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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]

DNA methylation is one important epigenetic modification that involves in regulating multiple cellular functions. DNA methyltransferases (MTases) maintain methylation patterns in genomic DNA, and their aberrant activities may lead to multiple diseases including cancers. However, the efficient sensing of multiple DNA MTases is limited by the diversity of catalytic substrates and the rareness of methylation-dependent restriction endonucleases. Herein, inspired by the intrinsic superiorities of light-up RNA aptamers and unique properties of T7-based transcription amplification, we construct a novel methylation-powered engineering of a dual-color light-up RNA nanosensor for label-free and ultrasensitive sensing of multiple DNA MTases. Taking advantage of ultrahigh specificity of MDREs toward methylated DNAs, high efficiency of bicyclic cascade amplifications and high signal-to-noise ratio of light-up RNA aptamer-fluorophore pairs, this nanosensor exhibits good specificity and high sensitivity with a detection limit of 3.77 × 10−3 U/mL for M.SssI and 7.21 × 10−5 U/mL for Dam. Furthermore, it can screen potential inhibitors and quantify M.SssI and Dam MTases activities in E. coli cells and human serum samples. Notably, this assay can be completed homogeneously within 2 h in a single tube in a label-free manner, providing a powerful platform for sensing multiple DNA MTases in biomedical research and clinical therapeutics. • A dual-color light-up RNA nanosensor is developed for label-free and ultrasensitive sensing of multiple DNA MTases. • This nanosensor relies on methylation-powered bicyclic cascade amplifications for generating two light-up aptamers. • The assay may be completed homogeneously within 2 h in single tube. • This nanosensor exhibits good specificity and high sensitivity. • This nanosensor can screen inhibitors and quantify DNA MTases activity from clinical samples. [ABSTRACT FROM AUTHOR]