No Structure-Switching Required: A Generalizable Exonuclease-Mediated Aptamer-Based Assay for Small-Molecule Detection

The binding of small molecules to double-stranded DNA can modulate its susceptibility to digestion by exonucleases. Here, we show that the digestion of aptamers by exonuclease III can likewise be inhibited upon binding of small molecule targets and exploit this finding for the first time to achieve sensitive, label-free small-molecule detection. This approach does not require any sequence engineering and employs prefolded aptamers which have higher target-binding affinities than structure-switching aptamers widely used in current small-molecule detecting assays. We first use a dehydroisoandrosterone-3-sulfate-binding aptamer to show that target binding halts exonuclease III digestion four bases prior to the binding site. This leaves behind a double-stranded product that retains strong target affinity, whereas digestion of nontarget-bound aptamer produces a single-stranded product incapable of target binding. Exonuclease I efficiently eliminates these single-stranded products but is unable to digest the target-bound double-stranded product. The remaining products can be fluorescently quantified with SYBR Gold to determine target concentrations. We demonstrate that this dual-exonuclease-mediated approach can be broadly applied to other aptamers with differing secondary structures to achieve sensitive detection of various targets, even in biological matrices. Importantly, each aptamer digestion product has a unique sequence, enabling the creation of multiplex assays, and we successfully demonstrate simultaneous detection of cocaine and ATP in a single microliter volume sample in 25 min via sequence-specific molecular beacons. Due to the generality and simplicity of this assay, we believe that different DNA signal-reporting or amplification strategies can be adopted into our assay for target detection in diverse analytical contexts.