dTAT1: An Unnatural Nucleoside Exhibiting Low Photocytotoxicity for Genetic Code Expansion

Synthetic biology's central goal is to create new forms and functions of genetic material by increasing the information a cell can store and retrieve. Wang and co-workers reached a significant milestone by engineering the dTAT1-dNaM unnatural base pair that is significantly more photo- and thermostable than dTPT3-dNaM while exhibiting nearly equal high efficiency and fidelity both in vitro and in vivo. Despite this breakthrough, the photophysical properties and cell viability of dTAT1 upon exposure to ultraviolet light, essential for cellular DNA integrity, have remained unexplored. This information is crucial to understanding the chemical and physical properties that set dTAT1 apart from other unnatural nucleosides in developing live semisynthetic organisms and therapeutic applications. Herein, we demonstrate that dTAT1 efficiently populates the reactive triplet state under near-visible light excitation. Surprisingly, however, cell viability assays indicate no significant cytotoxicity of dTAT1 in human epidermoid carcinoma cells upon 370 ± 9 nm irradiation or under dark conditions. Reactive Oxygen Species (ROS) analysis reveals that dTAT1 generates singlet oxygen with a relatively low quantum yield of 17% in aqueous phosphate buffer, while simultaneously generating superoxide, a significantly less toxic ROS. It is concluded that the 2.7-fold shorter triplet decay lifetime of dTAT1 compared to dTPT3, together with the generation of superoxide, holds the key to its superior reduction in photocytotoxicity. These findings rationalize the superior photochemical properties of dTAT1 for its use in synthetic biology and therapeutic applications, ensuring genetic expansion with minimal cellular disruption, and providing design principles to further optimize the development of unnatural nucleosides for expanding the genetic code.