Construction of bidirectional strand displacement-driven three-dimensional DNA walkers for single-molecule monitoring of multiple DNA glycosylases

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 x 10-10 U/mu L for hSMUG1 and 4.50 x 10-9 U/mu L 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.