A Topology-Programmed Circular DNAzyme Enables High-Precision Diagnosis of Metastatic Triple-Negative Breast Cancer.

DNAzymes capable of RNA-cleaving are attractive, protein-independent candidates for biosensing and gene regulation. However, precise modulation of their activity remains a significant obstacle to biomedical applications. Herein, we uncovered a cyclization-provoked, size-reliant topological barrier that suppresses the catalytic core folding of DNAzymes, thereby governing their catalytic activity. Leveraging this underlying topological insight, we engineered a catalytically inert, minimal-sequence circular DNAzyme bearing a cleavable apurinic/apyrimidinic (AP) site and revealed that its substrate-cleavage potential could be restored through miRNA-10b-responsive circular-to-linear switching. The smallest circular size confers the strongest topological barrier for catalytic activity self-blocking in the circular DNAzyme, providing the most effective means for stimuli-specific activity restoration. This topology-informed strategy facilitated the design of an orthogonally activated circular DNAzyme-metal nanohybrid via Zn2+-mediated coordination self-assembly of the circular DNAzyme with its substrates. The nanohybrid allowed accurate discrimination of metastatic triple-negative breast cancer in various complex biological settings, including a heterogeneous population of cell subtypes, an in vivo subcutaneous tumor model, an in vivo sentinel lymph node metastasis model, and clinical human breast tissue samples. Our findings provide a flexible and robust framework for conditional manipulation of DNAzyme activity, enabling more accurate, spatiotemporal regulation for future therapeutic interventions.