Detection of protein-DNA interaction with a DNA probe: distinction between single-strand and double-strand DNA-protein interaction.
A simple, direct method for the detection of DNA-protein interaction was developed with electrochemical methods. Single-stranded DNA (ss-DNA) probes were prepared through the chemical bonding of an oligonucleotide to a polymer film bearing carboxylic acid groups, and double-stranded DNA (ds-DNA) probes were prepared through hybridization of the complementary sequence DNA on the ss-DNA probe. Impedance spectroscopy and differential pulse voltammetry (DPV) distinguished the interaction between the DNA probes with mouse Purbeta (mPurbeta), an ss-DNA binding protein, and with Escherichia coli MutH, a ds-DNA binding protein. Impedance spectra obtained before and after the interaction of DNA probes with these proteins clearly showed the sequence-specific ss-DNA preference of mPurbeta and the sequence-specific ds-DNA preference of MutH. The concentration dependence of proteins on the response of the DNA probes was also investigated, and the detection limits of MutH and mPurbeta were 25 and 3 microg/ml, respectively. To confirm the impedance results, the variation of the current oxidation peak of adenine of the DNA probe was monitored with DPV. The formation constants of the complexes formed between the probe DNA and the proteins were estimated based on the DPV results.
Ban
Changill
C
Department of Chemistry and Center for Innovative Bio-Physio Sensor Technology, Pusan National University, Busan 609-735, Korea.
Chung
Suhman
S
Park
Deog-Su
DS
Shim
Yoon-Bo
YB
D004247:DNA / Q000378:metabolism*; D015342:DNA Probes; D045643:DNA Repair Enzymes / Q000378:metabolism; D004277:DNA, Single-Stranded / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D017097:Electric Impedance; D004563:Electrochemistry / Q000379:methods*; D004706:Endodeoxyribonucleases / Q000378:metabolism; D029968:Escherichia coli Proteins; D009693:Nucleic Acid Hybridization; D015345:Oligonucleotide Probes; D011485:Protein Binding; D013057:Spectrum Analysis
D015342:DNA Probes; D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D015345:Oligonucleotide Probes; D004247:DNA; D004706:Endodeoxyribonucleases; C059682:methyl-directed mismatch repair protein, E coli; D045643:DNA Repair Enzymes