Exonuclease III protection assay with FRET probe for detecting DNA-binding proteins.
Exonuclease III protection assay with FRET probe for detecting DNA-binding proteins.
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DOI：
10.1093/nar/gni021
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发表时间：
2005-02-01
影响因子：
14.9
通讯作者：
Lu Z
中科院分区：
文献类型：
--
作者：
Wang J;Li T;Guo X;Lu Z
在本文中，我们描述了一种用于检测序列特异性DNA结合蛋白的新方法。在该方法中，将灵敏的荧光共振能量转移（FRET）技术与常见的DNA足迹分析相结合，以开发一种简单、快速且高通量的定量检测序列特异性DNA结合蛋白的方法。我们将这种方法命名为带有FRET探针的核酸外切酶III（ExoIII）保护分析。该分析中使用的FRET探针是一种双链DNA，其设计为在中心包含一对FRET荧光对以及两个侧翼的蛋白结合位点。在蛋白检测过程中，如果存在目标蛋白，它将与FRET探针的两个蛋白结合位点结合，从而保护FRET荧光对不被ExoIII消化，导致较高的FRET值。然而，如果目标蛋白不存在，裸露的FRET探针上的FRET荧光对将被ExoIII降解，导致较低的FRET值。通过该分析成功检测了包括NF - κB、SP1和p50在内的三种重组转录因子以及HeLa细胞核提取物中NF - κB的目标蛋白。这种分析可广泛应用于针对DNA结合蛋白的生物医学研究。
We describe a new method for the assay of sequence-specific DNA-binding proteins in this paper. In this method, the sensitive fluorescence resonance energy transfer (FRET) technology is combined with the common DNA footprinting assay in order to develop a simple, rapid and high-throughput approach for quantitatively detecting the sequence-specific DNA-binding proteins. We named this method as exonuclease III (ExoIII) protection assay with FRET probe. The FRET probe used in this assay was a duplex DNA which was designed to contain one FRET pair in the center and two flanking protein-binding sites. During protein detection, if a target protein exists, it will bind to the two protein-binding sites of the FRET probe and thus protect the FRET pair from ExoIII digestion, resulting in high FRET. However, if the target protein does not exist, the FRET pair on the naked FRET probe will be degraded by ExoIII, resulting in low FRET. Three kinds of recombinant transcription factors including NF-κB, SP1 and p50, and the target protein of NF-κB in HeLa cell nuclear extracts, were successfully detected by the assay. This assay can be extensively used in biomedical research targeted at DNA-binding proteins.
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