Independent Control of the Thermodynamic and Kinetic Properties of Aptamer Switches

Abstract

AbstractMolecular switches that change their conformation upon target binding offer powerful capabilities for biotechnology and synthetic biology. In particular, aptamers have proven useful as molecular switches because they offer excellent binding properties, undergo reversible folding, and can be readily engineered into a wide range of nanostructures. Unfortunately, the thermodynamic and kinetic properties of the aptamer switches developed to date are intrinsically coupled, such that high temporal resolution (i.e., switching time) can only be achieved at the cost of lower sensitivity or high background. Here, we describe a general design strategy that decouples the thermodynamic and kinetic behavior of aptamer switches to achieve independent control of sensitivity and temporal resolution. We used this strategy to generate an array of aptamer switches with effective dissociation constants (KD) ranging from 10 μM to 40 mM and binding kinetics ranging from 170 ms to 3 s—all generated from the same parent ATP aptamer. Our strategy is broadly applicable to other aptamers, enabling the efficient development of switches with characteristics suitable for diverse range of biotechnology applications.