Article pubs.acs.org/Langmuir
Electrochemical Properties of Interstrand Cross-Linked DNA Duplexes Labeled with Nile Blue Yasuhiro Mie, Keiko Kowata, Naoshi Kojima, and Yasuo Komatsu* Bioproduction Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), 2-17-2-1 Tsukisamu-higashi, Toyohira, Sapporo 062-8517, Japan S Supporting Information *
ABSTRACT: DNA molecules have attracted considerable attention as functional materials in various fields such as electrochemical sensors with redox-labeled DNA. However, the recently developed interstrand cross-link (ICL) technique for doublestranded DNA can adequately modify the electronic properties inside the duplex. Hence, the electrochemical investigation of ICL-DNA helps us to understand the electron transfer of redox-labeled DNA at an electrode surface, which would develop useful sensors. In this study, the first insight into this matter is presented. We prepared 17-mer DNA duplexes incorporating Nile blue (NB-DNA) at one end as a redox marker and a disulfide tether at the other end for immobilization onto an electrode. The duplexes were covalently cross-linked by bifunctional cross-linkers that utilize either a propyl or naphthalene residue to replace a base pair. Their electrochemical responses at the electrode surface were compared to evaluate the effect of the ICL on the electron-transfer reactions of the redox-labeled DNA duplexes. A direct transfer of electrons between NB and the electrode was observed for a standard DNA, as previously reported, whereas interstrand cross-linked DNA (CL-DNA) strands showed a decrease in the direct electron-transfer pathway. This is expected to result from constraining the elastic bending/ flexibility of the duplex caused by the covalent cross-links. Interestingly, the CL-DNA incorporating naphthalene residues exhibited additional voltammetric peaks derived from DNA-mediated electron transfer (through base π stacking), which was not observed in the mismatched CL-DNA. The present results indicate that the ICL significantly affects electron transfer in the redox-labeled DNA at the electrode and can be an important determinant for electrochemical signaling in addition to its role in stabilizing the duplex structure.
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using a bifunctional cross-linker.22 This ICL is able to fix an aromatic or an aliphatic molecule within the double helix through covalent bond formation. The redox-labeled ICLDNA, in which electronic properties in the base stacking have been modified, is a good subject to be studied for understanding the electron transfer of DNA. However, this issue remains to be investigated. In this context, we prepared interstrand cross-linked NB-DNA duplexes, CL-N and CL-P, incorporating naphthalene residues and propyl groups, respectively, as shown in Figure 1, and then compared their electrochemical responses. The cross-linkers were installed at two base pair positions to induce a clear effect. We report our findings that CL-N and CL-P had features that were different from each other and from a standard perfectly matched NBDNA (PM). Very interestingly, whereas direct ET occurred using the standard NB-DNA as previously reported, DNAmediated ET was newly detected in CL-N. The present results offer significant insight into controlling the ET of redox-labeled DNA at the electrode surface by the ICLs.
INTRODUCTION Electrochemical sensors in which redox-marker-modified DNAs are immobilized on an electrode surface have been developed as simple, cost-effective analytical tools.1−10 Understanding and precisely controlling electron transfer (ET) between the redox marker and the electrode is crucial to such applications. DNAmediated ET (electron transfer via the DNA duplex) is generally reported using intercalators, such as Nile blue (NB) as a redox marker and alkyl thiols as end tethers for the immobilization of DNA onto the electrode surface. The importance of base π stacking in this process is revealed using mismatched sequences.1,11,12 However, direct ET between the redox marker and electrode is also reported using pendant-type markers. The effects of duplex rigidity/ flexibility and end-tether molecules on the electrochemical responses are clearly shown.13−15 Very recently, we also found that a cyclic disulfide (SS) tether (Figure 1a) connecting NBlabeled DNA (NB-DNA) to the electrode facilitates the direct ET pathway, whereas DNA-mediated ET takes place using a six-carbon alkyl thiol tether because of the alterations in duplex orientation at the surface.16 The interstrand cross-link (ICL) of DNA duplexes has been intensively studied with respect to cancer chemotherapy and gene manipulation.17−21 Fairly recently, we also reported a novel ICL reaction that covalently links a pair of apurinic/ apyrimidinic (AP) sites in complementary oligonucleotides © 2012 American Chemical Society
Received: September 11, 2012 Revised: November 13, 2012 Published: November 15, 2012 17211
dx.doi.org/10.1021/la3036538 | Langmuir 2012, 28, 17211−17216
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Article
Figure 1. (a) Schematic representation of NB-DNA cross-linked at two base pair positions at an electrode surface, potential ET pathways, and chemical structure of cross-linkers. (b) DNA sequences of standard perfectly matched NB-DNA (PM), NB-DNA cross-linked with aoPao (CL-P), NB-DNA cross-linked with aoNao (CL-N), and mismatched CL-N (CL-mm-N) used in the present study. Y, X, the bold lines, and the underlines indicate 5-[3-acrylate NHS ester]-2′-deoxyuridine for NB modification, an AP site, a cross-linker, and a mismatched base pair, respectively.
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EXPERIMENTAL SECTION
Γ=
Synthesis and Preparation of NB-DNA, Cross-Linkers, and Cross-Linked NB-DNA. All of the DNA sequences are shown in Figure 1. The oligodeoxyribonucleotides (ODNs) other than the NBcontaining ODNs were prepared on a solid support using standard phosphoramidite chemistry.23 Complementary ODN sequences used for the preparation of PM and the CL-duplexes were 5′-ssGAGATATAAAGCACGCA-3′(II)/5′-YGCGTGCTTTATATCTC-3′(I) and 5′-ss-GAUATATAAAGUACGCA-3′(IV)/5′-YGCGTUCTTTATATUTC-3′(III), where ss, Y, and the underlined U indicate a disulfide tether, 5-[3-acrylate NHS ester]-2′-deoxyuridine, and 2′-deoxyuridine, respectively. NB-modified ODNs were prepared according to a previous report.11 A cyclic disulfide (SS) tether was introduced at the 5′ terminus of the DNA strand as previously reported.16 Cross-linkers of bis(aminooxy) derivatives containing a naphthalene and propyl group (aoNao and aoPao, respectively) were synthesized according to a previous method.22 CL-N and CL-P were prepared by cross-linking reactions using aoNao and aoPao, respectively (Figure 1). These interstrand crosslinked DNA (CL-DNA) duplexes were formed at a pair of AP sites according to a previous report after treatment of the above-mentioned duplex with uracil DNA glycosylase.19 CL-mm-N, which contains a TT mismatch in CL-N, was also synthesized by the ICL reaction using the ODN pair 5′-ss-GAUATATTAAGUACGCA-3′(V)/5′-YGCGTUCTTTATATUTC-3′(III). All CL duplexes were purified by HPLC using a reverse-phase column, and the formation of the complex was confirmed using denaturing polyacrylamide gel electrophoresis (Figure S2). Preparation of NB-DNA-Modified Electrodes and Electrochemical Measurements. Commercially available gold disk electrodes (ø = 3 mm, Bioanalytical Systems, Inc.) were cleaned according to a previously reported procedure.24 DNA immobilization was performed by placing a 5 μM DNA-NB solution (buffer: 5 mM phosphate and 50 mM NaCl, pH 7.1) on the electrode surface and maintaining the association for 17 h in a humid environment. The electrode was subsequently washed with the same buffer several times. The prepared surface was backfilled with mercaptohexanol by soaking the electrode in a 1 mM solution of this compound for 90 min.4 Then, the modified electrode was again thoroughly washed with the buffer solution to ensure the removal of trace alkanethiols. Voltammetry was performed with a standard three-electrode configuration consisting of an Ag/AgCl reference electrode, a Pt auxiliary electrode, and a DNAmodified working electrode, typically in a 5 mM phosphate buffer (pH 7.1) containing 50 mM NaCl under an Ar atmosphere. Voltammograms were Fourier filtered to remove remaining noise, and then background subtraction was conducted using the SOSA program.25 The surface density of NB-DNA (Γ) was calculated from the voltammetric peak area (faradaic charges) at 10 mV/s (eq 1).
Q nFA
(1)
In eq 1, Q is the area of the faradaic CV signal, n is the number of electrons per redox event (n = 2 for NB), F is Faraday’s constant, and A is the electrode area. The electrode area was estimated from the gold surface oxide reduction peaks in H2SO4 according to the reported procedure.26 The surface density of NB-DNA was also estimated by chronocoulometry (Figure S6) in accordance with the reported method.27
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RESULTS AND DISCUSSION ICLs Alter the Electrochemical Responses of RedoxLabeled DNA. First, we briefly compared the electrochemical responses of PM, CL-P, and CL-N. Figure 2a−c shows the
Figure 2. Background-subtracted cyclic voltammograms of 17-mer DNA duplexes in a pH 7.1 buffer solution at a scan rate of 0.1 V/s: (a) PM, (b) CL-P, (c) CL-N, and (d) CL-mm-N. Black and gray arrows indicate different peak pairs. 17212
dx.doi.org/10.1021/la3036538 | Langmuir 2012, 28, 17211−17216
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direct ET, whereas neutral anthraquinone (AQ) does not, indicating that the electrostatic interaction between MB and a negatively charged electrode surface is important for the ET.29 We used positively charged NB, hence a similar interaction may also occur in the present system. Importantly, Ferapontova et al. have used the stand-up orientation of the duplex system with a longer linker chain between DNA and the redox probe, whereas we used the lying-down orientation with a shorter chain. Therefore, duplex flexibility appears to be important for ET in our system because the probe linker is less flexible. Considering these facts, the difference in the critical log(v) values between PM and CL-P can be explained by the rigid (less-flexible) conformation of the CL-P, which is induced by covalent bonding within the duplex that suppresses the interaction between NB and the electrode surface. ICLs with Naphthalene Residues Enable DNA-Mediated Electron Transfer between NB and the Electrode. Next, we focus on the two pairs of peaks in CL-N. The shapes of the voltammograms of CL-N were uniquely scan-ratedependent, as shown in Figure 4a−d, and were quite different from those of the other NB-DNA-modified electrodes. Although only one pair of peaks was seen in the voltammograms taken at slow scan rates (e.g., 0.01 V/s, Figure 4a), the
background-subtracted cyclic voltammograms of PM-, CL-P-, and CL-N-modified electrodes, respectively, measured at a potential scan rate of 0.1 V/s. All DNAs showed clear faradaic peaks at approximately −0.19 V (vs Ag/AgCl), and this potential is in good agreement with the reported formal potential for NB intercalated in DNA,1 suggesting that ET between the NB in the DNA and the electrode occurred. Two obvious features were recognized from the comparison of the voltammograms. First, the current intensity of CL-P was smaller than that of PM at this scan rate. Note that all NB-DNA duplexes had similar intensities at a slower scan rate (0.01 V/s), as described below. From the faradaic charges in the voltammograms at 0.01 V/s, the surface densities of duplexes were calculated to be 1.5 ± 0.3, 1.8 ± 0.1, 1.7 ± 0.2, and 1.6 ± 0.2 pmol/cm 2 for PM, CL-P, CL-N, and CL-mm-N, respectively. Chronocoulometric analysis (Figure S6) resulted in similar surface-density values. Second, CL-N very interestingly showed two pairs of peaks (black and gray arrows in Figure 2c) in the voltammograms, and PM- and CL-P-modified electrodes displayed only one pair of peaks. ICLs with Propyl Groups Show a Reduction in “Direct” ET between NB and the Electrode. To evaluate the ET mechanism of PM and CL-P, we analyzed the relationship between the peak currents (ip) obtained from the voltammograms and the scan rates (v), as shown in Figure 3. The ratio of
Figure 3. Peak current function of ip/v vs log(v) for the electrodes modified with PM (filled triangle symbols), CL-P (filled square symbols), and CL-mm-N (open circle symbols).
the peak currents to the scan rates (ip/v) for PM (filled triangles in Figure 3) was constant in the lower-v region (
