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Electrochemistry Communications 10 (2008) 438–442

Sequence dependence of cytochrome c electrochemistry onDNA modified electrodes: Effect of hydrogen bonding of a ligand

to nucleobases opposite an abasic site

Yong Shao, Kotaro Morita, Qing Dai, Seiichi Nishizawa, Norio Teramae *

Department of Chemistry, Graduate School of Science, Tohoku University, Aramaki, Aoba-ku, Sendai, Miyagi 980-8578, Japan

Received 4 December 2007; received in revised form 25 December 2007; accepted 7 January 2008Available online 12 January 2008

Abstract

The binding of a hydrogen bond-forming ligand to abasic (AP) site-containing DNA was electrochemically investigated for discrim-ination of a nucleotide opposite the AP site. The surface of a gold electrode was modified by AP site-containing DNA duplexes on whichcytochrome c (Cyto c) was attached electrostatically as a probe. Cyto c showed quasi-reversible electrochemical behavior depending onthe base opposite the AP site. When the base opposite the AP site was cytosine, much slower kinetics of Cyto c electron transfer wasobserved. This observation could be explained by previous reports that the base stacking was disturbed to a much greater extent becausethe cytosine base opposite the AP site was flipped out extra-helically. The binding of a hydrogen bond-forming ligand, 2-amino-7-methyl-1,8-naphthyridine (AMND), to cytosine opposite the AP site could significantly improve the electrochemical behavior of Cyto c, indi-cating effective base stacking due to the AMND binding. The present method demonstrates an easy way for investigating the binding of asmall ligand to the AP site through DNA-mediated charge transfer.� 2008 Elsevier B.V. All rights reserved.

Keywords: DNA; Abasic site; Electrochemistry; Interaction; Single nucleotide polymorphisms; Hydrogen bond-forming ligand

1. Introduction

A number of redox active species [1–5] have been used asprobes for DNA hybridization, reagents for mediation ofcharge transfer (CT) through a DNA duplex, and reagentsfor chemotherapy. As for the electrochemical response of aredox active reagent bound to DNA, current attenuation isusually observed due to the base stacking perturbations, ifa mismatched site, an abasic site, or other lesions in DNAare present between the probes and electrode surface. Thestructural distortions of base pairs within a DNA duplexcan have a significant effect on the electronic coupling inthe DNA p-stacks [6].

1388-2481/$ - see front matter � 2008 Elsevier B.V. All rights reserved.

doi:10.1016/j.elecom.2008.01.008

* Corresponding author. Tel./fax: +81 22 795 6549.E-mail address: teramae@mail.tains.tohoku.ac.jp (N. Teramae).

We have recently discovered a series of ligands that canbind to a base opposite an abasic (AP) site in DNAduplexes, and have proposed a ligand-based assay for sin-gle nucleotide polymorphisms (SNPs) based on fluores-cence and electrochemical responses [7–10]. In the presentstudy, we describe the voltammetric studies of cytochromec (Cyto c) adsorbed electrostatically onto a self-assembledmonolayer of AP site-containing DNA (AP-DNA)duplexes, and we examine the complexation effect of ahydrogen bond-forming ligand, 2-amino-7-methyl-1,8-naphthyridine (AMND), with the AP-DNA (Scheme 1).We expect that base stacking in the AP site-containingDNA duplex would be promoted when a hydrogen bond-forming ligand enters into the AP site. Of importance forthe selection of a probe to investigate its CT throughDNA is to keep the DNA structure intact within the poten-tial range investigated. In addition, it has been experimen-tally [11] and theoretically [12] proved that extremely

Scheme 1. Scheme for construction of AP site-containing DNA duplexes on a gold electrode.

Y. Shao et al. / Electrochemistry Communications 10 (2008) 438–442 439

negative potential will denaturize the prehybridized dupleximmobilized on an electrode because of electrostatic repul-sion between the DNA duplexes. Cyto c seems to be abetter probe because it has an appropriately positive redoxpotential compared with other probes [1–3]. In principle,the advantage of this strategy is that electro-inactiveligands can be utilized in detecting lesions in a DNAduplex. Another advantage is that greater sensitivity to per-turbations in the DNA sequence can be attained by cou-pling the DNA-mediated CT of the probe intocompensating base stacking perturbations by a ligand.

2. Experimental

AMND was synthesized according to the literature [13].The DNA strand containing the AP site with a thiolterminus at the 50 end (HS-(CH2)6-50-TCTGCGTC-CAGXGCAACGCACAC-30, X = Spacer C3 (propyleneresidue, Spacer phosphoramidite C3)) was mixed in anequimolar amount with its complementary strand contain-ing four different bases opposite the AP site (50-GTGTGCGTTGCNCTGGACGCAGA-30, N = A, C, G,or T) and annealed. Then 20 ll of 10 lM DNA duplex con-taining 0.1 M MgCl2 with or without 50 lM AMND weredropped onto a gold disk electrode (1.6 mm in diameter,BAS, Tokyo, Japan) to form a monolayer and this waskept under the saturated vapor pressure condition over-night. The electrode was again immersed into a 1 mM6-mercapto-1-hexanol (MCH) buffer solution (10 mMphosphate, pH 7.0) for 1 h. Finally, the electrode modifiedby AP-DNA duplexes was immersed into a 10 mM phos-phate buffer solution (pH 7.0) containing 50 lM horseheart Cyto c (99%, Sigma, St. Louis, USA) for 30 min(Scheme 1). Electrochemical experiments were performedusing CHI 650A (CHI, Austin, USA) at 25 �C. The goldelectrode modified by AP-DNA duplexes, Ag/AgCl (sat.KCl, BAS), and platinum wire (Nilaco, Japan) were usedas working, reference, and counter electrodes, respectively.The 10 mM phosphate buffer solution (pH 7.0) was deoxy-

genated by purging with purified nitrogen gas. For simplic-ity, the formed AP-DNA duplexes were referred to asAP-[N] (N = A, C, G, or T) according to the bases oppositethe AP site.

3. Results and discussion

It is well known that Cyto c can electrostatically adsorbonto self-assembled thiol monolayers terminated by nega-tively charged groups due to its positive charged state (iso-electric point of about 10) at physiological pH [14]. Hereinwe investigated the electrochemistry of adsorbed Cyto c atAP-[N]-modified electrodes. As shown in Fig. 1, clearredox peaks appeared, indicating quasi-reversible electro-chemical behaviors on the AP-[N]-modified electrodes.The peak currents remained constant within 8% deviationduring 3 h repetitive scans (data not shown). The possibil-ity of Cyto c adsorption on the MCH monolayer wasexcluded by the control experiment using the MCH self-assembled monolayer without AP-DNA duplexes.

Surface coverages of electrostatically adsorbed Cyto c

molecules, determined at low potential scan rate, were12.4 ± 1.0 pmol cm�2 in all cases, in spite of the DNAsequences and with or without AMND. These coverageswere comparable to those reported on the DNA monolayerwith a fully matched DNA duplex by Lisdat et al. [15,16].On the other hand, it was very interesting to find that thepeak separations (DEp) were sequence dependent (in revers-ibility A > T > G > C, as DEp increased, Fig. 1) in theabsence of AMND. A previous report [17] proposed thatthe stacking ability for unpaired bases was in the orderof A > G P T = C when they were located at the terminusin DNA as dangling residues. Additionally, the unpairedadenine in the center of a DNA strand also stacked wellwithin the helix as that occurred for intact A:T base pairs[18]. Compared with the data of peak separations (DEp

without AMND), the nucleobases opposite the AP siteseemed to show the same tendency in electrochemicalreversibility as their stacking abilities, indicating the crucial

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c d

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Fig. 1. CVs of AP-[N]-modified gold electrodes in a 10 mM phosphate buffer solution (pH 7.0) with (solid line) and without (dotted line) AMNDpretreatment before duplex assembly. Nucleobases opposite the AP site were (a) cytosine, (b) thymine, (c) adenine, and (d) guanine. Scan rate = 5 V/s.

440 Y. Shao et al. / Electrochemistry Communications 10 (2008) 438–442

effect of base stacking on the electrochemical behaviors ofCyto c. The sequence dependence of Cyto c electrochemis-try also supported the argument that the CT pathway foradsorbed Cyto c is through DNA molecules [16], and notthrough-space tunneling between Cyto c and the goldsurface.

In order to explore the effect of stacking integrity in theAP-[N] duplexes on the electrochemical behavior ofadsorbed Cyto c, an appropriate ligand should be identifiedfor its binding to the nucleobase opposite the AP site. Theprevious study done using thermodynamic stability andfluorescence spectroscopy proved that AMND bindingoccurred through hydrogen bonds to a target nucleobaseopposite the AP site with selectivity in the order ofC > T > G > A [7]. This base pairing process was alsobelieved to be accompanied by AMND stacking withnucleobases flanking the AP site. Here AMND was alsoused to investigate its effect on electrochemical behaviorof Cyto c for the electrode modified by AP-[N]. The effectof the AMND binding was more obvious for AP-[C] andAP-[T] compared to AP-[A] and AP-[G] (Fig. 1). Signifi-cant increases in peak currents were observed for AP-[C]and AP-[T], accompanied by a decrease in peak separations(DEp) from 0.104 V to 0.060 V for AP-[C] and from 0.050 Vto 0.038 V for AP-[T], respectively, although the AMNDbinding to AP-[N] made a slight shift in the formal poten-tials (E00) for all duplexes. These improvements in the vol-tammograms were in agreement with the binding abilityof AMND to the nucleobases opposite the AP site [7]and meant that a more reversible electrochemical behaviorof Cyto c was achieved by incorporation of AMND intothe AP site. Since the AP site is located at the middle ofa DNA strand, adsorbed Cyto c is separated from the

bound AMND by DNA base pairs. Accordingly, directinteraction between Cyto c and AMND is not expected.AMND is also electro-inactive under the present potentialrange. Based on these facts, we reasonably expected thatAMND binds to cytosine opposite the AP site throughhydrogen bonding and that favorable stacking interactionwith flanking bases takes place, which is in accordance witha decrease in peak separations in the presence of AMND.

The dependence of electrochemical behaviors on poten-tial scan rates for the AP-DNA electrodes was again eval-uated in order to assess the kinetics of electrode reactions.As shown in Fig. 2, AP-[C] was much more electrochemi-cally discrete from other duplexes in the absence ofAMND. However, upon binding with AMND, the electro-chemical behavior of Cyto c on the AP-[C] modified elec-trode was improved with positive shifting of the peakpotential especially for the cathode process at higher scanrates. The electrode kinetics constants changed from48.6 s�1 to 162.2 s�1 after binding of AMND to AP-[C][19]. For other duplexes, however, it was in the range of162.2 ± 30.5 s�1 with and without AMND. The presenceof an AP site in DNA would affect the duplex conforma-tion mainly at the site and at adjacent base pairs [20]. Whenthe unpaired bases are flanked by cytosine (that is, CNC),the unpaired cytosine is extra-helical [21–23] and thymine isin equilibrium between the intra- and extra-helical forms[23], while the unpaired guanine and adenine are foundto lie within the helix, even if the unpaired purines areflanked by pyrimidines [24] or purines [25]. Although thesestudies reported DNA structures based on deoxyribose andits analogues as a model for an AP site, the AP site ofSpacer C3 we used seemed to be comparable to these mod-els for its effect on DNA conformation at the AP site

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Fig. 2. Dependence of CV peak potentials on the scan rate for Cyto c adsorbed on the duplex-modified electrodes with (solid symbols) and without (opensymbols) AMND at the AP site. Nucleobases opposite the AP site were (a) cytosine, (b) thymine, (c) adenine, and (d) guanine. CV measurements werecarried out in a 10 mM phosphate buffer solution (pH 7.0).

Y. Shao et al. / Electrochemistry Communications 10 (2008) 438–442 441

[26,27] or thermodynamic stability [28]. Based on our elec-trochemical experimental results, we concluded that theAP-[C] must be very different in conformation comparedwith other duplexes. According to these reports, AP-[C]would adopt an extra-helical contribution for the unpairedcytosine. This extra-helical conformation for the unpairedcytosine should strongly weaken the base stacking on thestrand where the unpaired cytosine is located, as opposedto the other nucleobases opposite the AP site, resulting inslow electrode kinetics for the adsorbed Cyto c. AfterAMND binding, the electrochemical behaviors of theadsorbed Cyto c showed peak potential dependence similarto other duplexes, indicating the improved Cyto c electro-chemical behaviors resulted from the significance of basestacking. Therefore, AMND binding would make it possi-ble for the AP-[C] to recover its intra-helical conformationas reflected by recovery in the electrochemical behaviors ofadsorbed Cyto c (Fig. 2).

4. Conclusions

Electrochemical measurements revealed that Cyto c

showed sequence-dependant response on the AP-[N] mod-ified electrodes, especially for the case of cytosine oppositethe AP site. The difference in the electrode kinetics at AP-[C] could be attributed to the extra-helical conformation ofcytosine and intra-helical conformation of other nucleo-bases. The pairing of AMND with cytosine opposite theAP site through hydrogen bonding should keep AMNDin the AP site with a favorable orientation to induce awell-stacked base integrity for facilitating Cyto c electro-chemistry. Therefore, the electrochemical behavior ofprobe molecules on AP site-containing DNA duplex-mod-

ified electrodes can be easily modulated by introduction ofa suitable ligand into the AP site. Studies are underway toexamine the scope of this approach. We are, for example,beginning to explore the utility of this process to detect bio-logically active substances by utilizing AP site-containingDNA duplex as an aptamer.

Acknowledgments

This work was supported by Grant-in-Aid for ScientificResearch (No. 17205009) from the Ministry of Education,Culture, Sports, Science and Technology, Japan. Dr. Y.Shao acknowledges the financial support of the JSPS-PDfellowship for foreign researchers.

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