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4-Thiouracil is a photoactivatable probe designated for site-specific applications in detecting RNA structures and nucleic acid-nucleic acid contacts. Upon illumination with ultraviolet light exceeding 300 nm and in the presence of oxygen, it serves as an energy donor, facilitating the generation of singlet oxygen through triplet-triplet energy transfer. This process enables the highly reactive oxygen species to interact with 4-thiouracil, leading to the formation of uracil and uracil-6-sulfonate; the latter exhibits fluorescence around a wavelength of approximately 390 nm. Additionally, 4-Thiouracil functions as a substrate for T. gondii uracil phosphoribosyltransferase, allowing the synthesis of 4-thiouridine monophosphate for subsequent integration into RNA.