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Synthetic biology aims to expand the genetic code by increasing cellular information storage and retrieval. A recent advance is the dTAT1-dNaM unnatural base pair, which is more photo- and thermostable than dTPT3-dNaM while maintaining high efficiency and fidelity in vitro and in vivo. However, the photophysics and cytotoxicity behavior of dTAT1 under UV light have not been investigated. We demonstrate that dTAT1 populates the triplet state upon 390 nm excitation but exhibits minimal cytotoxicity in cells. Analysis of reactive oxygen species indicates that dTAT1 produces a low singlet oxygen quantum yield of 17% while it generates superoxide, a less harmful reactive oxygen species. Its triplet lifetime is 2.7 times shorter than that of dTPT3, contributing to its lower photocytotoxicity. These findings highlight the potential of dTAT1 for safe genetic code expansion and therapeutic applications, providing valuable insights for designing next-generation unnatural nucleosides with minimal impact on cellular health. 

Abstract The development of a suitable irradiation setup is essential for in vitro experiments in photodynamic therapy (PDT). While various irradiation systems have been developed for PDT, only a few offer practical and high‐quality setups for precise and reproducible results in cell culture experiments. This report introduces a cost‐effective illumination setup designed for in vitro photodynamic treatments. The setup consists of a commercially available light‐emitting diode (LED) lamp, a cooling unit, and a specially designed 3D‐printed enclosure to accommodate a multiwell plate insert. The LED lamp is versatile, supporting various irradiation wavelengths and adjustable illumination fields, ensuring consistent and reliable performance. The study evaluates the setup through various parameters, including photon flux density, illumination uniformity, photon distribution across the multiwell plate, and temperature changes during irradiation. In addition, the effectiveness of the LED‐based illumination system is tested by treating mouse mammary breast carcinoma cells (4T1) with Rose Bengal and LED irradiation at around 525 nm. The resulting IC₅₀of 5.2 ± 0.9 μM and a minimum media temperature change of ca. 1.2°C indicate a highly promising LED‐based setup that offers a cost‐effective and technically feasible solution for achieving consistent, reproducible, and uniform irradiation, enhancing research capabilities and potential applications. 

It is demonstrated that thieno[3,4-d]pyrimidin-4(3H)-thione is an effective, oxygenation independent, heavy-atom-free photosensitizer against mono-layer of melanoma and cervical cancer cells.