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

Abstract 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.