| Popis: |
The binding of small molecules to their DNA aptamers can modulate their susceptibility to digestion by exonucleases, however, absolute differentiation between digestion and inhibition has never been reported. Here, we show that the digestion of aptamers by T7 exonuclease can be completely inhibited upon binding of small-molecule targets and exploit this finding for the first time to achieve sensitive, label-free small-molecule detection. We use a quinine-binding aptamer to show that target binding entirely halts T7 exonuclease digestion, leaving behind an intact double-stranded product that retains strong target affinity. On the contrary, 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, giving a limit of detection of 100 nM with the linear range from 0 to 8 μM. We demonstrate the first example of a dual-exonuclease-mediated approach capable of producing a concentration-dependent response in terms of aptamer digestion modules, therefore improving performance of the current aptamer-based assay for small-molecule detection. |
