Electrochemical sensing of DNA damage

Miroslav Fojta ,  Jan Vacek ,  Petra Brázdilová ,  Pavel Kostečka ,  Luděk Havran ,  Emil Paleček 

Institute of Biophysics, v.v.i., Acad. Sci. Czech. Rep. (IBP), Kralovpolska 135, Brno CZ-61265, Czech Republic

Abstract

It has been known for decades that nucleic acids are electroactive and surface-active substances yielding at mercury and carbon electrodes redox and/or tensammetric signals. Some of these signals exhibit a remarkable sensitivity to alterations in the DNA structure (such as local unwinding of the DNA double helix, interruptions of the sugar-phosphate backbone or chemical modifications of the nucleobase residues). Hence, electrochemical techniques have proved useful in detecting DNA damage that is usually accompanied by changes in electrochemical activity of particular base residues, changes of the DNA conformation affecting DNA adsorption at the electrode surfaces, and/or may result in appearance of new electrochemical signals specific for the DNA lesions (reviewed in[1]). Behavior of double stranded DNA and mercury and some types of amalgam electrodes is strongly influenced by presence of DNA strand breaks (interruptions of the DNA sugar-phosphate backbone). Based on this finding we proposed a sensor for DNA damage consisting of supercoiled covalently closed circular DNA (scDNA), not containing any strand break, adsorbed at the surface of a mercury or silver amalgam electrode. Upon formation of the strand breaks (due to exposure to a sample containing DNA damaging species), a qualitative change in the sensor AC voltammetric response is observed. By this way, one DNA strand break per 2x105 nucleotides can be detected [1]. The technique has been adapted for detecting damage to DNA bases via application of enzymes recognizing specific nucleobase lesions and cleaving the DNA sugar-phosphate backbone at such sites [2]. The most common electrochemical techniques applied in the detection of DNA damage and/or in sensing of potentially genotoxic substances use various types of carbon electrodes and involve measurements of a signal due to electrochemical oxidation of guanine [1]. Guanine is a target for a variety of toxic compounds, including carcinogens and antineoplastic drugs, and its modification usually results in a loss of its electrochemical activity. Other approaches based on guanine signals employ soluble or surface-confined mediators of electrocatalytic guanine oxidation [3]. In addition to the label-free techniques utilizing intrinsic electrochemical activity of DNA, detection principles involving non-covalently bound redox indicators or covalently bound markers. For example, DNA intercalators such as [Co(bipy)3]2+/3+ bind efficiently to duplex (native) DNA adsorbed at surface of a carbon electrode, yielding an intense signal due to oxidation/reduction of the cobalt ion [4]. Upon damage to the DNA, its double-helical structure is disrupted, binding of the indicator is reduced and the signal decreases. Pyrimidine bases in single-stranded regions of damaged (and/or enzymatically pretreated) DNA can be modified by osmium tetroxide complexes followed by electrochemical detection of the osmium markers [5]. Voltammetric techniques can easily be combined with simple magnetoseparation protocols [6] to increase sensitivity and/or specificity of the analysis.
This work was supported by GACR (203/07/1195) and by Ministry of Education, Youth and Sports of the CR (LC06035, FRVS/1468/2007).

References

[1] M. Fojta, in: E. Palecek, F. Scheller, J. Wang, (Eds.), Electrochemistry of nucleic acids and proteins. Towards electrochemical sensors for genomics and proteomics Vol., Elsevier, Amsterdam, 2005.p. 386-431.

[2] K. Cahova-Kucharikova, M. Fojta, T. Mozga, E. Palecek, Anal. Chem. 77 (2005) 2920-2927.

[3] J.F. Rusling, Biosens. Bioelectron. 20 (2004) 1022-1028.

[4] J. Labuda, M. Buckova, L. Heilerova, A. Caniova-Ziakova, E. Brandsteterova, J. Mattusch, R. Wennrich, Sensors 2 (2002) 1-10.

[5] L. Havran, K. Cahová, M. Fojta, in preparation (2007).

[6] J. Vacek, T. Mozga, K. Cahova, H. Pivonkova, M. Fojta, Electroanal. in press (2007).

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Presentation: Keynote lecture at SMCBS'2007 International Workshop, by Miroslav Fojta
See On-line Journal of SMCBS'2007 International Workshop

Submitted: 2007-08-20 14:05
Revised:   2009-06-07 00:44
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