This journal is c the Owner Societies 2013 Phys. Chem. Chem. Phys. Cite this: DOI: 10.1039/c3cp50866h Self-assembled G-quadruplex nanostructures: AFM and voltammetric characterization Ana-Maria Chiorcea-Paquim, a Paulina Viegas Santos, a Ramon Eritja b and Ana Maria Oliveira-Brett* a G-rich oligodeoxynucleotides (ODNs) have great medical and nanotechnological potential, because they can self-assemble into G-quadruplexes and higher-order nanostructures. The folding properties of d(G) 10 , d(TG 9 ) and d(TG 8 T) ODNs were studied using atomic force microscopy (AFM) and voltammetry at carbon electrodes. Single-stranded ODNs, in Na + containing solutions and for short incubation times, were detected using AFM as network films and polymeric structures and using voltammetry by the occurrence of only the guanine oxidation peak. G-quadruplexes, in Na + containing solutions and long incubation times, or in K + containing solutions, were detected using AFM as spherical aggregates and using voltammetry by the decrease of the guanine oxidation peak and the occurrence of the G-quartet oxidation peak. Concerning the self-assembling into higher-order nanostructures, d(G) 10 was the only sequence forming G-nanowires observed using AFM, d(TG 9 ) formed short G-based super-structures that adsorbed as rod-like shape aggregates, and d(TG 8 T) formed no nanostructures, due to the presence of thymine residues at both 5 0 and 3 0 ends. Introduction Guanine (G) rich nucleic acid sequences that contain G repeats can form four-stranded nucleic acid secondary structures named G-quadruplexes (Scheme 1, left). 1–6 The building blocks of G-quadruplexes are structures known as G-quartets (G q ) (Scheme 1, right), which are planar association of four G bases held together by eight Hoogsteen hydrogen bonds. The G-quartets are stacked on top of each other by p–p hydrophobic interactions, being stabilized by monovalent cations, such as K + and Na + . The cations are located in between the G-quartet planes, and form cation–dipole interactions with the 8G of the two adjacent G-quartets, therefore enhancing the hydrogen bond strength and stabilizing the G-quartet stacking. A variety of G-quadruplex structures exist and can be classified in terms of their molecularity (i.e. the number of associated strands leading to the formation of monomer, dimer or tetramer G-quadruplexes), the strand polarity (i.e. the relative arrangement of adjacent strands in parallel or antiparallel orientations), the glycosidic torsion angle (anti or syn), and the orientation of the connecting loops (lateral, diagonal or both). 2,7–9 In the recent years, the G-quadruplexes have become the focus of attention because they are found in telomeric regions of chromosomes, oncogene promoter sequences, and other biologically relevant regions of the genome, such as immuno- globulin switch regions, ribosomal DNA and RNA. 1–3 In addition, G-rich oligodeoxynucleotides (ODNs) are able to self-assemble into higher-order nanostructures, such as two-dimensional reticulated networks and rigid, long nanowires, being excellent building blocks for the development of future DNA hybrid electronic devices in nanotechnology. 10–13 The importance of the G-quadruplexes for the development of novel devices, with medical and nanotechnology applica- tions, requires a comprehensive knowledge of the structural and folding properties of G-rich ODNs, the structural poly- morphism, and the stability and disruption of the DNA self-assemblies. In this context, atomic force microscopy (AFM) can resolve with extraordinary resolution and accuracy the surface morphological characteristics of nucleic acid nanostructures. Self-assembled G-quadruplex structure AFM images were obtained at insulating, hydrophilic mica surfaces. 13–17 However, the investigation of formation of the inter-molecular G-quadruplexes and their adsorption and stability on the surface of conducting hydrophobic carbon, such as HOPG, electrodes has not been studied and these complicated processes require special attention from the point view of nanotechnology and biosensor technology applications. a Departamento de Quı ´mica, Faculdade de Cie ˆncias e Tecnologia, Universidade de Coimbra, Coimbra, Portugal. E-mail: [email protected]; Fax: +351 239827703; Tel: +351 239854487 b Institute for Research in Biomedicine, IQAC-CSIC, CIBER-BBN Networking Centre on Bioengineering, Biomaterials and Nanomedicine, Barcelona, Spain. E-mail: [email protected]; Fax: +34 932045904; Tel: +34 934039942 Received 27th February 2013, Accepted 15th April 2013 DOI: 10.1039/c3cp50866h www.rsc.org/pccp PCCP PAPER Downloaded by Universidade de Coimbra on 03/05/2013 16:19:09. Published on 03 May 2013 on http://pubs.rsc.org | doi:10.1039/C3CP50866H View Article Online View Journal
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This journal is c the Owner Societies 2013 Phys. Chem. Chem. Phys.
Ana-Maria Chiorcea-Paquim,a Paulina Viegas Santos,a Ramon Eritjab andAna Maria Oliveira-Brett*a
G-rich oligodeoxynucleotides (ODNs) have great medical and nanotechnological potential, because they can
self-assemble into G-quadruplexes and higher-order nanostructures. The folding properties of d(G)10, d(TG9)
and d(TG8T) ODNs were studied using atomic force microscopy (AFM) and voltammetry at carbon electrodes.
Single-stranded ODNs, in Na+ containing solutions and for short incubation times, were detected using AFM
as network films and polymeric structures and using voltammetry by the occurrence of only the guanine
oxidation peak. G-quadruplexes, in Na+ containing solutions and long incubation times, or in K+ containing
solutions, were detected using AFM as spherical aggregates and using voltammetry by the decrease of the
guanine oxidation peak and the occurrence of the G-quartet oxidation peak. Concerning the self-assembling
into higher-order nanostructures, d(G)10 was the only sequence forming G-nanowires observed using AFM,
d(TG9) formed short G-based super-structures that adsorbed as rod-like shape aggregates, and d(TG8T)
formed no nanostructures, due to the presence of thymine residues at both 50 and 30 ends.
Introduction
Guanine (G) rich nucleic acid sequences that contain G repeatscan form four-stranded nucleic acid secondary structures namedG-quadruplexes (Scheme 1, left).1–6 The building blocks ofG-quadruplexes are structures known as G-quartets (Gq)(Scheme 1, right), which are planar association of four G basesheld together by eight Hoogsteen hydrogen bonds. The G-quartetsare stacked on top of each other by p–p hydrophobic interactions,being stabilized by monovalent cations, such as K+ and Na+. Thecations are located in between the G-quartet planes, and formcation–dipole interactions with the 8G of the two adjacentG-quartets, therefore enhancing the hydrogen bond strengthand stabilizing the G-quartet stacking.
A variety of G-quadruplex structures exist and can be classifiedin terms of their molecularity (i.e. the number of associatedstrands leading to the formation of monomer, dimer or tetramerG-quadruplexes), the strand polarity (i.e. the relative arrangementof adjacent strands in parallel or antiparallel orientations), theglycosidic torsion angle (anti or syn), and the orientation of theconnecting loops (lateral, diagonal or both).2,7–9
In the recent years, the G-quadruplexes have become thefocus of attention because they are found in telomeric regionsof chromosomes, oncogene promoter sequences, and otherbiologically relevant regions of the genome, such as immuno-globulin switch regions, ribosomal DNA and RNA.1–3 In addition,G-rich oligodeoxynucleotides (ODNs) are able to self-assemble intohigher-order nanostructures, such as two-dimensional reticulatednetworks and rigid, long nanowires, being excellent buildingblocks for the development of future DNA hybrid electronicdevices in nanotechnology.10–13
The importance of the G-quadruplexes for the developmentof novel devices, with medical and nanotechnology applica-tions, requires a comprehensive knowledge of the structuraland folding properties of G-rich ODNs, the structural poly-morphism, and the stability and disruption of the DNAself-assemblies. In this context, atomic force microscopy(AFM) can resolve with extraordinary resolution and accuracythe surface morphological characteristics of nucleic acidnanostructures.
However, the investigation of formation of the inter-molecularG-quadruplexes and their adsorption and stability on the surfaceof conducting hydrophobic carbon, such as HOPG, electrodeshas not been studied and these complicated processes requirespecial attention from the point view of nanotechnology andbiosensor technology applications.
a Departamento de Quımica, Faculdade de Ciencias e Tecnologia, Universidade de
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Differential pulse (DP) voltammetry is a powerful electro-chemical method that presents very high sensitivity and selectivityand can be successfully employed for the rapid detection of smallperturbations in the nucleic acid secondary structure, and onlyvery recently started to be used for the detection of G-quadruplexconfigurations.16,17
The present paper describes a systematic study performedusing AFM on highly oriented pyrolytic graphite (HOPG) andDP voltammetry on a glassy carbon (GC) electrode to elucidatethe adsorption mechanism and the redox behaviour of threeG-rich ODN sequences, d(G)10, d(TG9) and d(TG8T), with respectto their ability to form G-quadruplex secondary structures.
The formation of higher-order nanostructures, due to thepresence of a long contiguous G region, and the influence of thethymine (T) residues at the 50 and 30 molecular ends will bediscussed. DP voltammetry allowed the detection of the associationof single-strands into G-quadruplexes and G-based nanostructures,in freshly prepared solutions, at concentrations 10 times lowerthan usually detected using other techniques currently employedto study the formation of G-quadruplexes.
Materials and methodsReagents
The 10 mer single-stranded ODNs used in this study weresynthesized on an Applied Biosystems 380B automated DNAsynthesizer (USA) using reagents for ODN chemistry purchasedfrom Fluka (Germany). The purity of the ODN sequences wasverified using NMR and HPLC analyses. The base sequencesused were:
d(G)10: 50-GGGGGGGGGG-30
d(TG9): 50-TGGGGGGGGG-3 0
d(TG8T): 50-TGGGGGGGGT-3 0
Microvolumes were measured using EP-10 and EP-100 PlusMotorized Microliter Pipettes (Rainin Instruments Co. Inc.,Woburn, USA). The pH measurements were carried out usinga GLP 21 Crison pH meter.
The 0.1 M phosphate buffer pH = 7.0 (NaH2PO4/Na2HPO4)supporting electrolyte solution was prepared using analyticalgrade reagents and purified water from a Millipore Milli-Qsystem (conductivity o0.1 mS cm�1). Solutions of differentconcentrations were obtained by direct dilution of the appropriatevolume in a buffer electrolyte.
Atomic force microscopy
HOPG, grade ZYB of 15 � 15 � 2 mm3 dimensions, fromAdvanced Ceramics Co., USA, was used as a substrate in theAFM study. The HOPG was freshly cleaved using adhesive tapeprior to each experiment and imaged using AFM in order toestablish its cleanliness.
AFM was performed in the acoustic AC (AAC) mode using aPicoScan controller from Agilent Technologies, Tempe, AZ, USA. Allthe AFM experiments were performed using a CS AFM S scannerwith a scan range of 6 mm in x–y and 2 mm in z, from AgilentTechnologies. AppNano type FORT of 225 mm length, 3.0 N m�1
spring constants and 47–76 kHz resonant frequencies in air(Applied NanoStructures, Inc., USA) were used. All AFM imageswere topographical and were taken with 256 samples per line� 256lines and scan rates of 0.8–2.0 lines per s. When necessary, the AFMimages were processed by flattening in order to remove the back-ground slope and the contrast and brightness were adjusted.
Sample preparation for AFM
The solutions 0.3 mM ODNs, d(G)10, d(TG9) or d(TG8T), wereprepared in 0.1 M phosphate buffer pH = 7.0, and incubated inthe absence or the presence of 100 mM or 200 mM KCl, for 0 h,24 h and several days, at room temperature.
The ODN modified HOPG surfaces were obtained by sponta-neous adsorption, after depositing 200 mL of the appropriateODN solution onto the freshly cleaved HOPG surface, for 3 min.The excess of solution was gently cleaned with a jet of MilliporeMilli-Q water, and the HOPG with adsorbed ODN moleculeswas then dried in a N2 sterile atmosphere and imaged usingAAC Mode AFM in air.
Voltammetric parameters and electrochemical cells
Voltammetric experiments were carried out using a mAutolab typeII potentiostat running with GPES 4.9 software (Metrohm-Autolab,Utrecht, The Netherlands). The experimental conditions for DPvoltammetry were: pulse amplitude of 50 mV, pulse width of70 ms and scan rate of 5 mV s�1. Measurements were carriedout using a GC working electrode (d = 1 mm), a Pt wire counterelectrode, and an Ag/AgCl (3 M KCl) reference electrode, in aone-compartment 2 mL electrochemical cell.
The GC electrode was polished using diamond spray (particlesize 1 mm, Kemet International Ltd, UK) before every electrochemicalassay. After polishing, the electrode was rinsed thoroughly with
Scheme 1 Schematic representation of a tetrameric intermolecular G-quadruplex composed by two G-quartets and one cation, and the chemical structure of aG-quartet (Gq).
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Milli-Q water. Following this mechanical treatment, the GCelectrode was placed in the buffer supporting electrolyte andvarious DP voltammograms were recorded until a steady statebaseline voltammogram was obtained. This procedure ensuredvery reproducible experimental results.
Acquisition and presentation of voltammetric data
DP voltammograms were baseline corrected using the movingaverage with a step window of 2 mV included in GPES version 4.9software. This mathematical treatment improves the visualizationand identification of peaks over the baseline without introducingany artefact, the peak height is in some cases reduced (o10%)relative to that of the untreated curve. Nevertheless, this mathe-matical treatment of the original voltammograms was used in thepresentation of all experimental voltammograms for a better andclearer identification of the peaks. The peak currents presentedin all graphs were determined from the original untreatedvoltammograms after baseline subtraction.
ResultsAFM characterization
The capacity of d(G)10, d(TG9) and d(TG8T) to interact andadsorb spontaneously on the HOPG surface, forming differentmorphological films, was investigated by AFM, using solutionsof 0.3 mM ODNs in 0.1 M phosphate buffer pH = 7.0.
In order to establish the influence of the presence of K+ ionson the formation and stabilisation of G-quadruplexes, the
adsorption of d(G)10, d(TG9) and d(TG8T) after incubation with100 and 200 mM K+ ions for different periods of time was alsoinvestigated.
The HOPG surface was used as a substrate in the AFM study,because it is atomically flat with less than 0.06 nm of root-mean-square (r.m.s.) roughness for a 1000� 1000 nm2 surface area.The GC electrode used for the voltammetric characterisation ismuch rougher, with 2.10 nm r.m.s. roughness for the same surfacearea, therefore unsuitable for AFM surface characterisation.However, the electrochemical experiments showed similarredox behaviour using GC and HOPG electrodes.
AFM images of d(G)10 spontaneously adsorbed from freshlyprepared solutions (0 h incubation, Fig. 1A) and after 24 hincubation showed only tilted polymeric structures of 0.8 �0.2 nm height, due to the adsorption of single-stranded ODNs.After 14 days incubation (Fig. 1B) several adsorption patterns wereobserved: a densely packed, 1.0� 0.2 nm height network film dueto the adsorption of single-stranded molecules, 1.5–3.0 nm heightspherical aggregates (white arrow) due to the adsorption ofG-quadruplexes, close to the G-quadruplex diameter of B2.8 nmmeasured using X-ray crystallography,18,19 and 1.5–4.5 nm heightrod-like shape aggregates (black arrow) due to the adsorption ofG-based super-structures.
AFM images of d(G)10 incubated for 0 h and 24 h with 100 mMK+ ions (Fig. 1C) and 200 mM K+ ions (Fig. 1D and E) showed theformation of small, tilted polymeric structures of 1.0 � 0.3 nm and0.9 � 0.3 nm height due to the adsorption of single-strandedmolecules, as well as larger spherical (white arrow) and rod-like
Fig. 1 AFM images of (A–E) d(G)10, (F and G) d(TG9) and (H) d(TG8T) spontaneous adsorbed onto HOPG from 0.3 mM ODNs in pH = 7.0, (A and B) in the absence and(C–H) in the presence of (C and F) 100 mM and (D, E, G, and H) 200 mM K+ ions, after (A) 0 h, (C–H) 24 h and (B) 14 days incubation.
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shape (black arrow) aggregates of 1.5–3.5 nm height due to theadsorption of G-quadruplexes and G-based super-structures.The number of spherical and rod-like shape aggregatesincreased with increasing K+ ion concentration and incubationtime. After incubation of d(G)10 for 24 h with 200 mM K+ ions,besides the aggregates adsorbed on the HOPG plane terraces(Fig. 1E), G-nanowires of up to 100 nm length (red arrows,Fig. 1D) were observed on the HOPG step edges.
AFM images of d(TG9) and d(TG8T) after 0 h and 24 hshowed only tilted polymeric structures of B0.8–1.0 nm height,due to the adsorption of single-stranded molecules, as alsoobserved for d(G)10 (Fig. 1A).
AFM images of d(TG9) (Fig. 1F) and d(TG8T) in the presenceof 100 mM K+ ions showed tilted polymeric structures withB1.0 nm height, due to the adsorption of single-strandedmolecules, and 2.0–3.5 nm height spherical aggregates (whitearrows), due to the adsorption of G-quadruplexes.
Increasing the K+ ion concentration to 200 mM, the numberof G-quadruplexes formed by d(TG9) (Fig. 1G) and d(TG8T)(Fig. 1H) increased as expected and, rarely, for d(TG9), rod-likeshape aggregates (black arrow, Fig. 1G), due to the adsorptionof G-based super-structures, could also be observed.
Electrochemical characterisation
DP voltammograms were recorded in solutions of 3.0 mM d(G)10,d(TG9) or d(TG8T) in 0.1 M phosphate buffer pH = 7.0, in theabsence and in the presence of different K+ ion concentrationsand incubation times. Between measurements, the GC electrodesurface was always cleaned by polishing, in order to avoidmisleading results from the ODN adsorption.
Fig. 2 shows the d(G)10 voltammetric behaviour in theabsence (black voltammograms) and in the presence of 1 mM
K+ ions (red voltammograms), for different incubation times.Fig. 3–5 show the d(G)10 (Fig. 3), d(TG9) (Fig. 4) and d(TG8T)(Fig. 5) voltammetric behaviour after 0 h incubation at differentK+ ion concentrations.
DP voltammograms obtained in freshly prepared solutions(0 h incubation) of d(G)10 showed the occurrence of only the Goxidation peak, at Epa = +0.92 V (Fig. 2, and 3A, ),corresponding to the oxidation of guanine residues at theC8–H position, in a two-step mechanism involving the total lossof four electrons and four protons.20 DP voltammograms obtainedin the same solutions after 24 h incubation (Fig. 2, ) showedthe G oxidation peak, at Epa = +0.89 V, due to the guanineresidues in single-stranded ODNs, and the Gq oxidation peakoccurred, at Epa = +0.94 V, due to the oxidation of guanineresidues in the G-quartets.
Increasing the incubation time up to 14 days (Fig. 2,and ), a decrease of the G oxidation peak and an increase ofthe Gq oxidation peak current with a shift to more positivepotentials, in a time-dependent manner, were observed.
DP voltammograms in freshly prepared solutions of d(G)10
incubated with 100 mM K+ ions showed both the G oxidationpeak, at Epa = +0.90 V, due to the guanine residues in single-stranded ODNs, and the Gq oxidation peak, at Epa = +0.96 V(Fig. 3A, ), due to the oxidation of guanine residues in theG-quartets. Increasing the concentration to 5 mM K+, a decreaseof the G oxidation peak current and an increase of the Gq
oxidation peak current with a shift to more positive potentials,Epa = +1.00 V, (Fig. 3A, ) were observed.
A progressive increase of K+ ion concentration incubatedwith d(G)10 caused first an increase of the Gq oxidation peakcurrent (Fig. 3A), followed by a very substantial current decreasefor concentrations above 100 mM K+ ions (Fig. 3B).
After 24 h incubation in the presence of K+ ions (Fig. 2, ,1 mM K+ ions), the DP voltammograms showed the decrease ofthe G oxidation peak and the Gq oxidation peak shifted to morepositive potentials, when compared with 0 h incubation (Fig. 2,
, 1 mM K+ ions). The increase of K+ ion concentration ledfirst to an increase of the Gq oxidation peak current and then,for concentrations above 100 mM K+, to a current decrease.
DP voltammograms obtained in freshly prepared solutions(0 h incubation) of 3.0 mM d(TG9) (Fig. 4A, ) and d(TG8T)(Fig. 5, ) showed the occurrence of only the G oxidation peakof guanine residues in single-stranded ODNs, for d(TG9) atEpa = +0.91 V and for d(TG8T) at Epa = +0.93 V.
The oxidation of thymine residues in d(TG9) and d(TG8T) couldnot be detected using DP voltammetry, because it occurs at muchhigher positive potential near the potential of oxygen evolution.21
DP voltammograms immediately after the addition (0 hincubation) of 5 mM K+ ions to d(TG9) or d(TG8T) showed theoccurrence of two oxidation peaks, the G oxidation peak of theguanine residues in single-stranded ODNs, at Epa = +0.90 V, andthe Gq oxidation peak of the guanine residues in the G-quartets,at Epa = +0.97 V (Fig. 4A, and 5, ). After the addition of50 mM K+ ions the G oxidation peak decreases and the Gq
oxidation peak increases and shifts to more positive potentialsfor both sequences (Fig. 4A, and 5, ).
Fig. 2 DP voltammograms baseline corrected in 3.0 mM d(G)10 in pH = 7.0, inthe absence of K+ ions ( ) 0 h, ( ) 24 h, ( ) 48 h and ( ) 14 daysincubation, and in the presence of 1 mM K+ ions ( ) 0 h and ( ) 24 hincubation.
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A further increase of K+ ion concentration incubated withd(TG9) caused an increase of the Gq oxidation peak current(Fig. 4A), and only for very high, 1 M K+ ion concentration(Fig. 4B, ), a small decrease of the Gq oxidation peak currentoccurred. An increase of K+ ions concentration incubated withd(TG8T) always caused an increase of the Gq oxidation peakcurrent (Fig. 5). For all ODN sequences, the Gq oxidation peakshifted to more positive potentials with increasing K+ ionconcentration and incubation time.
Discussion
The 10 mer ODNs studied, d(G)10, d(TG9) and d(TG8T), containonly one block of contiguous 8–10 guanines, therefore they
are expected to form parallel tetramolecular G-quadruplexes(Scheme 1).8,9,22–24 However, recent studies demonstrated thatd(TGnT) sequences can also form monomer, dimer or tetramerG-quadruplexes.25 Apart from G-quadruplexes, the ODNs mayalso self-assemble into higher-order nanostructures.
The adsorption of the ODN sequences onto HOPG and GC ismainly driven by hydrophobic interactions.26 The constituent Gresidues influence the ODNs global hydrophobicity, directlythrough the intrinsic hydrophobic character of the aromaticrings, and indirectly, by allowing to establish G-quadruplexsecondary structures and G-based nanostructures. After theformation of G-quadruplexes, the ODN sequences interactand adsorb less on the hydrophobic HOPG or GC, because theyhave the bases protected by the sugar-phosphate backbones,
Fig. 3 DP voltammograms baseline corrected in 3.0 mM d(G)10 in pH = 7.0, (A, ) in the absence and in the presence of (A, ) 100 mM, (A, ) 5 mM, (A and B, )100 mM, (B, ) 200 mM, (B, ) 500 mM and (B, ) 1 M K+ ions, 0 h incubation.
Fig. 4 DP voltammograms baseline corrected in 3.0 mM d(TG9) in pH = 7.0, (A, ) in the absence and in the presence of (A, ) 5 mM, (A, ) 50 mM,(A and B, ) 100 mM, (A and B, ) 200 mM and (B, ) 1 M K+ ions, 0 h incubation.
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when compared with the single-stranded ODNs that have thebases more exposed and free to undergo hydrophobic interac-tions with the surface.
The AFM and voltammetric study showed d(G)10, d(TG9) andd(TG8T) spontaneous adsorption onto HOPG (Fig. 1) andoxidation at the GC electrode (Fig. 2–5), with the formationand stabilisation of G-quadruplexes and different higher-ordernanostructures. Considering the AFM and voltammetricresults, a model was proposed to describe the formation andstabilisation of G-quadruplexes and G-based nanostructures,depending on incubation time and K+ ion concentration.
After 0 h incubation with Na+ ions, d(G)10, d(TG9) and d(TG8T)formed only single-stranded configurations, detected using AFMas thin network films and polymeric structures and using DPvoltammetry by the occurrence of only the G oxidation peak.
After short incubation times (B24 h) with Na+ ions orimmediately after the addition (0 h incubation) of low K+ ionconcentration (100 mM), d(G)10, d(TG9) and d(TG8T) moleculesstart to interact with each other forming G-quartets, detected bythe occurrence of the Gq oxidation peak, but no G-quadruplexeswere observed using AFM.
Increasing the incubation time or the K+ ion concentration,the number of adjacent G-quartets increased and gets stabilisedby p–p hydrophobic interactions, leading to the formation of anumber of G-quadruplexes, detected using AFM as sphericalaggregates (Fig. 1, white arrows). DP voltammograms showed adecrease of the G oxidation peak, due to a diminished number offree guanine residues in single-stranded ODNs, an increase of theGq oxidation peak, due to an increased number of G-quartets, andthe Gq oxidation peak potential shift to more positive potentials,due to the formation and stabilisation of rigid G-quadruplexes(Scheme 2A, left) that are more difficult to oxidise.
An increase of K+ ion concentration above 100 mM incubatedwith d(G)10 led to the self-assembling of d(G)10 molecules intolong G-nanowires (Scheme 2A, right), which adsorb on the HOPGstep edge defects (Fig. 1D, red arrows). The transition of electrons
from the inside of these very rigid and stable G-nanowires tothe GC electrode surface is very difficult, which explains thedecrease of the Gq oxidation peak and its potential shift to evenmore positive values. Additionally, above 100 mM K+ ionconcentration, the G oxidation peak current was constant, becausethe G-nanowires were not self-assembled perfectly, their structureresembling a frayed G-nanowire with slipped-strands (Scheme 2B).The G-rich single-stranded hang-ups of the G-nanowires were easilyoxidized, therefore contributing to the G oxidation peak current,and also helped the G-nanowires adsorption onto HOPG.
In the case of d(TG9), AFM showed only spherical androd-like shape aggregates (Fig. 1E and F), and nanowires werenever observed. The presence of T residues at the 50 end inhibitsthe ability of d(TG9) to self-assemble into long G-nanowires.However, d(TG9) may self-assemble into G-based super-structures,detected by the small decrease of the Gq oxidation peak current,the largest G-based super-structure presenting a maximum of 18G-quartets (Scheme 2C).
AFM showed only spherical aggregates for sequence d(TG8T)(Fig. 1G), and the adsorption of rod-like-shape aggregates andnanowires was never detected. The presence of T residues atboth 50 and 30 ends completely inhibits the ability of d(TG8T) toself-assemble into G-based nanostructures, offering a greaterstability for the G-quadruplexes (Scheme 2D). The results are inagreement with absorbance spectroscopy of d(TGnTm) ODNs,indicating that the presence of T residues at the 50 and 30 endsinhibits the formation of higher-order nanostructures, butstabilizes the G-quadruplex.8
The presence of K+ ions strongly stabilises and acceleratesthe G-quadruplex formation. In the presence of only 1 mM of K+
ions, 0 h incubation, the same DP voltammetric response as inthe presence of Na+ ions after 72 h incubation was obtained,and in the presence of 1 mM of K+ ions after 24 h incubationthe same DP voltammetric response as in Na+ ions after 14 daysincubation was obtained (Fig. 2). Kinetic studies using absorbancespectroscopy of association and dissociation of tetramolecularG-quadruplexes formed by ODNs containing blocks of more than4 contiguous G residues also showed faster association time in thepresence of K+ ions.8
The detailed knowledge of the G-quadruplex formationmechanism is extremely important for the design and fabrica-tion of quadruplex-based therapeutic agents in medicine ornanostructures in nanotechnology. The AFM and voltammetricresults clearly demonstrated that the appropriate choice of theODN sequence base composition, d(G)10, d(TG9) or d(TG8T),monovalent cation concentration and incubation time can leadto the formation of tetramolecular G-quadruplexes, G-basedsuper-structures or G-nanowires.
Depending on the application, each one of the structurescan be desirable: the perfectly aligned short and compactG-quadruplexes can be used for screening cancer therapeuticagents, the perfectly aligned G-nanowires may represent buildingblocks of molecular nanowires for nanoelectronics and the G-basedsuper-structures and frayed G-nanowires with slipped-strands canwork as a nucleation platform for the addition of subsequentstrands and the formation of larger structures. DP voltammetry
Fig. 5 DP voltammograms baseline corrected in 3.0 mM d(TG8T) in pH = 7.0,( ) in the absence and in the presence of ( ) 5 mM, ( ) 50 mM and ( )1 M K+ ions, 0 h incubation.
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is an extremely useful technique to study the transformationof single-stranded ODNs into G-quadruplexes or G-basednanostructures, in freshly prepared solutions, at concentrations10 times lower than usually detected using other techniques,such as UV absorbance, circular dichroism or electrospray massspectroscopy.8,25
Conclusions
The redox behaviour and the adsorption morphology of G-richODN sequences, d(G)10, d(TG9), d(TG8T), containing only oneblock of guanines were studied using AFM on HOPG and DPvoltammetry at GC electrodes, revealing the ODNs ability toself-assemble into G-quadruplex secondary structures and
higher-order nanostructures, relevant for biomedical and nano-technological applications.
Single-stranded ODNs were present in Na+ containingsolutions for short incubation times and were detected usingAFM as thin polymeric structures and using DP voltammetry bythe occurrence of only the G oxidation peak.
G-quadruplex secondary structures were obtained eitherslowly in Na+ ions containing solutions or very fast infreshly prepared K+ ions containing solutions. AFM detectedthe adsorption of higher spherical aggregates, and DP voltam-metry the G oxidation peak decrease and the G-quartet oxida-tion peak occurrence/increase and shift to positive potentials,in a time dependent and K+ ion concentration-dependentmanner.
Scheme 2 Schematic representation of: (A) d(G)10 perfectly aligned G-quadruplex (left) and the formation of a G-nanowire (right); (B) frayed G-nanowire withslipped-strands; (C) d(TG9) perfectly aligned G-quadruplex (left) and the formation of a G-based super-structure with 18 G-quartets (right); (D) d(TG8T) perfectlyaligned G-quadruplex.
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AFM and DP voltammetry enabled the characterisation ofODNs self-assembling into higher-order nanostructures, in thepresence of high K+ ion concentration and/or long incubationtimes. The only ODN sequence with the ability to form longG-nanowires was d(G)10, but both d(G)10 and d(TG9) self-assembleinto short quadruplex super-structures, while d(TG8T) was notable to self-assemble into quadruplex super-structures, due to thepresence of T residues at both 50 and 30 ends, forming only shortG-quadruplexes.
Acknowledgements
Financial support from Fundaçao para a Ciencia e Tecnologia(FCT), Project Grants (P.V. Santos), projects PTDC/QUI-QUI/098562/2008 and PTDC/SAU-BMA/118531/2010, POPH (co-financed by theEuropean Community Funds FSE and FEDER/COMPETE), andCEMUC-R (Research Unit 285), is gratefully acknowledged.
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