Synthesis of oligonucleotides
The synthesis of the branched oligonucleotides designed to form a monomolecular G-quadruplex was performed in an automatic DNA synthesizer (Scheme 1). The sequence of the strands was chosen based on the hexamer d(TG4T), which is known to form a stable parallel tetramolecular quadruplex that has been well characterized in previous studies.16–18 In this study, an additional T was inserted to prevent steric hindrance of trebler with the nearest G-quartet. The main structural feature of these oligonucleotides is the attachment of four strand ends through a three-branched linker after the synthetic completion of one of the strands. The branched structure was incorporated into the molecule with commercially available trebler phosphoramidite (Scheme 1). By selecting standard or reversed phosphoramidites at different synthesis steps, three structures with different strand orientations were prepared (Scheme 1). The synthesis of oligonucleotide 1 started with a solid support carrying a T linked via a succinyl linker through its 5′ end (reversed-T). The first TG4TT strand was assembled using reversed phosphoramidites. Trebler phosphoramidite was then added followed by the assembly of the remaining three strands using standard phosphoramidites. After the assembly of the sequence, the final detritylation was not performed in order to facilitate reversed-phase HPLC purification. Ammonia deprotection generated a major eluted product containing three DMT groups. This product was collected and treated with acetic acid to obtain the desired compound, which was then characterized by MS. Oligonucleotide 1 has all four strands in the same orientation and was designed to form a monomolecular parallel G-quadruplex structure.
Oligonucleotide 2 was prepared in a similar way. This time, the first strand was assembled using a 3′-end T-linked solid support and standard phosphoramidite. Trebler phosphoramidite was then added, followed by the assembly of the remaining three strands using standard phosphoramidites. Oligonucleotide 2 had one of the strands in antiparallel polarity compared with the other three strands.
Oligonucleotide 3 was prepared using the standard 3′–5′ direction of synthesis as described for oligonucleotide 2, however, after the addition of the trebler, the assembly of the remaining three strands was performed using reversed phosphoramidites. Thus, oligonucleotide 3 had the four strands in the same orientation as oligonucleotide 1, but linked through the 5′-end, while in oligonucleotide 1 the strands were linked through the 3′-end. Two additional oligonucleotides carrying one single 8-aminoguanine in each position of the first strand (4 and 5) were prepared. Insertion of an 8-aminoguanine residue was performed with 8-amino-dG phosphoramidite protected with a dimethylaminomethylidine group.21 The synthesis of 8-amino-guanine oligonucleotides is straightforward and requires no changes from regular procedures, with the exception of the addition of 2-mercaptoethanol to the cleavage and deprotection solutions to prevent further oxidative damage.21 The addition of 8-aminoguanine to the parallel structure was performed to determine the effect of a single substitution in each position of the parallel quadruplex.
It is important to mention that during the synthesis of oligonucleotides 3–5, the removal of the DMT group after DMT-on HPLC purification was very slow. The usual treatment (80 % acetic acid, 30 min, RT) was not sufficient to remove the three DMT groups linked to sterically hindered secondary alcohols. Instead an increased temperature was required (80 % acetic acid, 30 min, 55 °C).
Analysis of the structure of oligonucleotides 1–3
CD spectra of aqueous solutions of oligonucleotide 1 show a weak positive band with a maximum around 260 nm and a negative band at 240 nm, thereby indicating the presence of residual parallel quadruplex (Figure 1). The addition of K+ (5 mm), Na+ (100 mm) and NH4+ (100 mm) enhanced the CD signal, indicating a strong stabilization of the parallel quadruplex. CD spectra of aqueous solutions of oligonucleotide 2 in water suggest that the sequence is unstructured. Upon addition of NH4+ (100 mM), two positive bands with maxima around 260 and 295 nm were enhanced. This spectrum resembles that expected for a quadruplex with three strands in one direction and one strand in an antiparallel direction (3+1 quadruplex).22 This observation suggests that for oligonucleotide 2 in NH4+, the trebler remains on one side of the G-quadruplex and that the four strands keep the 3+1 orientation given by the synthesis. In contrast, the positive band around 295 nm disappeared and the 260 nm band increased when K+ (5 mm) or Na+ (100 mm) is added instead of NH4+. This indicates the formation of a parallel quadruplex in the presence of K+ and Na+ ions, and it is consistent with the literature on similar tetra-end-linked quadruplexes.15a Our findings show that the branching unit has enough flexibility to allow the antiparallel strand to be antiparallel in water and NH4+ solutions or to be in the parallel orientation in the presence of K+ and Na+ ions. CD spectra of oligonucleotide 3 were very similar to those of oligonucleotide 1, thus indicating the formation of a parallel quadruplex under all conditions studied, including water, with strong stabilization by the addition of K+, Na+ and NH4+ ions.
Figure 1. CD spectra of oligonucleotide 1 (left), 2 (middle) and 3 (right) dissolved in water (—), 5 mM KCl (—), 100 mM NaCl (—) and 100 mM NH4OAc (—).
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The thermal denaturation of quadruplexes formed by oligonucleotides 1–3 and nonbranched [TG4T]4 was studied by UV spectroscopy (Table 1). In the presence of KCl (5 mM), all quadruplexes were stable at temperatures up to 80 °C. In the presence of Na+ ions (10 mM sodium cacodylate, 100 mM NaCl),23 tetra-end linked quadruplexes 1 and 3 were stable up to 80 °C. The tetramolecular [TG4T]4 quadruplex had a melting temperature (Tm) of 58 °C similar to the Tm value of oligonucleotide 2 (55 °C). This result indicates that the quadruplex formed by 2 in the presence of Na+ ions is the least stable. In the presence of NH4+ ions (100 mM NH4OAc), similar results were obtained. Only parallel tetra-end linked quadruplexes (1 and 3) were stable at temperatures up to 80 °C. The tetrameric [TG4T]4 quadruplexes had a Tm value of 67 °C equal to that of oligonucleotide 2.
Table 1. Melting temperatures (Tm).
ESI-MS was performed in order to study the stability of these quadruplexes in the gas phase, and hence without the boiling temperature restriction. However, this analysis is possible only with G-quadruplexes produced from ammonium solutions. In the gas phase, the number of trapped ammonium ions indicates the gas phase stability of these branched G-quadruplexes.19a Figure 2 (center) shows the bidimensional mass/mobility plots obtained for oligonucleotide 1. The 2D plot allows the discrimination of monomeric and dimeric structures (the dimer is discussed below). To analyze the monomer, we extracted the mass spectrum corresponding to the m/z region 1535–1550. The extracted mass spectra of oligonucleotides 1–3 are shown in Figure 2 a–c, and the numbers indicate the number of ammonium ions preserved. Oligonucleotide 2 is the least able to preserve the inner ammonium ions in the gas phase (Figure 2), and hence is the least stable, in agreement with melting experiments in solution. Interestingly, the gas phase data indicate that 1 is more stable than 3, although both oligonucleotides have solution Tm values above 80 °C. Overall, the relative stability of quadruplexes in the gas phase ranks 1>3>2.
Figure 2. Left: ESI-MS of a quadruplex formed by oligonucleotides 1 (a), 2 (b) and 3 (c) and the distribution of the number of NH4+ ions preserved in the G-quadruplex at −6 charge state; the mass spectra were smoothed using a mean function, 2*10 channels, using MassLynx 4.0. Center: 2D ESI-MS and drift time distribution of oligonucleotide 1. Right: ESI-MS of a dimer formed by oligonucleotides 1 (d), 2 (e) and 3 (f); the mass spectra were smoothed using a mean function, 2*30 channels, using MassLynx 4.0.
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The imino proton region of the NMR spectra of oligonucleotides 1–3 confirms the formation of a quadruplex structure (Figure 3). NMR spectra were acquired in Na+ and K+ buffers. Under both conditions, the spectra of the three oligonucleotides exhibited imino signals at δ values in the range of 10.4–11.5 ppm, characteristic of imino protons involved in the Hoogsteen N1HO6 hydrogen bonds of G-quartets. These imino signals were observed at high temperatures, indicating that the three quadruplexes were very stable, being more stable under K+ than Na+ conditions. The relative stability between the three oligonucleotides was in agreement with UV-melting experiments (1> 3> 2). At low temperatures, NMR signals were broad, suggesting the presence of more than one species in equilibrium. In quadruplex 1, signals became sharper upon temperature increase. This effect was more pronounced in K+ buffer and is probably due to the dissociation of multimeric species. To determine the oligomerization state of the samples, we performed native gel electrophoreses (see below).
Figure 3. Top: General scheme of a tetra-end-linked quadruplex showing the numeration of the residues as mentioned in the text. Bottom: exchangeable proton region of 1H NMR spectra at different temperatures of oligonucleotides 1, 2 and 3 in 10 mM sodium phosphate buffer (upper rows) or 10 mM potassium phosphate buffer (lower rows).
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NMR spectra of 1 in K+ buffer conditions were of sufficient quality to acquire 2D experiments. On the basis of NOESY and TOCSY spectra, four spin systems with relatively sharp signals were clearly identified, and they were sequentially assigned to residues 1–4. NOE cross-peak patterns for these residues indicate that the four chains are equivalent and the guanines adopt an anti-conformation. The remaining nucleotides (G5, T6 and T7) presented a broad signal and their NOE cross-peaks were almost invisible at low temperatures (Figure 4). At higher temperatures, these residues exhibited multiple cross-peaks in the NOESY spectra (Figure 4), suggesting the presence of several conformers in this region of the molecule. The presence of these conformers is possibly related to a conformational heterogeneity that affects the nucleotides near the linker. This is consistent with the number of signals and their relative intensities observed in the 1D spectra at this temperature (Figure 3).
Figure 4. Fragments of NOESY spectra (250 ms mixing time) of oligonucleotide 1 at 5 °C (bottom) and 45 °C (top) in 10 mM potassium phosphate (pH 7).
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Methyl-imino cross-peaks in the exchangeable proton region of the NOESY experiment allowed for the assignment of H1 of G2 (Figure S2 in the Supporting Information). Also an imino–imino sequential cross-peak between H1G2 and H1G3 was clearly observed. Other exchangeable protons corresponding to G4 and G5 were also observed, but could not be specifically assigned. The number of exchangeable proton signals and their cross-peak pattern indicated the formation of four guanine tetrads.
In summary, the NMR experiments indicate that at high temperature oligonucleotide 1 forms a symmetrical parallel quadruplex, most probably monomeric, where the four chains are equivalent and all guanines adopt an anti-conformation. Although oligonucleotide 1 exhibits a single global fold, some conformational heterogeneity occurs in residues close to the linker. This is probably due to steric constraints provoked by the linker, which impede the interconversion between different conformers. At low temperatures, NMR signals are broader suggesting an equilibrium with other species of higher molecular weight.
Presence of dimeric quadruplex structures
Native polyacrylamide gel electrophoresis (PAGE) has been widely used to detect oligomers and aggregates. Electrophoretic analysis was carried out using the tetramolecular quadruplex [d(TG4T)]4 as a reference. First, native PAGE was performed to assess the oligomerization state of the quadruplexes studied by NMR (oligonucleotides 1–3). In all cases oligonucleotides resulted in two major bands suggesting that they form not only monomeric species but also dimeric structures (Figure S1 in the Supporting Information). Also, in all cases the band corresponding to the monomer was the major band (60–70 %). ESI-MS showed that branched oligonucleotide sequences (1–3) to some extent form dimers in ammonium acetate (100 mM). The ESI-MS spectra recorded for three sequences are shown in Figure 2 d–f. Quadruplex 3 had a lower signal-to-noise ratio as a result of the presence of residual salts, but in all three cases, the presence of a dimeric quadruplex as a minor component was confirmed by mass spectrometry.
We propose two hypothetical dimeric structures for the dimer formed by these branched oligonucleotides. The association of two molecules allows the formation of two parallel quadruplexes, each containing one strand belonging to the adjacent molecule (e.g., interlocked structure B shown in Figure 5). The second model proposed is based on previous observations reported in the literature (structure A in Figure 5). Crystallographic studies of d(TG4T) quadruplexes performed by Cáceres et al.24 revealed the stacking of T tetrads between neighboring quadruplexes packed in a head-to-head fashion. Recent NMR studies on UG4U revealed the existence of a dimeric quadruplex structure in the presence of K+ and NH4+ but not Na+ ions.25 ESI-MS studies on telomeric sequences performed by Collie et al26 revealed that telomeric RNA form higher-order dimeric assemblies, initiated by cation-mediated stacking of two parallel G-quadruplex subunits.
This dimer, consistent with two G-quadruplex subunits, each with three NH4+ ions, plus one NH4+ ion stacked between the two subunits was observed by ESI-MS experiments. This result suggests a structural model for the dimer involving the cation-mediated stacking of G-quadruplex subunits (structure A in Figure 5).
Analysis of the stability of oligonucleotides carrying 8-aminoguanine
CD studies and thermal denaturation were performed in order to study the effect of 8-aminoguanine substitution on the stability of the quadruplex. As described above, in the presence of K+ ions (5 mM KCl), all quadruplexes were stable up to 80 °C as no changes in the UV spectra were observed. For this reason, oligonucleotides 3–5 were dissolved in water and CD thermal denaturation was performed (see the Supporting Information). Under these conditions, the samples contained some residual triethylammonium acetate (TEAA) from HPLC purification. Even in the absence of K+ and Na+ ions, the CD spectra of oligonucleotides 3–5 showed the presence of a parallel quadruplex structures: a positive band with a maximum at 260 nm and a negative band with a minimum at 240 nm. In the absence of K+ and Na+ ions, unmodified oligonucleotide 3 had a Tm value of 50 °C (Table 1). The introduction of an 8-aminoguanine residue in the external position of the quadruplex (oligonucleotide 4) produces a quadruplex with similar stability (52 °C). In contrast, the substitution of a single G by 8-aminoguanine in the internal quartet (oligonucleotide 5) induced the formation of a very stable quadruplex, as only a partial melting was observed at 80 °C.