Electrochemical Discrimination between G-Quadruplex and Duplex

Article pubs.acs.org/ac

Electrochemical Discrimination between G‑Quadruplex and Duplex DNA Aurore De Rache,§ Thomas Doneux, and Claudine Buess-Herman* Chimie Analytique et Chimie des Interfaces, Faculté des Sciences, Université Libre de Bruxelles, CP 255, Boulevard du Triomphe 2, B-1050 Bruxelles, Belgium S Supporting Information *

ABSTRACT: Analytical tools enabling the discrimination between duplex DNA and G-quadruplex DNA are necessary to unravel the biological function(s) of G-quadruplexes. A methodology relying on the electrochemical response of the electroactive hexaammineruthenium(III) cation at DNA-modified surfaces is presented. A characteristic voltammetric peak is evidenced for all the investigated G-quadruplex sequences, encompassing various types of folding and numbers of quartets. In contrast, no such peak is detected for dsDNA sequences. The occurrence of the voltammetric peak is the consequence of a strong association between the hexaammineruthenium ligand and the surfaceimmobilized G-quadruplexes. The peak potential points to a significant contribution of nonelectrostatic interactions between the electroactive ligand and G-quadruplexes. The very good efficiency of the discrimination methodology is demonstrated by comparing a G-quadruplex and its corresponding duplex.

T

and suggested that the peak potentials can be used for electrochemical screening purposes. In the present work, we propose an original electrochemical discrimination between Gquadruplexes and duplexes based on our recent finding that a positively charged redox marker, the [Ru(NH3)6]3+ complex, can be tightly confined at the surface of electrodes modified with the thrombin binding aptamer (TBA),10 a DNA sequence folding into an antiparallel G-quadruplex conformation. In the first step, we will demonstrate that this nonconventional interaction between a DNA sequence and [Ru(NH3)6]3+ is not limited to TBA but also takes place with other Gquadruplex folding sequences. Besides the TBA itself (herein Q1), two other G-quadruplexes were considered for this purpose, Q2 and Q3 (see Table 1). The sequence Q2 is composed of the TBA sequence elongated at its 5′-end by six supplementary bases. This sequence was shown to adopt a predominant parallel folding in the presence of [Ru(NH3)6]3+.11 To make our restricted set of sequences representative of the wide diversity of G-quadruplex structures, the human telomeric sequence was chosen because it contains three quartets, unlike TBA which has only two, and because it is polymorphic as attested by its reported foldings ranging from two different antiparallel ones to a parallel or a 3 + 1 one12 (see Table 1 for details and most recent references). In the second step, it will be shown that no confinement of the used redox marker is observed at duplex-modified

he G-quadruplexes are nucleic acid structures formed by the stacking of two or more G-quartets on top of each other. Each G-quartet is composed of 4 guanines interacting through Hoogsteen H-bonds. The evidence of the relevance of this structure type in vivo is growing.1 Bioinformatic studies identifying putative G-quadruplex structures in the human genome estimate their number to be around 370 000.2,3 In the human cell, DNA is mostly in duplex form with the notable exception of the G-rich telomeric region which consists mainly of thousands of TTAGGG repeats terminated with a 100−200 nucleotides 3′-end overhang. To effectively adopt a Gquadruplex folding in vivo, DNA sequences identified by algorithms would thus require the disruption of the double stranded form. The development of discrimination tools between G-quadruplexes and duplexes could offer new opportunities in studying this impressive number of sequences, and there is currently a great interest in developing new classes of ligands with a high selectivity toward G-quadruplexes. Among various analytical approaches, fluorescence-based methods are receiving the highest attention because they are usually considered as particularly adapted for the development of high throughput tests. To date, several fluorescent dyes have been shown to present a more intense fluorescence emission in the presence of G-quadruplexes than with duplexes.4−6 Besides fluorescence techniques, electrochemical methods also offer interesting perspectives in the field of nucleic acid analysis, owing to their high sensitivity, low costs, or miniaturization possibilities.7−9 For instance, Doménech-Carbó et al.8 have recently shown that the voltammetric response of canthin-6one, an electroactive ligand, is influenced by the type of nucleic acid (ssDNA, dsDNA, G-quadruplex, etc.) present in solution © 2014 American Chemical Society

Received: February 28, 2014 Accepted: July 21, 2014 Published: July 21, 2014 8057

dx.doi.org/10.1021/ac500791s | Anal. Chem. 2014, 86, 8057−8065

Analytical Chemistry

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Table 1. DNA Sequences Used in the Present Study and Corresponding Reported Structure Typesa short name

description

thiolated strand

structure type

DNA surface concentration/ mol cm−2

thrombin binding aptamer13 (TBA) (fibrinogen binding site) elongated TBA

5′-HS-(CH2)6−GGT TGG TGT GGT TGG-3′

antiparallel quadruplex14−20

1.9 × 10−11

5′-HS-(CH2)6−TTT TTT GGT TGG TGT GGT TGG-3′

2.0 × 10−11

Q3

human telomeric sequence

5′-HS-(CH2)6−AGG GTT AGG GTT AGG GTT AGG G-3′

Q4

elongated TBA 2

5′-HS-(CH2)6−GTA GGT GGT TGG TGT GGT TGG-3′

Q-hp

hairpin thrombin binding aptamer38 (heparin binding site) Helicobacter pylori duplex random duplex elongated TBA 2duplex form

5′-HS-(CH2)6−TTT TTT CGT CCG TGG TTG GGC AGG TTG GGG TGA CT-3′

antiparallel or parallel quadruplex11 antiparallel,21−25 parallel26−28 or 3 + 1 quadruplex24,29−36 antiparallel or parallel quadruplex11,37 hairpin antiparallel quadruplex38

5′-HS-(CH2)6−CAA GAC GGA AAG ACC C-3′ + 5′-GGG TCT TTC CGT CTT G-3′

duplex

7.0 × 10−12

5′-HS-(CH2)6−CGT GTC GTT GCA TCC C-3′ + 5′-GGG ATG CAA CGA CAC G-3′ 5′-HS-(CH2)6−GTA GGT GGT TGG TGT GGT TGG-3′ + 5′-CCA ACC ACA CCA ACC ACC TAC-3′

duplex duplex

6.4 × 10−12 5.2 × 10−12

Q1

Q2

TS1 TS2 Q4TS a

1.2 × 10−11 1.8 × 10−11 9.4 × 10−12

Various cationic conditions; see references for details. Q, hp, and TS stand for G-quadruplex, hairpin, and two strands, respectively.

method proposed by Tarlov and colleagues40 and illustrated in Section S-1 of the Supporting Information. Electrochemical Measurements. An Autolab PGSTAT 30 (Eco Chemie, The Netherlands) potentiostat equipped with Scangen and FRA modules was used to perform the electrochemical experiments. They were carried out in a three electrode cell thermostated at 20.0 ± 0.1 °C with a Julabo F10UC thermostat-cryostat. The working electrode was a polycrystalline gold disc electrode with a 1.6 mm diameter (Bioanalytical Systems). It was cleaned by mechanical polishing on a smooth cloth impregnated with 1 μm alumina particles, then sonicated for 10 min in ultrapure water, and finally submitted to numerous potential cycling at a 50 mV s−1 scan rate between −0.3 and +1.5 V in 0.1 M HClO4, until the voltammogram characteristic of a polycrystalline gold electrode was obtained. The real area of the electrode was determined from the integrated charge involved in the gold oxidation peaks, using an upper integration limit at +1.35 V and a theoretical charge density of 400 μC cm−2.41 The roughness factor (the ratio of the real area to the geometric area) never exceeded 2. A large area platinum grid was used as auxiliary electrode. A double-bridge saturated calomel electrode (SCE) was used as reference electrode (Radiometer REF 451). All potentials given in this work refer to this electrode. A water-saturated nitrogen purge of at least 15 min was performed for all electrolyte solutions before keeping them under a nitrogen blanket during the measurements. ac Voltammetry. ac voltammograms were recorded with a potential perturbation of 5 mV (rms) both at 37 Hz and at 10 kHz. One data point was recorded every 10 mV between +0.05 and −0.40 V, a full potential scan taking approximately 6 min. For each potential, the real and imaginary parts of the cell impedance, Z′tot and Z″tot, were determined. The interfacial components of the impedance, Z′el and Z″el, were obtained using the following relations: Z′el = Z′tot − Rs and Z″el = Z″tot. The real part of the cell impedance measured at 10 kHz was used to estimate the uncompensated cell resistance Rs. Finally, the interfacial admittance components were calculated from the interfacial impedance according to the equations:

electrodes. The more complex case of a hybrid structure consisting of a hairpin sequence with a G-quadruplex in the loop will be also considered. Eventually, it will be shown that our method is able to discriminate between a G-quadruplex (Q4) and its corresponding duplex.

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EXPERIMENTAL SECTION Reagents. The various DNA sequences investigated in the present study are listed in Table 1. All sequences were purchased from Eurogentec s.a. (Belgium) with a HPLC purification and used as received. The lyophilized samples were dissolved to a concentration of 1 mM (per strand) in 100 mM Tris buffer, pH 7.4, containing 10 mM EDTA. The stock solutions were kept frozen at −80 °C. All solutions were prepared with ultrapure water (Millipore Milli-Q system). Hexaammineruthenium(III) chloride (>32.1% Ru, Alfa Aesar) was used as received. The 4-mercaptobutan-1-ol (>97%, Fluka) was stored at 4 °C. All other chemicals were of analytical reagent grade. DNA Immobilization. For G-quadruplex DNA, the thiolmodified DNA probes were mixed with 4-mercaptobutan-1-ol (MCB) at 1−1 mole fraction to obtain a 20 μM thiol total concentration. To favor the formation of high DNA surface coverage monolayers39 and ensure the folding into Gquadruplex conformations, which are cation dependent and favored in K+-medium, a 1 M potassium phosphate buffer (pH 7.4) was used for immobilization. In a sealed plastic tube, a volume of 50 μL of the DNA/MCB buffered mixture was placed onto the electrochemically cleaned gold electrode surface (see below) and left overnight (16 h) at room temperature for chemisorption and self-assembly. In the case of the duplex DNA, the thiolated strand was mixed with MCB in the same proportions and in the same buffer, and two equivalents of the nonthiolated complementary strand were added. The mixture was allowed to equilibrate for at least 30 min to ensure complete hybridization of the probe, before the overnight immersion of the gold electrode. In both cases, after immersion, the electrode was thoroughly rinsed with the electrolyte, dried with nitrogen, and transferred in the electrochemical cell. The amount of DNA immobilized at the electrode surface was determined by the chronocoulometric 8058


Analytical Chemistry Y el′ = Y el″ =

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Zel′ 2

Zel′ + Zel″

2

through electrostatic interactions with the negatively charged DNA backbone. The involvement of adsorption in the voltammetric response is deduced from the very small peakto-peak separation between the cathodic and anodic peaks and from the linear relationship between the peak current and the scan rate (see Supporting Information Section S-2). The occurrence of this nonspecific interaction between [Ru(NH3)6]3+ and surface-immobilized DNA is well documented40,42−51 and can be described by a surface−solution ionic equilibrium.45 Because the adsorption of the electroactive complex is electrostatically driven, the adsorption strength is higher for the oxidized form [Ru(NH3)6]3+ than for its reduced form [Ru(NH3)6]2+. From simple thermodynamics arguments, the adsorption peak is thus found at potentials more negative than the formal potential of freely diffusing [Ru(NH3)6]3+/2+ couple,40,42,43,45,52,53 hence the denomination “postpeak” when the reduction process is considered.54 In cyclic voltammetry, there is a significant overlap between the tail of the diffusioncontrolled peak Pdiff and the postpeak Pads. Resorting to other electrochemical methods such as square-wave voltammetry44 or ac voltammetry,43,45 the adsorption peak can be more clearly evidenced. Figure 1b presents an ac voltammogram recorded in the same conditions as in Figure 1a. It displays a prominent peak centered at −0.32 V, at the exact potential where the postpeak is observed in cyclic voltammetry. This is to be expected, since for adsorbed species the peak potentials in cyclic voltammetry and ac voltammetry are identical (for fast electron transfer kinetics, see Supporting Information Section S-3). The intense peak in the ac voltammogram can thus be safely labeled “Pads” and attributed to the reduction of adsorbed [Ru(NH3)6]3+. Interestingly, the peak intensity is directly proportional to the surface concentration of adsorbed [Ru(NH3)6]3+ and can thus be used to compare the amount of immobilized DNA for the various sequences used in this study. The contribution of freely diffusing hexaammineruthenium(III) is almost negligible, as it appears only as a small hump (labeled “Pdiff”) in the ac voltammogram. This arises from the fact that, for the system under investigation and in the present experimental conditions, ac voltammetry is much more sensitive to adsorbed electroactive species than to dissolved ones (for a more detailed discussion and a comparison with cyclic voltammetry, see Supporting Information Section S-3). ac voltammetry is, therefore, ideally suited to detect changes occurring at the electrode surface even in the presence of fairly high concentrations of dissolved electroactive species and will be used below for this purpose. The adsorption postpeak is mostly observed at low ionic strength, since it is sensitive to the presence of other cations,45,55,56 and its amplitude is dependent on the [Ru(NH3)6]3+ concentration in the measurement solution.45,50 As a result, when a DNA-modified electrode previously immersed in a [Ru(NH3)6]3+ solution is transferred in a solution containing only a pure electrolyte, the recorded cyclic voltammograms display only the postpeak, whose intensity decreases upon scanning and the peak vanishes after a few cycles.42 This behavior, illustrated in Figure 2, is explained by the diffusion of the complex into the bulk, which is promoted after reduction of the [Ru(NH3)6]3+ in [Ru(NH3)6]2+ because the interaction of the latter with DNA is weaker. In this transfer experiment, the intensity of the postpeak observed in the first cycle is strongly dependent on the electrode rinsing process. Indeed, electrostatically adsorbed [Ru(NH3)6]3+ can be replaced by cations from the rinsing solution and dissociate

(1)

Zel″ 2

Zel′ + Zel″ 2

(2)

RESULTS AND DISCUSSION Conventional Electrostatic Interaction between [Ru(NH3)6]3+ and DNA-Modified Eelectrodes. The triply charged [Ru(NH3)6]3+ cation is known to interact with the DNA backbone through an electrostatic attraction with the negatively charged phosphate groups of the macromolecule. The [Ru(NH3)6]3+ complex is electroactive, and its interaction with DNA-modified electrodes is easily evidenced electrochemically by the appearance of an adsorption postpeak in cyclic,42,43 square wave,44 or ac voltammetry.43,45 This is illustrated in Figure 1, which presents a typical cyclic

Figure 1. Cyclic voltammogram (a) and ac voltammogram (b) recorded at a Q2-modified electrode in 10 mM Tris buffer (pH 7.4) + 100 μM [Ru(NH3)6]3+. υ = 50 mV s−1 in (a), f = 37 Hz in (b), T = 20 °C.

voltammogram and the corresponding ac voltammogram recorded at a DNA-modified electrode (Q2 in Figure 1) in the presence of [Ru(NH3)6]3+ in solution. In the cyclic voltammogram (Figure 1a), two pairs of redox peaks are apparent. The first one, at the less negative potentials, is labeled “Pdiff” because it reflects the diffusion-controlled reduction and subsequent reoxidation of [Ru(NH3)6]3+ dissolved in solution, as attested by the characteristic separation between the cathodic and anodic peaks and by the square-root dependence of the peak current on the scan rate (see Supporting Information Section S-2). The second pair of peaks, labeled “Pads”, corresponds to the reduction and subsequent reoxidation of hexaammineruthenium(III) cations adsorbed at the electrode 8059



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subjected to a certain experimental variability and will not be used for quantitative purposes. Confinement of [Ru(NH3)6]3+ at G-QuadruplexesModified Electrodes. In the previous paragraph, we have presented the general electrochemical behavior observed for all DNA-modified electrodes in the presence of hexaammineruthenium(III) cations. Recently, we reported a very different behavior for the interaction between [Ru(NH3)6]3+ and the thrombin binding aptamer, a G-quadruplex DNA sequence (herein, Q1).10 This distinct interaction was evidenced after recording about 75 successive ac voltammograms at 5 °C in the presence of [Ru(NH3)6]3+ on a Q1-modified electrode. This electrode was subsequently rinsed (with the electrolyte) and transferred to the same electrolyte devoid of [Ru(NH3)6]3+, where cyclic voltammograms were measured. A clear pair of redox peaks were present in the voltammograms, whose intensity remained unaffected upon continuous cycling. Both the reduction and the consecutive oxidation peak currents depended linearly on the scan rate,10 revealing the presence of a surface-confined species. In the present study, we applied the same methodology to different G-quadruplex and dsDNA sequences. A minor change from our initial report was the change of temperature from 5 to 20 °C, motivated by our finding that the confinement occurred much faster (ca. 20 successive voltammograms instead of 75) in the new conditions. Figure 3a shows 20 successive ac voltammograms recorded at 20 °C on a Q2-modified electrode. The first ac voltammogram presents a single pronounced peak with a maximum at −0.32 V which corresponds to the reduction of the electrostatically adsorbed [Ru(NH3)6]3+ and whose amplitude is proportional to the amount of DNA

Figure 2. Ten consecutive voltammograms recorded at a Q2-modified electrode in 10 mM Tris buffer (pH 7.4), after the electrode has been immersed in a solution containing the same electrolyte + 100 μM [Ru(NH3)6]3+. υ = 50 mV s−1, T = 20 °C.

from the negatively charged DNA. Yu and co-workers55,56 have studied the thermodynamics and kinetics of this dissociation process and showed that the competition is very efficient with divalent cations (Mg2+, Ca2+) at low concentrations (10 mM) of monovalent salts. In the experiment shown in Figure 2 and in the forthcoming transfer experiments presented in this work, the electrode was quickly rinsed by flowing a few milliliters of 10 mM Tris buffer (i.e., the electrolyte used both before and after the transfer step) onto the surface of the electrode. The intensity of the postpeak observed in the first cycle can thus be

Figure 3. Interactions between hexaammineruthenium(III) and the G-quadruplex Q2-modified electrode. (a) Successive ac voltammmograms recorded in 10 mM Tris buffer (pH 7.4) + 100 μM [Ru(NH3)6]3+. One voltammogram is plotted every two measurements, the arrows indicating the evolution. f = 37 Hz, T = 20 °C. (b) Ten consecutive cyclic voltammograms recorded after transfer in 10 mM Tris buffer (pH 7.4). υ = 50 mV s−1, T = 20 °C. Right: schematic representation of the whole procedure. 8060



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Figure 4. Interactions between hexaammineruthenium(III) and the G-quadruplexes Q1-modified (a, b) and Q3-modified (c, d) electrodes. (a, c) Successive ac voltammmograms recorded in 10 mM Tris buffer (pH 7.4) + 100 μM [Ru(NH3)6]3+. One voltammogram is plotted every two measurements, the arrows indicating the evolution. f = 37 Hz, T = 20 °C. (b, d) 10th cyclic voltammograms recorded after transfer in 10 mM Tris buffer (pH 7.4). υ = 50 mV s−1, T = 20 °C.

and characterized by ac voltammetry (Supporting Information Section S-5), cyclic voltammetry is more convenient to establish a quick electrochemical diagnostic, such as for instance the peak current vs scan rate dependences that showed a linear relationship (see Supporting Information Section S-5), confirming the adsorbed character of the redox compounds. The amplitudes of the cathodic and anodic peaks remain constant upon cycling, indicating that the adsorbed species do not diffuse back into the bulk and that the peaks correspond to the electrochemical response of [Ru(NH3)6]3+ confined at Q2-modified electrodes. This confinement appears to be rather strong, as we did not observe any significant difference in the peak intensities after 1 day of immersion in the electrolyte. Moreover, in contrast to the peak Pads, the peak Pconf is not affected when the electrode is rinsed with MgCl2 (see Supporting Information Section S-4). To evaluate whether this behavior can be observed with other G-quadruplexes, electrodes modified with Q1 and Q3 were prepared. When the same procedure is applied to these electrodes, the ac voltammograms of Figure 4a,c are recorded. In both cases, an evolution of the voltammetric features, similar to that of Q2, is again clearly discernible between successive measurements. The occurrence of these changes with all three G-quadruplexes under study suggests that the modification of the interfacial interactions between the immobilized DNA and the [Ru(NH3)6]3+ is not specific to the Q2 sequence. The cyclic voltammograms (the 10th cycle is presented in each case) of Figure 4b,d, measured after rinsing the quadruplexmodified electrodes and transferring them to a cell exempt of [Ru(NH3)6]3+ confirm that statement. Indeed, for these electrodes, the peak Pconf is clearly evidenced, at −0.19 V. The properties of this peak, such as the peak position, the scan

immobilized at the electrode. This peak is obviously the adsorption postpeak Pads described in the first section. In the subsequent potential scans, the morphology of these ac voltammograms changes markedly, in a manner similar to that previously described:10 the admittance at the maximum decreases while a new peak progressively appears around −0.14 V. The difference between consecutive scans decreases gradually until no more significant changes are observed. When the electrode is subsequently rinsed with the Tris buffer electrolyte and then transferred into a cell containing only the pure electrolyte, as schematically represented on the right side of Figure 3, the cyclic voltammograms of Figure 3b are recorded. Two distinct reduction/reoxidation responses are observed, one centered around −0.32 V whose intensity decreases upon continuous cycling and one centered around −0.20 V which remains unaffected during the course of the voltammetric measurement. The former can be reasonably assigned to Pads. Indeed, the decrease of the peaks located at −0.32 V is reminiscent of the behavior described in the first section for the electrostatically adsorbed [Ru(NH3)6]3+ (see Figure 2). A further confirmation of this assignment is provided by the nonappearance of Pads in the cyclic voltammogram when the electrode is rinsed with MgCl2 instead of Tris (shown in Supporting Information Section S-4), indicating the complete displacement of electrostatically bound [Ru(NH3)6]3+ from the surface. After ca. 10 scans, the peaks Pads have vanished and the characteristic voltammogram of a surface confined species is obtained, with the occurrence of a single reduction peak Pconf around −0.20 V and its corresponding reoxidation peak. Both peaks have an identical peak potential, which is characteristic of a surface-immobilized species undergoing a fast electron transfer kinetics. Although this peak could also be evidenced 8061



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rate dependence, and the stability during continuous cycling, are identical to those described with the Q2-modified electrode and thus correspond to the confined marker of interest here. It is clear that the evolution observed in the ac voltammograms recorded in the presence of hexaammineruthenium(III) can be directly connected with the presence of the peak Pconf after transfer in the pure electrolyte. The slight difference in the peak positions observed before and after transfer arises from the fact that the peak potential depends on the concentration of [Ru(NH3)6]3+ present in solution (see Supporting Information Section S-6). The simultaneous observation of Pads and Pconf in the ac voltammograms64 reveals that two distinct types of interaction between G-quadruplexes and [Ru(NH3)6]3+ exist and are not mutually exclusive. The first one is the nonspecific electrostatic interaction common to all DNA sequences and associated with the peak Pads. The second one, associated with Pconf, is a new type of interaction which is strong enough to withstand continuous potential cycling in pure electrolyte without detectable loss of marker. From an analytical perspective, it is interesting to remark that both interactions can be readily distinguished through the simple criterion of the peak position. The differences in the peak potentials are a manifestation that different interactions are involved for each case. Indeed, for a given type of interaction, the peak potential reflects the relative strengths of the association between the nucleic acid sequence and the oxidized or reduced forms of the redox complex:10,53,57 0′ Ep = E[Ru(NH − ) ]3 + /2 + 3 6

RT Kox ln F K red

Figure 5. Interactions between hexaammineruthenium(III) and a duplex sequence. (a) Successive ac voltammograms recorded at a TS1modified electrode in 10 mM Tris buffer (pH 7.4) + 100 μM [Ru(NH3)6]3+. One voltammogram is plotted every two measurements. f = 37 Hz, T = 20 °C. (b) 10th cyclic voltammograms recorded on the same electrode after transfer in 10 mM Tris buffer (pH 7.4). υ = 50 mV s−1, T = 20 °C.

(3)

where Ep is the peak potential, E0[Ru(NH ′ 3+/2+ is the formal 3)6] potential of the redox couple freely diffusing in solution, T is the temperature, R is the gas constant, F the Faraday constant, and Kox and Kred are the association constants between the nucleic acid strand and the oxidized and reduced forms, respectively, of the electroactive ligand. The fact that the peak Pconf is located at less negative potentials than Pads reveals that the electrostatic contributions to the association phenomena are less pronounced for the former interaction, in a way reminiscent of the behavior reported for intercalators.52,53,57−60 Selectivity of the Confined [Ru(NH3)6]3+ toward GQuadruplexes against ds-DNA-Modified Electrodes. The observation of confined [Ru(NH3)6]3+ with every studied Gquadruplex, independently of its folding, suggests that the corresponding interaction between this redox marker and the DNA possibly exists for all G-quadruplexes and that it could be selective for this type of folding. If this is the case, it should not be observed for duplex-modified electrodes, providing thus the basis for an electrochemical discrimination between DNA Gquadruplexes and duplexes. To address this potential selectivity, the confinement procedure was applied to electrodes modified with the TS1 duplex. Figure 5a shows the resulting successive ac voltammograms. As expected for any DNA-modified electrode, a pronounced peak attributed to the redox response of electrostatically adsorbed [Ru(NH3)6]3+ is seen in the first ac voltammogram, around −0.28 V. In comparison to the previous cases, the lower intensity of this peak indicates that the DNA coverage of this duplex is smaller than that of the Gquadruplexes, in agreement with the values determined by chronocoulometry and reported in Table 1. In stark contrast to the case of G-quadruplexes, no changes in the morphology of

the ac voltammograms are noticeable when successive potential scans are performed. Only one single peak is present from the first to the last measurement and its intensity does not vary in the course of the experiment. After rinsing and transfer into the pure electrolyte, no peak Pconf associated with confined [Ru(NH3)6]3+ is observed in the cyclic voltammogram (Figure 5b, 10th cycle). The absence of the peak Pconf when no alteration is observed in the morphology of the ac voltammograms confirms that the changes obtained at G-quadruplexmodified electrodes do correlate with the formation of confined [Ru(NH3)6]3+. Similar results were obtained with the duplex TS2 (see Supporting Information Section S-7). The fact that no confinement of [Ru(NH3)6]3+ is observed at duplex-modified electrodes while confinement of this redox marker is systematically obtained at G-quadruplex-modified ones effectively offers an elegant means to discriminate between these two types of DNA folding at the interface. To highlight the discrimination abilities of the proposed method, the sequence Q-hp was investigated. It consists of an antiparallel G-quadruplex whose 3′- and 5′-ends hybridize and form a duplex region. The global structure corresponds to the hairpin G-quadruplex represented in Scheme 1. The result of the confinement procedure is presented in Figure 6. Like in the case of the G-quadruplexes (Figures 3 and 4), the appearance of a peak at −0.15 V in the ac voltammograms (Figure 6a) is observed and the peak Pconf is present in the cyclic voltammogram (Figure 6b, 10th cycle) recorded after rinsing and transfer of the electrode into the pure electrolyte. This experiment shows that, when the immobilized structure contains both a G-quadruplex and a duplex part, the behavior 8062



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overhang of the telomeric region where the DNA is single stranded. Every sequence able to fold into a G-quadruplex may thus exist either in its G-quadruplex form or in its corresponding duplex one. The location of some of these sequences in promoter regions suggests that the G-quadruplex structures are involved in regulation processes61 whose study would benefit from a tool to discriminate between both structure types. To demonstrate the relevance of the proposed approach in this biological context, a key experiment was performed where the behavior of a G-quadruplex, Q4, was compared to that of a duplex, Q4-TS, formed by the same sequence Q4 and its complementary strand. Figure 7a,c, presents the successive ac voltammograms recorded with the Q4-modified and the Q4/TS-modified electrodes, respectively. The morphological changes previously described are observed for Q4 immobilized in the absence of its complementary strand (Figure 7a). In accordance with this evolution, the peak Pconf is present in the cyclic voltammogram of Figure 7b (10th cycle) recorded after transfer in a [Ru(NH3)6]3+-free solution. These observations provide an additional hint that the confinement of the [Ru(NH3)6]3+ might be observed for any G-quadruplex, independently of its exact sequence or specific folding. In contrast, no changes in the morphology of the initial ac voltammogram are observed with the corresponding duplex (Figure 7c) and no peak Pconf is detected in the cyclic voltammogram (Figure 7d, 10th cycle) after transfer, demonstrating once more that no confinement takes place at a duplex-modified electrode. This ability to distinguish between a G-quadruplex sequence and its corresponding duplex shows that our method for the discrimination between duplexes and G-quadruplexes relies effectively on the structure type and is not an artifact based on our choice of sequences. At present, we can only speculate about the exact nature of this rather unconventional association between hexaammineruthenium(III) and G-quadruplexes. Nevertheless, the peak potential obtained from our electrochemical data clearly shows that electrostatic attraction between the positively charged complex and the negatively charged DNA backbone is not the major driving force for the association. This might point to other contributions, for instance hydrogen bonding between the ammine ligands (H-donors) and H-acceptor sites of the nucleic acid sequences. Such bonding patterns have been demonstrated in the case of another noncanonical form of DNA, namely, Z-DNA.62,63

Scheme 1. Schematic Structure of the Q-hp Sequence

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CONCLUSIONS An electrochemical discrimination between G-quadruplexes and duplexes was developed, which relies on a selective interaction between the [Ru(NH3)6]3+ redox marker and Gquadruplexes. Even though the number of G-quadruplex sequences used in this study is rather limited, the interaction was evidenced for all of them, independently of their folding and number of quartets. This suggests that the proposed method could possibly be applicable to most, and perhaps all, G-quadruplexes. In the genome, folded G-quadruplexes are surrounded by duplex DNA. The ability to detect Gquadruplexes in a hairpin structure indicates that the approach is relevant in this biological context. This is further reinforced by the possibility to distinguish between a folded G-quadruplex and the same sequence in duplex conformation, which could be used to establish how G-quadruplexes located in promoter regions do impact the transcription process.

Figure 6. Interactions between hexaammineruthenium(III) and the hairpin G-quadruplex sequence. (a) Successive ac voltammograms recorded at a Q-hp-modified electrode in 10 mM Tris buffer (pH 7.4) + 100 μM [Ru(NH3)6]3+. One voltammogram is plotted every two measurements, the arrows indicating the evolution. f = 37 Hz, T = 20 °C. (b) 10th cyclic voltammograms recorded on the same electrode after transfer in 10 mM Tris buffer (pH 7.4). υ = 50 mV s−1, T = 20 °C.

expected for the G-quadruplex is still observed. This means that G-quadruplexes can be evidenced even when duplex DNA is also present, a situation encountered in the genome where most of the DNA is in duplex conformation. In biological cells, the formation of a G-quadruplex structure requires the disruption of the double helix, except in the 8063



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Figure 7. Comparison between the G-quadruplex Q4 (a, b) and its corresponding duplex Q4-TS (c, d). (a, c) Successive ac voltammmograms recorded in 10 mM Tris buffer (pH 7.4) + 100 μM [Ru(NH3)6]3+. One voltammogram is plotted every two measurements, the arrows indicating the evolution. f = 37 Hz, T = 20 °C. (b, d) 10th cyclic voltammograms recorded after transfer in 10 mM Tris buffer (pH 7.4). υ = 50 mV s−1, T = 20 °C.

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ACKNOWLEDGMENTS This work was supported by a grant from the Belgian National Science Foundation (FRFC Project).

From an analytical point of view, the method is very convenient because the potential of the reduction peak is a direct indication of the occurrence of the [Ru(NH3)6]3+−Gquadruplex interaction. Such a discrimination based on the peak position offers additional possibilities as compared to fluorescence methods, which usually rely on the sole variation of intensities. Finally, this unambiguous electrochemical discrimination between G-quadruplexes and duplexes immobilized on electrodes opens new perspectives involving electroactive G-quadruplex ligands.

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ASSOCIATED CONTENT

S Supporting Information *

Quantitation of immobilized DNA; scan rate dependences of the peaks Pdiff and Pads; comparisons between ac voltammetry and cyclic voltammetry; rinsing in transfer experiments; influence of the [Ru(NH3)6]3+ concentration on the peak potential of Pconf; results for the duplex TS2. This material is available free of charge via the Internet at http://pubs.acs.org.

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REFERENCES

AUTHOR INFORMATION

Corresponding Author

*Tel: +32 2 650 29 39. Fax: +32 2 650 29 34. E-mail: cbuess@ ulb.ac.be. Present Address §

A.D.R.: Univ. Bordeaux, INSERM, U869, ARNA Laboratory, European Institute of Chemistry and Biology, 2, Rue Robert Escarpit, 33600 Pessac, France. Notes

The authors declare no competing financial interest. 8064



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Electrochemical Discrimination between G-Quadruplex and Duplex

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