Facilitating aptamer selection and collection by capillary transient isotachophoresis with laser-induced fluorescence detection
Facilitating aptamer selection and collection by capillary transient isotachophoresis with laser-induced fluorescence detection
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DOI：
10.1016/j.chroma.2014.09.062
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发表时间：
2014-11-14
影响因子：
4.1
通讯作者：
Colyer, Christa L.
中科院分区：
文献类型：
--
作者：
Riley, Kathryn R.;Saito, Shingo;Colyer, Christa L.
建立了一种高效的毛细管瞬时等速电泳（ctITP）分离方法，用于结合配体的预选。为了从用于药物发现的大量文库中提供富集级分的最终目标，本文所述的预选过程需要三个不同的元件，其已经使用模型凝血酶蛋白（靶标）和凝血酶适体（配体）系统进行了验证。首先，一个高保真，采用非共价，荧光试剂SYBR金柱上标记计划被证明为单链DNA的灵敏度比柱前标记程序的11倍。其次，这种柱上标记被纳入具有激光诱导荧光（LIF）检测的新ctITP方法中，该方法大大提高了蛋白质-适体复合物和游离适体的分辨率（与传统的毛细管区带电泳（CZE）方法相比）。第三，这种增强的分辨率允许随后通过自动收集方法积累结合的适体级分，建立DNA积累的定量测量。预先选择的适体或配体样品可以作为后续珠上实验室或下一代测序技术的输入，从而加速药物发现。(C)2014爱思唯尔有限公司版权所有。
An efficient separation method that utilizes capillary transient isotachophoresis (ctITP) was developed for the preselection of binding ligands. With the ultimate goal of providing enriched fractions from vast libraries for drug discovery, the preselection process described herein entails three distinct elements, which have been validated using a model thrombin protein (target) and thrombin aptamer (ligand) system. First, a high fidelity, on-column labeling scheme employing the noncovalent, fluorescent reagent SYBR Gold was demonstrated for single-stranded DNA with an 11-fold greater sensitivity than pre-column labeling procedures. Second, this on-column labeling was incorporated into a new ctITP method with laser-induced fluorescence (LIF) detection, which provided greatly enhanced resolution of protein-aptamer complex and free aptamer (in comparison to traditional capillary zone electrophoresis (CZE) methods). Third, this enhanced resolution permitted the subsequent accumulation of bound aptamer fractions via an automated collection method, with the establishment of quantitative measures of DNA accumulation. Preselected aptamer or ligand samples such as these can serve as inputs for subsequent lab-on-bead or next-generation-sequencing technologies, enabling accelerated drug discovery. (C) 2014 Elsevier B.V. All rights reserved.