Real-Time PCR-Coupled CE-SELEX for DNA Aptamer Selection.
Real-Time PCR-Coupled CE-SELEX for DNA Aptamer Selection.
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DOI：
10.5402/2012/939083
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发表时间：
2012
期刊：
影响因子：
--
通讯作者：
Storici F
中科院分区：
文献类型：
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作者：
Ruff P;Pai RB;Storici F
适配体是一种短的核酸或肽序列，能够以高特异性和亲和力与靶分子结合。适体也被称为“人工抗体”，与抗体相比，适体具有许多优势。适体分离的主要障碍之一是选择所需的初始时间和精力。配体的指数富集系统进化（SELEX）是传统的适体生成方法，但该方法耗时长，需要大量的靶体和起始适体库。使用毛细管电泳（CE）生成适配体的一种相对较新的方法，被称为CE-SELEX，比SELEX更快、更有效，但需要激光诱导荧光（LIF）来检测适配体-靶复合物。在这里，我们使用实时（RT-） PCR实现了一个没有LIF的替代系统来间接测量适配体-靶标复合物。在三轮选择中，而不是在SELEX协议中常见的十轮或更多轮，获得了牛血清白蛋白（BSA）的特异性适配体。该适体对BSA的特异性通过电泳迁移迁移试验（EMSAs）、无标记竞争对手试验和超位移试验证实。这里使用的系统提供了一种具有成本效益和高效的产生适体的方法。
Aptamers are short nucleic acid or peptide sequences capable of binding to a target molecule with high specificity and affinity. Also known as “artificial antibodies,” aptamers provide many advantages over antibodies. One of the major hurdles to aptamer isolation is the initial time and effort needed for selection. The systematic evolution of ligands by exponential enrichment (SELEX) is the traditional procedure for generating aptamers, but this process is lengthy and requires a large quantity of target and starting aptamer library. A relatively new procedure for generating aptamers using capillary electrophoresis (CE), known as CE-SELEX, is faster and more efficient than SELEX but requires laser-induced fluorescence (LIF) to detect the aptamer-target complexes. Here, we implemented an alternative system without LIF using real-time- (RT-) PCR to indirectly measure aptamer-target complexes. In three rounds of selection, as opposed to ten or more rounds common in SELEX protocols, a specific aptamer for bovine serum albumin (BSA) was obtained. The specificity of the aptamer to BSA was confirmed by electrophoretic mobility shift assays (EMSAs), an unlabeled competitor assay, and by a supershift assay. The system used here provides a cost effective and a highly efficient means of generating aptamers.