DNA Nanotube-Confined 3D Walker Enables Endogenous Dual-Activated AND Gate Imaging of Tumor-Associated piRNA

Abstract Intracellular molecular imaging for tumor biomarkers faces significant challenges in achieving high specificity and sensitivity, primarily due to signal leakage and the low abundance of target molecules. Herein, we develop an endogenous dual-activation sensing platform (termed E-DWRB) that integrates an AND logic gate driven by endogenous apurinic/apyrimidinic endonuclease 1 (APE1) with the signal amplification of a 3D DNA walker and the spatial confinement of DNA nanotubes for highly sensitive detection of piRNA in living tumor cells. In this design, DNA nanotubes serve as multidirectional tracks that confine the 3D DNA walker, preventing the walking arms from dissociating and thereby enhancing operational efficiency. APE1 and piRNA function as dual inputs for the AND logic operation, enabling precise discrimination between normal and tumor cells and achieving highly specific piRNA imaging. Owing to the synergy of the dual-activation strategy and DNA nanotube architecture, the E-DWRB platform achieves a detection limit as low as 8.5 aM and completes detection within 60 min with an initial reaction rate of 1.68 × 10–9 M·s–1. This strategy enables rapid visual imaging of piRNA with high sensitivity and specificity, offering promising potential for early cancer diagnosis and tumor-specific detection.