Letter pubs.acs.org/JPCL
Biocompatible Xanthine-Quadruplex Scaffold for Ion-Transporting DNA Channels Jan Novotný, Petr Kulhánek,* and Radek Marek* CEITEC − Central European Institute of Technology, Masaryk University, Kamenice 5/A4, CZ-62500 Brno, Czech Republic S Supporting Information *
ABSTRACT: Molecular dynamics simulations and adaptive biasing force analysis of the quadruplex DNA dynamics in an explicit solvent reveal fundamentally different mechanisms of Na+ transport in xanthine- and guanine-based DNA systems. The barrier to the transport of K+ through the xanthine-based quadruplex is significantly lower than those reported for the guanine-based analogs.
SECTION: Biophysical Chemistry and Biomolecules
guanine tetrads are more stable than their xanthine analogs in the gas phase as well as in stacked systems (telomere-like structures) because of the strong cooperativity effects (−21 kcal mol−1) of the hydrogen bonds (HBs). However, this difference seems to be vanishingly small in an implicit water environment, where the synergy of the HB formation in guanine tetrads drops to nearly 0 kcal mol−1.15 In this work, we extended the elegant idea of a xanthine scaffold to the construction of artificial N3-xanthosine-modified DNA quadruplexes (XQs). We employed unrestrained molecular dynamics (MD) simulations to characterize the stability and dynamic behavior of the XQs as compared with the guanine-based DNA quadruplexes (GQs).16 We also investigated the mechanism of ion and water transport in both types of quadruplexes by means of free-energy calculations. First, we performed MD simulations using various starting models of d(X4)4·nM+ and d(G4)4·nM+ systems. These differ essentially in the arrangement of the cations and the water molecules inside the quadruplex channel, the bulk counterions, and the ionic strength. The simulations were performed using an ff99bsc0 force field17,18 and an explicit solvent (TIP3P model) on a time scale of 30−50 ns. Although this time scale could be insufficient to evaluate the long-term stability or folding processes of quadruplexes, it should be relevant for analyzing the structural fluctuations.19 The analysis of MD trajectories (rmsd plots in Supporting Information, Figures S1 and S2) points to rather similar degree of fluctuations of the
Noncovalent interactions are central to many processes in various fields ranging from supramolecular chemistry and nanosciences to biochemistry and structural biology.1 DNA quadruplex motifs based on hydrogen-bonded tetrads formed by cation-templated assemblies of guanines have been known since 1962.2 The guanine (G) quadruplexes present in the 3′ overhanging ends of chromosome (telomeres) represent an essential part of eukaryotic cells. Therefore, G-quadruplexes are considered to be potential therapeutic targets.3 In general, the structure of a guanine quadruplex is characterized by three principal noncovalent interactions: (a) the hydrogen bonding that is responsible for the self-assembly of guanine-based tetrads; (b) the stacking interactions that enable these tetrads to couple and form octameric, oligomeric, and polymeric assemblies; and (c) the ion−dipole interactions between monovalent cations (e.g., Na+ or K+ incorporated in the intertetrad regions)4 and the tetrads that provide additional stability to these supramolecular systems.5 Although DNA/ RNA G-quadruplexes were originally found to bind K+ ions more readily than Na+ ions, an effect attributed to the better fit of K+ into the interbase cage of the system,6 this selectivity was later shown to be caused mainly by the lower dehydration energy of K+ as compared with that of Na+.7 Nowadays, it is known that ions move relatively quickly in the inner channel of the G-quadruplex without disrupting the G-tetrad.8−10 This phenomenon is currently being explored extensively because of its potential applications in materials science,11 biosensor design, and nanotechnology.12,13 Very recently, Kovacs et al. have investigated N3methylxanthine as a novel building block forming the tetrad structures.14 The formation of tetrameric and octameric assemblies was detected by using mass spectrometry and NMR spectroscopy. Theoretical calculations have indicated that © 2012 American Chemical Society
Received: May 3, 2012 Accepted: June 19, 2012 Published: June 19, 2012 1788
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therefore crucially dependent on the presence of water molecules inside the XQ channel, and further analysis reveals the direct coordination of water molecules with the cation. On the contrary, the binding of K+ in the interplane region of the XQ is impervious to the applied solvent model. The distinctly preferred positions of the Na+ (in-plane) and K+ (interplane) ions, each coordinated with two water molecules in the pore of the d(X4)4 system, are shown in Figure 2. Na+ is trapped in the plane of the xanthine tetrad by the four oxygen atoms at the rim of the pore. This chelating effect induces a moderate compression of the pore (the radius is reduced from 2.7 to 2.5 Å, see Figure 3), which might be
XQ and GQ systems. All of the simulations showed that the XQs have significantly different structural parameters for the sugar−phosphate backbone as compared with the GQ systems (Supporting Information, Figures S3−S5). In addition, the average radius (∼2.6 Å) of the pores in the quadruplex system consisting of dX4 units was considerably larger than that of its GQ analog (∼2.2 Å) (Figure 1, Supporting Information,
Figure 1. Structures of GQ (left) and XQ (right) tetrads, highlighting the averaged diameters of their pores.
Figures S6 and S7). This might be one of several reasons for the different ion interactions with the quadruplex channel. In d(G4)4, the channel is occupied by three sodium or potassium ions staying permanently in the interplane regions. The absence of any of these three ions leads either to lower quadruplex stability or to its complete destruction, which is consistent with previous experimental and computational findings.20 In contrast, the channel of d(X4)4 is capable of accommodating only one sodium cation oscillating between the planes of the individual xanthine tetrads. Any attempt to put more sodium cations into the channel leads quickly to their expulsion from it. The d(X4)4 also appears to be stable in the presence of a single potassium, cesium, or ammonium cation. However, these are trapped in the central interplane area of the quadruplex. The rest of the xanthine channel is occupied by water molecules, which are retentively (for Na+) or transiently (for cations with a larger radius) coordinated with the cation. Simulations of the extended systems d(X6)4·1Na+ and d(X8)4·2Na+ support the ion stoichiometry as being one monovalent cation per four xanthine tetrads. It should be highlighted that in contrast with the GQ, the XQ binds the Na+ in the plane of the tetrad (i.e., the in-plane region). This preference for the in-plane over the more usual interplane position vanishes when the simulation is performed in an implicit solvent using the Generalized-Born model. This unprecedented in-plane stabilization of Na+ in the XQ is
Figure 3. Synchronization of the position of ion and the radius of the pore in the 2nd and 3rd tetrads of the d(X4)4 as revealed by MD simulation. (For details, see the Supporting Information.)
considered as another stabilizing factor favoring the in-plane arrangement. In contrast, the Na+ passing through the in-plane region forces the guanine tetrad to expand its pore. (The radius increases from 2.2 to 2.4 Å.) A comparative MD simulation of d(X4)4 in the absence of any cation in the channel but with Na+ in the bulk solvent resulted in 3′-terminal tetrad disintegrating after 1 ns with the rest of the system remaining folded throughout this simulation. An additional MD simulation of d(X4)4 with bulk K+ displaced the terminal pairing, thus providing another indication that the presence of one cation inside the tetrameric XQ is essential to its stability. To explore the scope of the XQ scaffold and its compatibility with the GQ analog, we investigated a few representatives of the mixed quadruplex systems, namely, d(GXG)4, d(GXGG)4,
Figure 2. Preferred locations of Na+ (left) and K+ (right) ions in the channel of the d(X4)4. Note the two water molecules coordinated with each ion. 1789
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Figure 4. Barriers to the transport of Na+, K+, and NH4+ through the 2nd tetrad of the d(X4)4 system calculated by using implicit (A) and explicit (B) solvent models. The dashed vertical line represents the position of the tetrad plane. (For details, see the Supporting Information.)
could potentially attach an XQ system to a membrane. For the stability reasons discussed above, we have limited our evaluation of ion passage to the central part of the quadruplex. An ABF simulation of the XQ using an implicit solvent model showed that all of the cations tested (Na+, K+, and NH4+) are most stable in interplane positions (see Figure 4A, ξ ≈ −1.7 Å, ξ ≈ + 1.5 Å). The increase in the free-energy barrier is consistent with an increase in the ionic radius (Na+ < K+ < NH4+). Interestingly, the positions of the maxima and minima are shifted slightly (Δξ ≈ −0.4 Å) from the expected values. A detailed analysis revealed that this shift is induced by dynamic distortions of the individual bases out of the plane of the tetrad. A different picture is obtained from simulations employing an explicit solvent model (Figure 4B), with the most pronounced changes in the position of the minimum and the barrier to the transport of the sodium ion. The minimum for Na+ (ξ ≈ −0.5 Å) in an explicit solvent is located in the in-plane area, whereas the other ions exhibit rather energy maxima in this region. This finding supports the higher affinity of the sodium ion for the inplane region of the XQ, as was already detected by the unbiased MD simulations. All of the barriers to transport are larger than those calculated using an implicit solvent, which points to the significant structural and energy consequences of the presence of water molecules in the quadruplex channel. To investigate further the role of the explicit water molecules in the XQ channel, we placed the sodium ion tightly coordinated with two water molecules (H2O·Na+·OH2) into the XQ channel, and the free-energy profile was then evaluated in the same way, as previously discussed. The profile obtained (Supporting Information, Figure S10) was almost identical with that obtained for the passage of Na+, which indicates the simultaneous transport of Na+ and the coordinated water molecules within the XQ. In the last section of this Letter, the ion-transporting capabilities of XQ and GQ will be compared. A similar study for the GQ has been reported very recently,9 however, using shorter time scales than those presented here. The transport of K+ through the GQ has been reported to require 13−15 kcal mol−1,9 whereas its passage through the XQ is less impeded (∼9 kcal mol−1, see Figure 4B). Because water molecules have a significant impact on the ion transport in the channel, we simulated the transfer of a single water molecule in the cation-free quadruplex by using implicit and explicit solvent models. The barrier to water transport through the XQ amounts to ∼5 kcal mol−1, whereas transport
d(GGXG)4, and d(GXXG)4. In all of the models, the original pairing within the individual tetrads was preserved despite the different requirements of the N3-xanthosine versus N9guanosine residues for the conformation of the backbone (Supporting Information, Figures S3−S5). The ability of the system to accommodate an ion in the channel is strongly correlated with the number of guanine tetrads incorporated. Nevertheless, the observed structural compatibility of both types of tetrads in forming the quadruplex DNA structures might suggest a potential application of N3-xanthine nucleotides as therapeutic agents interfering in vivo during the formation of the quadruplex. The behavior of all pure and mixed model systems with regard to the stability of the ions in the channel is shown schematically in Figures S8 and S9 (Supporting Information). The crucial effects of ions on the structure and stability of the quadruplex systems evoked the question of the potential of these systems for the trapping and transporting of monovalent ions through the channel of a quadruplex.9,21,22 In particular, we focused our simulations on investigating the ion-transporting properties with respect to the following phenomena: the freeenergy barriers, the structural alterations associated with the ion transport through the channel, the role of the water molecules occupying the quadruplex channel, and the mechanistic features (the synchronous or asynchronous transfer of the individual components). To compare the properties of the two quadruplex types in a straightforward manner, we employed four-tetrad models containing a single cation in the channel and evaluated the passage of this ion through the quadruplex channel using an Adaptive Biasing Force (ABF) approach.23 However, the presence of only a single cation in the channel presents several complications. First, the unoccupied space inside the channel is filled by water molecules from the bulk solvent. (For simulations in an explicit solvent, this happens soon after equilibration, within one nanosecond.) Second, the GQ of this arrangement represents a very unstable artificial model, which would in reality be saturated immediately by the corresponding number of monovalent ions. Because our models (mainly GQs) might be balancing on the edge of a base-pair ripping or other type of structural corruption, we employed geometrical restraints in the simulations, as needed. In most cases, only the terminal O5′ and O3′ atoms were restrained. This is partially substantiated by the fact that these atoms represent the points at which chemical modifications 1790
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pathway. This arrangement is further stabilized in the last step by recoupling the ion to a second water molecule from the preceding interbase region. In other words, whereas Na+ is transported through the XQ simultaneously with H2O, the transport of Na+ in the GQ is characterized by a pathway with several consecutive steps including the decoupling and recoupling of the Na+·H2O pair. To summarize, several facts should be highlighted. Being aware of the limitations of our approach (force field parameters, computational time scales), we investigated the ability of the xanthosine-based oligonucleotides to assemble stable structures analogous to guanine DNA quadruplexes. The stability of xanthine quadruplexes is significantly less dependent on the presence of cations bound inside the somewhat broader channel. Our simulations imply a high degree of structural complementarity of the xanthine and guanine tetrads in the DNA quadruplexes. Furthermore, our findings can be interpreted as preliminary qualitative evidence of much more suitable ion-transporting capabilities of xanthine-based quadruplexes. Whereas guaninebased DNA quadruplexes resemble ion containers or receptors, xanthine-based systems may represent prospective building blocks for ion-transporting devices.
through the GQ is an almost barrier-free process (
