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Human single-stranded-selective monofunctional uracil glycosylase (hSMUG1) and alkyladenine glycosylase (hAAG) are important base-excision repair (BER) glycosylases responsible for deamination and alkylation repair. Their dysfunctional activities have strong association with various multifactorial diseases and cancers. Herein, by integrating three-dimensional (3D) DNA walker with single-molecule technique, we construct bidirectional strand displacement-driven 3D DNA walkers for single-molecule monitoring of multiple DNA glycosylases. In the presence of hSMUG1 and hAAG, the damaged U and I in bifunctional dumbbell probe are removed by APE1, activating bidirectional self-priming strand displacement amplification (SP-SDA) to produce two trigger DNAs. Trigger DNAs hybridize with Cy5/Alexa Fluor 488 signal probes and subsequently serve as the walker DNAs to induce cyclic APE1-powered cleavage of two signal probes, liberating abundant Cy5 and Alexa Fluor 488 from AuNPs. Due to high precision of intracellular BER mechanisms, high efficiency of bidirectional SP-SDA-directed 3D DNA walkers and ultrahigh signal-to-noise ratio of single-molecule imaging, this nanosensor exhibits a detection limit of 8.14 × 10−10 U/μL for hSMUG1 and 4.50 × 10−9 U/μL for hAAG. Importantly, it can measure kinetic parameters, identify potential inhibitors, discriminate cancer from normal cells, and quantify glycosylases activities in various cancer cells at single-cell level, with promising potentials in biomedical and clinical applications. • We construct bidirectional strand displacement-driven 3D DNA walkers. • This nanosensor enables single-molecule monitoring of multiple human glycosylases. • This nanosensor can measure kinetic parameters and screen potential inhibitors. • This nanosensor can discriminate cancer cells from normal cells. [ABSTRACT FROM AUTHOR]

An exonuclease III-assisted recycling amplification (ERA) coupled with fluorescent DNA-scaffolded silver nanoclusters (Ag NCs) for sensitive determination of hepatitis B virus (HBV) DNA is proposed. The sensing mechanism is based on the employment of two different molecular beacons (MBs), MB1 and MB2. In the absence of targets, the MBs self-hybridize into stable stem-loop structures with exonuclease III (Exo III) cleaving resistance, and the MB2 containing a C-rich loop can be utilized to synthesize Ag NCs with high fluorescence signal. In the presence of targets, the targets can bind with MB1 to form partly duplex structure. With the cleaving of Exo III, the liberated target DNA can initiate the cycle of MB1-target hybridization, while the generated trigger DNA can hybridize with MB2, inducing a conformational change of MB2 from hairpin to single-stranded structure. The structural change of MB2 would influence the formation of Ag NCs, thus producing weak fluorescence signal, and the released trigger DNA could activate the cycle of MB2-trigger hybridization. The present method for HBV DNA analysis exhibits a detection limit of 0.97 nM ( S / N = 3) with high specificity, and demonstrates its applicability for the HBV detection in human serum. [ABSTRACT FROM AUTHOR]