To overcome these limitations, Wang and co-workers recently engineered several dTPT3 analogues based on the modification of its thiophene rings. This strategic design focused on improving the photostability of the unnatural dTPT3 nucleoside while maintaining its replication efficiency and fidelity. A promising UBP, dTAT1-dNaM, was developed, which exhibited enhanced photostability, superior thermostability, and significantly reduced dark cytotoxicity compared to the original dTPT3-dNaM pair. However, the high photostability reported for dTAT1 is surprising. This is so because oxygen-for-sulfur substitution of carbonyl groups in organic biomolecules and nucleobase derivatives has been demonstrated to generate long-lived, highly reactive triplet states and high singlet oxygen quantum yields upon light absorption, severely decreasing cell viability. To understand the high photostability of dTAT1, in this contribution, we investigated its electronic relaxation mechanism upon excitation at 390 nm using a comprehensive suite of techniques including steady-state absorption and emission spectroscopy, femtosecond broadband transient absorption, nanosecond-to-microsecond transient absorption measurements, paired with quantum chemical calculations. In addition, we performed cell viability experiments upon irradiation of dTAT1 in the presence of a monolayer of human epidermoid carcinoma (A-431) cells. Investigating these photochemical and cell biology properties for dTAT1 is crucial to demonstrate cellular DNA integrity under dark and ambient light conditions. Figure presents the absorption spectra of dTAT1 in aqueous phosphate buffered saline (PBS, pH 7.4) and acetonitrile (ACN) solutions. In PBS, dTAT1 shows absorption bands at 238, 267, and 362 nm, with absorption tail extending to the near-visible (ca. 415 nm), while these bands shift bathochromically to 240, 270, 294, and 375 nm in ACN, and the absorption tail extending to ca. 435 nm. The ~13 nm red shift observed in ACN compared to PBS is attributed to ACN's stronger electron pair-donating ability relative to water. This behavior is consistent with previous steady-state investigations of sulfur-substituted (thionated) nucleobases and is corroborated by quantum chemical calculations performed at the TD-X3LYP/CPCM/def2-TZVPD//B3LYPG-D3BJ/CPCM/def2-TZVPD level of theory (Figure ).