Aptamers are nucleic acid bioreceptors that have been widely utilized for a variety of biosensing applications, including in vivo 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 EcoRI 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.

中文翻译：

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高亲和力小分子适体的快速核酸酶辅助选择


适体是核酸生物受体，已广泛用于各种生物传感应用，包括基于抗体的系统无法实现的体内检测方法。然而，为小分子靶标生成高质量的适体仍然具有挑战性，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，利用核酸酶 EcoRI 在溶液相中去除非特异性或弱结合序列，在短短几轮选择内快速富集高亲和力靶标结合物。作为概念验证，我们使用核酸酶辅助 SELEX (NA-SELEX) 方法分离合成大麻素 AB-FUBINACA 的适体。在五轮内，我们鉴定了两种高度特异性的适体，它们在生理温度下表现出纳摩尔亲和力。我们还通过使用新鲜试剂和文库进行相同的选择实验，获得相同的两个适体以及其他两个高质量适体候选物，证明了 NA-SELEX 的稳健性和重现性。最后，我们使用相同的筛选条件将 NA-SELEX 与传统的文库固定化 SELEX 筛选 AB-FUBINACA 进行比较，在 11 轮选择后鉴定出亲和力弱 25-100 倍的适体。因此，NA-SELEX 可能是分离小分子靶点高质量适体的有效选择方法。