<br>使用核酸外切酶消化加速 SELEX 后适体工程,Journal of the American Chemical Society

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使用核酸外切酶消化加速 SELEX 后适体工程
Journal of the American Chemical Society ( IF 14.4 ) Pub Date : 2020-12-30 , DOI: 10.1021/jacs.0c09559
Juan Canoura ₁ , Haixiang Yu ₁ , Obtin Alkhamis ₁ , Daniel Roncancio ₁ , Rifat Farhana ₁ , Yi Xiao ₁

Affiliation

通过指数富集的配体系统进化 (SELEX) 过程能够从随机寡核苷酸文库中分离适体。然而，通常很难从所得序列中鉴定出最佳的适体，并且所选择的适体通常表现出次优的亲和力和特异性。 SELEX 后适体工程可以提高适体性能，但目前的方法表现出固有的偏差和可变的成功率，或者需要专门的仪器。在这里，我们描述了一种通用方法，该方法利用核酸外切酶 III 和核酸外切酶 I 在快速、无标记测定中询问小分子结合适体的结合特性。通过分析赭曲霉毒素结合 DNA 适体及其六个突变体，我们确定配体结合会改变核酸外切酶消化动力学，其程度与适体的配体亲和力密切相关。然后，我们利用该测定来增强 DNA 适体的结合特性，该适体不加区别地与 ATP、ADP、AMP 和腺苷结合。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，并鉴定了两种仅与腺苷结合的新的高亲和力适体。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% 血清中实现了 1 μM 腺苷的检测限。我们还证实了我们的方法来表征蛋白质结合适体的靶结合亲和力的通用性。我们相信，无论序列、结构和长度如何，我们的方法都适用于 DNA 适体，并且可以很容易地适应自动化格式，用于小分子结合适体的高通量工程，以获得适合各种应用的具有改进的结合特性的适体。

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Accelerating Post-SELEX Aptamer Engineering Using Exonuclease Digestion

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.

更新日期：2020-12-30

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