An Argonaute-Based Assay for Synchronous Quantification of Deaminase Catalytic Activity toward Cytidine and...

Precise enzymatic assays for cytidine deaminases (CDAs), which catalyze the deamination of cytidine (C) and 5-methylcytidine (5mC) into uridine (U) and thymidine (T), are essential tools in both epigenetics and genome editing. However, current biochemical methods have been hindered by cumbersome procedures and the challenge of concurrently assessing C and 5mC deamination. To address these limitations, we developed a novel Argonaute-based deaminase detection (ABDD) assay. This method employs guide-DNA-directed hyperthermophilic Pyrococcus furiosus Argonaute (PfAgo) to selectively cleave the fluorescence-labeled deamination products at U and T positions, respectively, enabling quantification of both C and 5mC deamination within 30 min by direct visualization of urea-PAGE. The ABDD assay was validated using APOBEC3A, APOBEC3H, and gain-of-function mutants A3H-RL18AA and A3A-Y130F, demonstrating concentration-dependent activity. The kinetic parameters k_cat and K_m for A3H are 8.6 × 10^-2 s^-1 and 4.7 μM, respectively, which align well with those from conventional glycosylase-based assays. ABDD also proved to be robust over a broad pH range of 5.5 to 8.5 and enabled accurate measurement in complex cellular lysates. In summary, the ABDD assay establishes a versatile and efficient platform for the functional and kinetic analysis of CDAs, offering a powerful tool with broad applicability in related research fields.