Endogenous and Exogenous Dual-Gated DNA Nanoactuator in Autonomous Two-Step Catalytic Amplification for Robust Biomolecular Sensing In Vitro and In Vivo

Although DNA nanoactuator-based biosensors have promising applications for fluorescence imaging of disease-related biomolecules in living biosamples, challenges persist regarding sensing sensitivity, initiation selectivity, and detection accuracy. Herein, we present an endogenous and exogenous dual-gated DNA nanoactuator for autonomous two-step catalytic amplification. This amplification course combines an upstream self-sustaining Mn²⁺-reliant DNAzyme (achieved using glutathione to reduce manganese dioxide nanoflakes) with downstream entropy-driven catalysis. Subsequently, endogenous TK1 mRNA, which is abundant in various cancerous cells, serves as one gate to selectively initiate the sensing route. Additionally, 365 nm ultraviolet upconversion luminescence transformed by exogenous 808 nm near-infrared light is used to power another gate, with one sensing module incorporating a photocleavage connector. As a conceptual study, the DNA nanoactuator demonstrated exceptional sensitivity and specificity in sensing a model biomolecule (microRNA-21, an overexpressed biomarker associated with multiple cancers). This analytical methodology enables robust biomolecular sensing of this low-abundance analyte, both in vitro and in vivo, advancing the diagnostic utility of DNA nanoactuators.