Department of Chemistry and Biochemistry, Florida International University, 11200 Southwest Eighth Street, Miami, Florida 33199, United States.
J Am Chem Soc. 2021 Jan 20;143(2):805-816. doi: 10.1021/jacs.0c09559. Epub 2020 Dec 30.
The systematic evolution of ligands by exponential enrichment (SELEX) process enables the isolation of aptamers from random oligonucleotide libraries. However, it is generally difficult to identify the best aptamer from the resulting sequences, and the selected aptamers often exhibit suboptimal affinity and specificity. Post-SELEX aptamer engineering can improve aptamer performance, but current methods exhibit inherent bias and variable rates of success or require specialized instruments. Here, we describe a generalizable method that utilizes exonuclease III and exonuclease I to interrogate the binding properties of small-molecule-binding aptamers in a rapid, label-free assay. By analyzing an ochratoxin-binding DNA aptamer and six of its mutants, we determined that ligand binding alters the exonuclease digestion kinetics to an extent that closely correlates with the aptamer's ligand affinity. We then utilized this assay to enhance the binding characteristics of a DNA aptamer which binds indiscriminately to ATP, ADP, AMP, and adenosine. We screened 13 mutants derived from this aptamer against all these analogues and identified two new high-affinity aptamers that solely bind to adenosine. We incorporated these two aptamers directly into an electrochemical aptamer-based sensor, which achieved a detection limit of 1 μM adenosine in 50% serum. We also confirmed the generality of our method to characterize target-binding affinities of protein-binding aptamers. We believe our approach is generalizable for DNA aptamers regardless of sequence, structure, and length and could be readily adapted into an automated format for high-throughput engineering of small-molecule-binding aptamers to acquire those with improved binding properties suitable for various applications.
通过指数富集的配体系统进化（SELEX）过程，可以从随机寡核苷酸文库中分离出适体。然而，从得到的序列中确定最佳适体通常很困难，并且所选适体的亲和力和特异性通常较差。SELEX 后适体工程可以改善适体的性能，但目前的方法存在固有偏差，成功率变化较大，或者需要专用仪器。在这里，我们描述了一种可推广的方法，该方法利用外切核酸酶 III 和外切核酸酶 I 在快速、无标记的测定中探究小分子结合适体的结合特性。通过分析一种赭曲霉毒素结合 DNA 适体及其六个突变体，我们确定配体结合会改变外切核酸酶消化动力学，使其与适体的配体亲和力密切相关。然后，我们利用该测定法增强了一种随机结合 ATP、ADP、AMP 和腺苷的 DNA 适体的结合特性。我们针对所有这些类似物筛选了源自该适体的 13 个突变体，并鉴定了两个仅与腺苷结合的新高亲和力适体。我们将这两个适体直接整合到基于电化学适体的传感器中，该传感器在 50%血清中对腺苷的检测限达到 1 μM。我们还证实了我们的方法可以用于表征蛋白质结合适体的靶标结合亲和力，具有普遍性。我们相信，无论序列、结构和长度如何，我们的方法都具有通用性，并且可以很容易地适应自动化格式，以高通量工程化小分子结合适体，从而获得具有改善的结合性能的适体，适用于各种应用。
