Hou Peng, Chen Zaozao, Ji Meiju, He Nongyue, Lu Zuhong
Chien-Shiung Wu Laboratory, Department of Biological Science and Medical Engineering, Southeast University, Nanjing, China.
Clin Chem. 2007 Apr;53(4):581-6. doi: 10.1373/clinchem.2006.077503. Epub 2007 Feb 8.
The specific binding of proteins to DNA is a key step for many cellular activities, such as transcription regulation, DNA replication, recombination, repair, and restriction. The detection of DNA-binding proteins, as well as the identification of specific binding sites, is therefore important to understand gene expression mechanisms and cellular function. We describe an ultrasensitive method for quantification of DNA-binding proteins.
We combined the common exonuclease III (ExoIII) footprinting assay and real-time PCR for quantification of DNA-binding proteins, for an assay that does not require antibodies against the target proteins. Double-strand DNA probes were designed to monitor the activities of DNA-binding protein. The protein-binding site is at the 5' end of the forward primer. When a target protein is present, it will specifically bind to the protein-binding site and produce a physical hindrance to ExoIII, which protects the reverse DNA strand from digestion by ExoIII. The remaining single-strand DNA template can be quantitatively detected by real-time PCR. Conversely, in the absence of the target protein, the naked primer regions will be degraded by ExoIII, which then cannot be amplified by real-time PCR.
We detected the binding of 10 different transcription factors in crude cell extracts. The assay quantitatively detected binding at femtomolar concentrations of protein.
This technique is customizable and easy to establish. It has potential applications in research, medical diagnosis, and drug discovery.
蛋白质与DNA的特异性结合是许多细胞活动的关键步骤，如转录调控、DNA复制、重组、修复和限制。因此，检测DNA结合蛋白以及鉴定特异性结合位点对于理解基因表达机制和细胞功能很重要。我们描述了一种用于定量DNA结合蛋白的超灵敏方法。
我们将常见的核酸外切酶III（ExoIII）足迹分析与实时PCR相结合，用于定量DNA结合蛋白，该分析不需要针对目标蛋白的抗体。设计双链DNA探针来监测DNA结合蛋白的活性。蛋白质结合位点位于正向引物的5'端。当存在目标蛋白时，它将特异性结合到蛋白质结合位点，并对ExoIII产生物理阻碍，从而保护反向DNA链不被ExoIII消化。剩余的单链DNA模板可以通过实时PCR进行定量检测。相反，在没有目标蛋白的情况下，裸露的引物区域将被ExoIII降解，然后不能通过实时PCR进行扩增。
我们在粗细胞提取物中检测到1了0种不同转录因子的结合。该分析在飞摩尔浓度的蛋白质下定量检测结合。
该技术可定制且易于建立。它在研究、医学诊断和药物发现中具有潜在应用。
