A novel method of screening thrombin-inhibiting DNA aptamers using an evolution-mimicking algorithm.
A novel method of screening thrombin-inhibiting DNA aptamers using an evolution-mimicking algorithm.
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DOI：
10.1093/nar/gni108
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发表时间：
2005-07-07
影响因子：
14.9
通讯作者：
Karube I
中科院分区：
文献类型：
--
作者：
Ikebukuro K;Okumura Y;Sumikura K;Karube I
凝血酶抑制性DNA适体已经通过指数富集配体系统进化（SELEX）获得。然而，SELEX是一种筛选与其靶分子结合的DNA适体的方法，它有时无法筛选出良好的抑制剂。因此，有必要开发一种基于其对靶分子的抑制作用来筛选DNA适体的方法。我们开发了一种新的方法来检测适体使用进化模拟算法，我们将其应用于寻找新的适体抑制凝血酶。首先，我们随机设计并合成了10个15聚体的寡核苷酸，推测它们可以形成G-四联体结构，然后测定它们的凝血酶抑制活性。选择显示高抑制活性的适体，并且我们在计算机上改组和突变这些序列以产生下一代适体的10个新序列。重复5次后，我们成功地获得了与先前报道的相同的适体，并且它们显示出高的抑制活性。此外，我们在所选择的15 mer适体的5′和3′端添加8 mer寡核苷酸，然后在计算机上重复进化。两个循环后，我们能够获得比15聚体适体具有更高抑制活性的适体。
Thrombin-inhibiting DNA aptamers have already been obtained through the systematic evolution of ligands by exponential enrichment (SELEX). However, SELEX is a method that screens DNA aptamers that bind to their target molecules, and it sometimes fails to screen good inhibitors. Therefore, it is necessary to develop a method of screening DNA aptamers based on their inhibitory effects on the target molecules. We developed a novel method of detecting aptamers using an evolution-mimicking algorithm, and we applied it to the search of new aptamers which inhibit thrombin. First, we randomly designed and synthesized ten 15mer oligonucleotides presumed to form G-quartet structures, and then measured their thrombin-inhibiting activities. The aptamers showing high inhibitory activity were selected, and we shuffled and mutated those sequences in silico to generate 10 new sequences of next-generation aptamers. After repeating the cycle five times, we successfully obtained the same aptamers reported previously, and they showed high inhibitory activity. In addition, we added 8mer oligonucleotides to both the 5′ and the 3′ end of the selected 15mer aptamers, and then repeated the evolution in silico. After two cycles, we were able to obtain aptamers with higher inhibitory activity than that of the 15mer aptamers.
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