AXES Research Group, Department of Bioscience Engineering, University of Antwerp, Antwerp, 2020, Belgium.
BAMS Research Group, Department of Chemistry, University of Antwerp, Antwerp, 2020, Belgium.
J Am Chem Soc. 2020 Nov 18;142(46):19622-19630. doi: 10.1021/jacs.0c08691. Epub 2020 Nov 9.
In this manuscript, we compare different analytical methodologies to validate or disprove the binding capabilities of aptamer sequences. This was prompted by the lack of a universally accepted and robust quality control protocol for the characterization of aptamer performances coupled with the observation of independent yet inconsistent data sets in the literature. As an example, we chose three aptamers with a reported affinity in the nanomolar range for ampicillin, a β-lactam antibiotic, used as biorecognition elements in several detection strategies described in the literature. Application of a well-known colorimetric assay based on aggregation of gold nanoparticles (AuNPs) yielded conflicting results with respect to the original report. Therefore, ampicillin binding was evaluated in solution using isothermal titration calorimetry (ITC), native nano-electrospray ionization mass spectrometry (native nESI-MS), and H-nuclear magnetic resonance spectroscopy (H NMR). By coupling the thermodynamic data obtained with ITC with the structural information on the binding event given by native nESI-MS and H NMR we could verify that none of the ampicillin aptamers show any specific binding with their intended target. The effect of AuNPs on the binding event was studied by both ITC and H NMR, again without providing positive evidence of ampicillin binding. To validate the performance of our analytical approach, we investigated two well-characterized aptamers for cocaine/quinine (MN4), chosen for its nanomolar range affinity, and l-argininamide (1OLD) to show the versatility of our approach. The results clearly indicate the need for a multifaceted analytical approach, to unequivocally establish the actual detection potential and performance of aptamers aimed at small organic molecules.
在本文中，我们比较了不同的分析方法，以验证或反驳适体序列的结合能力。这是由于缺乏普遍接受和稳健的质量控制协议来表征适体性能，并且观察到文献中独立但不一致的数据集。例如，我们选择了三个报道的亲和力在纳摩尔范围内的适体，用于氨苄西林，一种β-内酰胺抗生素，作为文献中描述的几种检测策略中的生物识别元件。应用一种基于金纳米粒子（AuNPs）聚集的著名比色测定法，与原始报道相比，得到了相互矛盾的结果。因此，使用等温滴定量热法（ITC）、天然纳喷电离质谱（native nESI-MS）和 H 核磁共振波谱（H NMR）在溶液中评估氨苄西林的结合。通过将 ITC 获得的热力学数据与 native nESI-MS 和 H NMR 给出的结合事件的结构信息耦合，我们可以验证没有一个氨苄西林适体与其预期的靶标表现出任何特异性结合。通过 ITC 和 H NMR 研究了 AuNPs 对结合事件的影响，再次没有提供氨苄西林结合的阳性证据。为了验证我们分析方法的性能，我们研究了两种经过良好表征的可卡因/奎宁（MN4）适体和 l-精氨酸酰胺（1OLD）适体，用于其纳摩尔范围内的亲和力，以展示我们方法的多功能性。结果清楚地表明需要一种多方面的分析方法，以明确建立针对小分子的适体的实际检测潜力和性能。
