Selection of DNA aptamers against insulin and construction of an aptameric enzyme subunit for insulin sensing
Selection of DNA aptamers against insulin and construction of an aptameric enzyme subunit for insulin sensing
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DOI：
10.1016/j.bios.2008.06.016
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发表时间：
2009-01-01
影响因子：
12.6
通讯作者：
Ikebukuro, Kazunori
中科院分区：
文献类型：
--
作者：
Yoshida, Wataru;Mochizuki, Eriko;Ikebukuro, Kazunori
我们选择了针对胰岛素的DNA适体，并开发了用于胰岛素传感的适体酶亚基（AES）。从单链DNA文库中鉴定出胰岛素结合适体，预期其形成各种G-四联体结构。通过适体印迹法进行体外选择，适体印迹法在每一轮显示与靶蛋白结合的寡核苷酸。在第6轮选择后，鉴定了胰岛素结合适体。这些鉴定的胰岛素结合适体具有比胰岛素连接多态性区域（ILPR）寡核苷酸更高的结合能力，其可被称为“天然”胰岛素结合DNA适体。对所鉴定的胰岛素结合DNA适体的圆二色性（CD）光谱测量表明，适体将折叠成G-四重体结构。我们还开发了一个AES通过连接最好的识别胰岛素结合适体与凝血酶抑制适体。使用该AES，我们能够通过测量凝血酶酶活性而无需结合/游离分离来检测胰岛素。(c)2008 Elsevier B. V.保留所有权利。
We selected DNA aptamers against insulin and developed an aptameric enzyme subunit (AES) for insulin sensing. The insulin-binding aptamers were identified from a single-strand DNA library which was expected to form various kinds of G-quartet structures. in vitro selection was carried out by means of aptamer blotting, which visualizes the oligonucleotides binding to the target protein at each round. After the 6th round of selection, insulin-binding aptamers were identified. These identified insulin-binding aptamers had a higher binding ability than the insulin-linked polymorphic region (ILPR) oligonucleotide, which can be called a "natural" insulin-binding DNA aptamer. The circular-dichroism (CD) spectrum measurement of the identified insulin-binding DNA aptamers indicated that the aptamers would fold into a G-quartet structure. We also developed an AES by connecting the best identified insulin-binding aptamer with the thrombin-inhibiting aptamer. Using this AES, we were able to detect insulin by measuring the thrombin enzymatic activity without bound/free separation. (c) 2008 Elsevier B.V. All rights reserved.