Stimulus-Responsive NOT Gate for Single-Rail DNA Logic Circuits and Biosensing

DNA molecular circuits offer significant promise for biomedical applications by combining computational functionality with inherent biocompatibility. However, their operational logic fundamentally differs from electronic systems as they utilize the physical presence or absence of DNA strands rather than voltage levels to encode information. This distinction creates a critical barrier for implementing single-rail NOT gates. Consequently, existing systems typically employ dual-rail architectures that increase the complexity and elevate leakage risks. To address this limitation, we developed optically and thermally controlled NOT gates that perform rapid logical inversion while maintaining compatibility with both polymerase-driven and toehold-mediated circuit systems. We validated these gates in multilayer computational networks and demonstrated their practical utility across diverse biosensing applications, including molecular diagnostics and live-cell imaging. This work establishes a robust platform for scalable DNA computing with direct translational potential in biological environments.