Improvement of the VEGF binding ability of DNA aptamers through in silico maturation and multimerization strategy
Improvement of the VEGF binding ability of DNA aptamers through in silico maturation and multimerization strategy
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DOI：
10.1016/j.jbiotec.2015.08.011
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发表时间：
2015-10-20
影响因子：
4.1
通讯作者：
Ikebukuro, Kazunori
中科院分区：
文献类型：
--
作者：
Fukaya, Takahiro;Abe, Koichi;Ikebukuro, Kazunori
主要通过使用随机核酸文库的体外选择来选择适体。这些适体通常显示出对生物医学应用的亲和力不足。我们通过计算机模拟成熟（ISM）和适体多聚化提高了DNA适体对血管内皮生长因子（VEGF）的结合亲和力。ISM是许多进化方法之一，适体多聚化是改善功能的几种半理性策略之一。我们首先使用部分随机化的DNA文库重新选择VEGF结合适体，并鉴定出两种具有比已知适体更高的结合能力的适体。我们使用重新选择的适体进行ISM，以优化由三向连接结构产生的关键环序列。在五轮ISM之后，我们将适体2G 19 [解离常数（Kd），52 nM]鉴定为限定搜索空间的局部最优。我们的适体的特点，并发现一个特定的茎环结构参与适体VEGF的识别。为了进一步提高其对VEGF的亲和力，我们将2G 19或其茎环结构多聚化。设计的具有三个结合基序的SL 5-三价适体（Kd，0.37 nM）显著增加了结合亲和力，代表了通过指数富集选择的适体从配体的系统进化的500倍改进。(C)2015爱思唯尔B. V.保留所有权利。
Aptamers are mainly selected by in vitro selection using random nucleic acid libraries. These aptamers have often shown insufficient affinity for biomedical applications. We improved DNA aptamer binding affinity for vascular endothelial growth factor (VEGF) through in silico maturation (ISM) and aptamer multimerization. ISM is one of a number of evolutionary approaches and aptamer multimerization is one of several semi-rational strategies to improve function. We first reselected VEGF-binding aptamers using a partially randomized DNA library and identified two aptamers with higher binding ability than that of a known aptamer. We conducted ISM using the re-selected aptamers to optimize the key loop sequences created by a three-way junction structure. After five ISM rounds, we identified aptamer 2G19 [dissociation constant (Kd), 52 nM] as a local optimum of the defined search space. We characterized the aptamer and found that a specific stem-loop structure was involved in aptamer VEGF recognition. To further improve its affinity for VEGF, we multimerized 2G19 or its stem-loop structure. The designed SL5-trivalent aptamer (Kd, 0.37 nM) with three binding motifs significantly increased binding affinity, representing a 500-fold improvement from systematic evolution of ligands by exponential enrichment-selected aptamers. (C) 2015 Elsevier B.V. All rights reserved.