8-Oxoguanine Forms Quartets with a Large Central Cavity

Oxidation of a guanine nucleotide in DNA yields an 8-oxoguanine nucleotide (oxoG) and is a mutagenic event in the genome. Due to different arrangements of hydrogen-bond donors and acceptors, oxoG can affect the secondary structure of nucleic acids. We have investigated base pairing preferences of oxoG in the core of a tetrahelical G-quadruplex structure, adopted by analogues of d(TG4T). Using spectroscopic methods, we have shown that G-quartets can be fully substituted with oxoG nucleobases to form an oxoG-quartet with a revamped hydrogen-bonding scheme. While an oxoG-quartet can be incorporated into the G-quadruplex core without distorting the phosphodiester backbone, larger dimensions of the central cavity change the cation localization and exchange properties.


■ INTRODUCTION
Guanine-rich repeats are abundant in telomeres and promoter regions of genomes, where they can form non-B DNA structures. 1 Four guanines can form a G-quartet, stabilized by hydrogen bonds in the Hoogsteen geometry. G-quartets stack with each other to form the core of a G-quadruplex. In tetrameric G-quadruplexes, four oligonucleotides adopt parallel or antiparallel directionalities, and guanine nucleotides adopt either anti or syn conformations. 2 Guanine has the lowest redox potential of the four nucleobases found in vivo and is thus most likely to get oxidized. G-rich repeats are even more susceptible to oxidation. 3 Reactive oxygen species (ROS), which are a byproduct of cellular respiration, can induce oxidation of the guanine moiety to yield 8-oxoguanine nucleotides ( oxo G), among other oxidation products. 4,5 It is believed that G-rich regions act as oxidation sinks, thus serving as DNA damage reservoirs. 6,7 oxo G can pair with both adenine and cytosine in a DNA duplex and thus cause G to T transverse mutations. 8 When paired with adenine, oxo G adopts the syn conformation in B-DNA duplexes as shown by NMR, 9 X-ray diffraction, 10 and molecular dynamics. 11 Similarly, oxo G adopts the syn conformation in G-quadruplexes originating from human telomeres, leading to structure reorganization. 12,13 oxo G has recently been found to accumulate in enhancer regions of the human genome, with the oxidized enhancers also being enriched with G-quadruplex structures. 14 However, we are far from understanding how the incorporation of oxo G into Grich oligonucleotides affects the formation of G-quadruplexes and their thermal stability. 12,13,15−17 Since G-quadruplex forming sequences are found in regulatory regions of the genome and in telomeres, a change in the structure and/or stability of G-quadruplexes can affect cellular processes such as replication, transcription, and telomere maintenance. 18−20 Our recent studies revealed that the introduction of a single oxo G into G-rich constructs originating from human telomeres and oncogene promoter regions does not necessarily prevent G-quadruplex formation. 13,21 Three scenarios were found to accompany oxo G incorporation. Certain positions were found to tolerate oxo G substitutions while retaining the original Gquadruplex topology. Alternatively, substitutions with oxo G induced changes in strand directionality and/or rearrangements in G-quadruplex loops. 13 Both scenarios resulted in suboptimal hydrogen bonding of oxo G and a substantial reduction of thermal stability. 21 However, in the third scenario, oxo G was displaced from the G-quadruplex core and formed well-stacked (wobble) base pairs with loop nucleotides, which enhanced the thermal stability of the G-quadruplex structure.
This work focuses on the evaluation of structural effects and changes in thermal stability caused by the incorporation of oxo G in a simple G-quadruplex model system without interference of loop interactions. A model system was chosen to discern the effects of oxo G incorporation that originate from specific stacking and/or hydrogen bonding between oxo G and adjacent G nucleotides. For this purpose, we utilized the d(TG 4 T) oligonucleotide, which forms a parallel tetrameric Gquadruplex with four G-quartet planes and thymine overhangs on 5′ and 3′ ends. 22,23 It was previously shown that G to oxo G substitutions within d(TG 4 T) generally decrease the thermal stability of G-quadruplex structures in a Na + cation solution; however, when substituting the third guanine position in d(TG 4 T), a slight increase in thermal stability of the Gquadruplex structure was observed. 15 Mixed oxo G-G quartets could be achieved either by reversal of directionality of two strands resulting in antiparallel G-quadruplexes or by slipping of two strands, while retaining the parallel topology. Our initial hypothesis was that mixed quartets, including both G and oxo G nucleotides, would induce less perturbations in the structure and be preferred over quartets composed exclusively of oxo G. The affinity of oxo G for the syn glycosidic conformation could favor strand reversal and influence stacking interactions within individual strands.

Oligonucleotide Synthesis and Sample Preparation.
Oligonucleotides were synthesized using a DNA/RNA H-8 Synthesizer (K&A Laborgeraẗe) operating on the phosphoramidite chemistry principle and using nucleotide phosphoramidites obtained from Glen Research. All oligonucleotides were synthesized with DMT protecting group. Deprotection and deblocking were achieved with ammonium hydroxide and methylamine in a 1:1 (v/v) ratio for 30 min at room temperature and 30 min at 65°C. Samples were purified using GlenPak cartridges and desalted on FPLC with a HiPrep 26/ 10 Desalting column (GE Healthcare). Samples were dried using a vacuum centrifuge and dissolved in 1 mL of deionized water and dialyzed against 110 mM NaCl or KCl overnight. After dialysis, 2 H 2 O was added to a final 10% concentration. Solutions were buffered at pH 7 using either NaPi for Na + solution or KPi for K + solution. The oligonucleotide solutions were heated to 95°C for 5 min and left to cool at room temperature. For mixed Na + /K + cation samples, 100 mM KCl samples were diluted 2-fold and sodium chloride solution and 2 H 2 O were added to a final concentration of 50 mM NaCl, 50 mM KCl, 5 mM KPi pH 7, and 90%/10% 1 H 2 O/ 2 H 2 O. Concentration of monomers in solution was determined by UV spectrophotometry using a Varian Cary 100 Bio UV/VIS spectrophotometer at a wavelength of 260 nm, using the extinction coefficient ε = 57,800 M −1 cm −1 for all oligonucleotides. The extinction coefficient was determined by the nearest neighbor method. The concentration of oligonucleotides per strand was between 0.5 and 1.0 mM. CD Spectroscopy. NMR samples of ODN2-5 in a mixture of 50 mM NaCl and 50 mM KCl were kept at NMR concentration and their CD spectra were recorded on a Chirascan CD spectrometer (Applied Photophysics) at 25°C. Spectra were recorded at wavelengths from 220 to 320 nm with a 0.1 mm cell length in 10 parallels.
UV Melting. We diluted 30 μL of Na + /K + NMR sample in 970 μL of buffer solution (50 mM NaCl, 50 mM KCl, 5 mM potassium phosphate, pH 7). We recorded absorbance at 260 nm wavelength from 15 to 95°C, with a temperature gradient of 0.5°C/min and sampling at every 0.1°C. We determined the first-order derivatives using Origin 2018.
NMR Spectroscopy. NMR data were collected on Bruker Avance NEO 600 and 800 MHz NMR spectrometers. Onedimensional (1D) and two-dimensional (2D) NMR spectra were acquired using excitation sculpting to achieve solvent suppression. Molecular Dynamics. Molecular dynamics calculations were performed with the AMBER 20 software using the ff99bsc0 force field and ε/ζOL1 and χOL4 modifications. Force field parameters for oxo G nucleotides were obtained from the R.E.D. Server. Calculations were started from initial linear structures obtained with the LEAP module of AMBER 20. A total of 100 structures were obtained in 1 ns restrained simulated annealing simulations using the Born implicit solvent model with random starting velocities. Restraints included hydrogen-bond distances in G-and oxo G-quartets and χ torsion angles. Force constants were 20 kcal·mol −1 ·Å −2 for hydrogen bonds and 200 kcal·mol −1 ·rad −2 for torsion angles. In the first 200 ps, the temperature was held at 1000 K. The temperature was decreased to 300 K in the following 400 ps and further decreased to 0 K in the last 400 ps. Ten structures were selected based on lowest energy and used for further analysis.
DFT Geometry Optimization. DFT geometry optimization was performed with ORCA utilizing gCP geometrical counterpoise correction, 24 D3BJ atom-pairwise dispersion correction with the Becke−Johnson damping scheme, 25,26 the def2-mSVP basis set, def2/J auxiliary basis set, 27 and employing the PBEh-3c composite approach. 28 An implicit water model was included using the CPCM method. 29 The oxo G-quartet was first generated in Avogadro and converted to an ORCA input file. The ORCA output structures were analyzed using UCSF Chimera. 30 ■ RESULTS AND DISCUSSION oxo G Analogues of d(TG 4 T) Form Parallel G-Quadruplexes. Analysis of imino regions of 1 H NMR spectra characteristic for Hoogsteen hydrogen bonding of oxo G analogues of d(TG 4 T) (designated as ODN2-5) reveals their folding into G-quadruplex structures in the presence of Na + and/or K + ions (Figures 1 and 2). Notably, an oxo G nucleobase gives rise to a pair of imino resonances due to protons attached to both N1 and N7. H7 resonances of oxo Gs are generally found downfield from the corresponding H1 resonances most likely due to the deshielding effect of the adjacent carbonyl group. Folding in the presence of 100 mM NaCl is slow and Figure 1. Schematic representation of the topology of the Gquadruplex adopted by d(TG 4 T). White spheres depict thymine nucleotides. Gray rectangles and black spheres are used to depict guanine nucleotides involved in G-quartets. Numbering of nucleotides is indicated along the right side of the scheme. Sequences, names used in this study, and temperatures of unfolding of oxo G analogues are presented in the table. T unf is the apparent midpoint of the thermal unfolding absorbance curve of a tetramolecular G-quadruplex. Oligonucleotides were dissolved in 50 mM KCl, 50 mM NaCl, 5 mM KPi, pH 7. The concentration of oligonucleotides was between 8 and 12 μM per strand. significant amounts of oligonucleotides remained unfolded even after several days of incubation at room temperature ( Figure S1). On the other hand, folding of ODN2-5 in the presence of 100 mM KCl is fast (within minutes) ( Figure S2). Interestingly, spectra of ODN3 and ODN4 in the presence of K + cations give rise to two sets of some imino, aromatic and deoxyribose 1 H resonances (Figures 2 and S2), which suggests that two major species coexist in solution and are involved in slow exchange on the NMR chemical shift timescale ( Figure  S3). At 25°C the ratio of signal integrals between imino resonances of the two species in ODN3 is 1:2, while it is 1:1 in ODN4. However, at higher temperatures, the resolved sets of two resonances coalesce as the exchange between the two species is accelerated ( Figure S4).
Dissolving ODN2-5 in a mixed salt solution of 50 mM NaCl and 50 mM KCl gives best NMR spectral quality with complete and fast folding into G-quadruplex structures. Furthermore, the split resonances in ODN3 and ODN4 coalesced ( Figure 2). Therefore, we have attributed the signal splitting in ODN3 and ODN4 to two long-lived localization modes of K + cations in the vicinity of oxo G nucleotides. It is noteworthy that broadening of oxo G3′s imino resonances is observed in spectra of ODN3 under these conditions. Interestingly, the effect of Na + cations could not be replicated by adding NH 4 + cations as no resolving of the split resonances was observed in ODN3 and ODN4 ( Figure S5). Furthermore, resolving of the split resonances was also achieved with the addition of Cs + cations to ODN3 and ODN4 G-quadruplexes in a K + cation solution. Only imino resonances belonging to oxo G protons show considerable chemical shift differences after addition of Cs + , indicating that Cs + must localize in the vicinity of oxo Gs. ( Figure S6). Due to their favorable spectral properties, G-quadruplexes of ODN2-5 dissolved in a mixture of 50 mM NaCl and 50 mM KCl were chosen for further characterization. Low-intensity imino resonances (<5%) were also noted in NMR spectra of ODN2-5, but could not be characterized in detail due to the low population of these minor species.
The stability of G-quadruplexes was evaluated with UV melting experiments of ODN2-5 in a mixture of 50 mM NaCl and 50 mM KCl (Figures 1 and S7). The UV melting profile of ODN2 does not exhibit signs of unfolding at up to 95°C. Similarly, only initial indications of unfolding can be observed close to 95°C for ODN5. This is consistent with the high thermal stability of the parent d(TG 4 T) 4 G-quadruplex, which showed no signs of unfolding up to 90°C in a 110 mM KCl solution. 15 On the other hand, the thermal stabilities of ODN3 and ODN4 are lower with T unf values of 91 and 82°C, respectively.
A detailed structural analysis of G-quadruplexes adopted by ODN2-5 was undertaken with a set of NMR experiments. Imino, aromatic, methyl, and well-resolved deoxyribose proton resonances in NOESY spectra of ODN2-5 in the presence of 100 mM NaCl, 100 mM KCl, and a mixture of 50 mM NaCl and 50 mM KCl were assigned ( Figure 3). Sequential walks can be traced in aromatic-anomeric regions of NOESY spectra for ODN2-5 regardless of the type of cations present in solution and exhibit similar connectivity patterns suggesting that all G-quadruplexes adopt the same topology. Furthermore, the two major species observed in solutions of ODN3 and ODN4 in the presence of K + cations exhibit identical NOE cross-peak patterns, which is in agreement with our rationale that the two species differ only in K + cation localization.
Intensities of intra-residual cross-peaks in NOESY spectra of ODN2-5 correspond to all G and T nucleotides adopting the anti conformation, which is typical for parallel G-quadruplexes (data for ODN4 is shown as an example in Figure 3). As expected, sequential walks for ODN2-5 are interrupted at oxo G positions due to absence of aromatic H8 protons. Consequently, we were not able to directly determine the preference for syn or anti conformations of oxo G based on the intensity of oxo G (i) H1′-H8 cross-peaks in NOESY spectra alone. Therefore, we resorted to the analysis of chemical shifts  . This is in agreement with chemical shifts for 8-substituted purine nucleosides in the syn conformation, 31 and suggests that all oxo G nucleotides in ODN2-5 are in the syn conformation. Interestingly, intense cross-peaks can be observed in NOESY spectra of ODN2-5 between anomeric protons of oxo Gs and aromatic protons of subsequent nucleotides [i.e., oxo G (i) H1′-G (i+1) H8] ( Figure 3A). Furthermore, amino protons of oxo Gs are found to be isochronous at 25°C and with their chemical shifts in the range from δ 6.1 to 6.5 ppm ( Figure S10), suggesting they are not involved in hydrogen bonds.
In the imino-imino regions of NOESY spectra only sequential connectivities are observed ( Figure 3B) and the imino-aromatic regions exhibit G (i) H1-G (i) H8 cross-peaks ( Figure 3C), both of which are in agreement with the parallel G-quadruplex topology. Additionally, oxo G (i) NH 2 -G (i−1) H8 ( Figure 3A) and oxo G (i) H1-G (i−1) H8 ( Figure 3C) cross-peaks are observed in NOESY spectra of ODN2-5. However, due to the symmetry of ODN2-5 G-quadruplexes, it is ambiguous if these cross-peaks are of intra-or interstrand nature. oxo G nucleobases Exhibit a Distinct Intra-Quartet Hydrogen-Bonding Scheme. The hydrogen-bonding network of oxo G nucleotides within G-quadruplex structures was determined via analysis of NOE connectivities. Upfield chemical shifts of amino protons of oxo Gs (vide supra) suggest that they are not hydrogen-bond donors. On the other hand, downfield shifted narrow H1 and H7 resonances of oxo G suggest that both protons are protected from exchange with bulk solvent and involved in hydrogen bonds. We observe intense NOE cross-peaks between H1 and H7 of oxo Gs ( Figure  3B). Such cross-peaks cannot arise from intra-nucleotide correlations due to the large distance between H1 and H7 within the oxo G nucleobase (cca. 5.0 Å). oxo Gs also cannot be positioned in different G-quartet planes as this would result in a minimum plane separation distance of 3.4 Å. Furthermore, oxo Gs in different quartet planes is not in agreement with the NMR data, which shows the formation of symmetrical parallel G-quadruplexes. However, a simple model with a planar arrangement of four oxo G nucleobases exhibits short H1 to H7 distances (cca. 2.3 Å), which is in agreement with collected NMR data. Their interpretation led us to propose the formation of an oxo G-quartet, comprised of four oxo G nucleobases, connected via hydrogen bonds N1−H1···O8 and N7−H7···O6 ( Figure 4A). The same hydrogen-bonding arrangement was already proposed for helix-forming lipophilic 8-oxoguanine derivatives. 32 A detailed model of an oxo G-quartet, formed by four 9methyl-8-oxoguanines, was obtained via DFT optimization (using the def2-mSVP basis set and PBEh-3c method). Simulations were carried out without cations and with either a Na + or a K + cation in-plane and in-line with the ion cavity of the oxo G-quartet. During the optimization planarity of the oxo Gquartet was constrained to prevent any out-of-plane movement of the nucleobases. Selected distances in the energy-optimized oxo G-quartet geometries are summarized in Table 1. To assess the size of the central cavity of the oxo G-quartet, we measured the distances between carbonyl group O6 atoms of the neighboring 9-methyl-8-oxoguanines ( Figure 4A). The distance ranges from 3.72 Å, when a Na + cation is positioned in the plane of the oxo G-quartet, to 4.04 Å, when no cation is present.
The distance between diagonally opposite O6 atoms in the oxo G-quartet ranges from 5.27 to 5.72 Å. In comparison, the average distance between diagonally opposite O6 atoms in Gquartets (PDB: 352D 33 ) amounts to 4.50 Å. Taking into account the van der Waals radii of the O6 atoms, a sphere with a maximum diameter of 2.68 and 1.46 Å is able to fit into the oxo G-quartet and G-quartet cavity, respectively ( Figure 4B,C). Hydrogen-bond distances are less variable between the optimized geometries, with a maximum difference between the distances of the N1−H1···O8 hydrogen bond amounting to 0.04 Å. A comparison of space-filling models of a G-quartet (PDB: 352D 33 ) and a DFT-optimized oxo G-quartet is shown in Figure 4, demonstrating the larger central cavity of the oxo Gquartet.
Using the optimized geometry of the oxo G-quartet, we have performed atomistic simulations of ODN2-5 G-quadruplexes using a simulated annealing protocol. Since ODN2-5 Gquadruplexes exhibit fourfold symmetry, we were unable to unambiguously determine inter-or intra-nucleotide nature of certain NMR distance constraints. Therefore, we only included hydrogen-bond distance and glycosidic torsion angle restraints in simulated annealing calculations. Good convergence was achieved over 100 runs of simulated annealing ( Figure 4D and Table S1). Calculations resulted in parallel, right-handed Gquadruplex structures, with G and oxo G nucleotides in anti and syn conformations, respectively. Efficient stacking of six- Biochemistry pubs.acs.org/biochemistry Article membered rings of oxo G (i) and G (i−1) nucleobases is observed in all structures ( Figure 4E and Table 2). On the other hand, only partial stacking of five-membered rings can be observed between oxo G (i) and G (i+1) nucleobases, resulting in a much lower stacking surface ( Figure 4F and Table 2). oxo G has an effect on the rise and twist, with the oxo G (i) -G (i+1) step exhibiting a larger rise parameter compared to the rest of the structure and the twist being considerably larger for the steps which include the oxo G nucleotide (Table 2). Specific stacking of oxo G nucleobases is reflected in CD spectra of ODN2-5 ( Figure 5). ODN2 and ODN5 with terminal oxo G-quartets exhibit CD maxima at 265 and 270 nm, respectively. On the other hand, CD spectra of ODN3 and ODN4, with oxo G-quartets sandwiched between G-quartets, exhibit two maxima at 240 and 275 nm. Interestingly, a  Distances in DFT-optimized oxo G-quartets without cations and with a Na + or K + cation positioned in the center of the oxo G-quartet plane. DFT optimization was done using the PBEh-3c method and the def2-mSVP basis set. Distance between hydrogen atom and hydrogen-bond acceptor. b Distance between hydrogen-bond donor and hydrogenbond acceptor. c Distance between the O6 atoms of two neighboring 9-methyl-8-oxoguanines. d Distance between the O6 atoms of the two diagonally opposite 9-methyl-8-oxoguanines.  Biochemistry pubs.acs.org/biochemistry Article shoulder is present in the CD spectrum of ODN2 above 300 nm, which may point to DNA condensation into higherordered structures. 34 Oxidation of G to oxo G leads to a rearrangement of hydrogen-bond donors and acceptors on the Hoogsteen edge of the nucleobase. The newly protonated N7 precludes the formation of the N2−H2···N7 hydrogen bond found in canonical G-quartets. Our previous study showed that a single oxo G lesion in a G-quartet can be tolerated, with the O6 of oxo G serving as a hydrogen-bond acceptor for a bifurcated hydrogen bond with the neighboring guanine. 13 Furthermore, G-quartet formation was not hindered when an oxo G was hydrogenbonded with a neighboring xanthine. 17 Here, we show that an oxo G-quartet can also be a stable structural element within the G-quadruplex core with H7 as a hydrogen-bond donor.
While the planar oxo G-quartet stacks well with an adjacent 5′ G-quartet, stacking with a 3′ G-quartet is less efficient, as conferred through analysis of stacking surfaces. Nevertheless, G-quadruplexes with 5′-and 3′-terminal oxo G-quartets in ODN2 and ODN5, respectively, are more thermally stable than ODN3 and ODN4 with an oxo G-quartet sandwiched between two G-quartets. This is also reflected in differences in CD spectra of ODN2 and ODN5 versus ODN3 and ODN4 and is likely related to less constrained positioning of oxo G at 5′-and 3′-termini of the G-quadruplex core. 35 Since the anti conformation is unfavorable for nucleotides with a (bulky) substituent at position 8 (e.g., oxo G), it is not surprising that the oxo G-quartet features an all-syn arrangement. Interestingly, d(TG 4 T) 4 was reported to exhibit 15% of 5′-end G-quartets in all-syn orientation. 36 A study has shown that a slow dynamic interconversion between all-syn and all-anti Gquartets is possible without disrupting the whole G-quadruplex core. 37 This is not the case here with ODN2-5 where no synanti flipping of nucleobases could be detected. However, perturbations in the hydrogen-bond network cause mutual repositioning of oxo G nucleobases. Using DFT optimization, we showed that oxo G-quartets exhibit a central cavity, with a 4.04 Å distance between O6 atoms of two neighboring 9-methyl-8oxoguanines. For comparison, the average O6−O6 distance in G-quartets is 3.15 Å, which makes the central cavity in oxo Gquartets considerably larger. Studies of crystal structures showed that K + cations localize equidistantly between two G-quartet planes. 38,39 On the other hand, due to their smaller ionic radius, Na + cations can localize in a G-quartet plane or any distance between two planes. 33 However, due to the larger cavity in the center of an oxo G-quartet and only minor differences in H-bond lengths and neighboring carbonyl distances the absence or presence of different cations, an inplane localization of Na + as well as K + cations is feasible. Furthermore, due to reduced steric restrictions cation movement through the oxo G-quartet plane is expected to be faster (i.e., exchange between binding sites).
We have observed that in the presence of K + cations alone, two structures were present in solution for ODN3 and ODN4 at 25°C, which were shown with 2D ROESY spectra to be in slow exchange (Figure S3). At 25°C, the maximum chemical shift difference between a pair of doubled resonances ( oxo G3H7 in ODN3) at a magnetic field of an 800 MHz spectrometer is 0.34 ppm, which corresponds to a lifetime of 3.6 ms. We have eliminated the possibility of switching of the glycosidic conformation or sugar-repuckering of oxo G nucleotides, since no relevant cross-peaks could be observed in NOESY or DQF-COSY/TOCSY experiments. We have also eliminated the possibility of tautomerism of the 8-oxoguanine moiety. Chemical shifts and intensities of the imino proton resonances are pH-independent, which does not support the formation of an 8-hydroxy tautomer ( Figure S11). In full agreement, quantum mechanical studies showed that the 8keto tautomer is predominant. 40 Instead, we propose that the two species of ODN3 and ODN4 differ in K + cation localization. This is in agreement with the millisecond lifetimes of ammonium ions bound within the d(TG 4 T) G-quadruplex. 23 K + cations may localize either between an oxo G and a G-quartet or in an oxo G-quartet plane ( Figure 6). Due to mutual Coulombic repulsion, one K + cation could be ejected from the G-quadruplex structure. Nevertheless, in-plane K + localization appears to be suboptimal since the addition of smaller size Na + cations to existing solutions of ODN3 and ODN4 with K + leads to resolution of doubled resonances in 1 H spectra. The addition of Na + cations likely eliminates K + movement by preferential localization of Na + in-plane of oxo Gquartets. Interestingly, the same effect was observed with the addition of Cs + cations. However, binding of the considerably larger (with respect to Na + and K + ) Cs + cation is expected between a G-quartet and oxo G-quartet plane (vide supra). In ODN2 and ODN5, where oxo G-quartets are positioned at the 5′-and 3′-termini of the G-quadruplex core, K + cation movement is fast at temperatures as low as 5°C and only single sets of NMR resonances can be observed ( Figure S12). NMR spectra of ODN2 and ODN5 did not show any significant differences after the addition of Na + cations, except for minor changes in imino proton chemical shifts and resonance linewidths (Figure 2).
Based on the results reported herein and on previous studies of the effect of oxo G incorporation on the structure and stability of G-quadruplexes, 13,21 oxo G may act as a cryptic lesion in the context of G-rich regions without the presence of a complementary strand. Proposed pathways of oxo G repair in double-stranded G-rich DNA also stipulate that oxo G is not destabilizing enough to promote a duplex-to-quadruplex transition. Destabilization of the duplex and transition to a G-quadruplex is thought to occur after excision of oxo G via the action of the glycosylase OGG1, which yields an abasic site. 41,42 Oxidative damage in vivo is highly unlikely to introduce more than a single oxo G lesion within short DNA stretches needed for one G-quadruplex unit. 43 However, the d(TG 4 T) 4 model system demonstrates how redistribution of hydrogen- Figure 6. Proposed localization of Na + and K + cations within the ODN4 G-quadruplex. K + cation may localize in between the quartet planes (A) or in an oxo G-quartet plane (B). Mixed Na + /K + form (C) could exhibit in-plane bound Na + cations. Unbroken and broken lines represent G-quartets and oxo G-quartets, respectively. Biochemistry pubs.acs.org/biochemistry Article bond donors and acceptors affects the structure of nucleic acids, which can also have a functional effect. For instance, siphoviruses use aminoadenine instead of adenine in their genome, therefore having three hydrogen bonds in the aminoadenine−thymine base pair, and have a DNA polymerase that preferentially selects aminoadenine for a thymine template. 44 Since oxo G is a DNA lesion frequently occurring alongside pathophysiological changes, 45,46 insights into the effect of guanine oxidation on structural changes of nucleic acids in functionally important genome regions could reveal possible disease origins.
■ ASSOCIATED CONTENT * sı Supporting Information