Three-dimensional selection of leptin aptamers using capillary electrophoresis and implications for clone validation
Three-dimensional selection of leptin aptamers using capillary electrophoresis and implications for clone validation
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DOI：
10.1016/j.ab.2012.11.024
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发表时间：
2013-03-01
影响因子：
2.9
通讯作者：
Li, Sam F. Y.
中科院分区：
文献类型：
--
作者：
Ashley, Jon;Li, Sam F. Y.
毛细管电泳-指数富集配体系统进化（CE-SELEX）已被用作一种快速有效的方法来选择针对蛋白质靶标的适体，并且提供了在自由溶液三维环境中将结合的DNA与未结合的DNA分离的优点。CE-SELEX用于选择针对人瘦素蛋白的适体。进行了用于验证适体对靶标的结合亲和力的两种方法，并给出了不同的结果。平衡混合物的非平衡毛细管电泳（NECEEM）产生的K-D值在高纳摩尔范围内，而荧光强度法得到的K-D值在低微摩尔范围内。这些结果可能表明，适体验证必须在与选择分配步骤和预期使用适体的环境相似的环境中进行。我们还注意到，使用酶标仪通过荧光强度进行的亲和力结合可能限于具有相对低的k（off）速率的靶标，当使用两种不同技术验证适体时可能发生系统误差，并且将较小靶标固定到板威尔斯孔上可能影响DNA的结合，从而导致较低的结合亲和力。(c)2012 Elsevier Inc. All rights reserved.
Capillary electrophoresis-systematic evolution of ligands by exponential enrichment (CE-SELEX) has been used as a fast and efficient way to select aptamers against protein targets and offers the advantage of separating bound DNA from unbound DNA in a free solution three-dimensional environment. CE-SELEX was used to select aptamers against human leptin protein. Two methods used to validate the aptamers' binding affinity against the target were performed and gave differing results. Nonequilibrium capillary electrophoresis of equilibrium mixtures (NECEEM) yielded K-D values in the high nanomolar range, whereas the fluorescence intensity method gave K-D values in the low micromolar range. These results may suggest that aptamer validation must be carried out in a similar environment to that of the selection partitioning step and the environment in which the aptamer is intended to be used. We also note that affinity binding by fluorescence intensity using microplate readers may be limited to targets that have relatively low k(off) rates, systematic errors may occur when aptamers are validated using two different techniques, and the immobilization of smaller targets onto plate wells can affect the binding of the DNA, giving rise to lower binding affinities. (c) 2012 Elsevier Inc. All rights reserved.