Lou Beilei, Chen Erning, Zhao Xinying, Qu Feng, Yan Jieying
School of Life Science, Beijing Institute of Technology, Beijing 100081, China.
School of Life Science, Beijing Institute of Technology, Beijing 100081, China; Beijing Centre for Physical and Chemical Analysis, Beijing 100089, China.
J Chromatogr A. 2016 Mar 11;1437:203-209. doi: 10.1016/j.chroma.2016.01.073. Epub 2016 Feb 2.
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.
全细胞指数富集的配体系统进化技术（Whole-cell SELEX）比针对纯化分子靶标的SELEX面临更多困难。在这项工作中，我们通过表征完整的单链DNA分布，证明了毛细管电泳在辅助全细胞适配体筛选中的应用。我们选择了三种癌细胞系U251、Hela和PC3作为靶标，用荧光素标记的Sgc8c（一种41聚体适配体）和荧光素标记的41聚体随机单链DNA文库作为单链DNA模型。对运行缓冲液、毛细管长度和内径以及紫外和激光诱导荧光检测的毛细管电泳条件进行了优化。展示了Sgc8c和单链DNA文库针对U251、Hela和PC3的分布百分比，其复合物的相对峰面积对于Sgc8c分别为8.94%、1.05%和0.44%，对于单链DNA文库分别为9.03%、1.04%和0.12%。在所选实验条件下，三种细胞系的结合能力比较为U251>Hela>PC3，这通过激光共聚焦显微镜得到了验证。对于每种细胞，将单链DNA文库的分布百分比与Sgc8c的分布百分比进行了比较。最后，通过延长孵育时间和片段毛细管电泳分析证实了U251 - Sgc8c的全细胞复合物。
