Kinetic-Gated Signal Transduction Decouples Binding Affinity in a Dual-Pocket Riboswitch RNA

RNA has emerged beyond its traditional biological role to become a premier biorecognition element in biosensor design. While single-target RNA sensors demonstrate high specificity, multi-pocket variants, such as the tetrahydrofolate (THF) riboswitch, enable complex signal integration and cooperative allosteric regulation. Intriguingly, the THF riboswitch binds both folinic acid and purine derivatives with high affinity, yet exhibits different signal transduction outputs. The mechanistic basis for this decoupling of molecular recognition from sensor-proper folding remains a critical hurdle. Herein, we established a comprehensive multi-modal analytical platform, including engineered fluorescent sensors, position-specific RNA labeling, stopped-flow kinetics, microscale thermophoresis (MST), and single-molecule FRET, to decipher this decoupling mechanism. Our investigations support that the functional activation of the THF riboswitch is dictated decisively by transient binding kinetics (target residence time) rather than overall affinity alone. Furthermore, we demonstrate that concentration-dependent heterotropic cooperativity among distinct ligands functions as an allosteric amplifier, broadening the dynamic range and lowering the detection threshold of the sensor. By elucidating these intricate kinetic principles, this work is valuable for engineering next-generation, logic-gated RNA aptasensors for multiplexed diagnostics.