A Unimolecular Entropy-Driven DNA Circuit for Orthogonal Logic-Gated Imaging of Cancer Cells

Abstract DNA logic gates integrated with signal amplification networks offer powerful tools for the highly specific, logic-gated imaging of cancer cells. However, the intracellular efficacy of standard entropy-driven circuits (EDCs) is severely hindered by the diffusion limits of their multiple components, namely the three-strand duplex (TD), fuel (F), and target strands, which must simultaneously colocalize in cells at equimolar ratios within complex cellular environments. To overcome this spatiotemporal barrier, we engineered a unimolecular integrated EDC (iEDC) that executes an orthogonal AND logic operation. This structural integration enforces an exact stoichiometric ratio of the reaction components and leverages nanoscale unimolecular spatial proximity effect to dramatically accelerate logic operation efficiency compared to traditional multi-strand EDCs. The iEDC system is orthogonally activated by a “double key” mechanism requiring two endogenous cancer biomarkers: apurinic/apyrimidinic endonuclease 1 (APE1) and microRNA-155 (miR-155). This dual-target activation ensures a highly specific AND logic response, minimizing background signal leakage and substantially enhancing the accuracy and sensitivity of live-cell imaging. Ultimately, this unimolecular architecture provides a highly efficient framework for constructing integrated DNA circuits, holding great promise for reliable imaging of intracellular biomarkers and the advancement of precise cancer diagnosis.