Published online 25 July 2004
Nucleic Acids Research, Vol. 32 No. 13 © Oxford University Press 2004; all rights reserved
Detection of protein–DNA interaction with a DNA probe: distinction between single-strand and double-strand DNA–protein interaction
Department of Chemistry and Center for Innovative Bio-Physio Sensor Technology, Pusan National University, Busan 609-735, Korea and 1 Department of Chemistry and Division of Molecular & Life Sciences (BK21), Pohang University of Science and Technology, Kyungbuk 780-784, Korea
* To whom correspondence should be addressed. Tel: +82 51 510 2244; Fax: +82 51 514 2430; Email: ybshim{at}pusan.ac.kr
Correspondence may also be addressed to Changill Ban. Tel: +82 54 279 2127; Fax: +82 54 279 3399; Email: ciban{at}postech.ac.kr
Received April 28, 2004; Revised and Accepted July 12, 2004
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 Purß (mPurß), 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 mPurß 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 mPurß were 25 and 3 µg/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.
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