Accelerating Post-SELEX Aptamer Engineering Using Exonuclease Digestion.
Accelerating Post-SELEX Aptamer Engineering Using Exonuclease Digestion.
复制标题
使用核酸外切酶消化加速 SELEX 后适体工程。
DOI：
10.1021/jacs.0c09559
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发表时间：
2021-01-20
影响因子：
15
通讯作者：
Xiao Y
中科院分区：
文献类型：
--
作者：
Canoura J;Yu H;Alkhamis O;Roncancio D;Farhana R;Xiao Y
通过指数富集的配体系统进化（SELEX）过程使得能够从随机寡核苷酸文库中分离适体。然而，通常难以从所得序列中鉴定最佳适体，并且所选择的适体通常表现出次优的亲和力和特异性。后SELEX适体工程可以提高适体的性能，但目前的方法表现出固有的偏差和可变的成功率或需要专门的仪器。在这里，我们描述了一种可推广的方法，利用核酸外切酶III和核酸外切酶I在快速，无标记的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适体和其突变体中的六个，我们确定配体结合改变核酸外切酶消化动力学的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，所述DNA适体不加区别地结合ATP、ADP、AMP和腺苷。我们筛选了13个突变体，从这个适配体对所有这些类似物，并确定了两个新的高亲和力的适配体，仅结合腺苷。我们将这两种适体直接结合到基于电化学适体的传感器中，该传感器在50%血清中达到1 μM腺苷的检测限。我们还证实了我们的方法表征蛋白结合适体的靶结合亲和力的一般性。我们相信，我们的方法是可推广的DNA适体，无论序列，结构和长度，并可以很容易地适应到一个自动化的格式，高通量工程的小分子结合适体，以获得那些具有改善的结合特性，适合于各种应用。
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.
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影响因子：
8.8
作者：
Eltzschig HK
通讯作者：
Eltzschig HK
影响因子：
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FAMULOK, M
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作者：
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Krylov, Sergey N.