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Mendel-Brno 2000

category image Volume: 17
Issue Number 6, Part 2
June 2000

Electrodes Modified with Biomacromolecules. Studies of DNA Interactions and DNA Damage.

Nucleic acids (NA) produce redox and tensammetric signals at the mercury electrodes and oxidation signals at carbon electrodes (reviewed in 1). These signals can be utilized in NA analysis. Especially the signals obtained with the mercury electrodesare highly sensitive to minor changes in the structure of the double-helical DNA and can be used in determination of RNA traces in DNA samples. Peptides and proteins (depending on the amino acid content and composition of the medium) yield peaks due to cysteine/cystine and several catalytic signals at the mercury electrodes, and oxidation peaks due to tyrosine and tryptophan at the carbon ones (3). Modern approaches to electroanalysis of biomolecules utilize (i) biopolymer-modified electrodes. The latter can easily be prepared via strong adsorption of DNA, RNA or a protein at mercury or carbon electrodes upon immersing the electrode into a small drop of sample solution. This technique (called adsorptive transfer stripping) strongly decrease the amounts of biomolecules required for the analysis and possess a number of other advantages (e.g., easy preparation of an electrochemical DNA biodetectors). (ii) Modern electrochemical techniques (such as constant current chronopotentiometric stripping analysis (CPSA) or square-wave voltammetry supplemented with a sophisticated data processing) allowed to attain remarkably low detection limits of DNA, RNA as well as peptides and proteins. In particular, the oxidation voltammetric signals obtained with carbon electrodes were little utilized in biomolecule analysis before the application of CPSA. Using this method, about 4x10-16 mol of tRNA was detected, and subnanogram amounts of bioactive peptides containing Tyr and/or Trp could be determined.

Biopolymer-modified electrodes can be utilized in studies of specific molecular interactions including DNA hybridization, DNA interactions with small molecules, DNA-protein interactions and DNA damage (reviewed in 2). In the DNA hybridization studies we have shown that surface-attached peptide nucleic acid (PNA) exhibit unique hybridization features (4). Adsorption of PNA at electrodes shows some differences from the known behavior of DNA or RNA due to its electrically neutral backbone. We have studied the electrochemical behavior of DNA complexes with intercalators (which are frequently used as DNA hybridization indicators) at the mercury electrodes (5). We proposed a supercoiled DNA-modified mercury electrode-based detector for DNA damage. This detector is capable to recognize a single interruption of the DNA sugar-phosphate backbone among >2.105 phosphodiesteric bonds. The sensor can be utilized both in the DNA research [including studies of the interactions of surface-attached DNA with DNase I and influence of the electrode potential on these interactions (6), or of electrochemically modulated DNA cleavage by chemical nucleases (7)] as well as for the detection of the DNA-damaging environmental pollutants. Recently a mercury film electrode has been applied (8).

This work was supported by grants of the Grant Agency of the Academy of Sciences of the Czech Republic No. A4004801, and of the Grant Agency of the Czech Republic Nos. 204/97/K084 and 204/98/P091.

References

1. E. Palecek, Electroanalysis 8, 7-14 (1996).
2. E. Palecek, M. Fojta, M.Tomschik and J. Wang, Biosen. Bioelectron. 13, 621-628 (1998).
3. M. Tomschik, L. Havran, M. Fojta and E. Palecek, Electroanalysis 10, 403-409 (1998).
4. J. Wang, E. Palecek, P.E. Nielsen, G. Rivas, X.H. Cai, H. Shiraishi, N. Dontha, D. Luo and P.A.M. Farias, J. Am. Chem. Soc. 118, 7667-7670 (1996)
5. M. Fojta, L. Havran, T. Kubicarova and J. Fulneckova, Electroanalysis in press(2000).
6. M. Fojta, T. Kubicarova and E. Palecek, Electroanalysis 11, 1005-1012 (1999).
7. M. Fojta, T. Kubicarova and E. Palecek, Biosens. & Bioelectron in press (2000).
8. T. Kubicarova, M. Fojta, J. Vidic, D. Suznjevic, M. Tomschik and E. Palecek, Electroanalysis in press (2000).

M. Fojta, L. Havran, F. Jelen, T. Kubicarova, M. Tomschik and E. Palecek

Institute of Biophysics of the Academy of Sciences of the Czech Republic,
612 65 Brno

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