Photoaffinity electrophoretic mobility shift assay using photoreactive DNA bearing 3-trifluoromethyl-3-phenyldiazirine in its phosphate backbone.
Photoaffinity electrophoretic mobility shift assay using photoreactive DNA bearing 3-trifluoromethyl-3-phenyldiazirine in its phosphate backbone.
复制标题
使用磷酸主链中带有 3-三氟甲基-3-苯基二氮丙啶的光反应 DNA 进行光亲和电泳迁移率变动测定。
DOI：
10.1016/j.ab.2016.04.016
复制
发表时间：
2016
期刊：
影响因子：
--
通讯作者：
Yasumaru Hatanaka
中科院分区：
文献类型：
--
作者：
Yutaka Sadakane;Yasumaru Hatanaka
光亲和交联使得即使在变性条件下也能够分析DNA和蛋白质之间的相互作用。本文提出了一种结合光亲和电泳迁移率变动分析（EMSA）的方法，该方法采用了两种异质性分析技术，即光亲和交联法和十二烷基硫酸钠聚丙烯酰胺凝胶电泳（SDS-PAGE）分析。为了制备光反应性DNA，这是光亲和EMSA的重要工具，我们首先确定了将4-（3-三氟甲基-3H-二氮杂环丙烯-3-基）苄基溴整合到寡核苷酸的特定位点的最佳条件，其中磷酸二酯键被硫代磷酸酯键取代。利用Oct-1蛋白的POU（Pit-1、Oct-1，2和unc-86三个基因的首字母）结构域区域特异性结合八聚体DNA基序（ATGCAAAT），建立了光亲和EMSA。亲和纯化的重组POU结构域蛋白与谷胱甘肽-S-转移酶（GST）的共轭含有三个不同的蛋白质的分子量为34，36和45 kDa。光亲和EMSA可以在一条泳道上清楚地区分三种蛋白的结合能力，并通过竞争实验表明整个POU结构域蛋白与八聚体DNA基序特异性结合。利用HeLa细胞核提取物，光亲和EMSA显示，至少有五个特定的蛋白质可以结合到八聚体DNA基序。这些结果表明，使用3-三氟甲基-3-苯基二氮杂环丙烯的光亲和EMSA可以提供DNA结合蛋白的高性能分析。
Photoaffinity cross-linking enables the analysis of interactions between DNA and proteins even under denaturing conditions. We present a photoaffinity electrophoretic mobility shiftassay (EMSA) in which two heterogeneous techniques―photoaffinity cross-linking using DNA bearing 3-trifluoromethyl-3-phenyldiazirine and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS–PAGE) analysis—are combined. To prepare the photoreactive DNA, which is an essential tool for photoaffinity EMSA, we first determined the optimal conditions for the integration of 4-(3-trifluoromethyl-3H-diazirin-3-yl)benzyl bromide to the specific site of oligonucleotide where phosphodiester linkage was replaced with phosphorothioate linkage. The photoaffinity EMSA was developed using the POU (initial letters of three genes: Pit-l, Oct-1,2, and unc-86) domain region of Oct-1 protein, which specifically bound to octamer DNA motif (ATGCAAAT). The affinity-purified recombinant POU domain proteins conjugated with glutathione-S-transferase (GST) contained three distinct proteins with molecular weights of 34, 36, and 45 kDa. The photoaffinity EMSA could clearly distinguish the individual binding abilities of three proteins on a single lane and showed that the whole POU domain protein specifically bound to octamer DNA motif by competition experiments. Using the nuclear extract of HeLa cells, the photoaffinity EMSA revealed that at least five specific proteins could bind to the octamer DNA motif. These results show that photoaffinity EMSA using 3-trifluoromethyl-3-phenyldiazirine can provide high-performance analysis of DNA-binding proteins.