Aptamers are short sequences of DNA (or RNA) that are engineered to have high affinity and specificity for proteins and small molecules of interest. Aptamer sensors frequently rely on a significant shift in DNA secondary structure when binding to targets, which leads to changes in the response of an attached label. Unfortunately, some monolithic aptamers show little structural change when binding to small molecule targets. Splitting the aptamer in a strategic location, typically a hairpin loop, maintains high affinity and specificity while inducing a large, easily detectable secondary structure shift as two strands come together. Currently, the dynamics of this complex, ternary assembly are unknown, which can hinder optimization of their structure and response as biosensors. Addressing this gap in knowledge, we measure the assembly kinetics of the cocaine split-aptamer in real time using single-molecule fluorescence imaging. We tethered one strand of the split aptamer (capture probe) to a glass substrate and exposed it to a buffered solution containing the other aptamer strand tagged with a fluorescent label (detection strand) and varying concentrations of the cocaine analyte. Total-internal-reflection fluorescence (TIRF) imaging was used to detect individual aptamer assembly events at a glass-solution interface. Dissociation constants were extracted by tracking molecules and measuring their individual bound lifetimes. Bi-exponential dissociation was observed at all concentrations of cocaine (including blanks), thought to be from the capture probe interacting with the detection strand in different folded states. The lifetime of the longer-lived complex increases with cocaine concentration, which indicates that cocaine further stabilizes the aptamer complex, decreasing its dissociation rate. The sensitivity of the dissociation rate to the solution concentration of cocaine provides evidence that cocaine is exchanging during the life of the aptamer complex.

Single-molecule kinetics study of a small-molecule-dependent split aptamer assembly

251st National Meeting (2016)

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