Aptamers
are nucleic acid-based affinity reagents that are isolated
via an in vitro process known as systematic evolution
of ligands by exponential enrichment (SELEX). Despite their great
potential for a wide range of analytical applications, there are relatively
few high-quality small-molecule binding aptamers, especially for “challenging”
targets that have low water solubility and/or limited moieties for
aptamer recognition. The use of libraries containing chemically modified
bases may improve the outcome of some SELEX experiments, but this
approach is costly and yields inconsistent results. Here, we demonstrate
that a thoughtfully designed SELEX procedure with natural DNA libraries
can isolate aptamers with high affinity and specificity for challenging
small molecules, including targets for which such selections have
previously failed. We first isolate a DNA aptamer with nanomolar affinity
and high specificity for (−)-trans-Δ9-tetrahydrocannabinol (THC), a target previously thought to
be unsuitable for SELEX with natural DNA libraries. We subsequently
isolate aptamers that exhibit high affinity and cross-reactivity to
two other challenging targets, synthetic cannabinoids UR-144 and XLR-11,
while maintaining excellent specificity against a wide range of non-target
interferents. Our findings demonstrate that natural nucleic acid libraries
can yield high-quality aptamers for small-molecule targets, and we
outline a robust workflow for isolating other such aptamers in future
selection efforts.
Colorimetric aptamer-based sensors offer a simple means of on-site or point-of-care analyte detection. However, these sensors are largely incapable of achieving naked-eye detection, because of the poor performance of the target-recognition and signal-reporting elements employed. To address this problem, we report a generalizable strategy for engineering novel multimodule split DNA constructs termed “CBSAzymes” that utilize a cooperative binding split aptamer (CBSA) as a highly target-responsive bioreceptor and a new, highly active split DNAzyme as an efficient signal reporter. CBSAzymes consist of two fragments that remain separate in the absence of target, but effectively assemble in the presence of the target to form a complex that catalyzes the oxidation of 2,2′-azino-bis(3-ethylbenzthiazoline)-6-sulfonic acid, developing a dark green color within 5 min. Such assay enables rapid, sensitive, and visual detection of small molecules, which has not been achieved with any previously reported split-aptamer-DNAzyme conjugates. In an initial demonstration, we generate a cocaine-binding CBSAzyme that enables naked-eye detection of cocaine at concentrations as low as 10 μM. Notably, CBSAzyme engineering is straightforward and generalizable. We demonstrate this by developing a methylenedioxypyrovalerone (MDPV)-binding CBSAzyme for visual detection of MDPV and 10 other synthetic cathinones at low micromolar concentrations, even in biological samples. Given that CBSAzyme-based assays are simple, label-free, rapid, robust, and instrument-free, we believe that such assays should be readily applicable for on-site visual detection of various important small molecules such as illicit drugs, medical biomarkers, and toxins in various sample matrices.
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