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

[Image: see text] The base excision repair (BER) pathway is a frontline defender of genomic integrity and plays a central role in epigenetic regulation through its involvement in the erasure of 5-methylcytosine. This biological and clinical significance has led to a demand for analytical methods capable of monitoring BER activities, especially in living cells. Unfortunately, prevailing methods, which are primarily derived from nucleic acids, are mostly incompatible with intracellular use due to their susceptibility to nuclease degradation and other off-target interactions. These limitations preclude important biological studies of BER enzymes and many clinical applications. Herein, we report a straightforward approach for constructing biostable BER probes using a unique chimeric d/l-DNA architecture that exploits the bioorthogonal properties of mirror-image l-DNA. We show that 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 study enzyme mutants. Notably, we report the first example of a fluorescent probe for real-time monitoring of thymine DNA glycosylase (TDG)-mediated BER of 5-formylcytosine and 5-carboxylcytosine in living cells, providing a much-needed tool for studying DNA (de)methylation biology. Chimeric probes offer a robust and highly generalizable approach for real-time monitoring of BER activity in living cells, which should enable a broad spectrum of basic research and clinical applications.