Advances in enzyme-free nucleic acid amplification-based fluorescent biosensors for real-time imaging of DNA repair enzymes in living cells

To maintain the stability and integrity of cellular genomic DNA, DNA repair enzymes catalyze the repair of DNA damage caused by exogenous and endogenous factors through various DNA repair pathways (e.g., base excision repair pathway). Abnormal DNA repair enzyme activity hampers the timely repair of DNA damage, inducing cell growth stagnation, cell death, transformation of normal cells into tumor cells and eventually the occurrence of human diseases including cancer. DNA repair enzymes have become the valuable biomarkers and potential therapeutic targets in biomedical research and clinical diagnosis. To accurately monitor DNA repair enzymes with low expression levels in cells, signal amplification strategies have been introduced to achieve reliable signal sensing and signal gain, facilitating diagnosis and treatment of malignant diseases. Conventional strategies for amplified detection of DNA repair enzymes involve multiple enzymes, washing/separation steps, and thermal cycling, and they are unable to provide spatiotemporal information of intracellular DNA repair enzymes. In recent years, some enzyme-free nucleic acid amplification approaches have been introduced for real-time imaging of intracellular DNA repair enzymes, providing new insights into the dynamic changes of DNA repair enzymes at the single-cell level. Herein, we review the recent advances in enzyme-free nucleic acid amplificationbased fluorescent biosensors for the imaging of intracellular DNA repair enzymes. We highlight the mechanisms and applications of these enzyme-free nucleic acid amplification approaches, and further compare the performances of different enzyme-free nucleic acid amplification approaches for DNA repair enzyme imaging. Furthermore, we discuss the potential challenges and future perspectives in this field.