Journal of Inorganic Biochemistry 126 (2013) 84–90

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Journal of Inorganic Biochemistry

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On the interaction between [Ru(NH3)6]3+ and the G-quadruplexforming thrombin binding aptamer sequence

Aurore De Rache a, Thomas Doneux a, Iva Kejnovská b, Claudine Buess-Herman a,⁎a Chimie Analytique et Chimie des Interfaces, Faculté des Sciences, Université Libre de Bruxelles, CP 255, Boulevard du Triomphe 2, B-1050 Bruxelles, Belgiumb Institute of Biophysics, Academy of Sciences of the Czech Republic v.v.i., Královopolská 135, 612 65 Brno, Czech Republic

⁎ Corresponding author. Tel.: +32 2 650 29 39; fax: +E-mail address: cbuess@ulb.ac.be (C. Buess-Herman

0162-0134/$ – see front matter © 2013 Elsevier Inc. Allhttp://dx.doi.org/10.1016/j.jinorgbio.2013.05.014

a b s t r a c t

a r t i c l e i n f o

Article history:Received 19 November 2012Received in revised form 23 May 2013Accepted 24 May 2013Available online 30 May 2013

Keywords:BiosensorsThrombin binding aptamerHexaamminerutheniumG-quadruplexCircular dichroismVoltammetry

The interaction between the thrombin binding aptamer (TBA), a G-quadruplex forming DNA sequence, andthe electroactive hexaammineruthenium(III) cation has been studied by electrochemical methods and circu-lar dichroism spectroscopy. When TBA is immobilised on a gold surface in a typical aptasensor configuration,the [Ru(NH3)6]

3+ cation can be bound to the electrode surface through its interaction with the TBA sequence.This interaction is strong enough to enable the rutheniumcomplex to remain at the surfacewhen the electrode isimmersed in an electrolyte free of [Ru(NH3)6]3+, meaning that the complex does not diffuse back into thesolution. A stoichiometry of 2 [Ru(NH3)6]3+ per TBA strand has been determined, indicating that the inter-action differs from the conventional, non-specific electrostatic charge compensation, for which a 5 to 1 ratiowould be expected between the triply charged cation and the 15 bases sequence. It is shown that this inter-action takes place not only at the surface, but also when both TBA and hexaammineruthenium(III) aredissolved in solution. Under such conditions, a similar stoichiometry of 2 [Ru(NH3)6]

3+ per TBA strandhas been evidenced by two independent methods, namely circular dichroism spectroscopy and differentialpulse voltammetry.

© 2013 Elsevier Inc. All rights reserved.

1. Introduction

Aptamers are a new class of affinity probes made of nucleic acid se-quences selected by an in vitro combinatorial process for their selectiv-ity against a given target [1–3]. Aptamers attract a lot of interest aspossible substitutes to antibodies in the construction of protein biosen-sors [4]. The first aptamer selected against a specific protein was thethrombin binding aptamer (TBA) [5], which has been intensively stud-ied and is nowadays often used as a model system for the developmentof new aptamer-based biosensing strategies. In a biosensing device, thenucleic acid probe is immobilised on a surface and a convenient and ef-ficient transduction of the binding event is required. In this perspective,electrochemical methods are particularly attractive in terms of cost,miniaturisation and sensitivity [3,6]. The electrochemical detection isfrequently evidenced by changes in the faradaic responses of dissolvedelectroactive compounds such as [Fe(CN)6]3−/4−or [Ru(NH3)6]3+, cho-sen because they undergo a simple one electron transfer reaction. Inthe case of [Fe(CN)6]3−/4−, the binding is followed through the varia-tions of the electron transfer kinetics, which are dependent on the elec-trostatic repulsions between the redox marker and DNA. By contrast,

32 2 650 29 34.).

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electrostatic attractions exist between DNA and [Ru(NH3)6]3+, formingthe basis of a popular method devised by Tarlov and coworkers [7]for quantifying the amount of single-stranded and duplex DNAimmobilised at electrode surfaces. According to that publication [7],the faradaic response of the “redox marker is directly proportional tothe number of phosphate groups present at the surface” [7], one triplycharged hexaammineruthenium cation being able to compensate ex-actly three singly negatively charged phosphate moiety. The methodol-ogy of Tarlov and colleagues has been employed in numerous instances[7–10], under the assumption that the measurement is valid regardlessof the investigated nucleic acid sequence (base composition), strandmultiplicity (single strand, double strand,…) or conformation. Suchan assumption implies that hexaammineruthenium(III) is not sensitiveto any nucleic acid characteristic except the negative charges borne bythe phosphate groups and binds to DNA only through non-specific,purely electrostatic interactions. Although it is likely the case in manysituations, this hypothesis disregards the significant diversity of nucleicacid structures. As an example, it is well known in the chemical biologycommunity that the hexaammineruthenium(III) cation, like its closeanalog [Co(NH3)6]3+, is a coordination complex which can influencethe conformation of DNA at low concentrations, being for instance agood Z-DNA stabiliser [11,12]. Crystallographic studies have shownthat the stabilisation is promoted not only by non-specific electrostaticinteractions, but also by the formation of hydrogen bonds between themetal complex and the nucleic acid sequences [13,14].

85A. De Rache et al. / Journal of Inorganic Biochemistry 126 (2013) 84–90

It appears therefore legitimate to challenge the assumptionthat the simple charge compensation scheme is valid for all DNAsequences. The question is particularly relevant to G-quadruplexstructures, which involve non-Watson–Crick interactions and arecharacterised by high charge densities. The interaction of [Ru(NH3)6]3+

withG-quadruplexes has not been investigated so far, except for a recentpublication from our group [15], where we have investigated the influ-ence of various cations on the folding of TBA itself, and of twoelongated sequences containing each a set of six additional bases atthe 5′-extremity of TBA. While these three sequences fold in ananti-parallel G-quadruplex structure in the presence of high concentra-tions (millimolar range) of K+, circular dichroism results showed thatthe hexaammineruthenium(III) cation in particular has a markedinfluence on the quadruplex structures, being able to stabilise at verylow concentrations (micromolar range) the folding of the elongatedsequences in a parallel quadruplex. Such finding suggests that, at leastfor TBA, the interaction with [Ru(NH3)6]3+ might be different from asimple charge compensation effect.

Addressing this issue is important for the development of electro-chemical aptasensors, and the present work is thus devoted to thestudy of the interaction between the electrochemically importanthexaammineruthenium(III) cation and the G-quadruplex formingTBA sequence, taken as a model aptamer. In a typical biosensor con-figuration, the probe is immobilised at the electrode surface toallow the electrochemical transduction, therefore the interaction be-tween TBA and [Ru(NH3)6]3+ has to be studied “at the surface”, i.e.with TBA chemically attached to the electrode. It will be shown thatthe interaction departs markedly from the simple charge compensa-tion assumption, and that [Ru(NH3)6]3+ can be strongly bound toTBA at the surface. Such strong interaction is however not limited tosurface-attached TBA, but also takes place when both TBA and[Ru(NH3)6]3+ are dissolved in solution. Beside the obvious implica-tions in the field of electrochemical biosensing, our work bears rele-vance to the ongoing field of G-quadruplex–ligand interactions, wheremetal complexes are particularly interesting due to their versatilestructural, optical, and/or electrochemical properties [16,17].

2. Material and methods

2.1. Reagents

Two Thrombin Binding Aptamer sequences were investigated inthe present study: the unmodified TBA (Sequence 1) and a 5′-thiolmodified TBA (Sequence 2):

Sequence 1: 5′-GGT TGG TGT GGT TGG-3′Sequence 2: 5′-HS-(CH2)6-GGT TGG TGT GGT TGG-3′

Sequence 1 was used for the experiments carried out in solutionwhile Sequence 2 was used to prepare mixed self-assembled mono-layers (SAMs). The sequences, purified by HPLC, were purchased fromEurogentec s.a. (Belgium) and used as received. The lyophilised sampleswere dissolved to a concentration of 1 mM (per strand) in 10 mM Trisbuffer pH 7.4. The stock solutions were kept frozen below −20 °C.

All solutions were prepared with ultrapure water from a MilliporeMilli-Q system. The hexaammineruthenium(III) chloride was obtainedeither from Alfa Aesar (>32.1% Ru, for electrochemical measurements)or from Sigma (98%, CD measurements) and was used as received.The 4-mercaptobutan-1-ol (>97%, Fluka) was stored at 4 °C. All otherchemicals were of analytical reagent grade.

2.2. Electrochemical measurements

The electrochemical experiments were performed in a three-electrode cell connected to an Autolab PGSTAT 30 (Eco Chemie, The

Netherlands) potentiostat equipped with Scangen and FRA modules.The working electrodes were polycrystalline Au disc electrodes witha 1.6 mm (Bioanalytical Systems, UK) or 5.0 mm diameter (EDI,Tacussel, France). A platinum grid or a gold wire, with large areas,was used as auxiliary electrodes. All potentials given in this workrefer to the double-bridge saturated calomel electrode (SCE) used asreference electrode (Radiometer REF451).

The temperature was fixed at 5.0 ± 0.1 °C with a Julabo F10-UCthermostat-cryostat. This temperature was chosen to permit thedirect comparison between electrochemical and spectroscopic re-sults. Electrolyte solutions were purged with water-saturated nitro-gen for at least 15 min and then kept under nitrogen during themeasurements.

2.2.1. Electrode preparation and modificationThe preparation of the gold polycrystalline electrode surface be-

fore each modification consisted in polishing with 1.0 μm alumina–water slurry on a smooth polishing cloth (Struers) followed by soni-cation for 10 min and abundant rinsing with Milli-Q water. Finally,the electrode was electrochemically cleaned by cycling the potentialbetween −0.3 V and +1.5 V in a 0.1 M HClO4 solution at a scanrate of 50 mV s−1, until reproducible cyclic voltammograms wererecorded. The gold electrode surface was rinsed with Milli-Q waterand dried under a nitrogen stream before the SAM formation.

In the case of surface measurements, the thiol-modified DNAprobes were mixed with 4-mercaptobutan-1-ol (MCB) at 1–1 molefraction to obtain a 20 μM total concentration in 1 M potassium phos-phate buffer. A volume of 50 μL of this mixture was then placed, in asealed plastic tube, onto the clean gold electrode surface, and leftovernight (16 h) for chemisorption by self-assembly.

For the solution measurements, the clean gold electrode wasimmersed overnight in 1 mM MCB (in water). After immersion, theelectrode was thoroughly rinsed with the electrolyte, dried withnitrogen and transferred in the electrochemical cell. This surfacemodification was necessary to avoid DNA adsorption. Due to itssmall length, MCB does not significantly hinder the electron transferbetween the electrode and the [Ru(NH3)6]3+.

2.2.2. ChronocoulometryBefore each measurement, the electrolyte solution containing

[Ru(NH3)6]3+ was stirred for 2 min to favour the accumulation ofthe cationic redox complex at the modified electrode surface. Longerstirring times did not increase the amount of accumulated complex.Immediately prior to the measurement, the stirring was stopped for30 s to reach mechanical equilibrium. The potential was steppedfrom +0.05 V to −0.45 V. The current was measured during 2 swith a 0.0005 s sampling interval time, and the resulting transientwas integrated to obtain the charge transient. A similar potentialstep was performed in the absence of [Ru(NH3)6]3+, to account forthe double layer charging. The data were analysed according to theprocedure of Steel et al. [7] (described in the Supplementary data)to evaluate the surface concentration of [Ru(NH3)6]3+.

2.2.3. Ac voltammetryAc voltammograms were recorded at 37 Hz and 10 kHz with a po-

tential perturbation of 5 mV (root mean square). A low frequencywas chosen to maximise the capacitive and the reversible faradaiccontributions and the high frequency to obtain an estimation of theuncompensated resistance of the cell. One data point was recordedevery 10 mV in the potential range +0.05 V to−0.40 V, a full poten-tial scan taking approximately 6 min. At each potential, the in-phaseand out-of-phase components of the alternating current were mea-sured, and the data converted to the real and imaginary parts of thecell impedance, Z′tot and Z″tot. To obtain the interfacial componentsof the impedance, Z′el and Z″el, the following relations wereemployed: Z′el = Z′tot − Rs and Z″el = Z″tot. The ohmic resistance Rs

86 A. De Rache et al. / Journal of Inorganic Biochemistry 126 (2013) 84–90

was estimated from the real part of the cell impedance measuredat 10 kHz. Finally, the interfacial admittance components were calcu-lated from the interfacial impedance by:

Y ′el ¼

Z′el

Z′2el þ Z″2

el

Y″el ¼

Z″el

Z′2el þ Z″2

el

:

2.2.4. Differential pulse voltammetry (DPV)Differential pulse voltammograms were recorded between +0.10

and −0.45 V with a 10 mV step and a modulation amplitude of25 mV. The pulse (modulation) time and rest (interval) time were0.06 and 1 s, respectively. The current was sampled in the last 20 msof these periods.

2.3. Circular dichroism (CD)

The sample was prepared by dilution of the stock solution in10 mM Tris buffer pH 7.4 to a final concentration giving an optimalabsorbance between 0.7 and 0.8 at 260 nm. The exact concentrationof each sample was determined using a Unicam 5626 UV/visiblespectrometer (with ε = 143,300 M−1 cm−1). The CD-spectra weremeasured at 5 °C with a Jobin-Yvon Mark VI dichrograph in 1 cmpathlength quartz Hellma cells and converted into Δε according tothe Beer–Lambert equation.

Fig. 1. Cyclic voltammogram recorded with a TBA-modified electrode in 10 mM Trisbuffer (pH 7.4) + 50 μM [Ru(NH3)6]3+. T = 5.0 °C; scan rate 50 mV s−1. Inset: Influ-ence of the scan rate on the peak currents. Top: Representation of a TBA-modifiedelectrode. Mixed self-assembled monolayer composed of thiolated TBA (sequence 2)and 4-mercaptobutan-1-ol (MCB).

3. Results and discussion

3.1. Interaction of [Ru(NH3)6]3+ with surface-attached TBA

Fig. 1 shows a cyclic voltammogram of the TBA-modified electrode(situation depicted in the scheme of Fig. 1) recorded in the presenceof [Ru(NH3)6]3+. The reduction of the redox marker gives rise totwo distinct peaks P1 and P2 located at −0.19 V and −0.32 V,respectively. The influence of the scan rate on the peak intensitiesreveals that P1 corresponds to the diffusion-controlled reduction of[Ru(NH3)6]3+ present in solution, as attested by the square-rootdependence of the peak current on the scan rate [18]. By contrast,P2 is associated with the reduction of the redox species adsorbed atthe electrode surface, as inferred from the linear relationship be-tween the peak current and the scan rate (inset in Fig. 1) and fromthe identical values of the peak potentials observed for P2 and for itsanodic counterpart [18]. Such adsorption post-peak P2 originatesfrom the non-specific electrostatic interactions between the positive-ly charged hexaammineruthenium(III) complex and the negativelycharged backbone of TBA. This behaviour is typical of negativelycharged SAMs [8,19], and the charge associated with the peak P2can be used to evaluate the amount of DNA present at the surfaceassuming a total charge compensation hypothesis (i.e. a 1 to 3 ratiobetween the marker and the phosphate groups of the DNA backbone)[7,8].

Because of the overlap between the two peaks, the determinationof the charge of the post-peak from the voltammetric curves is notstraightforward. Chronocoulometry is however better suited for thispurpose (see Section 2.2.2 and Supplementary data). Fig. 2 presentsthe results of chronocoulometric experiments conducted in thepresence of increasing concentrations of [Ru(NH3)6]3+ in solution.The charge corresponding to the reduction of adsorbed [Ru(NH3)6]3+,σads, obtained from the chronocoulometric data, increases with thebulk concentration until it reaches a plateau. This curve is similar toan adsorption isotherm. The plateaumay be assigned here to a total sur-face charge compensation, which in the present case of a 15 bases DNAsequence leads to 5 adsorbed [Ru(NH3)6]3+ per TBA strand. The chargedensity, σads, and the aptamer surface concentration, ΓTBA, are thuslinked by the relation ΓTBA = σads / 5 nF (see Supporting data), wheren is the number of electron transferred in the reduction reaction (1 inthe present case) and F is the Faraday constant (96,485 C mol−1).From the charge density at saturation of (8.7 ± 0.2) μC cm−2, anaptamer surface concentration of (1.80 ± 0.04) × 10−11 mol cm−2 isobtained.

Fig. 2. Charge density of hexaammineruthenium electrostatically adsorbed at theTBA-modified electrode, determined by chronocoulometry, as a function of the ruthe-nium complex concentration in 10 mM Tris buffer (pH 7.4). T = 5 °C.

Fig. 4. Cyclic voltammograms recorded with a TBA-modified electrode in 10 mM Trisbuffer (pH 7.4), at scan rates of 20, 30, 40, 50, 60, 70, 80, 90 and 100 mV s−1. T = 5.0 °C.Inset: Influence of the scan rate on the peak currents.

87A. De Rache et al. / Journal of Inorganic Biochemistry 126 (2013) 84–90

While the above behaviour is quite usual for negatively chargedlayers, a different interaction between TBA and [Ru(NH3)6]3+ wasevidenced by ac voltammetry. The ac method is particularly sensitiveto the presence of adsorbed species, especially when monitoring theimaginary component of the interfacial admittance, Yel″ [20,21]. Fig. 3presents a series of successive ac voltammograms recorded with aTBA-modified electrode in the presence of [Ru(NH3)6]3+ in solution.The very first ac voltammogram (dotted line) exhibits a maximumat −0.32 V corresponding to the reduction of the electrostaticallyadsorbed [Ru(NH3)6]3+, in agreement with the behaviour observed bycyclic voltammetry, and a small shoulder around −0.16 V. Significantchanges in the voltammogrammorphology are noticeable upon record-ing successive ac voltammograms: the admittance at the maximumdecreases markedly while at less negative potentials a peak progres-sively replaces the initial shoulder. The changes between two successivescans are large at the beginning, later they progressively becomeless pronounced, until a steady-state ac voltammogram is finallyobtained after ca 75 scans. This ac voltammogram now displays twomaxima located at −0.30 V and −0.14 V. Such evolution of the acvoltammograms was very reproducible, being systematically observedwith the TBA-modified electrodes. The temperature was howeverfound to influence the process; at 20 °C, around 20 scans are neededto reach a steady-state response. The differences between the initialand final voltammograms indicate that the interfacial interactions be-tween TBA and the redox marker have been modified upon scanning.To obtain some insights into these modifications, the TBA-modifiedelectrodes were, at this stage, abundantly rinsed with the buffer andtransferred to a cell containing only the pure electrolyte (i.e. in the com-plete absence of [Ru(NH3)6]3+ in solution) and cyclic voltammogramswere recorded.

Fig. 4 presents the typical electrochemical behaviour observedunder these conditions. A time-stable voltammogram, showing a re-duction process in the forward scan and its subsequent re-oxidationduring the reverse scan is obtained. The linear dependence of thepeak currents on the scan rate (inset in Fig. 4) demonstrates thatthe peaks are associated with adsorbed electroactive species. Thevalues of the peak potentials, −0.196 V and −0.189 V for the reduc-tion and oxidation peaks, respectively, markedly differ from the valueof the post-peak P2 observed in Fig. 1. These results, together withthe fact that the voltammograms are recorded in the absence of[Ru(NH3)6]3+ in solution, indicate that the voltammetric responsesdo not involve [Ru(NH3)6]3+ in interaction with the DNA aptamerthrough the usual non-specific electrostatic attraction. Indeed, such

Fig. 3. Successive ac voltammograms recorded with a TBA-modified electrode in10 mM Tris buffer (pH 7.4) + 100 μM [Ru(NH3)6]3+. Starting from the dotted curve,one voltammogram is plotted every 5 measurements. T = 5.0 °C; f = 37 Hz.

interaction implies an equilibrium between the adsorbed and dissolvedspecies [8,19] and hence requires a certain amount of [Ru(NH3)6]3+

dissolved in solution, as shown above in Fig. 2. The possibility of anincomplete rinsing with some remaining traces of electrostaticallyadsorbed hexaammineruthenium(III) can also be ruled out, because inthat case the peaks would vanish after a few cycles [8], contrary to thepresent observations of time-stable voltammograms. As a controlexperiment, an electrode modified with MCB alone (no TBA) wassubjected to the same treatment. No peak could be seen in thevoltammogramafter the transfer into the pure electrolyte. For the inter-pretation of the data, we have to consider that the peaks result from astrong association between the TBA strand and the [Ru(NH3)6]3+

complex. In such situation, the redox complex will be referred toas “confined” [Ru(NH3)6]3+ to avoid any confusion with the usualbehaviour. This confined redox complex is strongly adsorbed to theTBA-modified surface. No loss is observed upon continuous cycling forat least 4 h or after overnight immersion into the electrolyte.

The amount of confined marker, Γconf, was calculated from theaverage of the reduction and oxidation charges determined at differ-ent scan rates, σconf, using the relation Γconf = σconf / nF. A chargedensity of (3.8 ± 0.3) μC cm−2 was measured, corresponding to a[Ru(NH3)6]3+ surface coverage of (4.0 ± 0.3) × 10−11 mol cm−2.Comparing this value to the aptamer surface coverage estimatedabove, (1.80 ± 0.04) × 10−11 mol cm−2, an interaction stoichiome-try of 2.2 ± 0.2 [Ru(NH3)6]3+ per TBA strand is found when theconfined marker is detected. Such stoichiometry is significantlydifferent from the 5:1 ratio expected for a random electrostaticcharge compensation. This fact gives further credit to the hypothesisthat the interaction between TBA and the metal complex, evidencedby ac voltammetry, differs from the usual non-specific electrostaticinteractions.

3.2. Interaction between [Ru(NH3)6]3+ and TBA in solution

The above results have shown that the [Ru(NH3)6]3+ can be tightlybound to TBA-modified surfaces. However, the interaction between[Ru(NH3)6]3+ and TBA which underlies this behaviour should not belimited to surface-bound TBA, but should occur as well when both theredox complex and the aptamer are dissolved in solution (situationdepicted in the scheme of Fig. 5). This statement was verified byperforming voltammetric titration experiments in solution (i.e. without

Fig. 5. Differential pulse voltammetric titrations of [Ru(NH3)6]3+ by TBA ((a), (b)), and of TBA by [Ru(NH3)6]3+ ((c), (d)). DPV was recorded with a MCB-modified electrode in10 mM Tris buffer (pH 7.4) containing: (a) 40 μM [Ru(NH3)6]3+ and 0, 0.5, 1.0, 1.5, 2.0, 2.5, 5.0, 7.5, 10, 12.5, 15, 17.5, 20, 22.5, 25, 27.5, 30, 32.5, 35, 37.5 and 40 μM TBA (increasingconcentrations indicated by the arrow); the dotted line is recorded in the absence of both TBA and [Ru(NH3)6]3+; (b) 10 μM TBA and 0, 2.5, 5.0, 10, 12.5, 15, 17.5, 20, 25, 30, 35, 40,50, 60, 65, 70, 75, 80, 85, 90, 95, 120 and 145 μM [Ru(NH3)6]3+ (increasing concentrations indicated by the arrow). The corresponding evolutions of the peak currents with thetitrant concentrations are shown in (b) and (d). T = 5 °C. Top: Representation of a MCB-modified electrode, used to prevent the non-specific adsorption of dissolved TBA (sequence1) to the electrode surface.

88 A. De Rache et al. / Journal of Inorganic Biochemistry 126 (2013) 84–90

attachment of TBA to the electrode surface), whose results are shown inFig. 5a. The first curve, measured in the absence of TBA in solution, re-flects the electrochemical reduction of the free [Ru(NH3)6]3+, presentin solution at a 40 μM concentration. The peak potential is locatedat −0.16 V, in agreement with the peak P1 observed above in Fig. 1.Upon addition of TBA, the peak current decreases and the peak po-tential shifts towards more negative potentials, reaching a final valueof−0.23 V at a 1:1 ratio between the TBA and hexaamminerutheniumconcentrations. This behaviour is consistent with the simple squarescheme mechanism described in Scheme 1 [22–24].

The reduction of the free [Ru(NH3)6]3+ is characterised by the

formal potential E0′

Ru NH3ð Þ6½ �3þ=2þ . Upon interaction with TBA, the formal

potential takes the form:

E0′

Ru·TBA ¼ E0′

Ru NH3ð Þ6½ �3þ=2þ−RTF

lnK3

K2

where K3 and K2 are the association constants between TBA and theoxidised or reduced complex, respectively. The shift towards morenegative potentials thus indicates that the association constantbetween TBA and the marker is higher for the oxidised form (K3)than for the reduced one (K2). Regarding the decrease of the peakcurrent, it reveals a lower diffusion coefficient for [Ru(NH3)6]3+ inthe presence of TBA, which is in agreement with the formation of alarger entity.

The interaction stoichiometry can be deduced from the titration ex-periment by plotting the peak current as a function of the added quanti-ty of TBA [25]. The equivalence point is found for a TBA concentration of(18.6 ± 0.7) μM,which corresponds to a stoichiometry of (2.15 ± 0.08)[Ru(NH3)6]3+ interactingwith each TBA strand (Fig. 5b). A similar resultwas obtained by performing the reverse titration, i.e. by addition of in-creasing amounts of [Ru(NH3)6]3+ to a fixed 10 μM TBA concentration(Fig. 5c). In this case, as expected, the peak current increases with theredox marker concentration and the peak potential moves towards

Scheme 1. Square-scheme representing the chemical equilibria involving hexaammineruthenium in the presence or absence of TBA. The rectangles symbolise the whole coordina-tion complex and the dots (●) the association of TBA with the complex. Vertical arrows indicates electron transfer reactions of the free or bound [Ru(NH3)6]3+, characterised bytheir respective formal potentials, and horizontal arrows are associated with the association/dissociation of the TBA/[Ru(NH3)6]3+ entity, characterised by the association constantsK3 (for the oxidised complex) and K2 (for the reduced complex).

89A. De Rache et al. / Journal of Inorganic Biochemistry 126 (2013) 84–90

less negative potentials. The equivalence occurs at a (20.0 ± 1.2) μM[Ru(NH3)6]3+ concentration and leads to (2.0 ± 0.1) redox markersper strand (Fig. 5d), confirming the stoichiometry found earlier.

A final evidence of this interaction between [Ru(NH3)6]3+ and TBAwas obtained by a non-electrochemical method, namely circular di-chroism (CD) spectroscopy. While the electrochemical data gatheredabove reflected the response of the redox complex, the CD-spectracharacteristics originate from the folding adoptedby the nucleic acid se-quence. Different G-quadruplex conformations (parallel, anti-parallel,3 + 1) can be distinguished according to their CD signature [26–28].Although CD spectroscopy alone is sometimes not sufficient to assignreliably a particular quadruplex conformation [29], TBA has been thesubject of numerous investigations [15,30–33] and its spectral proper-ties are now well-established. Here, we monitored the changes in theCD-spectra while increasing the ratio between [Ru(NH3)6]3+ and TBAconcentrations (Fig. 6).

In the absence of the redox marker (dotted line), two maxima lo-cated at 247 and 293 nm and one minimum at 267 nm are observed.This shape of the CD-spectrum is in agreement with the literature[34] and was attributed to an anti-parallel quadruplex. When increas-ing quantities of [Ru(NH3)6]3+ are added, the intensities of the peakcharacteristic of this folding decrease, reflecting a modification inthe relative orientations of the electronic transition dipole momentsof stacked guanines [29,35]. The shape of the curve after addition of

Fig. 6. Circular dichroism spectra of TBA recorded in 10 mM Tris buffer (pH 7.4), in thepresence of increasing concentrations of [Ru(NH3)6]3+, expressed in equivalents perTBA strand (0, 0.5, 1, 2, 3, 4, 5, 6, 10). T = 5.0 °C Inset: Evolution of Δε at 293 nmwith the ruthenium complex concentration.

10 [Ru(NH3)6]3+ per TBA strand is in agreement with the results ofour recent study [15].

The evolution of the peaks at 247 nm and 267 nm correlates withthat of the positive peak at 293 nm. The interaction between TBA andthe redox marker is followed through the intensity of this third peak,more intense. The inset in Fig. 6 shows that the values of Δε decrease,before reaching a plateau. The intercept between the plateau valueand the linear decrease at low marker concentrations reveals a stoi-chiometry of 2 [Ru(NH3)6]3+ per strand for the interaction betweenthe marker and the TBA sequence, which is perfectly consistentwith the electrochemical results.

At present, the exact nature of the binding is unclear but it is obviousthat it differs from the usual random electrostatic interaction. At thisstage, it is worth drawing an analogy with the abundant literatureregarding the interaction of [Co(NH3)6]3+ and, to a lesser extent[Ru(NH3)6]3+, with DNA structures such as Z-DNA or A-DNA[11,13,14,36–44]. Those hexaammine complexes have been identifieda long time ago as very efficient inducers of the Z-DNA conformation[11,37,40]. Although the electrostatic free energy is certainly an impor-tant contribution to the stabilisation of Z-DNA [36], crystallographicstudies have demonstrated the occurrence of specific hydrogen bond-ing between the ammine ligands of the complexes and the H-bondacceptor sites of DNA, which are the N7 and O6 of guanine bases andthe oxygen atoms of the phosphate groups [13,38,40]. For other nucleicacid sequences, similar hydrogen bond patterns have been also ob-served in solution by FTIR [44] and NMR [43] experiments. Braunlinand coworkers [41,42] have revealed by 59Co NMR a significant hetero-geneity in the binding modes of [Co(NH3)6]3+ to DNA. For instance,they showed that for GC-rich (72%) DNA sequences a class of slowlyexchanging hexaamminecobalt(III) cations prevails at low bindingdensities, whereas with lower GC contents only rapidly exchangingcomplexes and high binding densities were obtained [41]. In anotherwork, it was demonstrated that in the presence of [Co(NH3)6]3+ theCD spectra of certain oligonucleotides were affected, while it was notthe case for other sequences. Large chemical shifts in the NMR responseof the cobalt complexwere observedwith the former sequences but notwith the latter ones [42].

Our recent CD results [15] showing that [Ru(NH3)6]3+ stabilises theparallel folding of some G-quadruplexes suggest that the quadruplexcharacter might be essential in the formation of the TBA/[Ru(NH3)6]3+

entity reported here. Like Z-DNA, G-quadruplexes have H-bondacceptors (phosphate and guanine N3 atoms) located in the externalgrooves, and these atoms have been already identified as binding sitesfor water, ions and amines [45]. More work is certainly needed toclarify the exact nature of the binding, and other DNA sequences areunder investigation in order to identify whether key structural featuresare required for this interaction.

90 A. De Rache et al. / Journal of Inorganic Biochemistry 126 (2013) 84–90

4. Conclusions

While it is generally assumed that [Ru(NH3)6]3+ interacts withDNA in a non-specific, purely electrostatic manner, we have demon-strated in the present work that this is not the case with TBA, forwhich a different interaction is reported for the first time. Regardingthe use of aptamers in biosensing, a proper evaluation of the electro-chemical response of an aptasensor to a given redox marker is ofutmost importance for assessing and improving its analytical perfor-mances. The present finding is thus significant in this respect.

Acknowledgements

A.D.R. acknowledges Wallonie-Bruxelles International for fundingher stays at the Institute of Biophysics of Brnö. This work was sup-ported by a grant from the Belgian National Science Foundation(FRFC Project).

Appendix A. Supplementary data

Supplementary data to this article can be found online at http://dx.doi.org/10.1016/j.jinorgbio.2013.05.014.

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