A Triple-Input AND-Logic System Employing an APE1-Propelled Bipedal DNA Walker for Highly Sensitive Detection of Dual miRNAs
Haoping Zhang, Min Pan, Xinyi Wang, Jingyu Tao, Zhaolong Tang, Hong Wang, Yingwei Zhang
Abstract
The escalating global cancer burden necessitates the development of precise tumor-specific microRNA (miRNA) detection strategies. However, the low-abundance, short-length, and high-sequence homology of miRNAs significantly hinder their accurate detection. Moreover, growing evidence of miRNA codysregulation in tumorigenesis underscores the diagnostic advantage of multiplexed analysis over single-marker approaches. Here, we present a novel bipedal DNA walker-based biosensor (BDWA) assembled on gold nanoparticles for the simultaneous detection of dual-miRNA targets. This system uniquely integrates dual-miRNA responsiveness with tumor-endogenous enzyme-driven signal amplification operating within a triple-input orthogonal AND-logic gate framework to enhance diagnostic specificity. Specifically, the concurrent presence of miR-21 and miR-155 displaces blocking strands to activate the walker, while the tumor-associated endonuclease APE1 serves as an intracellular trigger that propels the walker along engineered hairpin tracks (H1) on AuNPs. APE1-mediated cleavage of abasic (AP) sites results in the release and amplification of fluorescence signals initiated by the dual-targets. The bipedal architecture of the walker ensures a high processivity and efficient signal amplification. Critically, this design produces output signals only when all three inputs─miR-21, miR-155, and APE1─are present, thereby minimizing background interference and false positives. The BDWA system demonstrates high sensitivity and specificity for dual-miRNA detection in tumor cell lysates and intracellular imaging, offering a robust, tumor-activated, and programmable molecular platform for next-generation cancer diagnostics.