4-Thiouracil is a site-specific, photoactivatable probe used to detect RNA structures and nucleic acid-nucleic acid contacts. It absorbs ultraviolet light >300 nm and, in the presence of oxygen, acts as an energy donor to produce singlet oxygen by triplet-triplet energy transfer. The highly reactive oxygen species then reacts readily with 4-thiouracil, leading to the production of uracil and uracil-6-sulfonate, which is fluorescent at a wavelength of ~390 nm. 4-Thiouracil is used as a T. gondii uracil phosphoribosyltransferase substrate to produce 4-thiouridine monophosphate, which can ultimately be incorporated into RNA.