-driven electron transfer from the excited state of the fully-reduced form of FAD to the DNA lesions induces rearrangement of the covalent bonds, leading to the restoration of intact nucleobases. Since DNA repair by photolyases is independent on other DNA repair pathways, it has been considered as a potential molecule for gene therapy of diseases related to repair of UV-induced DNA damage. However, permeability of blue light to organs is low. If the DNA repair activity of photolyases can be artificially enhanced, such an artificial DNA repair system will be applicable to the gene therapy.
In addition to the catalytic chromophore, some photolyases contain a secondary chromophore with better light absorption capability than FAD, acting as a light-harvesting chromophore that harvests photons in sunlight efficiently and transfers light energy to the catalytic center, as observed in natural photoreceptor proteins. Inspired by nature, a synthetic fluorescent chromophore was attached to the surface of photolyase using oligonucleotides containing a modified nucleoside and a cyclobutane-type DNA lesion. The modified enzymes successfully enhanced its enzymatic activity in the light-driven DNA repair. This first-generation strategy gave us a clue for appropriate amino acid side chains to be modified, and therefore site-selective conjugation of fluorophores was performed. The results indicate that one of the fluorophore-modified enzymes exhibited further enhancement of the activity.