Development of a DNAzyme Walker for the Detection of APE1 in Living Cancer Cells

DNAzyme walker technology is a compelling option for bioanalytical and drug delivery applications. While nucleic acid and protein targets have been used to activate DNAzyme walkers, investigations into enzyme-triggered DNAzyme walkers in living cells are still in their early stages. The base excision repair (BER) pathway presents an array of enzymes that are overexpressed in cancer cells. Here, we introduce a DNAzyme walker system that sensitively and specifically detects the BER enzyme apurinic/apyrimidinic endodeoxyribonuclease 1 (APE1). We constructed the DNAzyme walker on the surface of 20 nm-diameter gold nanoparticles. We achieved a detection limit of 160 fM of APE1 in a buffer and in whole cell lysate equivalent to the amount of APE1 in a single HeLa cell in a sample volume of 100 μL. Confocal imaging of the DNAzyme walking reveals a cytoplasmic distribution of APE1 in HeLa cells. Walking activity is tunable to exogenous Mn²⁺ concentrations and the uptake of the DNAzyme walker system does not require transfection assistance. We demonstrate the investigative potential of the DNAzyme walker for up-regulated or overactive enzyme biomarkers of the BER pathway in cancer cells.