Lock‐Open: An AND Logic‐Gated, Entropy‐Driven DNA Nanowalker for Highly Sensitive Detection of UDG and APE1 Activities in Hepatocellular Carcinoma

Early diagnosis of HCC remains a clinical challenge, with survival strongly dependent on reliable biomarkers and translatable detection tools. Herein, we establish uracil-DNA glycosylase (UDG) and apurinic/apyrimidinic endonuclease 1 (APE1) as diagnostic biomarkers for early-stage HCC and develop a lock-open strategy based on an AND logic-gated, entropy-driven DNA nanowalker (ALEN) for highly sensitive detection of their enzymatic activities. ALEN integrates sequential UDG/APE1 processing with miR-21 gating to achieve on-track signal amplification. In the locked state, circuit leakage is minimized. Upon UDG/APE1 processing and miR-21 gating, ALEN is unlocked to initiate walker propagation, reactant regeneration, and spatially confined amplification selectively in tumor cells. This ability overcomes critical limitations of conventional entropy-driven DNA circuits, including low efficiency, limited turnover, and excessive leakage. When encapsulated in aptamer-functionalized lipid nanoparticles, ALEN gains robust biostability, biocompatibility, and HCC-specific accumulation. Using in vitro cellular models and in vivo mouse models of HCC, we confirmed that ALEN enables highly sensitive, cell type-specific discrimination of the activities of UDG and APE1, as well as timely and sensitive sensing of their dynamic fluctuations. Our results demonstrate that ALEN facilitates HCC diagnosis and monitoring of tumor progression, highlighting its promise as a clinical management tool.