A hybrid RNA-based reporter assay for robust quantification of cytidine deaminase activity.

We developed a robust, hybrid RNA-based reporter assay for the quantification of cytidine deaminase (CDA) activity, overcoming limitations of current methods often compromised by interferences in crude biological samples. Our two-step method physically separates the enzymatic reaction in cell lysates from a highly specific detection step in a selected and validated CDA-deficient reporter cell line (143B cell line). This design confers exceptional robustness, eliminating the need for sample purification. In the first step, a cell lysate converts 5-fluorocytidine to 5-fluorouridine. After heat inactivation of lysate proteins, 5-fluorouridine is quantified by its incorporation into reporter cell RNA, yielding a highly specific fluorescent signal. The detection limit of a formed product in the assay achieves 2.7 µM. The assay shows good correlation with LC-MS data, and its adaptability to a multi-well plate format makes it an ideal and cost-effective tool for high-throughput screening. We further applied this assay to reveal that CDA-positive cells actively secrete 5-fluorouridine, providing quantitative evidence for a potential therapeutic bystander effect.