A protein-based electrochemical method for label-free characterization of sequence-specific protein-DNA interactions

摘要

Interactions between DNA binding proteins and specific DNA elements are the fundamental basis of many biological pathways during gene expression and regulation. The sequence diversity of DNA elements leads to affinity variation, which could play important roles in regulatory and/or pathogenic processes. Therefore detection and analysis of such interactions, particularly the interactions between essential protein factors and their various DNA targets, are crucial for unveiling the molecular mechanisms behind these processes. For this purpose, a simple electrochemical method based on protein-modified electrode was developed. Transcription factor Sp1, an important and well-studied DNA binding protein, was used as a proof example and immobilized onto a glass carbon electrode (GCE) surface, with the wild type Sp1 consensus binding sequence (wtDNA) and a mutant sequence carrying two point mutations (mutDNA) within the Sp1 binding site served as testing samples. Binding of DNA samples to GCE-immobilized Sp1 was analyzed by electrochemical impedance spectroscopy (EIS), and the wtDNA showed a 3-fold higher change of the charge-transfer resistance (R_(ct)) value than that of the mutDNA, correlating well with the known high affinity of wtDNA to Sp1. The reversible disassociation and re-association of Sp1-DNA complexes were also achieved and monitored closely by EIS. Further detection of a series of concentrations of wtDNA using difference pulse voltammetry (DPV) showed that as low as 1 × 10~(-10) mol/L wtDNA can be selectively detected in a DNA pool containing 0.05 g/L DNA fragments derived from salmon sperm. Exhibiting high affinity resolution, excellent selectivity, and low detection limit, this functionalized and reusable electrode provides a promising and convenient electrochemical approach for label-free, rapid monitoring, and comprehensive study of the specific interactions and/or affinity changes of DNA binding proteins with their various target DNAs under different conditions.