In Vitro Analysis of DNA-Protein Interactions in Gene Transcription Using DNAzyme-Based Electrochemical Assay

Anal Chem. 2017 May 2;89(9):5003-5007. doi: 10.1021/acs.analchem.7b00329. Epub 2017 Apr 12.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Base Sequence
  • DNA / metabolism*
  • DNA, Catalytic / chemistry*
  • DNA-Binding Proteins / analysis*
  • DNA-Binding Proteins / metabolism*
  • DNA-Directed RNA Polymerases / analysis
  • DNA-Directed RNA Polymerases / metabolism
  • Electrochemical Techniques / methods*
  • Hep G2 Cells
  • Hepatocyte Nuclear Factor 4 / analysis
  • Hepatocyte Nuclear Factor 4 / metabolism
  • Humans
  • NF-kappa B / analysis
  • NF-kappa B / metabolism
  • Protein Binding

Substances

  • DNA, Catalytic
  • DNA-Binding Proteins
  • Hepatocyte Nuclear Factor 4
  • NF-kappa B
  • DNA
  • RNA polymerase SP6
  • DNA-Directed RNA Polymerases