Endogenous APE1-mediated confined catalytic hairpin assembly for high-contrast imaging of miRNA in living cell enables tumor cell-specific recognition.

Precise imaging of intracellular biomarkers is crucial for accurate disease diagnosis and mechanistic insight. Although confined catalytic hairpin assembly (CCHA) can accelerate reaction kinetics, its performance is frequently compromised by nonspecific amplification, resulting in high background signals and limited imaging contrast. Here, we develop a tumor-upregulated endogenous apurinic/apyrimidinic endonuclease 1 (APE1)-triggered CCHA (AP-CCHA) system based on a DNA nanowire for selective microRNA (miRNA) imaging in living tumor cells. Using miR-21 as a proof-of-concept model, the mechanistic analyses demonstrated that conventional CCHA suffers from significant nonspecific signal amplification during both in vitro assembly and storage. For the proposed AP-CCHA system, we incorporated an APE1-activatable AP-H1 probe that improves molecular stability, suppresses nonspecific amplification, and consequently enhances the signal-to-noise ratio while preserving high reaction kinetics. As a result, the AP-CCHA system delivers a 3.5-fold improvement in sensitivity compared with CCHA and achieves an 18.2 pM detection limit for miR-21. Leveraging the elevated cytoplasmic APE1 levels in tumor cells, AP-CCHA remains quiescent in normal cells but is selectively activated in cancer cells, enabling high-contrast miRNA discrimination and precise in vivo tumor imaging, and effectively minimizing false positives. By integrating confinement-enhanced kinetics with endogenous enzyme activation, this strategy enables rapid, highly sensitive, and high-contrast biomarker imaging. The AP-CCHA platform thus provides a versatile analytical tool for nucleic acid-based diagnostics with strong potential for precision therapeutics and drug development applications.