Abstract：
Aptamers are oligonucleotide receptors with great potential for sensing and therapeutic applications. They are isolated from random libraries through an in vitro method termed systematic evolution of ligands by exponential enrichment (SELEX). Although SELEX-based methods have been widely employed over several decades, many aspects of the experimental process remain poorly understood in terms of how to adjust the selection conditions to obtain aptamers with the desired set of binding characteristics. As a result, SELEX is often performed with arbitrary parameters that tend to produce aptamers with insufficient affinity and/or specificity. Having a better understanding of these basic principles could increase the likelihood of obtaining high-quality aptamers. Here, we have systematically investigated how altering the selection stringency in terms of target concentration─which is essentially the root source of selection pressure for aptamer isolation─affects the outcome of SELEX. By performing four separate trials of SELEX for the same small-molecule target, we experimentally prove that the use of excessively high target concentrations promotes enrichment of low-affinity binders while also suppressing the enrichment of high-affinity aptamers. These findings should be broadly applicable across SELEX methods, given that they share the same core operating principle, and will be crucial for guiding selections to obtain high-quality aptamers in the future.
摘要：
适体是具有用于感测和治疗应用的巨大潜力的寡核苷酸受体。它们通过称为通过指数富集(SELEX)的配体系统进化的体外方法从随机文库中分离。尽管基于SELEX的方法已经被广泛使用了几十年，在如何调整选择条件以获得具有所需结合特性的适体方面，实验过程的许多方面仍然知之甚少。因此,SELEX通常以任意参数进行,所述参数倾向于产生具有不足亲和力和/或特异性的适体。更好地理解这些基本原理可以增加获得高质量适体的可能性。这里,我们已经系统地研究了在靶浓度方面改变选择严格性如何影响SELEX的结果。靶浓度本质上是适体分离选择压力的根源。通过对相同的小分子靶标进行SELEX的四次单独试验，我们通过实验证明，使用过高的靶浓度促进低亲和力结合剂的富集，同时也抑制高亲和力适体的富集。这些发现应该广泛适用于SELEX方法，鉴于它们具有相同的核心操作原理，并将在未来指导选择以获得高质量的适体。
