Single-Molecule Kinetic Investigation of Cocaine-Dependent Split-Aptamer Assembly

Aptamers are short nucleic-acid biopolymers selected to have high affinity and specificity for protein or small-molecule target analytes. Aptamers can be engineered into split-aptamer biosensors comprising two nucleic acid strands that coassemble as they bind to a target, resulting in a large signal change from attached molecular probes (e.g., molecular beacons). The kinetics of split-aptamer assembly and their dependence on target recognition are largely unknown; knowledge of these kinetics could help in design and optimization of split-aptamer biosensors. In this work, we measure assembly kinetics of cocaine-dependent split-aptamer molecules using single-molecule fluorescence imaging. Assembly is monitored between a DNA strand tethered to a glass substrate and solutions containing the other strand tagged with a fluorescent label, with varying concentrations of the cocaine analyte. Dissociation rates are measured by tracking individual molecules and measuring their bound lifetimes. Dissociation- time distributions are biexponential, possibly indicating different folded states of the aptamer. The dissociation rate of only the longer-lived complex decreases with cocaine concentration, suggesting that cocaine stabilizes the long-lived aptamer complex. The variation in the slow dissociation rate with cocaine concentration is well described with an equilibrium-binding model, where the dissociation rate approaches a saturation limit consistent with the dissociation-equilibrium constant for cocaine-binding to the split aptamer. This single-molecule methodology provides a sensitive readout of cocaine-binding based on the dissociation kinetics of the split aptamer, allowing one to distinguish target-dependent aptamer assembly from background assembly. This methodology could be used to study other systems where target association affects the stability of aptamer duplexes.