Analytical Chemistry Chemical Cytometry Quantitates Superoxide
Analytical Chemistry Chemical Cytometry Quantitates Superoxide
Analytical Chemistry Chemical Cytometry Quantitates Superoxide
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Anal. Chem. 2010, 82, 6807–6813<br />
Direct Voltammetric Analysis of DNA Modified with<br />
Enzymatically Incorporated 7-Deazapurines<br />
Hana Pivoňková, † Petra Horáková, †,‡ Miloslava Fojtová, †,§ and Miroslav Fojta* ,†<br />
Institute of Biophysics, v.v.i., Academy of Sciences of the Czech Republic, Královopolská 135,<br />
CZ-612 65 Brno, Czech Republic, Department of <strong>Analytical</strong> <strong>Chemistry</strong>, Faculty of <strong>Chemical</strong> Technology, University of<br />
Pardubice, Studentská 573, CZ-532 10 Pardubice, Czech Republic, and Department of Functional Genomics and<br />
Proteomics, Institute of Experimental Biology, Faculty of Science, Masaryk University, Kotlárˇská 2,<br />
CZ-611 37 Brno, Czech Republic<br />
Nucleic acids studies use 7-deazaguanine (G*) and 7-deazaadenine<br />
(A*) as analogues of natural purine bases<br />
incapable of forming Hoogsteen base pairs, which prevents<br />
them from being involved in DNA triplexes and<br />
tetraplexes. Reduced propensity of the G*- and/or A*modified<br />
DNA to form alternative DNA structures is<br />
utilized, for example, in PCR amplification of guanine-rich<br />
sequences. Both G* and A* exhibit significantly lower<br />
potentials of their oxidation, compared to the respective<br />
natural nucleobases. At carbon electrodes, A* yields an<br />
oxidation peak which is by about 200-250 mV less<br />
positive than the peak due to adenine, but coincides with<br />
oxidation peak produced by natural guanine residues. On<br />
the other hand, oxidation signal of G* occurs at a potential<br />
by about 300 mV less positive than the peak due to<br />
guanine, being well separated from electrochemical signals<br />
of any natural DNA component. We show that<br />
enzymatic incorporation of G* and A* can easily be<br />
monitored by simple ex situ voltammetric analysis of the<br />
modified DNA at carbon electrodes. Particularly G* is<br />
shown as an attractive electroactive marker for DNA,<br />
efficiently incorporable by PCR. While densely G*-modified<br />
DNA fragments exhibit strong quenching of fluorescence<br />
of SYBR dyes, commonly used as fluorescent<br />
indicators in both gel staining and real time PCR applications,<br />
the electrochemical detection provides G*-specific<br />
signal suitable for the quantitation of the amplified DNA<br />
as well as for the determination of the DNA modification<br />
extent. Determination of DNA amplicons based on the<br />
measurement of peak G* ox is not affected by signals<br />
produced by residual oligonucleotide primers or primary<br />
templates containing natural purines.<br />
Electrochemical techniques are increasingly applied in the area<br />
of nucleic acids sensing (reviewed in refs 1-4). Nucleic acids<br />
* To whom correspondence should be addressed. E-mail: fojta@ibp.cz .<br />
† Academy of Sciences of the Czech Republic.<br />
‡ University of Pardubice.<br />
§ Masaryk University.<br />
(1) Fojta, M. In Electrochemistry of Nucleic Acids and Proteins. Towards<br />
Electrochemical Sensors for Genomics and Proteomics; Palecek, E., Scheller,<br />
F., Wang, J., Eds.; Elsevier: Amsterdam, 2005, pp 386-431.<br />
(2) Fojta, M.; Jelen, F.; Havran, L.; Palecek, E. Curr Anal Chem 2008, 4, 250–<br />
262.<br />
possess intrinsic electroactivity due to the presence of electrochemically<br />
oxidizable or reducible nucleobases, 2,4 making it<br />
possible to analyze them electrochemically without any labeling.<br />
Indeed, various label-free electrochemical techniques were proposed<br />
for the detection of DNA damage 1 or DNA hybridization. 3<br />
In spite of these efforts, application of various redox indicators<br />
and labels proved useful particularly in sequence-specific DNA<br />
sensing requiring reliable discrimination between two complementary<br />
strands (e.g., target DNA and hybridization probe 5,6 ),<br />
specific determination of newly synthesized pieces or fragments<br />
of DNA (in primer extension or PCR-based assays 7-9 ) or even<br />
identification of a single nucleobase incorporated at a specific<br />
position (in SNP typing 9-11 ). Generally, introducing electroactive<br />
tags producing “new” specific electrochemical responses (not<br />
yielded by natural DNA components) increases specificity of the<br />
assays considerably. Electrochemically active moieties can be<br />
incorporated into nucleic acids during chemical oligonucleotide<br />
synthesis, postsynthetically by chemical modification of “natural”<br />
nucleic acids 5,6 or using modified nucleoside triphosphates<br />
(dNTPs) and DNA polymerases. 7-12 The latter approach represents<br />
a versatile way to facile construction of labeled or otherwise<br />
functionalized nucleic acids and to efficient sequence-specific DNA<br />
sensing. 12 A critical prerequisite for these applications is the ability<br />
of a DNA polymerase to use modified dNTPs as substrates for<br />
efficient incorporation without losing sequence-specificity. C7substituted<br />
7-deazapurines and C5-substituted pyrimidines are<br />
usually acceptable substrates for (at least some) DNA polymerases<br />
(3) Palecek, E.; Fojta, M. Talanta 2007, 74, 276–290.<br />
(4) Palecek, E.; Jelen, F. In Electrochemistry of Nucleic Acids and Proteins.<br />
Towards Electrochemical Sensors for Genomics and Proteomics.; Palecek, E.,<br />
Scheller, F., Wang, J., Eds.; Elsevier: Amsterdam, 2005, pp 74-174.<br />
(5) Flechsig, G. U.; Reske, T. Anal. Chem. 2007, 79, 2125–2130.<br />
(6) Fojta, M.; Kostecka, P.; Trefulka, M.; Havran, L.; Palecek, E. Anal. Chem.<br />
2007, 79, 1022–1029.<br />
(7) Brazdilova, P.; Vrabel, M.; Pohl, R.; Pivonkova, H.; Havran, L.; Hocek, M.;<br />
Fojta, M. Chem.-Eur. J. 2007, 13, 9527–9533.<br />
(8) Patolsky, F.; Weizmann, Y.; Willner, I. J. Am. Chem. Soc. 2002, 124, 770–<br />
772.<br />
(9) Vrabel, M.; Horakova, P.; Pivonkova, H.; Kalachova, L.; Cernocka, H.;<br />
Cahova, H.; Pohl, R.; Sebest, P.; Havran, L.; Hocek, M.; Fojta, M. Chem.-<br />
Eur. J. 2009, 15, 1144–1154.<br />
(10) Cahova, H.; Havran, L.; Brazdilova, P.; Pivonkova, H.; Pohl, R.; Fojta, M.;<br />
Hocek, M. Angew. Chem., Int. Ed. 2008, 47, 2059–2062.<br />
(11) Horakova, P.; Simkova, E.; Vychodilova, Z.; Brazdova, M.; Fojta, M.<br />
Electroanalysis 2009, 21, 1723–1729.<br />
(12) Hocek, M.; Fojta, M. Org. Biomol. Chem. 2008, 6, 2233–2241.<br />
10.1021/ac100757v © 2010 American <strong>Chemical</strong> Society 6807<br />
<strong>Analytical</strong> <strong>Chemistry</strong>, Vol. 82, No. 16, August 15, 2010<br />
Published on Web 07/23/2010