Chimeric d/l-DNA Probes of Base Excision Repair Enable Real-Time Monitoring of Thymine DNA Glycosylase Activity in Live Cells

The base excisionrepair (BER) pathway is a frontline defenderof genomic integrity and plays a central role in epigenetic regulationthrough its involvement in the erasure of 5-methylcytosine. This biologicaland clinical significance has led to a demand for analytical methodscapable of monitoring BER activities, especially in living cells.Unfortunately, prevailing methods, which are primarily derived fromnucleic acids, are mostly incompatible with intracellular use dueto their susceptibility to nuclease degradation and other off-targetinteractions. These limitations preclude important biological studiesof BER enzymes and many clinical applications. Herein, we report astraightforward approach for constructing biostable BER probes usinga unique chimeric d/l-DNA architecture that exploitsthe bioorthogonal properties of mirror-image l-DNA. We showthat chimeric BER probes have excellent stability within living cells,where they were successfully employed to monitor relative BER activity,evaluate the efficiency of small molecule BER inhibitors, and studyenzyme mutants. Notably, we report the first example of a fluorescentprobe for real-time monitoring of thymine DNA glycosylase (TDG)-mediatedBER of 5-formylcytosine and 5-carboxylcytosine in living cells, providinga much-needed tool for studying DNA (de)methylation biology. Chimericprobes offer a robust and highly generalizable approach for real-timemonitoring of BER activity in living cells, which should enable abroad spectrum of basic research and clinical applications.