Expanding the genetic alphabet beyond the canonical nucleobases (adenine, thymine, cytosine, and guanine) has garnered significant attention for its potential to create new biological systems and introduce novel functionalities into living organisms. By integrating unnatural base pairs (UBPs) into DNA, researchers can effectively expand the range of amino acids encoded by the genetic code, offering the possibility to design proteins with unnatural or enhanced properties. One crucial feature that enables the applicability of UBPs is their ability to pair and replicate within cells without triggering significant cell destruction. Unfortunately, many unnatural nucleobases fail in synthetic biology due to high cytotoxicity or inefficiencies in replication fidelity, leading to compromised cell viability. Therefore, developing UBPs that exhibit low toxicity and retain stability in cellular environments is essential for their practical use in synthetic biology and genetic engineering. Indeed, several UBPs have been developed to date, each contributing to advancements in synthetic biology. Among them, dTPT3-dNaM stands out as a widely used UBP, renowned for its ability to expand the genetic code, enhance genetic information storage, and produce therapeutic proteins. However, despite its successes, dTPT3 has limitations, including significant photosensitivity and photocytotoxicity, which poses challenges for its broader application.