In vitro selection of high-affinity DNA aptamers for streptavidin
In vitro selection of high-affinity DNA aptamers for streptavidin
复制标题
链霉亲和素高亲和力 DNA 适体的体外筛选
DOI：
10.1093/abbs/gmp022
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发表时间：
2009-04-01
影响因子：
3.7
通讯作者：
Ding, Hongmei
中科院分区：
文献类型：
--
作者：
Wang, Chenglong;Yang, Guang;Ding, Hongmei
在这项研究中，我们利用磁珠固定和流式细胞术相结合的方法，通过指数富集（SELEX）方法开发了配体的系统进化。以链霉亲和素特异性适配体的选择为例。在本方案中，对传统的SELEX程序进行了优化，首先使用磁珠进行靶固定，以促进结合的单链DNA （ssDNA）适配体与未结合的ssDNA的高效分离，其次使用流式细胞术和荧光素标记来监测富集。在SELEX过程中，流式细胞术的灵敏度足以用于ssDNA的定量。本研究获得的链霉亲和素特异性适配体可作为表征链霉亲和素修饰表面与生物素化靶分子占用的工具。本研究描述的方法也普遍适用于链霉亲和素以外的靶分子。
In this study, we developed a systematic evolution of ligands by exponential enrichment (SELEX) method using a combination of magnetic beads immobilization and flow cytometric measurement. As an example, the selection of streptavidin-specific aptamers was performed. In this protocol, the conventional SELEX procedure was optimized, first using magnetic beads for target immobilization to facilitate highly efficient separation of the binding single-stranded DNA (ssDNA) aptamers from the unbound ssDNAs, and second using flow cytometry and fluorescein labeling to monitor the enrichment. The sensitivity of flow cytometry was adequate for ssDNA quantification during the SELEX procedures. The streptavidin-specific aptamers obtained in this work can be used as tools for characterization of the occupancy of streptavidin-modified surfaces with biotinylated target molecules. The method described in the study is also generally applicable to target molecules other than streptavidin.