Department of Chemistry, North Carolina State University, 2620 Yarbrough Dr., Raleigh, North Carolina 27695, United States.
Department of Surgery, Duke University Medical Center, Durham, North Carolina 27710, United States.
J Am Chem Soc. 2024 Aug 7;146(31):21296-21307. doi: 10.1021/jacs.4c00748. Epub 2024 Jul 23.
Aptamers are nucleic acid bioreceptors that have been widely utilized for a variety of biosensing applications, including detection methods that would not be possible with antibody-based systems. However, it remains challenging to generate high-quality aptamers for small molecule targets, particularly for use under physiological conditions. We present a highly effective aptamer selection technology for small-molecule targets that utilizes the nuclease RI to remove nonspecific or weakly binding sequences in solution phase, rapidly enriching high-affinity target binders within just a few rounds of selection. As proof-of-concept, we used our nuclease-assisted SELEX (NA-SELEX) method to isolate aptamers for a synthetic cannabinoid, AB-FUBINACA. Within five rounds, we identified two highly specific aptamers that exhibit nanomolar affinity at physiological temperature. We also demonstrate the robustness and reproducibility of NA-SELEX by performing the same selection experiment with fresh reagents and libraries, obtaining the same two aptamers as well as two other high-quality aptamer candidates. Finally, we compare NA-SELEX against a conventional library-immobilized SELEX screen for AB-FUBINACA using the same screening conditions, identifying aptamers with 25-100-fold weaker affinity after 11 rounds of selection. NA-SELEX therefore could be an effective selection method for the isolation of high-quality aptamers for small-molecule targets.
适体是核酸生物受体，已广泛应用于各种生物传感应用，包括基于抗体的系统无法实现的检测方法。然而，对于小分子靶标，仍然难以生成高质量的适体，特别是在生理条件下使用。我们提出了一种针对小分子靶标的高效适体选择技术，该技术利用核酸内切酶 RI 在溶液相中去除非特异性或弱结合序列，仅通过几轮选择就可快速富集高亲和力的靶标结合物。作为概念验证，我们使用我们的核酸内切酶辅助 SELEX（NA-SELEX）方法来分离合成大麻素 AB-FUBINACA 的适体。在五轮选择中，我们鉴定了两个具有纳摩尔亲和力的高度特异性适体，在生理温度下。我们还通过使用新鲜试剂和文库进行相同的选择实验，证明了 NA-SELEX 的稳健性和可重复性，得到了相同的两个适体以及另外两个高质量的适体候选物。最后，我们在相同的筛选条件下，将 NA-SELEX 与常规文库固定化 SELEX 筛选进行了比较，在 11 轮筛选后，鉴定出亲和力弱 25-100 倍的适体。因此，NA-SELEX 可能是一种针对小分子靶标分离高质量适体的有效选择方法。
