Electrochemical sensing of tumor suppressor protein p53-deoxyribonucleic acid complex stability at an electrified interface.
Electrochemical biosensors have the unique ability to convert biological events directly into electrical signals suitable for parallel analysis. Here we utilize specific properties of constant current chronopotentiometric stripping (CPS) in the analysis of protein and DNA-protein complex nanolayers. Rapid potential changes at high negative current intensities (Istr) in CPS are utilized in the analysis of DNA-protein interactions at thiol-modified mercury electrodes. P53 core domain (p53CD) sequence-specific binding to DNA results in a striking decrease in the electrocatalytic signal of free p53. This decrease is related to changes in the accessibility of the electroactive amino acid residues in the p53CD-DNA complex. By adjusting Istr and temperature, weaker non-specific binding can be eliminated or distinguished from the sequence-specific binding. The method also reflects differences in the stabilities of different sequence-specific complexes, including those containing spacers between half-sites of the DNA consensus sequence. The high resolving power of this method is based on the disintegration of the p53CD-DNA complex by the electric field effects at a negatively charged surface and fine adjustment of the millisecond time intervals for which the complex is exposed to these effects. Picomole amounts of p53 proteins and DNA were used for the analysis at full electrode coverage but we show that even 10-20-fold smaller amounts can be analyzed. Our method cannot however take advantage of very low detection limits of the protein CPS detection because low I(str) intensities are deleterious to the p53CD-DNA complex stability at the electrode surface. These data highlight the utility of developing biosensors offering novel approaches for studying real-time macromolecular protein dynamics.
Institute of Biophysics, Academy of Sciences of the Czech Republic, v.v.i., Královopolská 135, 612 65 Brno, Czech Republic. Electronic address: palecek@ibp.cz.
Emil
E
Paleček
Institute of Biophysics, Academy of Sciences of the Czech Republic, v.v.i., Královopolská 135, 612 65 Brno, Czech Republic.
Hana
H
Cernocká
Institute of Biophysics, Academy of Sciences of the Czech Republic, v.v.i., Královopolská 135, 612 65 Brno, Czech Republic.
Veronika
V
Ostatná
Institute of Biophysics, Academy of Sciences of the Czech Republic, v.v.i., Královopolská 135, 612 65 Brno, Czech Republic.
Lucie
L
Navrátilová
Institute of Biophysics, Academy of Sciences of the Czech Republic, v.v.i., Královopolská 135, 612 65 Brno, Czech Republic.
Marie
M
Brázdová
D015374:Biosensing Techniques; D004247:DNA / Q000032:analysis* / Q000737:chemistry*; D055664:Electrochemical Techniques; D004566:Electrodes; D006801:Humans; D008628:Mercury / Q000737:chemistry; D013438:Sulfhydryl Compounds / Q000737:chemistry; D016159:Tumor Suppressor Protein p53 / Q000032:analysis* / Q000737:chemistry*
D013438:Sulfhydryl Compounds; D016159:Tumor Suppressor Protein p53; D004247:DNA; D008628:Mercury
Constant current chronopotentiometry; Deoxyribonucleic acid–protein binding; Electrochemical sensing; Mercury containing electrodes; Tumor suppressor protein p53