Dichromatically Encoded DNA Nanodevice for High-Resolution Molecular Subtyping of Triple-Negative Breast Cancer

Accurate cancer subtyping is essential for personalized medicine, yet existing diagnostic methods lack the multiplexing capability to decode complex biomarker signatures. Herein, we report a modular and dynamic DNA nanodevice, termed dichromatically routed hierarchically responsive DNA encoder (DRIVE), that enables the high-resolution molecular subtyping of triple-negative breast cancer (TNBC). Specifically, DRIVE integrates a tetrahedral DNA scaffold that is functionalized with two pairs of recognition and output modules responsive to apurinic/apyrimidinic endonuclease 1 (APE1) activity and specific microRNA (miRNA) expression. In the presence of APE1 and miRNA-21 (which are widely recognized as breast cancer biomarkers), the orthogonal recognition initiates a catalytic hairpin assembly (CHA) reaction that links a single DRIVE into a linear DNA nanostructure, thus significantly amplifying a monochromatic FAM signal. In TNBC subtypes that are characterized by the coexpression of APE1, miRNA-21, and miRNA-210, the cross-CHA makes a single DRIVE-form network DNA nanostructure, achieving the dichromatic FAM/Cy5 signal output. It is demonstrated that an approximately 4-fold enhancement in reaction kinetics of DRIVE is observed in comparison with that of individually dispersed probes. The dual-signal output enables a statistically significant differentiation of TNBC cells from other breast cancer subtypes. Together, this advance facilitates precise TNBC subtyping and provides great potential for accurate cancer diagnostics and personalized therapeutic strategies.