An endogenous enzyme-gated DNA nanomachine for spatially controlled imaging of intracellular microRNA and in situ evaluation of drug response

• An endogenous enzyme-gated DNA nanomachine (EGDN) is developed for spatially controlled imaging of miRNA in living cells and animals. • EGDN enables high-fidelity and robust miRNA imaging by reducing the undesired off-site signal leakage. • EGDN allows for effective identification of tumor cells with enhanced signal contrast. • EGDN is able to evaluate the biological processes of tumor cells under drug treatment by tracking the target miRNA. In situ imaging of tumor-associated microRNAs (miRNAs) is crucial for evaluating the risk of tumor occurrence and progression. However, traditional biosensors face challenges such as uncontrolled signal leakage and limited tumor specificity. Herein, we propose a novel strategy to construct an endogenous enzyme-gated DNA nanomachine (EGDN) for spatially controlled sensitive imaging of intracellular miRNA and in situ evaluation of drug response. The EGDN is established via rational engineering of traditional catalytic hairpin assembly with the incorporation of enzyme-activatable sites and further anchoring on a DNA tetrahedron scaffold. The sensing mechanism is selectively activated by the overexpressed apurinic/apyrimidinic endonuclease 1 in the cytoplasm of tumor cells instead of normal cells, thereby achieving tumor-specific miRNA imaging with improved spatial resolution. Furthermore, this system has been applied for in situ visualization of the miRNA-based drug response in tumor cells, which provides valuable insights for therapeutic evaluation and screening of chemotherapeutic agents. This study demonstrates a powerful biosensing platform with great potential for applications in cancer diagnosis and precision medicine.