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DNA-modifying enzymes act as critical regulators in a wide range of genetic functions (e.g., DNA damage & repair, DNA replication), and their aberrant expression may interfere with regular genetic functions and induce various malignant diseases including cancers. DNA-modifying enzymes have emerged as the potential biomarkers in early diagnosis of diseases and new therapeutic targets in genomic research. Consequently, the development of highly specific and sensitive biosensors for the detection of DNA-modifying enzymes is of great importance for basic biomedical research, disease diagnosis, and drug discovery. Single-molecule fluorescence detection has been widely implemented in the field of molecular diagnosis due to its simplicity, high sensitivity, visualization capability, and low sample consumption. In this paper, we summarize the recent advances in single-molecule counting-based biosensors for DNA-modifying enzyme (i.e, alkaline phosphatase, DNA methyltransferase, DNA glycosylase, flap endonuclease 1, and telomerase) assays in the past four years (2019 − 2023). We highlight the principles and applications of these biosensors, and give new insight into the future challenges and perspectives in the development of single-molecule counting-based biosensors. [Display omitted] • We review single-molecule counting-based biosensors for DNA-modifying enzyme detection. • The principles and applications of these biosensors are highlighted. • The performances of different biosensors are compared. • We discuss the future challenges and perspectives in this field. [ABSTRACT FROM AUTHOR]

The METTL3/14 complex is an important RNA N6-Methyladenosine (m6A) methyltransferase in organisms, and the abnormal METTL3/14 complex activity is associated with the pathogenesis and various cancers. Sensitive detection of METTL3/14 complex is essential to tumor pathogenesis study, cancer diagnosis, and anti-cancer drug discovery. However, traditional methods for METTL3/14 complex assay suffer from poor specificity, costly antibodies, unstable RNA substrates, and low sensitivity. Herein, we construct a single quantum dot (QD)-based förster resonance energy transfer (FRET) biosensor for sensitive detection of METTL3/14 complex activity. In the presence of METTL3/14 complex, it catalyzes the methylation of adenine in the substrate probe, leading to the formation of m6A that protects the substrate probes from MazF-mediated cleavage. The hybridization of methylated DNA substrate with biotinylated capture probe initiates polymerization reaction to obtain a biotinylated double-stranded DNA (dsDNA) with the incorporation of numerous Cy5 fluorophores. Subsequently, the Cy5-incorporated dsDNA can self-assembly onto the 605QD surface to form the 605QD-dsDNA-Cy5 nanostructure, causing FRET between 605QD donor and Cy5 acceptor. This biosensor has excellent sensitivity with a limit of detection (LOD) of 3.11 × 10−17 M, and it can measure the METTL3/14 complex activity in a single cell. Moreover, this biosensor can be used to evaluate the METTL3/14 complex kinetic parameters and screen potential inhibitors. Furthermore, it can differentiate the METTL3/14 complex expression in healthy human tissues and breast cancer patient tissues, providing a powerful tool for cancer pathogenesis study, clinical diagnosis, prognosis monitoring, and drug discovery. We develop a single quantum dot-based förster resonance energy transfer (FRET) biosensor for sensitive detection of METTL3/14 complex activity in breast cancer tissues. [Display omitted] • We develop a single quantum dot-based FRET biosensor. • This biosensor can sensitively detect the METTL3/14 complex activity. • It can measure the METTL3/14 complex activity in a single cell. • This biosensor can evaluate kinetic parameters and screen inhibitors. • It can discriminate breast cancer patient tissues from healthy tissues. [ABSTRACT FROM AUTHOR]