Riley Kathryn R, Saito Shingo, Gagliano Jason, Colyer Christa L
Department of Chemistry, Wake Forest University, Winston-Salem, NC, 27109, USA.
Graduate School of Science and Engineering, Saitama University, 255 Shimo-Okubo, Sakura-ku, Saitama 338-8570, Japan.
J Chromatogr A. 2014 Nov 14;1368:183-9. doi: 10.1016/j.chroma.2014.09.062. Epub 2014 Oct 2.
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.
开发了一种利用毛细管瞬态等速电泳（ctITP）的高效分离方法，用于结合配体的预选。本文所述的预选过程以从大量文库中提供富集级分用于药物发现为最终目标，它包含三个不同的要素，这些要素已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，证明了一种采用非共价荧光试剂SYBR Gold的高保真柱上标记方案，用于单链DNA，其灵敏度比柱前标记程序高11倍。其次，这种柱上标记被纳入一种新的具有激光诱导荧光（LIF）检测的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（与传统毛细管区带电泳（CZE）方法相比）。第三，这种提高的分辨率允许通过自动收集方法随后积累结合的适体级分，并建立DNA积累的定量测量方法。这样预选的适体或配体样品可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。
