Analytical Chemistry Chemical Cytometry Quantitates Superoxide

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