Department of Chemistry and Biochemistry, Florida International University, 11200 SW 8th Street, Miami, FL 33199, USA.
Nucleic Acids Res. 2020 Nov 18;48(20):e120. doi: 10.1093/nar/gkaa849.
In vitro aptamer isolation methods can yield hundreds of potential candidates, but selecting the optimal aptamer for a given application is challenging and laborious. Existing aptamer characterization methods either entail low-throughput analysis with sophisticated instrumentation, or offer the potential for higher throughput at the cost of providing a relatively increased risk of false-positive or -negative results. Here, we describe a novel method for accurately and sensitively evaluating the binding between DNA aptamers and small-molecule ligands in a high-throughput format without any aptamer engineering or labeling requirements. This approach is based on our new finding that ligand binding inhibits aptamer digestion by T5 exonuclease, where the extent of this inhibition correlates closely with the strength of aptamer-ligand binding. Our assay enables accurate and efficient screening of the ligand-binding profiles of individual aptamers, as well as the identification of the best target binders from a batch of aptamer candidates, independent of the ligands in question or the aptamer sequence and structure. We demonstrate the general applicability of this assay with a total of 106 aptamer-ligand pairs and validate these results with a gold-standard method. We expect that our assay can be readily expanded to characterize small-molecule-binding aptamers in an automated, high-throughput fashion.
体外适体分离方法可以产生数百种潜在的候选物，但选择最适合特定应用的适体是具有挑战性和繁琐的。现有的适体表征方法要么需要使用复杂的仪器进行低通量分析，要么可以提供更高的通量，但代价是增加了假阳性或假阴性结果的风险。在这里，我们描述了一种新的方法，可以在不进行适体工程或标记的情况下，以高通量的格式准确而灵敏地评估 DNA 适体与小分子配体之间的结合，而无需任何适体工程或标记要求。这种方法基于我们的新发现，即配体结合抑制 T5 外切核酸酶对适体的消化，这种抑制的程度与适体-配体结合的强度密切相关。我们的测定方法能够准确、有效地筛选单个适体的配体结合谱，以及从一批适体候选物中鉴定出最佳的靶标结合物，而无需考虑所涉及的配体或适体序列和结构。我们总共用 106 对适体-配体对验证了该测定方法的通用性，并通过金标准方法验证了这些结果。我们预计，我们的测定方法可以很容易地扩展到以自动化、高通量的方式对小分子结合适体进行表征。
