The application of capillary electrophoresis for assisting whole-cell aptamers selection by characterizing complete ssDNA distribution
The application of capillary electrophoresis for assisting whole-cell aptamers selection by characterizing complete ssDNA distribution
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应用毛细管电泳通过表征完整的 ssDNA 分布来辅助全细胞适体选择
DOI：
10.1016/j.chroma.2016.01.073
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发表时间：
2016
影响因子：
4.1
通讯作者：
Yan Jieying
中科院分区：
文献类型：
--
作者：
Lou Beilei;Chen Erning;Zhao Xinying;Qu Feng;Yan Jieying
全细胞SELEX比针对纯化分子靶点的SELEX面临更多困难。在这项工作中，我们展示了毛细管电泳的应用，以协助全细胞适配体的选择，表征完整的ssDNA分布。我们选择了U251、Hela和PC 3三种肿瘤细胞系作为靶细胞，以FAM标记的Sgc 8 c（一种41聚体适体）和FAM标记的41聚体随机ssDNA文库作为ssDNA模型。优化了毛细管电泳的运行缓冲液、毛细管长度和内径以及紫外检测和激光诱导荧光检测的条件。Sgc 8 c和ssDNA文库对U251、Hela和PC 3的分布百分比分别为8.94%、1.05%和0.44%; ssDNA文库对U251、Hela和PC 3的分布百分比分别为9.03%、1.04%和0.12%。在选定的实验条件下，三种细胞系的结合能力比较为U251 > Hela > PC 3，并通过激光共聚焦显微镜进行验证。对于每个细胞，将ssDNA文库的分布百分比与Sgc 8 c的分布百分比进行比较。最后，通过增加孵育时间和组分CE分析证实了U251-Sgc 8 c的全细胞复合物。
Whole-cell SELEX faces more difficulties than SELEX against purified molecules target. In this work, we demonstrate the application of capillary electrophoresis for assisting whole-cell aptamers selection by characterizing complete ssDNA distribution. We chose three cancer cell lines U251, Hela and PC3 as target, FAM labeled Sgc8c (a 41mer aptamer) and FAM labeled 41mer random ssDNA library as ssDNA model. CE conditions of running buffer and capillary length and inner diameter as well as UV and LIF detection were optimized. The distribution percentage of Sgc8c and ssDNA library against U251, Hela and PC3 was demonstrated, the relative peak area of their complex is 8.94%, 1.05% and 0.44% for Sgc8c and 9.03%, 1.04% and 0.12% for ssDNA library respectively. Under the chosen experimental conditions, binding ability comparison of three cell lines was U251 > Hela > PC3, which was validated by laser confocol microscope. For each cell, distribution percentage of ssDNA library was compared with that of Sgc8c. Finally, whole-cell complex of U251–Sgc8c was confirmed by increase incubation time and fraction CE analysis.