The low photocytotoxicity of dTAT1 to cells is surprising given the ultrafast and efficient population of the T 1 state demonstrated in this study. To provide further understanding of the low photocytotoxicity of dTAT1, we examined the potential generation of superoxide as a competitive mechanism of T 1 quenching by O 2 in cells. The fluorescent probe, dihydroethidium (DHE), was used to measure the intracellular superoxide production in A-431 cells treated with dTAT1 following UVA photoactivation. This probe fluoresces blue but emits red fluorescence (ex 500-530 nm/em 590-620 nm) when oxidized by superoxide to produce 2hydroxyethidium (2-OH-E+). Control experiments were conducted using Antimycin A and N-acetyl Cysteine as positive and negative controls, respectively (Figure ). Additionally, experiments were conducted without UVA exposure. The results presented in Figures and Finally, Table collects the photochemical properties of dTAT1, dTPT3, and d5SICS under similar experimental conditions. Notably, all three nucleosides undergo ultrafast intersystem crossing within sub-0.3 ps to populate long-lived triplet states upon excitation with UVA radiation. In addition, the singlet oxygen quantum yield under O 2 -saturated solutions is practically the same for dTAT1, dTPT3, and d5SICS within experimental uncertainties. However, the singlet oxygen yields of dTAT1 and d5SICS are ca. 45% and 32% lower than that of dTPT3 under air-saturated, ambient conditions (Table ). Hence, dTPT3 produces nearly twice as much singlet oxygen yield than dTAT1 in PBS under ambient conditions. Furthermore, the magnitude of the triplet decay lifetime of dTAT1 is 2.7(1.8)-and 1.6(1.2)-fold smaller than those of dTPT3 and d5SICS, respectively, under N 2 (air)-saturated conditions. Taken together, the generation of superoxide and the shorter triplet lifetime of dTAT1 compared to those of dTPT3 and d5SICS explains its lower photocytotoxicity. In a nutshell, the experimental and computational results explain the superior photochemical properties of dTAT1 over dTPT3 (and d5SICS) for its use in synthetic biology and therapeutic applications. The low toxicity of dTAT1 under both light and dark conditions, the relatively short triplet lifetime, and its lower singlet oxygen quantum yield under air-saturated conditions explain its safety profile relative to the dTPT3 and d5SICS counterparts. The insights gained from this study provide design principles to enhance the development of unnatural nucleosides.