A Trinity DNAzyme Circuit with Dual-Confinement Effect for Imaging-Guided Combined Gene/Chemo-Dynamic Therapy

Synthetic catalytic DNAzyme circuits have gained great attention as versatile theranostic toolboxes, but their accuracy and efficiency is restricted by uncontrolled signal leakage and inefficient circuitry activation. Here, a trinity DNAzyme circuit (TriDC) is constructed using a unique logic gate bioswitchable DNA tetrahedral walker@MnO 2 nanosheets structure. The AND logic gate bioswitchable DNA tetrahedral walker, composed of dual allosteric DNAzyme (termed “commander” and “soldier”) and tetrahedral scaffold, is used to execute imaging-guided gene therapy by avoiding undesired signal leakage and cascade “walking” in a confined space. Glutathione-mediated reduction of MnO 2 nanosheets provides secondary confinement to stack the DNA tetrahedral walker in situ, offering abundant Mn 2+ as a cofactor to achieve self-sufficiency of DNAzyme circuits and induce chemo-dynamic therapy. The structure increases local reaction concentrations by ≈100-fold according to the collision frequency model, enabling the AND logic detection of microRNA-10b and microRNA-155 at a limit of detection of 98.97 p m , thereby facilitating the precise imaging of breast cancer cells. The combined gene/chemo-dynamic therapy achieves 66.2% tumor growth inhibition efficiency. The TriDC offers novel insights into tumor theranostics and also presents a paradigm for the in situ implementation of higher-order DNAzyme circuits.