Highly sensitive detection of DNA-binding proteins based on a cationic conjugated polymervia a target-mediated fluorescence resonance energy transfer (TMFRET) strategy
Highly sensitive detection of DNA-binding proteins based on a cationic conjugated polymervia a target-mediated fluorescence resonance energy transfer (TMFRET) strategy
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DOI：
10.1039/c2py00499b
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发表时间：
2012-02
影响因子：
4.6
通讯作者：
Xingfen Liu;Ouyang Lan;Yan-Qin Huang;Xiaomiao Feng;Quli Fan;Wei Huang
中科院分区：
文献类型：
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作者：
Xingfen Liu;Ouyang Lan;Yan-Qin Huang;Xiaomiao Feng;Quli Fan;Wei Huang
对序列特异性dna结合蛋白进行灵敏、比例和简便的检测，对蛋白质组学、基因组学和生物医学具有重要意义。我们在此描述了一种新的靶向介导的荧光共振能量转移（TMFRET）策略，用于均匀和视觉检测序列特异性dna结合蛋白。这种基于共轭聚合物的光学生物传感器使用模型靶dna结合蛋白核因子-κB （NF-κB）进行了验证，NF-κB是参与许多重要转录调控网络的关键转录因子。设计了一种带有NF-κ b结合位点的荧光标记双链DNA探针来鉴定目标蛋白。将蛋白质与探针结合，可以保护探针免受外切酶III的酶切，产生从阳离子共轭聚合物到探针上的荧光基团标记的高效FRET。结果表明，纯化的重组NF-κB和HeLa细胞核提取物具有高度的灵敏度、选择性、比色性和开启性。对核提取物中目标蛋白的检出限估计为1 × 10−5 μL−1，灵敏度比以往报道的方法高5000倍。该方法的平均相对标准偏差小于5%，为重复性、低成本和简单的dna结合蛋白检测提供了一种新的方法。在透光器的帮助下，实验结果也可以用肉眼看到，这有望用于检测人体样本中的dna结合蛋白，以进行低成本的医学诊断，并用于高通量筛选针对dna结合蛋白的新药。
The sensitive, ratiometric and simple detection of sequence-specific DNA-binding proteins is of paramount importance for proteomics, genomics and biomedicine. We describe herein a novel target-mediated fluorescence resonance energy transfer (TMFRET) strategy for the homogeneous and visual detection of sequence-specific DNA-binding proteins. This conjugated polymer-based optical biosensor was demonstrated using a model target DNA-binding protein nuclear factor-kappaB (NF-κB), which is a key transcription factor involved in a number of important transcriptional regulatory networks. A fluorophore-labeled, double-stranded DNA probe bearing an NF-κB-binding site was designed to identify the target protein. Combining the protein with the probe could protect the probe from digestion by exonuclease III, yielding a high-efficiency FRET from the cationic conjugated polymer to the fluorophore label on the probe. The results revealed a highly sensitive, selective, colorimetric and turn-on detection of the purified recombinant NF-κB and HeLa cell nuclear extracts. The limit of detection for the target protein in nuclear extracts was estimated to be 1 × 10−5 μg μL−1, which is 5000-fold more sensitive than previously reported methods. This strategy offers a new method for the reproducible, low-cost and simple detection of DNA-binding proteins because an average percent relative standard deviation of less than 5% was obtained. The results of the assay can also be seen by the naked eye with the help of a transilluminator, which is promising for detecting DNA-binding proteins in human samples for low-cost medical diagnoses and for the high-throughput screening of new drugs targeted to DNA-binding proteins.