Topology-Informed Design of Circularly Locked DNAzymes Enables Orthogonally Controlled Gene Regulation

RNA-cleaving DNAzymes represent promising, protein-independent catalysts for gene silencing; yet achieving precise control over their activity remains a major challenge for biomedical applications. Here, we identify a cyclization-induced, size-dependent topological barrier that suppresses substrate binding, catalytic-core folding, and substrate cleavage of DNAzymes. Leveraging these underlying insights, we establish a modular strategy for the orthogonal control of DNAzyme activity via topological regulation. Specifically, we engineer catalytically inactive, circular DNAzyme precursors (termed circularly locked DNAzymes) bearing a cleavable linker and demonstrate that their substrate-cleavage activity can be reactivated through stimulus-responsive circular-to-linear switching. This topology-based design is broadly adaptable to diverse triggers (e.g., light, reductants, or enzymes), offering a simple and versatile route for conditional DNAzyme activation. Moreover, circularly locked DNAzymes exhibit enhanced biostability and maintain prolonged dormancy until on-demand activation, enabling precise, spatiotemporal control for potential therapeutic applications.