Spatial decoupling of recognition and signaling in aptamer switches via tri-strand intermolecular competition

Despite decades of progress in aptamer engineering, the development of a single, modular signaling architecture capable of transducing target recognition into a measurable output across structurally diverse analytes remains an unmet challenge in biosensing. We report the Competitor Bridge Aptamer (CoBrA) framework: a modular DNA tridomain architecture that functionally isolates target recognition from signal transduction. Unlike conventional sensors where signal output depends on unpredictable aptamer folding, the CoBrA system uses an aptamer switch to trigger a predictable, concerted spatial displacement between signaling end points. By leveraging computational simulations and energetic balancing, we systematically optimized the CoBrA framework to consolidate recognition, displacement, and ratiometric signaling within a single, minimal-component system. Using the dopamine and cortisol aptamers as models, we demonstrate strong thermodynamic and kinetic synchronization between the Cy3 and Cy5 channels across both targets for dopamine (Cy3: K_d = 13.3 ± 3.2 μM, τ = 447 ± 31 seconds; Cy5: K_d = 24.0 ± 3.3 μM, τ = 537 ± 22 seconds) and cortisol (Cy3: K_d = 36.6 ± 3.9 μM, τ = 205 ± 7 seconds; Cy5: K_d = 30.7 ± 5.5 μM, τ = 259 ± 4 seconds) in buffer, serum, and artificial cerebrospinal fluid. Because the signaling event is a predictable, geometry-defined change rather than a fluorescence-specific phenomenon, this architecture is in principle adaptable to surface-based transduction modalities, including electrochemical, field-effect transistor, and plasmonic readouts.