The adsorption and the redox behaviour of thrombin-binding aptamer (TBA) and extended TBA (eTBA) were studied using atomic force microscopy and voltammetry at highly oriented pyrolytic graphite and glassy carbon. The different adsorption patterns and degree of surface coverage were correlated with the sequence base composition, presence/absence of K+, and voltammetric behaviour of TBA and eTBA. In the presence of K+, only a few single-stranded sequences present adsorption, while the majority of the molecules forms stable and rigid quadruplexes with no adsorption. Both TBA and eTBA are oxidized and the only anodic peak corresponds to guanine oxidation. Upon addition of K+ ions, TBA and eTBA fold into a quadruplex, causing the decrease of guanine oxidation peak and occurrence of a new peak at a higher potential due to the oxidation of G-quartets. The higher oxidation potential of G-quartets is due to the greater difficulty of electron transfer from the inside of the quadruplex to the electrode surface than electron transfer from the more flexible single strands.
Since it has been recognized that G-rich nucleic acid sequences can adopt intermolecular and intramolecular quadruplex structures stabilized by G-quartets [
Schematic representation of the G-quartet.
Aptamers are nucleic acid sequences (DNA or RNA) selected in vitro from large combinatorial pools to bind to specific targets [
One of the most preeminent examples of the in vitro selection of DNA oligonucleotides for targeting a specific protein is the thrombin-binding aptamer (TBA), Scheme
Schematic representation of the antiparallel G-quadruplex structures of (a) TBA and (b) eTBA.
The present paper is the first report of a voltammetric and atomic force microscopy (AFM) study of two different thrombin-binding aptamer sequences. The process of adsorption of TBA (5
The ODN sequences TBA (5
Microvolumes were measured using EP-10 and EP-100 Plus Motorized Microliter Pippettes (Rainin Instrument Co. Inc., Woburn, USA). The pH measurements were carried out with a Crison micropH 2001 pH-meter with an Ingold combined glass electrode. All experiments were done at room temperature (25 ± 1
HOPG, grade ZYB of dimensions
AFM was performed in the AAC mode AFM, with a PicoScan controller from Agilent Technologies, Tempe, AZ, USA. All the AFM experiments were performed with a CS AFM S scanner with a scan range of 6
The TBA and eTBA modified HOPG surfaces were obtained by spontaneous adsorption, by depositing 200
Voltammetric experiments were carried out using a
The GC electrode was polished using diamond spray (particle size 1
The DP voltammograms were baseline-corrected using the moving average with a step window of 2 mV included in GPES version 4.9 software. This mathematical treatment improves the visualization and identification of peaks over the baseline without introducing any artefact, although the peak height is in some cases reduced (
The capacity of TBA and eTBA molecules to interact and adsorb spontaneously on the HOPG electrode forming different morphological films was investigated by AFM in air. The spontaneous adsorption of the ODN sequences was obtained using concentrations of 1
An atomically flat HOPG electrode was used as a substrate with less than 0.06 nm of root-mean-square (
AFM topographical images in air of the TBA modified HOPG electrode showed that the TBA adsorbs spontaneously onto HOPG, showing the formation of two different adsorbed structures: a thin and incomplete network film, Figure
AFM topographical images in air of TBA molecules, immobilized onto HOPG by spontaneous adsorption during 3 minutes, from solutions of 1
Similar results were obtained for the eTBA molecules, which present four extra guanines in the base sequence. The AFM images in air also showed a densely packed 0.8 ± 0.1 nm height self-assembled network with looped arms, Figure
AFM topographical images in air of eTBA molecules, immobilized onto HOPG by spontaneous adsorption during 3 minutes, from solutions of 1
The adsorption mechanism of the nucleic acid molecules onto HOPG is mainly driven by hydrophobic interactions [
Additionally, G-rich ODN sequences that contain sections of guanines can form G-quadruplex structures comprised of stacked tetrads, each one of the tetrads formed by a planar association of four guanines by Hoogsteen hydrogen bonding. Therefore, the guanine bases that enter in the TBA and eTBA composition influence the ODN hydrophobicity directly, through the intrinsic hydrophobic character of the aromatic ring, and indirectly, by allowing the ODN sequences to establish quadruplex conformations. In this context, the interaction of the single-stranded molecules with HOPG is facilitated because they have the bases exposed to the solution and free to undergo hydrophobic interactions, when compared to the ODNs with quadruplex morphology that have the bases protected by the sugar-phosphate backbones. Indeed, the AFM images of the TBA and eTBA modified HOPG surfaces show the formation of 1.5–1.9 nm height spherical and rod-like shape aggregates due to the adsorption of ODNs presenting G-quartet configurations, as also observed for telomeric G-rich sequences on mica [
The stabilisation of the quadruplex structures requires the presence of metal ions, in particular alkali metals, and the order of preference is K+
AFM images of the TBA modified HOPG obtained after incubation of TBA with 100 mM K+ during 1 hour showed tilted polymeric structures of 0.8 ± 0.2 nm height, Figure
AFM images of the eTBA modified HOPG obtained from solutions of eTBA incubated with 100 mM K+ during 1 hour, Figure
AFM images demonstrated that, after the interaction with K+ ions during long incubation times, the adsorption pattern of both TBA and eTBA molecules corresponded only to the spontaneous adsorption of a small number of single-stranded molecules present in solution, while no quadruplex TBA/eTBA was observed. This is due to the fact that, in the presence of K+ cations, TBA and eTBA form very stable and rigid intramolecular quadruplex configurations, which prevent the interaction of the hydrophobic bases with the HOPG. On the contrary, the presence of only Na+ cations from the solutions of TBA/eTBA in pH 7.0 0.1 M phosphate buffer led to the formation of less stable quadruplex TBA/eTBA morphologies that are locally destabilised by the HOPG hydrophobic surface, inducing their consequent adsorption.
DP voltammograms were recorded in solutions containing 1
Baseline corrected DPV obtained with the GC electrode in a solution of 1
A similar experiment was carried out in which 1
DP voltammograms were recorded in solutions containing 1
Baseline corrected DPV obtained with the GC electrode in a solution of 1
On the other hand, on the voltammograms obtained after 1 hour of incubation of eTBA with K+ ions, a new small peak
In another experiment, 1
The redox behaviour and adsorption process of two thrombin-binding aptamer sequences were studied at room temperature, using AFM and voltammetry at HOPG and GC electrodes, in the presence/absence of Na+ and K+ cations.
Due to the formation of very stable and rigid intramolecular quadruplex configurations in the presence of K+ cations, both TBA and eTBA adsorb less onto HOPG, compared to the adsorption in the presence of only Na+ cations present in the pH 7.0 0.1 M phosphate buffer. This is due to the fact that a larger number of intramolecular quadruplexes is formed, which were more stable due to the incorporation of K+, which prevents their hydrophobic interaction with HOPG. On the contrary, the presence of only Na+ cations led to the formation of less stable quadruplexes that are locally destabilised by the HOPG hydrophobic surface, inducing their adsorption together with single-stranded sequences.
Voltammetric studies showed that both TBA and eTBA are oxidized at GC electrode and the only electrochemical signal is due to the oxidation of guanine residues. Upon addition of K+ ions, both TBA and eTBA folded into G-quadruplex structures and this process was observed by the decreasing of the guanine oxidation peak and the occurrence of a new peak at higher potential values due to the oxidation of G-quartets. The difference in the oxidation potential of guanine and G-quartets is attributed to the difficulty of the transition of electrons from the inside of the rigid quadruplex to the GC electrode surface than from the more flexible form of single-stranded ODNs whose guanine residues can reach the surface leading to higher peak currents.
Financial support from Fundação para a Ciência e Tecnologia (FCT), Post-Doctoral Grant SFRH/BPD/36110/2007 (V. C. Diculescu), projects PTDC/QUI/65255/2006 and PTDC/QUI/098562/2008, POCI 2010 (cofinanced by the European Community Fund FEDER), CEMUC-R (Research Unit 285), and COST Action MP0802 (Self-assembled guanosine structures for molecular electronic devices (G4-net)) is gratefully acknowledged.