In Vitro Analysis of DNA-Protein Interactions in Gene Transcription Using DNAzyme-Based Electrochemical Assay
In Vitro Analysis of DNA-Protein Interactions in Gene Transcription Using DNAzyme-Based Electrochemical Assay
复制标题
使用基于 DNAzyme 的电化学测定对基因转录中 DNA-蛋白质相互作用进行体外分析
DOI：
10.1021/acs.analchem.7b00329
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发表时间：
2017-05-02
影响因子：
7.4
通讯作者：
Li, Genxi
中科院分区：
文献类型：
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作者：
Li, Chao;Tao, Yaqin;Li, Genxi
蛋白质和DNA元件之间的相互作用控制着基因组的多种功能。开发一种方便和经济的方法来研究dna结合蛋白的序列特异性是一个重要的挑战。作为回应，我们在这项工作中引入了一种电化学方法，基于蛋白质诱导的DNA脱氧核酶（DNAzyme）在电极表面上的独特运动行为，对核酸提取物中蛋白质和核酸之间的相互作用进行了特异性和敏感的分析。作为原理证明，我们还提出了快速、敏感和选择性检测三种转录因子（NF-kappa B、SP6 RNA聚合酶和HNF-4 α）的方法，以及突变蛋白结合序列的结合亲和力分析，甚至在体外筛选HNF-4 α蛋白的结合序列。这项工作可能为深入分析dna结合蛋白的序列特异性和研究已知蛋白质结合位点的核苷酸多态性开辟新的机会。
The interaction between protein and DNA elements controls a variety of functions of genomes. The development of a convenient and cost-effective method for investigating the sequence specificity of DNA-binding proteins represents an important challenge. In response, we have introduced an electrochemical assay in this work for specific and sensitive analysis of interaction between protein and nucleic acid in nucleic extracts, based on the protein-induced distinctive motion behavior of DNA deoxyribozyme (DNAzyme) on an electrode surface. As a proof of principle, we have also presented assays for the rapid, sensitive, and selective detection of three transcription factors (NF-kappa B, SP6 RNA polymerase, and HNF-4 alpha), as well as the analysis of binding affinity of the mutated protein-binding sequence, and even screening of the binding sequence of HNF-4 alpha protein in vitro. This work may open new opportunity for in-depth profiling of the sequence specificity of DNA-binding proteins and study of nucleotide polymorphisms in known protein-binding sites.