Direct Voltammetric Analysis of DNA Modified with Enzymatically

Jul 23, 2010 - E-mail: [email protected] ., †. Academy of Sciences of the Czech Republic. , ‡ ... Citation data is made available by participants in Cr...
0 downloads 10 Views 2MB Size
Download PDF
Anal. Chem. 2010, 82, 6807–6813

Direct Voltammetric Analysis of DNA Modified with Enzymatically Incorporated 7-Deazapurines Hana Pivonˇkova´,† Petra Hora´kova´,†,‡ Miloslava Fojtova´,†,§ and Miroslav Fojta*,† Institute of Biophysics, v.v.i., Academy of Sciences of the Czech Republic, Kra´lovopolska´ 135, CZ-612 65 Brno, Czech Republic, Department of Analytical Chemistry, Faculty of Chemical Technology, University of Pardubice, Studentska´ 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: [email protected] . † 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. 10.1021/ac100757v  2010 American Chemical Society Published on Web 07/23/2010

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 damage1 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 probe5,6), specific determination of newly synthesized pieces or fragments of DNA (in primer extension or PCR-based assays7-9) or even identification of a single nucleobase incorporated at a specific position (in SNP typing9-11). 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 acids5,6 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.

Analytical Chemistry, Vol. 82, No. 16, August 15, 2010

6807

Scheme 1. Top: Formulas of Purine Nucleobases (A, G) and Their 7-Deaza Analogues (A*, G*)a

Table 1. Oligonucleotides Used in This Worka primrnd primnoG temprnd16 tempnoG p53-for p53-rev

5′-CATGGGCGGCATGGG-3′ 5′-TACTCATCATATCAA-3′ 5′-CTAGCATGAGCTCAGTCCCATGCCGCCCATG-3′ 5′-AATATAAATATATTGATATGATGAGTA-3′ 5′-GAGGTTGTGAGGCGCTGCCC-3′ 5′-TCCTCTGTGCGCCGGTCTCT-3′

a The template strands (temprnd16, tempnoG) used in PEX experiments were 5′end-biotinylated.

a Atoms at 7-position are highlighted in red. Bottom: Watson-Crick and Hoogsteen base pairing. N7 is involved in the Hoogsteen pairing of natural purines.

in primer extension (PEX) experiments.12,13 Nevertheless, many of the nucleobase conjugates have displayed less facile incorporation at adjacent positions7,9,10 and only some of them have been efficiently used in PCR.13,14 The 7-deazapurines, 7-deazaguanine (G*), or 7-deazaadenine (A*) (Scheme 1) are substitutes of standard purine nucleobases incorporable into DNA by PCR. Although the rate of G* and A* incorporation by DNA polymerases has been reported15 to be lower, compared to the “parent” purines, both of these nucleobase analogues allow efficient sequence-specific DNA amplification by PCR and it has been possible to prepare PCR products with a high density of the corresponding modification. The 7-deazapurines are able to form Watson-Crick base pairs, maintaining pairing specificity of the respective natural nucleobases, but cannot form Hoogsteen pairs due to absence of the N7 atom (which is substituted by CH group, Scheme 1). Thus, the 7-deazapurines cannot be involved in triplex and tetraplex DNA structures.16 Reduced tendency of the “deaza-modified” DNA to adopting multistranded conformations has been utilized to improve PCR amplification of G-rich sequences such as (CGG)n repeat,17 the length of which is analyzed during molecular diagnostics of fragile X syndrome. Similarly, 7-deaza-8-azaguanine has been used to create G-rich DNA probes with reduced propensity to aggregate and improved specificity.18 Absence of the nitrogen atom at the 7-position in G* was also utilized in a study of sequence-specificity of DNA modification with cisplatin19 and in studies of echinomycin-DNA interaction modes.20 Moreover, DNA substituted with G* or A* has been shown to resist cleavage with certain endonucleases.15 (13) Cahova, H.; Pohl, R.; Bednarova, L.; Novakova, K.; Cvacka, J.; Hocek, M. Org. Biomol. Chem. 2008, 6, 3657–3660. (14) Raindlova, V.; Pohl, R.; Sanda, M.; Hocek, M. Angew. Chem., Int. Ed. 2010, 49, 1064–1066. (15) Seela, F.; Roling, A. Nucleic Acids Res. 1992, 20, 55–61. (16) Palecek, E. Crit. Rev. Biochem. Mol. Biol. 1991, 26, 151–226. (17) Cao, J.; Tarleton, J.; Barberio, D.; Davidow, L. S. Mol. Cell. Probes 1994, 8, 177–180. (18) Kutyavin, I. V.; Lokhov, S. G.; Afonina, I. A.; Dempcy, R.; Gall, A. A.; Gorn, V. V.; Lukhtanov, E.; Metcalf, M.; Mills, A.; Reed, M. W.; Sanders, S.; Shishkina, I.; Vermeulen, N. M. J. Nucleic Acids Res. 2002, 30, 4952–4959. (19) Cairns, M. J.; Murray, V. Biochim. Biophys. Acta 1994, 1218, 315–321. (20) Sayers, E. W.; Waring, M. J. Biochemistry 1993, 32, 9094–9107.

6808

Analytical Chemistry, Vol. 82, No. 16, August 15, 2010

Compared to the standard purines, 7-deazapurines exhibit significantly lower potentials of their oxidation.9,21,22 G* is thus easily photooxidized by intercalated ethidium and this photooxidation has been interrogated as a function of distance, nucleotide sequence and integrity of π-stacking in studies of DNA-mediated charge transfer.21 The G* ability of being selectively oxidized by a redox mediator with relatively low redox potential, such as Ru(dmb)33+/2+ (dmb )4,4′-dimethyl-2,2′-bipyridine), compared to A* together with natural G requiring stronger oxidants, such as Ru(bpy)33+/2+ (bpy )2,2′-bipyridine), has been utilized in a PCR-coupled electrochemical technique proposed for parallel detection of two genes.22 Mediated electrooxidation of G* (or G) was also applied in an indirect electrochemical real time monitoring of PCR via measuring consumption of the respective dNTPs.23 To our best knowledge, direct electrochemical analysis of DNA with incorporated G* or A* as electrochemically oxidizable tags has not been reported to date. In this paper we report on adsorptive transfer stripping voltammetric analysis of DNA with enzymatically incorporated G* or A* residues at a carbon electrode. We show that, particularly G* producing a specific signal separated from those yielded by natural DNA components, can be utilized as an excellent electroactive label suitable for monitoring of DNA amplification by PCR. MATERIALS AND METHODS Material. Synthetic ODNs (Table 1) were purchased from VBC genomics (Austria). Templates used in experiments involving the magnetoseparation procedure were biotinylated at their 5′ ends. Plasmid pT77 bearing wild type p53 cDNA insert24 (used as primary template for PCR amplification of the 347-bp fragment) was isolated from E. coli cells using Qiagen Plasmid Purification Kit and linearized with EcoR I restrictase (Takara). Streptavidincoated magnetic beads (MBstv) were purchased from Novagen (Germany), DyNAzyme II DNA Polymerase from Finnzymes (Finland), Pfu DNA Polymerase from Promega (U.S.), unmodified nucleoside triphosphates (dATP, dTTP, dCTP and dGTP), SYBR Green I, SYBR Gold and Stains-All reagent from Sigma, 7-deaza-dGTP and 7-deaza-dATP from Jena Bioscience. Other chemicals were of analytical grade. Primer Extension (PEX). The primer (0.7 µM) was mixed with corresponding template ODN (0.7 µM), dNTPs (100 µM each; composition of the dNTP is specified in the text and Figure legends for individual experiments) and the DyNAzyme II DNA (21) Kelley, S. O.; Barton, J. K. Chem. Biol. 1998, 5, 413–425. (22) Yang, I. V.; Ropp, P. A.; Thorp, H. H. Anal. Chem. 2002, 74, 347–354. (23) Defever, T.; Druet, M.; Rochelet-Dequaire, M.; Joannes, M.; Grossiord, C.; Limoges, B.; Marchal, D. J. Am. Chem. Soc. 2009, 131, 11433–11441. (24) Hupp, T. R.; Meek, D. W.; Midgley, C. A.; Lane, D. P. Cell 1992, 71, 875– 886.

Polymerase (1 U per sample). Reactions were carried out at 60 °C for 30 min. MBstv Magnetoseparation Procedure. The PEX products were captured at MBstv via biotin tags. Then, 50 µL aliquots of the PEX reaction mixtures were added to the MBstv (25 µL of the stock suspension washed twice with 100 µL of 0.3 M NaCl, 10 mM Tris-HCl, pH 7.4, buffer H). The mixture was incubated on a shaker for 30 min at 20 °C. Then the beads were washed three times with 100 µL of PBS (0.14 M NaCl, 3 mM KCl, 4 mM sodium phosphate, pH 7.4) containing 0.01% Tween20, three times with 100 µL of the buffer H and resuspended in deionized water (50 µL). The extended primers were released by heating at 75 °C for 2 min. Prior to the electrochemical measurements, NaCl in total concentration of 0.2 M was added to the samples. Preparative PCR. Amplification of the DNA fragment: 500 ng of the pT77 template was mixed with p53-for and p53-rev primers (0.5 µM each), Pfu DNA Polymerase (3 U) and mix of dNTPs (125 µM each) in total volume of 100 µL. The PCR involved 30 cycles if not stated otherwise (denaturation 94 °C/90 s, annealing 60 °C/120 s, polymerization 72 °C/180 s) and was run using C1000 Thermal Cycler (BioRad). The PCR products were purified using QIAquick PCR Purification Kit (Qiagen) and their concentrations were determined spectrophotometrically using NanoDrop ND-1000 Spectrophotometer (NanoDrop Technologies, U.S.). Real Time PCR. Reactions were prepared in duplicates in 20 µL handmade PCR mix consisting of 30 ng of pT77 template, p53for and p53-rev primers at concentrations 0.5 µM, 1 × DyNAzyme II buffer, 1.25 mM MgCl2, 20 000 × diluted SYBR Green I, each dNTP (or sum of dGTP+dG*TP) 125 µM, 1 U of DyNAzyme II DNA polymerase. PCR involved 20 cycles (denaturation 94 °C/30 s, annealing 56 °C/30 s, polymerization 72 °C/30 s, fluorescence measurement in SYBR Green I channel 15 s/78 °C, wavelength of the source 470 nm, wavelength of the detection filter 585 nm; final extension 72 °C/3 min) and was run and evaluated using the RotorGene-3000 (Qiagen, Germany). Native PAGE. The PCR products (1 µL each) were mixed with loading buffer (0.1% SDS, 5% glycerol, 5 mg mL-1 bromophenol blue) and subjected to electrophoresis in 5% native gel containing 1 × TBE buffer (pH 8) at 150 V, 4 °C for 35 min. Gels stained with ethidium bromide or SYBR dyes were visualized using LAS-3000 (FUJIFILM Corporation), those stained with the Stains-All reagent were scanned. Electrochemical Analysis. The PEX and PCR products were analyzed by using ex situ (adsorptive transfer stripping, AdTS) square-wave voltammetry (SWV). The DNA was accumulated at the basal-plane pyrolytic graphite electrode (PGE; prepared and pretreated as described in ref 25) surface from 5 µL aliquots containing 0.3 M NaCl for 60 s. Then the electrode was rinsed by deionized water and was placed into the electrochemical cell. SWV settings: initial potential -1.0 V, final potential +1.5 V, pulse amplitude 25 mV, frequency 200 Hz, potential step 5 mV. The measurements were performed at ambient temperature in 0.2 M acetate buffer pH 5 by using CHI440 Electrochemical Workstation (CH Instruments, Inc., U.S.) in a three-electrode setup (with the PGE as working electrode, Ag/AgCl/3 M KCl as reference, and (25) Fojta, M.; Havran, L.; Kizek, R.; Billova, S. Talanta 2002, 56, 867–874.

platinum wire as counter electrode). Baseline correction of the voltammograms was performed by means of a moving average algorithm (GPES 4 software, EcoChemie). RESULTS AND DISCUSSION Primer extension (PEX) has recently been used for the introduction of labeled nucleobases into oligodeoxynucleotides (ODNs).7-13 Using nucleobase-modified deoxynucleotide triphosphates (dNTPs) and DNA polymerases, we prepared ODNs bearing various tags producing analytically useful electrochemical responses. Since 7-deazapurines are electrochemically oxidized at considerably less positive potentials, compared to the respective natural purine nucleobases,9,21,22 it is in principle possible to apply these nucleobase analogues themselves (without introducing any extra label groups) as electroactive markers of the in vitro synthesized DNA. We performed PEX with primrnd primer and 5′-end-biotinylated temprnd16 template, the nucleotide sequence of which was designed to accommodate all four DNA bases (four-times each) in the synthesized DNA stretch (Figure 1A). The doublestranded PEX products were pulled-down from the reaction mixture using streptavidin coated magnetic beads (MBstv) and, after magnetic separation, the captured duplex ODN was thermally denatured to release the extended primer strand. The latter was finally analyzed using adsorptive transfer stripping square wave voltammetry (AdTS SWV). As shown in Figure 1B,C, voltammetric responses of the pexrnd16 products reflected the composition of dNTP mix used. For a standard dNTP mix (dATP, dGTP, dCTP, and dTTP), we observed two anodic signals corresponding to electrochemical oxidation of guanine (peak Gox at 1.15 V) and adenine (peak Aox at 1.35 V, Figure 1B,C). When dGTP was replaced by 7-deaza-dGTP (dG*TP), peak Gox was decreased and a new anodic peak appeared at 0.80 V (peak G*ox, Figure 1B,C) which has been ascribed to electrochemical oxidation of G* introduced into the extended ODN stretch. Lower intensity of the peak Gox corresponded to lower number of G residues in the G*-modified PEX product, compared to the PEX product composed of standard nucleotides (note that after PEX in the presence of dG*TP, there are still natural guanines present in the primer stretch, but not in the extended stretch, decreasing the total number of G residues from 12 to 8). When dATP was replaced by 7-deazadATP (dA*TP), we observed a decrease of the peak Aox intensity (due to lowering of the total number of adenines in the entire extended primer strand) and increase of the peak at 1.15 V. The potential of electrochemical oxidation of A* was shown9 to coincide with the potential of peak Gox, and thus currents due to A* oxidation were responsible for the increase of the apparent signal intensity. To verify this hypothesis, we prepared another PEX product using primnoG primer and tempnoG template (Table 1). Neither of these ODNs contains G residues and no G is incorporated during PEX, which is reflected in the absence of peak Gox at the voltammogram of unmodified pexnoG product, showing only peak Aox (black curve in Figure 1C, inset). After performing PEX with the same primer-template pair but with dATP replaced by dA*TP, the peak Aox was decreased and another signal appeared at 1.15 V, corresponding to the A* oxidation (peak A*ox; green curve in Figure 1C, inset). Analytical Chemistry, Vol. 82, No. 16, August 15, 2010

6809

Figure 1. Electrochemical responses of oligonucleotides containing 7-deazapurines incorporated by primer extension (PEX). (A) Scheme of the experiment. The PEX was performed using a 5′-terminally biotinylated template. After extension of the primer using either standard dNTP mix, a mixture with dGTP replaced by dG*TP, or a mixture with dATP replaced by dA*TP, the duplex ODN was captured at magnetic beads covered with streptavidin, the beads were washed and the modified strand released by thermal denaturation, followed by AdTS SWV measurements at the PGE (shown for temprnd16 and primrnd sequences; blue and red letters are used to highlight A and G positions, respectively). (B) SWV voltammograms of pexrnd16: standard nucleobases (black); A* instead of A (blue); G* instead of G (red); background electrolyte (dotted); (C) baseline-corrected curves taken from (B). Inset: PEX products obtained with tempnoG and primnoG for dTTP+dATP mix (black) or dTTP+dA*TP mix (green).

In next experiment, we prepared a 347-bp DNA fragment by the polymerase chain reaction (PCR) using the pT77 template and various dNTP mixtures. The PCR products were isolated from the reaction mixture using Qiagen columns and analyzed by AdTS SWV as above. During thermal cycling in the PCR, forward and backward primers (Table 1) are elongated on templates of both DNA strands, resulting in exponential amplification of the fragment delimited by the two primers (see Figure 2A). Thus, each strand of the double-stranded PCR product begins with the primer at its 5′-terminus, followed by newly synthesized stretch, the nucleotide composition of which (i.e., presence or absence of the 7-deazapurines) is dictated by the composition of the dNTP mix (Figure 2A). Accordingly, when using mix of four standard dNTPs (in Figure 2B denoted as G+A), we observed peak Gox and peak Aox corresponding to natural purine bases. Replacement of dGTP with dG*TP resulted in appearance of an intense peak G*ox (due to G* incorporated into the polymerase-synthesized strands) and strong decrease of the intensity of peak Gox which, however, never reached zero. This peak Gox was due to G residues in the primer stretches of the G*-modified amplicons (Figure 2A). In addition, certain amount of unconsumed primers and primary templates remaining in the samples after the 6810

Analytical Chemistry, Vol. 82, No. 16, August 15, 2010

purification step might contribute to the peak Gox intensity, as indicated by negative PCR control experiment (PCR mixture not subjected to thermal cycling, dotted curve in Figure 2B). (It should be however noted that contributions to the peak Gox from ODN primers in the negative PCR control, as apparent from Figure 2B, and those from primer stretches in the G*modified amplicon are not simply additive, because concentration of residual free primers after the PCR cycling is much lower than their initial concentration). When dATP was replaced with dA*TP, we observed increase of the signal close to 1.15 V (peak Gox + peak A*ox) and decrease of peak Aox (again, the residual peak Aox was yielded by A residues in the primer stretches). Further, we focused our attention on analysis of DNA amplicons containing G* as an independently detectable marker and performed PCR experiments using dNTP mixes containing both dGTP and dG*TP at different ratios (while keeping the total dGTP + dG*TP concentration constant). The resulting PCR products were analyzed electrochemically as above. As shown in Figure 3, changes in the relative abundances of G and G* in the PCR product (given by the molar fraction of the respective dNTP) were reflected in changes of corresponding electrochemical signals (peak Gox and peak G*ox). Peak Aox due to adenine, the content

Figure 3. Dependence of the heights of peak G*ox (red), peak Gox (black) and peak Aox (empty triangles) on the [dG*TP]/[dGTP]+[dG*TP] ratio in the PCR reaction used for amplification of the 347-bp DNA fragment. Other conditions as in Figure 2.

Figure 2. Electrochemical responses of a PCR-amplified 347-bp DNA fragment modified with 7-deazapurines. (A) Scheme of the PCR. Primers at the 5′-ends of both strands of the PCR product (black) contain only standard nucleobases, regardless of the dNTP composition. The synthesized stretches contain modified nucleobases depending on the dNTP mix. (B) Baseline-corrected voltammograms obtained for DNA fragment resulting from PCR in the presence of standard dNTP mix (black), for a mix with G* instead of G (red) and for a mix with A* instead of A (blue). The PCR was conducted in 30 cycles and the products were purified using Qiagen PCR Purification Kit. Dotted curve corresponds to control PCR mixture (with G*+A) which was not subjected to thermal cycling.

of which was constant for all amplicons analyzed in this experiment, did not show any significant trend, suggesting approximately constant yields of the PCR products obtained for any dGTP/dG*TP ratio after the 30 PCR cycles (which was also confirmed by UV-vis spectrophotometry, not shown). Dependences of the heights of peak Gox (yielded by the unmodified 347-bp PCR product) and peak G*ox (yielded by the same DNA fragment amplified with 100% of dG*TP) on concentrations of the amplicons followed similar trends (Figure 4A), showing approximately linear regions below 10 µg mL-1 and sublinear trends, suggesting saturation of the electrode surface, at higher DNA concentrations. The lowest detectable DNA concentrations were around 1 µg mL-1 in both cases. Electrochemical determination of the modified and unmodified 347bp amplicons was compared to other commonly used techniques, based on gel electrophoresis followed by DNA staining (Figure 4B). The amplicons were separated in native 5% polyacrylamide gel (1 µL of the reaction mixture, containing about 30 µg mL-1 of the amplicon, and three binary dilutions). After electrophoresis, the gels were stained with ethidium bromide,

SYBR Green I or Stains-All reagent (Figure 4B). Ethidiumstained bands of the G*-modified PCR products were considerably weaker than those of the unmodified amplicon (even when only 50% of Gs were substituted by G*, see Figure 4B). Such observation was in agreement with literature data26 showing that ethidium fluorescence is quenched when the dye is intercalated next to G*. Notably, we observed even stronger quenching effect of G* on the fluorescence of SYBR Green I (Figure 4B) and SYBR Gold (not shown) dyes. Thus, results of the fluorescent DNA staining might be misinterpreted, for the densely G*-modified DNA, in terms of (strongly) decreasing amount of the PCR products with increasing G/G* ratio. On the other hand, staining of the polyacrylamide gel with the Stains-All reagent (Figure 4B) did not reveal significant differences in the amounts of the unmodified and G*-substituted amplicons, in agreement with the electrochemical data. Despite the approximately same yields of the PCR products after 30 cycles, we were interested whether we are able to follow electrochemically differences in the kinetics of the PCR reactions through analysis of the amplicons after lower number of amplification cycles. Previous data15 revealed less efficient DNA amplification by PCR in the presence of dG*TPs, compared to PCR with standard dNTPs only. We followed electrochemical signals of the unmodified and fully G*-substituted PCR products after 5, 10, 20, and 30 cycles (Figure 5A). For five cycles, peak Gox produced by the unmodified amplicon was considerably higher than peak G*ox of the G*-modified PCR product. Even after subtraction of signal intensity produced by the control PCR mix not subjected to the thermal cycling (containing initial concentrations of ODN primers and the primary template), the peak Gox was at least twice higher than peak G*ox produced by the G*modified amplicon after the same number of cycles. Large differences between the signal intensities were also observed after 10 amplification cycles, while after 20 and 30 cycles both peaks reached their limiting values. Hence, more cycles were required to reach the limiting amount of the G*-modified PCR (26) Latimer, L. J. P.; Lee, J. S. J. Biol. Chem. 1991, 266, 13849–13851.

Analytical Chemistry, Vol. 82, No. 16, August 15, 2010

6811

Figure 4. (A) Dependence of the intensities of peak Gox (measured for unmodified 347-bp amplicon, black) and peak G*ox (measured for the same DNA fragment amplified in the presence of dG*TP instead of dGTP, red) on DNA concentration. Other conditions as in Figure 2. (B) Staining of the 347-bp amplicon in polyacryalmide gels with (from top to bottom) ethidium bromide, SYBR Green I and StainsAll. The PCR reaction contained either four standard dNTPs, G/G* ) 1, or dGTP fully replaced with dG*TP as indicated on the top. Loading of the PCR products: 1 µL of the undiluted reaction mixture (about 30 ng of the amplicon), followed by binary dilutions as indicated.

product, compared to PCR with standard dNTP mix. To support this conclusion, we performed a real-time PCR experiment using standard and dG*TP-containing dNTP mixes. Figure 5B shows increase of the fluorescence of SYBR Green I (which was used here as fluorescent dye indicating progression of DNA amplification) as a function of the number of PCR cycles for dNTP mixes containing 0, 10, 20, 30, 50, and 100% of dG*TP (of dGTP+dG*TP total). The steepest increase of the signal was observed for the unmodified amplicon and the amplification rate decreased with the G* content, showing clear difference even for 10% G*. For 100% of G* the apparent amplification rate was remarkably depressed. However, it should be taken into consideration that SYBR Green I fluorescence is quenched by G* incorporated (see above), and thus the weak signal detected for the G*-substituted amplicon can have reflected the dense DNA modification rather than the amount of the PCR product. We therefore focused on the evaluation of the shapes of the amplification curves rather than the absolute signal magnitudes. RotorGene3000 software enables to compare reactions parameters in the 6812

Analytical Chemistry, Vol. 82, No. 16, August 15, 2010

Figure 5. (A) Effects of the number of PCR cycles on intensities of peak Gox (measured for unmodified 347-bp amplicon, gray columns) and peak G*ox (measured for the same DNA fragment amplified in the presence of dG*TP instead of dGTP, red columns). Other conditions as in Figure 2. (B) Quantitative (real time) PCR amplification of the 347-bp DNA fragment: standard dNTP mix (no G*) (1); 10% G* (2); 20% G*(3); 30% G* (4); 50% G (5); G fully replaced with G* (6); negative “no template” control for standard dNTP mix (7). The graph shows fluorescence of SYBR Green I complexes with the DNA amplicons as a function of the number of PCR cycles. Inset, takeoff graphs derived from the real time PCR data (colors correspond to the raw data plot). All samples are plotted in duplicate.

comparative quantification mode. The takeoff points for each reaction (sample) are calculated from the second derivatives of raw data (Figure 5B, inset). Generally, the takeoff value is not possible to determine exactly and it is taken as 80% below the peak of second derivative (i.e., below the point where the amplification curve is increasing most steeply). In the same mode, reaction efficiencies of particular reactions are reporting the amplificability of the template under the given conditions. For example, amplification factors of 1.72, 1.65, and 1.61 obtained for reactions with 100% G, G/G* ) 1 and 100% G*, respectively, clearly show less efficient amplification in the dG*TP-substituted reactions. Similar effects of G* on the PCR kinetics were obtained using Taqman hydrolytic probes which release fluorescent indicators into solution as the PCR progresses, and thus the fluorescence intensity was not affected by the incorporated G* as it was in the case of the intercalative dyes (not shown). Hence, results of the real time PCR experiment were in a qualitative accordance with the above electrochemical data indicating lower amplification rate for the reaction with dG*TP. CONCLUSIONS 7-deazapurines G* and A* enzymatically incorporated into DNA are electrochemically oxidizable at carbon electrodes, producing

analytically useful signals at less positive potentials, compared to the corresponding natural purine nucleobases. Peak A*ox due to electrooxidation of A* occurs at the same potential as the peak Gox due to oxidation of natural G, preventing qualitative discrimination of A* incorporated into DNA containing guanine. On the other hand, total or partial substitution of G with G* results in appearance of a new anodic signal, peak G*ox, at potential less positive than potentials of oxidation of any natural component of DNA, allowing independent determination of G* incorporated. G* can thus be utilized as an inexpensive, commercially available electroactive label for easy monitoring of primer extension or polymerase chain reactions via simple direct electrochemistry using cheap, widely accessible carbon electrodes. In turn, considering applications of 7-deazaguanine as DNA modifications preventing formation of multistranded alternative structures in PCR analysis of G-rich sequences17 and problems with quenching of fluorescence of ethidium26 or “SYBR” (to our knowledge, for the first time reported in this paper) dyes, electrochemical analysis appears an attractive complementary approach providing modification-specific signal suitable for quantitation of the fully modified amplified DNA as well as for the determination of the DNA modification extent. Specifically for the G*-modified DNA, the voltammetric analysis represents a simple and direct way to differentiate between the

natural G and G* residues and to determine relative content of both. In contrast to using peak Gox or peak Aox, produced by natural purines, determination of DNA amplicons based on the measurement of peak G*ox is not affected by signals produced by residual ODN primers and/or the primary template. It has to be naturally taken into consideration that peak G*ox intensity must depend on the G + C content within the amplified region; alternatively, this feature may potentially be useful for the determination of the G + C content or estimation of the length of the amplified DNA fragment (e.g., triplet repeat expansion27,28) provided that a proper normalization of the signal intensity (such as fragment ends “counting” through the intensity of natural G in primers) is used. Besides the PCR applications, the G* electroactive is also potentially useful for taillabeling of DNA probes for electrochemical hybridization assays and other bioanalytical applications which are metter of our ongoing research and will be reported elsewhere.

(27) Fojta, M.; Brazdilova, P.; Cahova, K.; Pecinka, P. Electroanalysis 2006, 18, 141–151. (28) Fojta, M.; Havran, L.; Vojtı´sˇkova´, M.; Palecek, E. J. Am. Chem. Soc. 2004, 126, 6532–6533.

Received for review March 24, 2010. Accepted July 10, 2010.

ACKNOWLEDGMENT This work was supported by Grant Agency of the ASCR (grant IAA400040901), by the ASCR (AV0Z50040507 and AV0Z50040702) and by the MEYS CR (LC06035, MSM0021622415). H.P. and P.H. contributed equally to this work.

AC100757V

Analytical Chemistry, Vol. 82, No. 16, August 15, 2010

6813