Selection of smart aptamers by equilibrium capillary electrophoresis of equilibrium mixtures (ECEEM)
Selection of smart aptamers by equilibrium capillary electrophoresis of equilibrium mixtures (ECEEM)
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DOI：
10.1021/ja0530016
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发表时间：
2005-08-17
影响因子：
15
通讯作者：
Krylov, SN
中科院分区：
文献类型：
--
作者：
Drabovich, A;Berezovski, M;Krylov, SN
我们创造了一个术语“智能适体”，它描述了具有与靶点相互作用的预定义结合参数的适体。本文介绍了一种选择具有预定Kd值的智能适体的方法：平衡混合物平衡毛细管电泳法（ECEEM）。概念上，制备并平衡靶与DNA（RNA）文库的混合物。将平衡混合物的塞注入预先填充有运行缓冲液的毛细管中，所述运行缓冲液含有浓度与平衡混合物中的目标浓度相同的目标。平衡混合物的组分通过毛细管电泳分离，同时保持目标和适体之间的平衡。ECEEM的独特之处在于，具有不同Kd值的适体以不同且可预测的迁移率迁移。因此，收集具有不同迁移率的级分导致具有不同和预定义Kd值的智能适体。在这项原理验证工作中，我们使用ECEEM来选择MutS蛋白的智能适体，以前从未选择过适体。三轮基于ECEEM的选择足以获得Kd值接近理论预测值的智能适体。ECEEM是第一种用于适体选择的方法，其产生智能适体的能力已被实验证明。
We coin a term of “smart aptamers”, which describes aptamers with predefined binding parameters of their interaction with the target. Here, we introduce a method for selection of smart aptamers with predefined values ofKd: equilibrium capillary electrophoresis of equilibrium mixtures (ECEEM). Conceptually, a mixture of a target with a DNA (RNA) library is prepared and equilibrated. A plug of the equilibrium mixture is injected into a capillary prefilled with a run buffer containing the target at the concentration identical to the target concentration in the equilibrium mixture. The components of the equilibrium mixture are separated by capillary electrophoresis while equilibrium is maintained between the target and aptamers. The unique feature of ECEEM is that aptamers with differentKdvalues migrate with different and predictable mobilities. Thus, collecting fractions with different mobilities results in smart aptamers with different and predefinedKdvalues. In this proof-of-principle work, we used ECEEM to select smart aptamers for MutS protein, for which aptamers have never been previously selected. Three rounds of ECEEM-based selection were sufficient to obtain smart aptamers withKdvalues approaching theoretically predicted ones. ECEEM is the first method for aptamer selection whose ability to generate smart aptamers has been experimentally proven.