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

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.