Chen Zaozao, Ji Meiju, Hou Peng, Lu Zuhong
Chien-Shiung Wu Laboratory, Department of Biological Science and Medical Engineering, Southeast University, Nanjing 210096, China.
Biochem Biophys Res Commun. 2006 Jul 7;345(3):1254-63. doi: 10.1016/j.bbrc.2006.05.012. Epub 2006 May 11.
We reported herein a rapid, inexpensive, and sensitive technique for detecting sequence-specific DNA-binding proteins. In this technique, the common exonuclease III (ExoIII) footprinting assay is coupled with simple SYBR Green I staining for monitoring the activities of DNA-binding proteins. We named this technique as ExoIII-Dye-based assay. In this assay, a duplex probe was designed to detect DNA-binding protein. One side of the probe contains one protein-binding site, and another side of it contains five protruding bases at 3' end for protection from ExoIII digestion. If a target protein is present, it will bind to binding sites of probe and produce a physical hindrance to ExoIII, which protects the duplex probe from digestion of ExoIII. SYBR Green I will bind to probe, which results in high fluorescence intensity. On the contrary, in the absence of the target protein, the naked duplex probe will be degraded by ExoIII. SYBR Green I will be released, which results in a low fluorescence intensity. In this study, we employed this technique to successfully detect transcription factor NF-kappaB in crude cell extracts. Moreover, it could also be used to evaluate the binding affinity of NF-kappaB. This technique has therefore wide potential application in research, medical diagnosis, and drug discovery.
我们在此报告了一种用于检测序列特异性DNA结合蛋白的快速、廉价且灵敏的技术。在该技术中，常见的核酸外切酶III（ExoIII）足迹分析与简单的SYBR Green I染色相结合，以监测DNA结合蛋白的活性。我们将此技术命名为基于ExoIII-染料的分析方法。在该分析中，设计了一种双链探针来检测DNA结合蛋白。探针的一侧包含一个蛋白质结合位点，另一侧在3'端含有五个突出碱基以防止被ExoIII消化。如果存在靶蛋白，它将与探针的结合位点结合，并对ExoIII产生物理阻碍，从而保护双链探针不被ExoIII消化。SYBR Green I将与探针结合，导致荧光强度高。相反，在没有靶蛋白的情况下，裸露的双链探针将被ExoIII降解。SYBR Green I将被释放，导致荧光强度低。在本研究中，我们使用该技术成功检测了粗细胞提取物中的转录因子NF-κB。此外，它还可用于评估NF-κB的结合亲和力。因此，该技术在研究、医学诊断和药物发现中具有广泛的潜在应用。
