Real-Time, Light-Activated, and Multiplexed Monitoring of Base Excision Repair in Living Cells Using Chimeric <scp>d</scp>/<scp>l</scp>-DNA Molecular Beacons

Base excision repair (BER) is a biologically and biomedically important cellular pathway responsible for repairing common DNA lesions. As a central member of the BER pathway, apurinic/apyrimidinic endonuclease 1 (APE1) is important in DNA repair and has been identified as a diagnostic and predictive biomarker for several diseases, motivating the development of analytical methods. However, the current repertoire of APE1 probes, the majority of which are derived from nucleic acids, are poorly suited for use in living cells and organisms, putting many promising biomedical applications of APE1 out of reach. Here, we exploit the bio-orthogonal properties of mirror-image l-DNA, together with a novel chimeric d/l-DNA molecular beacon architecture, to develop a highly versatile probe for intracellular BER, which we apply to the detection of APE1. The chimeric probe is simple to use, biostable, fast, and permits both real-time and light-controlled monitoring of APE1 activity in the nucleus of living cells, making it well suited for diverse intracellular applications. For example, we show that the probe can rapidly distinguish cells based on different APE1 expression levels and can monitor dynamic APE1 activity at single-cell resolution. Moreover, the generality of the probe design allowed for the development of a multiplexed assay for simultaneous imaging of APE1 and DNA glycosylase activities in living cells, which we used to reveal new insights into the efficacy of several prominent APE1 inhibitors. Overall, the chimeric d/l-DNA beacon probe presented in this work will be highly useful for researchers studying BER and provides a versatile toolkit for the development of improved BER-targeted therapies.