title: In Vitro Analysis of DNA-Protein Interactions in Gene Transcription Using DNAzyme-Based Electrochemical Assay.
abstract: 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-κB, SP6 RNA polymerase, and HNF-4α), as well as the analysis of binding affinity of the mutated protein-binding sequence, and even screening of the binding sequence of HNF-4α 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.
authors.affiliation: State Key Laboratory of Pharmaceutical Biotechnology and Collaborative Innovation Center of Chemistry for Life Sciences, Department of Biochemistry, Nanjing University , Nanjing 210093, People's Republic of China.
authors.forename: Chao
authors.initials: C
authors.lastname: Li
authors.affiliation: State Key Laboratory of Pharmaceutical Biotechnology and Collaborative Innovation Center of Chemistry for Life Sciences, Department of Biochemistry, Nanjing University , Nanjing 210093, People's Republic of China.
authors.forename: Yaqin
authors.initials: Y
authors.lastname: Tao
authors.affiliation: State Key Laboratory of Pharmaceutical Biotechnology and Collaborative Innovation Center of Chemistry for Life Sciences, Department of Biochemistry, Nanjing University , Nanjing 210093, People's Republic of China.
authors.forename: Yi
authors.initials: Y
authors.lastname: Yang
authors.affiliation: State Key Laboratory of Pharmaceutical Biotechnology and Collaborative Innovation Center of Chemistry for Life Sciences, Department of Biochemistry, Nanjing University , Nanjing 210093, People's Republic of China.
authors.forename: Yang
authors.initials: Y
authors.lastname: Xiang
authors.affiliation: State Key Laboratory of Pharmaceutical Biotechnology and Collaborative Innovation Center of Chemistry for Life Sciences, Department of Biochemistry, Nanjing University , Nanjing 210093, People's Republic of China.|Center for Molecular Recognition and Biosensing, School of Life Sciences, Shanghai University , Shanghai 200444, People's Republic of China.
authors.forename: Genxi
authors.identifier: 0000-0001-9663-9914
authors.initials: G
authors.lastname: Li
mesh_terms: D001483:Base Sequence; D004247:DNA / Q000378:metabolism*; D021881:DNA, Catalytic / Q000737:chemistry*; D004268:DNA-Binding Proteins / Q000032:analysis* / Q000378:metabolism*; D012321:DNA-Directed RNA Polymerases / Q000032:analysis / Q000378:metabolism; D055664:Electrochemical Techniques / Q000379:methods*; D056945:Hep G2 Cells; D051557:Hepatocyte Nuclear Factor 4 / Q000032:analysis / Q000378:metabolism; D006801:Humans; D016328:NF-kappa B / Q000032:analysis / Q000378:metabolism; D011485:Protein Binding
chemical_list: D021881:DNA, Catalytic; D004268:DNA-Binding Proteins; D051557:Hepatocyte Nuclear Factor 4; D016328:NF-kappa B; D004247:DNA; C070852:RNA polymerase SP6; D012321:DNA-Directed RNA Polymerases