Self‐Assembly of G‐Rich Oligonucleotides Incorporating a 3′–3′ Inversion of Polarity Site: A New Route Towards G‐Wire DNA Nanostructures

Abstract Obtaining DNA nanostructures with potential applications in drug discovery, diagnostics, and electronics in a simple and affordable way represents one of the hottest topics in nanotechnological and medical sciences. Herein, we report a novel strategy to obtain structurally homogeneous DNA G‐wire nanostructures of known length, starting from the short unmodified G‐rich oligonucleotide d(5′‐CGGT‐3′–3′‐GGC‐5′) (1) incorporating a 3’–3′ inversion of polarity site. The reported approach allowed us to obtain long G‐wire assemblies through 5′–5′ π–π stacking interactions in between the tetramolecular G‐quadruplex building blocks that form when 1 is annealed in the presence of potassium ions. Our results expand the repertoire of synthetic methodologies to obtain new tailored DNA G‐wire nanostructures.


Introduction
In the development of new nanotechnologies and biomaterials, the possibility of assembling ordered supramolecular structures starting from small building blocks and the exploitation of self-assembly driving forces are very important and much studied topics. [1][2][3] In the bottom-up approach, DNA strands have been demonstrated to be very useful building blocks for the assembly of supramolecular structures of various dimensions and shapes. For example, the DNA nanotechnology known as DNA origami allows the design and the construction of tailored three-dimensional superstructures by exploiting the Watson-Crick base-pairing scheme. [4][5][6][7] Besides the Watson-Crick base-pairing scheme, also the Hoogsteen hydrogen bondingo ne, which allows the formation of G-quadruplex DNA, can be exploited to obtain DNA supramolecular struc-tures. G-Quadruplexes are unusual secondarys tructures of DNA, which form when guanine-rich DNA strandsa re annealed in the presence of suitable monovalent or divalent cations.
The building block of G-quadruplexesi st he G-quartet (Figure 1A), ap lanar arrangement of four guaninesh eld together by ac yclic array of eight Hoogsteen hydrogen bonds. [8][9][10] The onset of p-p interactions among the stackedG -quartets greatly stabilizes the G-quadruplex assembly,w hich is generally more stable than aD NA duplex of the same length. AG -quadruplex can be formed by one, two, or four G-rich DNA strands and can be classified as either parallel, antiparallel,o rh ybrid type depending on the mutual orientation of the strands in-Obtaining DNA nanostructures with potential applicationsi n drug discovery, diagnostics, and electronicsi nasimple and affordable way represents one of the hottest topics in nanotechnological and medical sciences.Herein, we report an ovel strategy to obtain structurally homogeneous DNA G-wire nanostructures of known length, starting from the short unmodified G-rich oligonucleotide d(5'-CGGT-3'-3'-GGC-5')( 1)i ncorporat-ing a3 '-3' inversion of polarity site. The reported approacha llowed us to obtain long G-wire assembliest hrough 5'-5' p-p stacking interactions in between the tetramolecular G-quadruplex building blocks that form when 1 is annealed in the presence of potassium ions. Our results expand the repertoire of synthetic methodologiest oo btain new tailoredD NA G-wire nanostructures. volved in the G-quartets formation. [11,12] Further factorst hat contribute to the wide polymorphism of G-quadruplexes are the length and the base composition of the loops (when present), as well as the nature of the cationsu sed to stabilize the quadrupleh elix structure. [13][14][15] The biological implications of Gquadruplexes in cellular processes [16][17][18] and their use as promising drugs [19][20][21][22] or in drugs delivery [23] and diagnostics [23][24][25] are well known. In addition, G-quadruplexes possess greater conductivityt han DNA duplexes, thus suggesting their use for the obtainment of electronic nano-biomaterials and nanodevices. [26][27][28] These properties indicate the G-quadruplex scaffold as au seful structuralm otif to obtain supramolecular self-assemblies, including the so-called G-wires. [29][30][31][32] G-wire superstructures can reach the length of thousands of nanometers along the axis perpendicular to the G-tetrad planes. Depending on the topological arrangement of the G-rich strandsp articipating in their formation, G-wires can be classified into two categories:i )interlocked G-wires,c haracterizedb yt he cooperative assembly of interlocked slipped strands( Figure 1B)a nd ii) stacked G-wires,c haracterized by the multimerization of Gquadruplexb uilding blocks held together by end-to-end p-p stacking ( Figure 1C). Besides the well-known interlocked Gwires [29][30][31][32][33][34][35] and G-wires containing both topological motifs, [36][37][38] only af ew examples of exclusively stacked G-wires have been reported so far. [39][40][41] In ap revious study,w er eported that in the presenceo fK + cations the 7-mer d(CGGTGGT)c an assemble into the octameric higher-ordered G-quadruplex complex d(CGGTGGT) 8 (Figure S1 in the Supporting Information) through p-p stacking of two unusualG (:C):G(:C):G(:C):G(:C) planar octadsb elonging to two identicalt etramolecularp arallel G-quadruplexes. [42] Later, we demonstrated that the same dimerization pathway is also possible for all the other d(CGGXGGT) DNA strands, with X = A, G, or C. [43] Herein, we report on the achievement of an ew type of stacked G-wire, here indicated as Q n (Figure 2) obtained by exploiting the 5'-5' p-p stacking interactions between the G(:C):G(:C):G(:C):G(:C) octads formed at both ends of the tetramolecular G-quadruplex building block Q 1 (Figure 2). The last forms when the d(5'-CGGT-3'-3'-GGC-5')D NA strand (1, Figure 2), incorporating a3 '-3' inversion of polarity site, is annealed in the presence of K + ions. The resulting G-wires were obtained as ad istribution of quadruplexm ultimers of different length.
The n subscript in Q n indicates the number of tetramolecular G-quadruplex building blocks participatingi nt he G-wirese longation.T he actual formation of the target G-wires was confirmed by polyacrylamide gel electrophoresis( PAGE), HPLC size exclusion chromatography (HPLC-SEC),c ircular dichroism (CD), 1 Hn uclear magnetic resonance (NMR) spectroscopy,a nd atomic force microscopy (AFM) studies. The analytical results allowed us also to gatheri nformation on the G-wires assembly and stability,a nd to isolate and characterize the three shortest Q n species (n = 2-4).

Synthesis of d(5'-CGGT-3'-3'-GGC-5')a nd G-Wires
The d(5'-CGGT-3'-3'-GGC-5')( 1)i ncorporating a3 '-3' inversion of polarity site was synthesized by using as olid-phase automated DNA synthesizer as described in the Experimental Section. The inversion of polarity site wasa chieved by performing the first four coupling cycles with 5'-phosphoramidites and the remaining three with standard 3'-phosphoramidites. The Q n species were obtained by heating 1-dissolved in 1.0 m K + containing buffer at the single strand concentration of 0.1 or 1.6 mm-at 90 8Cf or 10 min and then rapidlyc ooling it to 4 8C (fast annealing procedure). All samples were stored at 4 8Cf or 24 hb efore furtherinvestigation.

PAGE Studies
The annealed 1.6 mm 1 was analyzed by PAGE to obtain information on its propensity to form the tetramolecular G-quadruplex building block Q 1 and/ort he target Q n multimers. For this purpose, we compared the electrophoretic mobility of 1 (lane 3i nF igure 3) with those of the G-quadruplexes d(TGGGGT) 4 (lane 1) and d(CGGTGGT) 8 (lane 2), used as size  markersf or the tetramolecular G-quadruplex building block Q 1 and for its eight-stranded dimer Q 2 ,respectively.Atthe studied conditions, the annealed 1 migrated as al adder of bands, thus confirmingi ts propensity to form G-quadruplex multimers. The electrophoretic mobility of the two fastest bands matched almostp erfectly with that of the size markersi nl anes 1a nd 2, thus confirming the formation of at etra-stranded Q 1 and octastranded Q 2 ;w hereas the remaining slower bands in lane 3 were likely aresult of longer Q n species(n ! 3). The electrophoretic mobility of the isolated Q 1-4 species, obtained by HPLC-SEC fractionation (see the following section), was also assessed (lanes 4-7). Taken together, the PAGE data suggested that the oligodeoxynucleotide( ODN) 1 in the presence of K + ions is capable of self-assembling in ad istribution of G-wire species of differentl engths (Q n )o btained by the sequential stacking of the tetramolecular G-quadruplex buildingb lock Q 1 presenting two "sticky" G(:C):G(:C):G(:C):G(:C) planar octadsa tb oth 5'ends.

SEC Studies
The Q n G-wire population wasa nalyzed by HPLC-SECa tr oom temperature on aR eproSil 200 SEC column 24 ha fter annealing and storage at 4 8C. The HPLC profile of the annealed 0.1 mm 1 showed ad istribution of peaks in which low molecular weight speciesh ad greater retention times ( Figure S2 Ai n the Supporting Information). To obtain information on the M W of the observedp roduct peaks, we used the same HPLC-SEC conditions (see the Experimental Section) to determine the retention times (R t )o fi )the single-stranded dT 7 (panel B), ii)the tetramolecular quadruplexd (TGGGGT) 4 (panel C), and iii)the dimeric quadruplex d(CGGTGGT) 8 (panel D). Based on the HPLC-SEC data, the peak at the highest retention time (R t = 21.45 min) was attributedt ot he single-stranded 1,w hereas the two preceding peaks (at R t = 19.89 and 19.02 min) were assigned to the G-quadruplex buildingb lock Q 1 and to its dimer Q 2 ,r espectively.F rom these data, it was reasonable to hypothesize that each peak in the HPLC-SEC profile belongs to as peciesd iffering from those of the adjacent peaks foraM W corresponding to the tetramolecularG -quadruplexb uilding block Q 1 (i.e. 28 nucleotides). Exploiting the good peak resolution on the HPLC-SEC column, the speciesl ikely corresponding to Q 1-4 were recovered and their purity and electrophoretic mobility assessed by PAGE (Figure3,l anes 4-7). PAGE results confirmed the purity and allowed us to match the speciesr esponsible for the four fast-moving bands in the PAGE (i.e. Q 4 , Q 3 , Q 2 ,a nd Q 1 from the slowest to the fastest band in Figure 3) with the four corresponding peaks in the HPLC-SEC distribution (i.e. Q 4 , Q 3 , Q 2 ,a nd Q 1 from the least to the most retained in Figure S2 Ai nt he Supporting Information). Furthermore, HPLC-SEC re-injection of the isolated Q 1-4 species, performed 24 ha fter their isolation and storage at 4 8C( Figure S3 in the Supporting Information), disclosed that the G-quadruplex buildingb lock Q 1 and the first three Q n species (Q 2-4 )d o not interconverte ach other and can be stored as single G-wire segmentsofknown length for at least 24 h. However,considering the contribution of entropy to the formation of Q n G-wires, different behaviors could be anticipated for samples of 1 annealed at differentO DN concentrations. Thus, we exploited the good chromatographic separation of Q n species to assess the effect of the ODN annealingc oncentration on the formation and size distribution of Q n species. As expected, the HPLC-SEC profile of Q n obtained 24 ha fter the annealing of 1 at 1.6 mm concentration( Figure 4) confirmedt hat the formation of longerG -wiresi ss trongly dependent on the ODN concentration during the annealingp rocedure. Indeed, in Figure 4a ll Q n speciesw ith n 8a re clearly distinguishable and almost equally populated, and an intense envelope peak attributable to longerG -wiresi sp resent at R t = 13.6 min. Conversely,i nt he HPLC-SECp rofile obtained from the sample annealed at 0.1 mm ODN concentration ( Figure S2 Ai nt he Supporting Information), the envelope peak is absenta nd only the Q n species with n 3a re significantly populated.
The effect of the temperature on the distribution of Q n species was investigated by HPLC-SEC2 4h after the annealing of 0.1 mm 1 in 1.0 m K + buffer.H PLC-SEC analyses were performed by injecting the sample 30 min after heating and equilibration at 25, 45, 65, and 85 8C( Figure S4 in the Supporting Information). The profile obtained from the sample injected at 25 8Cs howed almost the same Q n distribution as the sample injecteda t48C, but showed that at room temperature the dimeric Q 2 speciesn ot only is more abundantt han the building block Q 1 (whereas the opposite was observed in the sample stored and injected at 4 8C), but represents the most abundant species. The progressive heatingo ft he sample determined the reduction of the amount of Q n speciesa nd the contextuali ncreaseo ft he single-stranded 1,w hich resulted in the only observable peak at 85 8C. However,t wo significant outcomes emerged from the profiles obtained at 45 and 65 8C: 1) at 45 8C, although the most abundant species was represented by the single-stranded 1,w eobserved that the distribution of Q n speciess hiftedt owards longer G-wires, with the appearance of an ew peak at R t = 13.3 min and with the Q 3 species being even more abundantt han Q 2 ;2 )the analysiso ft he pro- file obtained at 65 8Cs uggested that the Q n speciesm elt throughacooperative process,w hich drives directly to the random coil 1 (the peak of the G-quadruplex building block Q 1 was even less abundant than that of Q 5 species). The higher thermals tability of Q n species relative to Q 1 was also confirmed by CD meltingd ata (see the following section).

CD Studies
CD is aw ell-established diagnostic technique to provide preliminary informationa bout the topologyo fG -quadruplexes in solution.G enerally,t he CD profile of parallel G-quadruplexes, in which all guanosines in the G-tetrads are in the anti glycosidic bond conformation (Type 1stem), is characterized by apositive signal at around 264 nm and an egative signal at around 240 nm;whereas that of antiparallel G-quadruplexes with alternating syn-anti glycosidicb onds along the stem (Type 3s tem) shows ap ositive signal at 245 nm, at rough at around 260 nm, and as econd positive signal at 290 nm. [41,[44][45][46] Considering the formation of stacking interactions between the terminal G-tetrads of Q 1 building blocks (required to obtain the Q n G-wires) and the presence of the 3'-3' inversion of polarity site within the stem of Q 1 and its multimers, the CD profile obtained for the annealed 1 (containing am ixture of Q n of different lengths), showing positives ignals at 246 nm and 300 nm and an egative signal at 270 nm ( Figure 5, dashedl ine), was somewhat unexpected. In fact, although the latter structuralf eatures anticipated for aT ype 2C Dp rofile, [44] the experimental result resembled that of aT ype 3stem. AType 3s tem could be obtainablef rom 1 only if the thymines of two ODN 1 strands would participate in the formationo fapropeller loop, thusa llowing the formation of ab imolecular antiparallel quadruplex stem. However,t his structuralh ypothesis hasb een ruled out by PAGE and HPLC-SECevidence, which indicated the exclusive formationo ft he tetra-stranded quadruplex building block and its multimers.
Indeed, the CD profile of Q n resembles those reported for the G-quadruplexes formed by d(5'TG3'-3'GGT) 4 [47] and d(TGG3'-3'GGT) 4 [48] incorporating a3 '-3' inversion of polarity site and characterizedb yt he presence of all anti G-tetrads. The occurrence of the same system of uninterrupted stacked G-tetrads, including both clockwise and counter-clockwise directionality,i nQ 1 and Q n could explain the CD profiles shown in Figure 5. The resulting structural hypothesis requires that the thymines are projected outside the quadruplex stem to form bulge loops, [49] as depicted in Figure 2. Unfortunately,t he strongp ositive signal at 300 nm induced by the presence of the 3'-3' inversion of polarity site precluded the observation of the negative band at 290 nm, which, in our previous studies, provedd iagnostic for the formation of higher-ordered G-quadruplexa ssemblies obtained by non-covalent p-p stacking of planarG (:C):G(:C):G(:C):G(:C) octads. [42,43] Similar CD profiles were obtained for the isolated Q 1-4 ( Figure 5, colored lines), thus confirming that the CD profile of Q n was the result of the presence of quadruplex assemblies of different lengths incorporatingt he 3'-3' inversion of polarity site. The overall thermal stabilityo ft he species populating the Q n distribution and that of the isolated Q 1-4 complexes was assessedb ym eanso fC D denaturation experiments ( Figure S5 in the Supporting Information). The resulting apparent melting curves clearly provided evidencet hat the thermals tability of the quadruplexm ultimers was higher than that of the parent quadruplex building block Q 1 .

NMR Spectroscopy
The formation of G-quadruplex assemblies from suitable G-rich ODN sequences is usually confirmed by water-suppressed 1 HNMR spectroscopy.W hen the N-1 imino protons of guanine bases are engaged in Hoogsteen-type hydrogen bondingw ith the O-6 carbonyl oxygen atoms of flankingg uaninesi ne ach G-tetrad, they are protectedf rom exchange with the hydrogen atoms of the solvent and are thus visible in the 1 HNMR spectrum as quadruplex-diagnostic slightly broad signals resonating between1 0a nd 12 ppm. [50,51] Dependingo nt he orientation of the strands and on the glycosidic torsiona ngle of guanosines participating in G-tetrads, the four iminop rotons in each G-tetrad resonate as one, two, or four NMR signals. [52] The downfield region of the water-suppressed 1 HNMR spectra of the Q n distribution-obtained by annealing 1 in 1 m K + buffer at 1.6 mm singles trand concentration-recorded at 25, 45, 65, and 85 8Ca re shown in Figure S6 in the Supporting Information. In agreement with the CD evidence,t he observation of the G-quadruplex diagnostic iminop rotons signals in the 11-12 ppm region confirmed the presence in solution of G-quadruplexs pecies at temperatures lower than 65 8C. However,t he intensity and the shape of imino and anomeric protons signals differed significantly from those we observed in the 1 HNMR spectra of the dimeric higher-ordered G-quadruplexes formed by CGGTGGT [42] and CGGAGGT. [43] In the two latter,f our well-resolved imino protons (11.0-12.0 ppm) and intensea nomeric protons signals (5.5-6.5 ppm) werev isible at temperatures up to 65 8C. Conversely,i nt he NMR spectra of 1 recorded at 25 and 45 8C, all iminoa nd anomerics ignals appeared severely broadened, thus suggesting the involvement of imino and anomericp rotons in chemical or conformational exchange phenomena. The line broadening of the imino protonsw as so intense as to require 10 softwarea mplification for signal observation (see insets in Figure S6 in the SupportingI nformation). As far as the low intensity of the imino proton signals is concerned, we hypothesize that the presence of the bulge loops connecting the stacked G3 and G5 tetrads destabilizes the G-quadruplex stem, thusspeeding up the imino protonexchange with water.The anomeric protonsignal broadening, instead, could be the result of side-by-side aggregation of Gquadruplexu nits, as seen by Hu et al. in G-wire assemblies formed by dGMP annealedi nt he presenceo fS r 2 + . [53] As we have showni no ur previousp apers, [42,43] the anomeric protons are located at the outer shell of 2Q-like quadruplexes, thus it is conceivable that lateralc ontacts between flanking G-quadruplex structures participating in the Q n G-wire distribution could be responsible for the observed anomeric signal broadening. As we will see in the followings ection, AFM evidences upports the formation of extended quadruplex layers by lateral aggregation of quadruplex units. AlthoughAFM gives us apictureo f quadruplex aggregates deposited on mica surfaces, we believe that such aggregates could also form in solutionc onsidering the relativelyh igh ODN concentration used for the preparation of the NMR samples (6.0 mm). In the light of this hypothesis, the comparison of the NMR spectra recorded in the 25-85 8C temperature range indicates that the breakdown of quadruplex aggregates and the melting of quadruplex units to the random coil 1 are cooperative processes, which start at temperatures between 45 and6 58Ca nd completeatt emperatures lower than 85 8C.  Figure S7 in the SupportingI nformation). The AFM results are compatible with reported SEC results considering that, differently from heightm easurements, the length and width measurements are affected by the curvature radius of the AFM tip. The preferred orientation can be ascribed to the interaction between G-quadruplexes and the mica surface. The homogeneous values of heights recorded suggested the formation of self-assembled monolayers in the x,y plane owing to the evaporation of the buffer solution, which promoted laterala nd longitudinal aggregation of Gquadruplexes, the first one being favored by side-packing. A1 00-times diluted solution gave different results, as can be noted in Figure S8 in the Supporting Information. Even if apreferred orientation could still be envisaged, the structures seemed sparser and less ordered (see panel A);l engths are in the same intervala sb efore,w hereas the widths are smaller, never exceeding 30 nm, as evident from the measurements in panel B. Also in this case, at ip radius of curvature of about 10 nm must be taken in account. This result confirmed as idepacking mechanism in the assembly of G-structures during evaporation of the solution. The heights are alwaysa bout 2nma sb efore (see measurements in panel C), again endorsing the hypothesis of spontaneousf ormation of am onolayer. In the case of diluted starting solution, the longitudinal aggregation is competitive compared with the lateral one, owing to steric conditions during the evaporation.

Conclusions
In this paper,w er eported the successful results of our study aimed at the obtainmento fanew kind of G-wireD NA by exploitation of p-p stacking interactions between tetramolecular G-quadruplex building blocks incorporating a3 '-3' inversion of polarity site and exposing G(:C):G(:C):G(:C):G(:C) planar "sticky" octads at both 5'-ends. The main findings of the study can be summarized as follows:1 )the obtained G-wiresa re unprecedented and are achievable by using the short 5'CGGT3'-3'GGC5' G-rich ODN as the startingm ateriali nafacile and affordable way;2 )the use of at etramolecularG -quadruplex buildingb lock to obtain DNA G-wire polymers represents am ajor improvement over the use of monomolecular G-quadruplexes, which, possessing differente nd-facesa nd lateral nucleotide loops, could induce the formationo fd ifferent kinds of aggregates thus reducing the amount of target G-wires; 3) the reported G-wiresc an be easily monitored in their formation and length-growth by PAGE and HPLC-size exclusion chromatography.W ea lso demonstrated that the HPLC-SEC technique can be used to determine the number of quadruplex building blocks in each Q n G-wire,a sw ell as to isolatea nd recover the three shortest Q n species (n = 2-4). systems, USA) using au niversal CPG support purchased from Glen Research. The syntheses were performed by adopting the standard b-cyanoethyl phosphoramidite chemistry at 10-15 mm scale and the products were purified as previously described. [54] The synthesis of d(5'-CGGT-3'-3'-GGC-5')( 1)w as performed with the same DNA synthesizer.T he inversion of polarity site within the sequence was achieved by initially assembling the 5'-CGGT-3' tract by using 5'-phorphoramidites and then the 3'-GGC-5' tract with standard 3'phosphoramidites. After completion of the ODN sequence, the support was treated with concentrated aqueous ammonia at 55 8C for 15 h. The combined filtrates and washings were concentrated under reduced pressure and purified through HPLC (JASCO PU2089 pumps equipped with the JASCO 2075 UV detector) with an anion exchange column (Macherey-Nagel, 1000-8/46, 4.4 50 mm, 5 mm) using al inear gradient from 0t o1 00 %Bin 30 min, flow rate = 1mLmin À1 and detection at 260 nm (buffer A: 20 mm NaH 2 PO 4 aq. solution pH 7.0, containing 20 %( v/v) CH 3 CN;b uffer B: 20 mm NaH 2 PO 4 aq. solution pH 7.0, containing 1 m NaCl and 20 %(v/v) CH 3 CN).

Annealing Procedure
The ODN concentrations were determined in water by measuring the absorbance at 260 nm at 90 8Cb yu sing the nearest-neighbor calculated molar extinction coefficient of 5'-CGGTGGC-3' (e = 63 100 m À1 cm À1 ). The 0.1 and 1.6 mm solutions of 1 were obtained by dissolving the lyophilized sample in 900 mm KCl and 100 mm KH 2 PO 4 .T he samples were annealed by heating at 90 8Cf or 10 min and then quickly cooling to 4 8C. After the annealing procedure, the samples were stored at 4 8Cb efore measurements.

PAGE
Native gel electrophoresis experiments were performed on 20 % polyacrylamide gels containing TBE (8.9 mm Tris, 8.9 mm borate, 0.2 mm EDTA, from BIORAD) and 30 mm KCl, at room temperature, 120 Vf or 2h.T he ODN samples, annealed at 1.6 mm single strand concentration in 1.0 m K + buffer,w ere diluted at 0.6 mm loading concentration just before the PAGE runs. Glycerol was added (10 % final) to facilitate sample loading in the wells. The bands were finally visualized by ethidium bromide staining in aB io-Rad Laboratories Gel Doc TM XR + image system. CD CD spectra and CD melting profiles were recorded with aJ asco 715 CD spectrophotometer (Jasco, To kyo, Japan) equipped with aJ asco JPT423S Peltier temperature controller in 1mmo ptical path quartz cuvettes (100 nm min À1 scanning speed, 1s response time). The spectra were recorded in triplicate at 4 8Cf rom 220 to 320 nm. CD samples were prepared in potassium buffer (90 mm KH 2 PO 4 and 10 mm KCl) at 20 mm final single strand concentration. The buffer baseline was subtracted from each spectrum and the spectra were normalized to have zero at 320 nm. CD melting curves were registered at 268 nm, 1 8Cmin À1 heating rate, temperature range 5-90 8C.

NMR Spectroscopy
NMR data were recorded with aV arian UNITY INOVA5 00 MHz spectrometer equipped with ab roadband inverse probe with z-field gradient. The data were processed by using the iNMR software package (http://www.inmr.net). One-dimensional NMR spectra were acquired as 16384 data points with ar ecycle delay of 1.0 sa t 25, 45, 65, and 85 8Ca nd the spectra were apodized with as hifted sine bell squared window function. Water suppression was achieved by including ad ouble pulsed-field gradient spin-echo (DPFGSE) module [55,56] in the pulse sequence prior to acquisition. NMR samples were prepared at the concentration of 1.6 mm single strand in 200 mLo fH 2 O/D 2 O9 :1 containing 900 mm KCl and 100 mm KH 2 PO 4 .
AFM AX E-100 Park Systems instrument was used for the AFM imaging of Q n G-wires. Surface imaging was obtained in non-contact mode by using 125 mml ong silicon/aluminium-coated cantilevers (PPP-NCHR 10 m;Park Systems;tip radius lower than 10 nm), with aresonance frequency of 200 to 400 kHz and nominal force constant of 42 Nm À1 .T he scan frequency was typically 0.5 Hz per line. When necessary,t he AFM images were processed by flattening to remove the background slope, and the contrast and brightness were adjusted. Muscovite mica of about 1cm 2 surface was used as the substrate in the AFM study.M uscovite mica surfaces are typically used as AFM substrates owing to their perfect cleavage along a < 001 > plane, yielding large atomically flat areas. Mica consists of layers of an aluminium phyllosilicate lattice ionically bonded through interstitial K + ions. Upon cleavage, the K + ions are highly mobile and are readily exchanged with divalent cation species at the solid-liquid interface. This exchange results in ap ositive overcharging of the mica surface, which enables the deposition of molecules that hold an et negative charge, such as DNA. [57] Moreover, the positive charge distribution after cleavage enables as uper-hydrophilic surface that guarantees al ower interaction between suspended biomolecules during the evaporation of aqueous solvent. Mica was freshly cleaved by using adhesive tape prior to each deposition to establish its cleanliness. Aliquots (2 mL) of the DNA/imaging buffer were directly deposited by casting onto freshly cleaved muscovite mica. After 2min, every sample was gently washed with deionized water and then dried by evaporation at room temperature under av entilated fume hood.