Isolation of Natural DNA Aptamers for Challenging Small-Molecule Targets, Cannabinoids

Abstract

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.

Figure 1.

Identification and characterization of THC-binding aptamer THC1.2. (A) Sequence logo for the 44 clones obtained from the round 11 pool showing the nucleotide diversity at each position of the random domain. A larger font size represents higher frequency. (B–D) Strand-displacement fluorescence assay for determining the affinity and specificity of THC1.2. (B) K_D1 was determined by titrating different concentrations of Iowa Black RQ-labeled complementary DNA strand (Q-cDNA) into Cy5-labeled THC1.2 (F-THC1.2) and measuring fluorescence quenching at 668 nm. (C) K_D2 was determined from fluorescence recovery at 668 nm of F-THC1.2–Q-cDNA complexes combined with varying concentrations of THC or THC-OH. (D) Signal gains produced by cannabinoids and interferents and their cross-reactivity relative to THC. Error bars represent the standard deviation of measurements from three individual experiments.

Figure 2.

Dye-displacement assay for colorimetric detection of THC. (A) Spectra of ETC at various concentrations of THC (0, 0.25, 0.5, 1, 1.5, 2, 2.5, 3.5, 5, 10, and 20 μM) and (B) corresponding calibration curves. Inset shows response at low concentrations of target. (C) Cross-reactivity of 5 μM THC-COOH, CBN and THCA; 25 μM CBD, CBDA, CBG, CBGA, UR-144, and XLR-11; and other small-molecule drugs at a concentration of 100 μM. Cross-reactivity was calculated relative to the signal gain produced by 5 μM THC. Inset shows photographs of the assay for selected specificity tests. Error bars represent the standard deviation of measurements from three individual experiments. Abbreviation: α-PVP = α-pyrrolidinopentiophenone, Meth = methamphetamine.

Figure 3.

Identification and characterization of synthetic cannabinoid aptamers XA1 and XA2. (A) Population of sequences in the XA1 family (purple), XA2 family (yellow), and other sequences (gray) in the round 16 and 17 pools based on high-throughput sequencing. (B) Secondary structures of XA1 and XA2, and their free energy as predicted by Mfold. (C, D) Affinity and (E) specificity of XA1 as determined by strand-displacement fluorescence assay. Bar plot shows signal gains produced by different compounds and their cross-reactivity relative to UR-144. Inset shows structures of UR-144, XLR-11, and the metabolite UR-144 pentanoic acid (UR-144M).