Abstract
Enzymes that catalyze DNA modifying activities including cytidine deamination and cytosine methylation play important biological roles and have been implicated pathologically in diseases such as cancer. Here, we report Direct Resolution of ONE dalton difference (DRONE), an ultra high performance liquid chromatography (UHPLC)-based analytical method to track a single dalton change in the cytosine-to-uracil conversion catalyzed by the human apolipoprotein B m-RNA editing catalytic polypeptide-like 3 (APOBEC3) cytidine deaminases, implicated in cancer and antiviral defense. Additionally, we demonstrate broad applicability by tracking other important DNA modifications and assessing epigenetic enzyme inhibition. We have extended our methodology to obtain data on two distinct deamination events in the same oligonucleotide substrate designed from a putative APOBEC substrate, diversifying the utility of the described method. DRONE provides an important foundation for in-depth analysis of DNA-modifying enzymes and versatile detection of novel DNA modifications of interest.