Epigenetic DNA modifications, such as 5-methylcytosine, 5-hydroxymethylcytosine, and 5-formylcytosine, are associated with a variety of diseases and potential biomarkers for cancer diagnosis and therapy. Liquid chromatography−tandem mass spectrometry (LC−MS/MS) assays are considered to be the gold standard for qualitative and quantitative detection of DNA modifications. DNA digestion for converting long DNA polymer into 2′-deoxynucleosides is an important preprocessing step to achieve sensitive and accurate LC−MS/MS quantification. Here, we showed that, as stimulated by divalent metal ions, Mg 2+ and Mn 2+ , the engineered human DNase I Q9R:E13R:N74K mutant can efficiently digest DNA in the presence of monovalent metal ions at a high concentration (e.g., 1 M NaCl), showing hyperactivity on DNA cutting. We also found that the engineered DNase I mutants display exceptional DNA-cutting activity over a wider pH range (5.5−9.5). Due to their hyperactivity and high salt tolerance, the engineered DNase I mutants cut DNA 5mC and dC efficiently. Benefitting from this DNA-cutting hyperactivity, we demonstrated an LC−MS/MS assay for unbiased and accurate quantification of DNA 5mC.
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