Biocompatible Xanthine-Quadruplex Scaffold for Ion-TransportingDNA ChannelsJan Novotny, Petr Kulhanek,* 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 adaptivebiasing force analysis of the quadruplex DNA dynamics in anexplicit solvent reveal fundamentally different mechanisms ofNa+ transport in xanthine- and guanine-based DNA systems.The barrier to the transport of K+ through the xanthine-basedquadruplex is significantly lower than those reported for theguanine-based analogs.

SECTION: Biophysical Chemistry and Biomolecules

Noncovalent interactions are central to many processes invarious fields ranging from supramolecular chemistry andnanosciences to biochemistry and structural biology.1 DNAquadruplex motifs based on hydrogen-bonded tetrads formedby cation-templated assemblies of guanines have been knownsince 1962.2 The guanine (G) quadruplexes present in the 3′overhanging ends of chromosome (telomeres) represent anessential part of eukaryotic cells. Therefore, G-quadruplexes areconsidered to be potential therapeutic targets.3

In general, the structure of a guanine quadruplex ischaracterized by three principal noncovalent interactions: (a)the hydrogen bonding that is responsible for the self-assemblyof guanine-based tetrads; (b) the stacking interactions thatenable these tetrads to couple and form octameric, oligomeric,and polymeric assemblies; and (c) the ion−dipole interactionsbetween monovalent cations (e.g., Na+ or K+ incorporated inthe intertetrad regions)4 and the tetrads that provide additionalstability to these supramolecular systems.5 Although DNA/RNA G-quadruplexes were originally found to bind K+ ionsmore readily than Na+ ions, an effect attributed to the better fitof K+ into the interbase cage of the system,6 this selectivity waslater shown to be caused mainly by the lower dehydrationenergy of K+ as compared with that of Na+.7 Nowadays, it isknown that ions move relatively quickly in the inner channel ofthe G-quadruplex without disrupting the G-tetrad.8−10 Thisphenomenon is currently being explored extensively because ofits potential applications in materials science,11 biosensordesign, and nanotechnology.12,13

Very recently, Kovacs et al. have investigated N3-methylxanthine as a novel building block forming the tetradstructures.14 The formation of tetrameric and octamericassemblies was detected by using mass spectrometry andNMR spectroscopy. Theoretical calculations have indicated that

guanine tetrads are more stable than their xanthine analogs inthe gas phase as well as in stacked systems (telomere-likestructures) because of the strong cooperativity effects (−21 kcalmol−1) of the hydrogen bonds (HBs). However, this differenceseems to be vanishingly small in an implicit water environment,where the synergy of the HB formation in guanine tetradsdrops to nearly 0 kcal mol−1.15

In this work, we extended the elegant idea of a xanthinescaffold to the construction of artificial N3-xanthosine-modifiedDNA quadruplexes (XQs). We employed unrestrainedmolecular dynamics (MD) simulations to characterize thestability and dynamic behavior of the XQs as compared withthe guanine-based DNA quadruplexes (GQs).16 We alsoinvestigated the mechanism of ion and water transport inboth types of quadruplexes by means of free-energycalculations.First, we performed MD simulations using various starting

models of d(X4)4·nM+ and d(G4)4·nM

+ systems. These differessentially in the arrangement of the cations and the watermolecules inside the quadruplex channel, the bulk counterions,and the ionic strength. The simulations were performed usingan ff99bsc0 force field17,18 and an explicit solvent (TIP3Pmodel) on a time scale of 30−50 ns. Although this time scalecould be insufficient to evaluate the long-term stability orfolding processes of quadruplexes, it should be relevant foranalyzing the structural fluctuations.19 The analysis of MDtrajectories (rmsd plots in Supporting Information, Figures S1and S2) points to rather similar degree of fluctuations of the

Received: May 3, 2012Accepted: June 19, 2012Published: June 19, 2012

Letter

pubs.acs.org/JPCL

© 2012 American Chemical Society 1788 dx.doi.org/10.1021/jz300559w | J. Phys. Chem. Lett. 2012, 3, 1788−1792

XQ and GQ systems. All of the simulations showed that theXQs have significantly different structural parameters for thesugar−phosphate backbone as compared with the GQ systems(Supporting Information, Figures S3−S5). In addition, theaverage radius (∼2.6 Å) of the pores in the quadruplex systemconsisting of dX4 units was considerably larger than that of itsGQ analog (∼2.2 Å) (Figure 1, Supporting Information,

Figures S6 and S7). This might be one of several reasons forthe different ion interactions with the quadruplex channel. Ind(G4)4, the channel is occupied by three sodium or potassiumions staying permanently in the interplane regions. The absenceof any of these three ions leads either to lower quadruplexstability or to its complete destruction, which is consistent withprevious experimental and computational findings.20 Incontrast, the channel of d(X4)4 is capable of accommodatingonly one sodium cation oscillating between the planes of theindividual xanthine tetrads. Any attempt to put more sodiumcations into the channel leads quickly to their expulsion from it.The d(X4)4 also appears to be stable in the presence of a singlepotassium, cesium, or ammonium cation. However, these aretrapped in the central interplane area of the quadruplex. Therest of the xanthine channel is occupied by water molecules,which are retentively (for Na+) or transiently (for cations with alarger radius) coordinated with the cation. Simulations of theextended systems d(X6)4·1Na

+ and d(X8)4·2Na+ support the

ion stoichiometry as being one monovalent cation per fourxanthine 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-planeregion). This preference for the in-plane over the more usualinterplane position vanishes when the simulation is performedin an implicit solvent using the Generalized-Born model. Thisunprecedented in-plane stabilization of Na+ in the XQ is

therefore crucially dependent on the presence of watermolecules inside the XQ channel, and further analysis revealsthe direct coordination of water molecules with the cation. Onthe contrary, the binding of K+ in the interplane region of theXQ is impervious to the applied solvent model. The distinctlypreferred positions of the Na+ (in-plane) and K+ (interplane)ions, each coordinated with two water molecules in the pore ofthe 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 effectinduces a moderate compression of the pore (the radius isreduced from 2.7 to 2.5 Å, see Figure 3), which might be

considered as another stabilizing factor favoring the in-planearrangement. In contrast, the Na+ passing through the in-planeregion forces the guanine tetrad to expand its pore. (The radiusincreases 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 solventresulted in 3′-terminal tetrad disintegrating after 1 ns with therest of the system remaining folded throughout this simulation.An additional MD simulation of d(X4)4 with bulk K+ displacedthe terminal pairing, thus providing another indication that thepresence of one cation inside the tetrameric XQ is essential toits stability.To explore the scope of the XQ scaffold and its compatibility

with the GQ analog, we investigated a few representatives ofthe mixed quadruplex systems, namely, d(GXG)4, d(GXGG)4,

Figure 1. Structures of GQ (left) and XQ (right) tetrads, highlightingthe averaged diameters of their pores.

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.

Figure 3. Synchronization of the position of ion and the radius of thepore in the 2nd and 3rd tetrads of the d(X4)4 as revealed by MDsimulation. (For details, see the Supporting Information.)

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d(GGXG)4, and d(GXXG)4. In all of the models, the originalpairing within the individual tetrads was preserved despite thedifferent requirements of the N3-xanthosine versus N9-guanosine residues for the conformation of the backbone(Supporting Information, Figures S3−S5). The ability of thesystem to accommodate an ion in the channel is stronglycorrelated with the number of guanine tetrads incorporated.Nevertheless, the observed structural compatibility of bothtypes of tetrads in forming the quadruplex DNA structuresmight suggest a potential application of N3-xanthine nucleo-tides as therapeutic agents interfering in vivo during theformation of the quadruplex. The behavior of all pure andmixed model systems with regard to the stability of the ions inthe 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 ofthese systems for the trapping and transporting of monovalentions through the channel of a quadruplex.9,21,22 In particular, wefocused our simulations on investigating the ion-transportingproperties with respect to the following phenomena: the free-energy barriers, the structural alterations associated with the iontransport through the channel, the role of the water moleculesoccupying the quadruplex channel, and the mechanistic features(the synchronous or asynchronous transfer of the individualcomponents). To compare the properties of the twoquadruplex types in a straightforward manner, we employedfour-tetrad models containing a single cation in the channel andevaluated the passage of this ion through the quadruplexchannel using an Adaptive Biasing Force (ABF) approach.23

However, the presence of only a single cation in the channelpresents several complications. First, the unoccupied spaceinside the channel is filled by water molecules from the bulksolvent. (For simulations in an explicit solvent, this happenssoon after equilibration, within one nanosecond.) Second, theGQ of this arrangement represents a very unstable artificialmodel, which would in reality be saturated immediately by thecorresponding number of monovalent ions. Because ourmodels (mainly GQs) might be balancing on the edge of abase-pair ripping or other type of structural corruption, weemployed geometrical restraints in the simulations, as needed.In most cases, only the terminal O5′ and O3′ atoms wererestrained. This is partially substantiated by the fact that theseatoms represent the points at which chemical modifications

could potentially attach an XQ system to a membrane. For thestability reasons discussed above, we have limited ourevaluation 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+, andNH4

+) are most stable in interplane positions (see Figure 4A, ξ≈ −1.7 Å, ξ ≈ + 1.5 Å). The increase in the free-energy barrieris consistent with an increase in the ionic radius (Na+ < K+ <NH4

+). Interestingly, the positions of the maxima and minimaare shifted slightly (Δξ ≈ −0.4 Å) from the expected values. Adetailed analysis revealed that this shift is induced by dynamicdistortions 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 pronouncedchanges in the position of the minimum and the barrier to thetransport of the sodium ion. The minimum for Na+ (ξ ≈ −0.5Å) in an explicit solvent is located in the in-plane area, whereasthe other ions exhibit rather energy maxima in this region. Thisfinding supports the higher affinity of the sodium ion for the in-plane region of the XQ, as was already detected by the unbiasedMD simulations. All of the barriers to transport are larger thanthose calculated using an implicit solvent, which points to thesignificant structural and energy consequences of the presenceof water molecules in the quadruplex channel. To investigatefurther the role of the explicit water molecules in the XQchannel, we placed the sodium ion tightly coordinated with twowater molecules (H2O·Na

+·OH2) into the XQ channel, and thefree-energy profile was then evaluated in the same way, aspreviously discussed. The profile obtained (SupportingInformation, Figure S10) was almost identical with thatobtained for the passage of Na+, which indicates thesimultaneous transport of Na+ and the coordinated watermolecules within the XQ. In the last section of this Letter, theion-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 torequire 13−15 kcal mol−1,9 whereas its passage through the XQis 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 singlewater molecule in the cation-free quadruplex by using implicitand explicit solvent models. The barrier to water transportthrough the XQ amounts to ∼5 kcal mol−1, whereas transport

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.)

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through the GQ is an almost barrier-free process (<1.5 kcalmol−1). The latter, however, should be considered aconsequence of the expansion of the pore of the guaninetetrad resulting from the uncompensated repulsion of thecarbonyl rims. This behavior is supported by additionalcomputational experiments simulating evacuation of the Na+

or K+ out of the channel via the 5′ portal (SupportingInformation, Figure S11). Whereas only marginal modulationof the size of the pores is observed in d(X4)4·Na+/K+

(fluctuating around 2.7 Å), evacuation of the ion from thed(G4)4·Na

+/K+ complex leads to a significant increase in theradius of its pores (from 2.2 up to 3.0 Å).In contrast to the above-mentioned ion-free situation, the

radius of the pore in the GQ occupied by a cation issignificantly reduced as compared with that in the XQ. Thehigher flexibility of the guanine pore could be responsible forthe problems we experienced during the ABF simulations of theion transport in the GQ system. Preliminary tests showed badsampling of the ABF forces, probably caused by compression ofthe pore induced by the presence of the ion in the interplaneregion. In this case, the size of the pore was reduced below thecritical minimum required for water molecules to pass throughthe in-plane region during the ABF simulation. This motivatedus to use a more general approach based on two collectivevariables: the coordination numbers of the water molecules andions toward the interplane and in-plane positions. Two-dimensional free-energy plots for the Na+-H2O transport inthe XQ and GQ channels are shown in Figure 5.

A pathway apparent from Figure 5 clearly confirms thesynchronous mechanism of the Na+-H2O transport in XQrevealed already by the 1D ABF simulations of the Na+ andH2O·Na

+·OH2 systems. The corresponding barrier to transportis ∼4 kcal mol−1. Good agreement between the values obtainedfrom various simulations points to the relative invariance of thissynchronous process to external factors.In contrast with the XQ system, the transport of Na+ in the

presence of explicit water in the GQ channel is a much morecomplex process. The consecutive ion transport in the GQ (seeFigure 5) requires decoupling of the Na+·H2O pair by slidingthe water molecule away from its original position. This initialstep is followed by the penetration of Na+ through the in-planearea, which in fact represents a local minimum on the free-energy surface. A further shift of Na+ to the next interplaneregion represents another local minimum on the transport

pathway. This arrangement is further stabilized in the last stepby recoupling the ion to a second water molecule from thepreceding interbase region. In other words, whereas Na+ istransported through the XQ simultaneously with H2O, thetransport of Na+ in the GQ is characterized by a pathway withseveral consecutive steps including the decoupling andrecoupling 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 thexanthosine-based oligonucleotides to assemble stable structuresanalogous to guanine DNA quadruplexes. The stability ofxanthine quadruplexes is significantly less dependent on thepresence of cations bound inside the somewhat broaderchannel. Our simulations imply a high degree of structuralcomplementarity of the xanthine and guanine tetrads in theDNA quadruplexes.Furthermore, our findings can be interpreted as preliminary

qualitative evidence of much more suitable ion-transportingcapabilities of xanthine-based quadruplexes. Whereas guanine-based DNA quadruplexes resemble ion containers or receptors,xanthine-based systems may represent prospective buildingblocks for ion-transporting devices.

■ ASSOCIATED CONTENT*S Supporting InformationForce-field parameters, rmsd values as functions of MD time,distributions of structural parameters extracted from unbiasedMD simulations, schematic representations of structuralchanges and stabilities of the investigated models, and detailsof free-energy calculations. This material is available free ofcharge via the Internet at http://pubs.acs.org.

■ AUTHOR INFORMATIONCorresponding Author*E-mail: kulhanek@chemi.muni.cz and rmarek@chemi.muni.cz. Fax: +420549492556.NotesThe authors declare no competing financial interest.

■ ACKNOWLEDGMENTSThis work was supported by the project “CEITEC − CentralEuropean Institute of Technology” (CZ.1.05/1.1.00/02.0068)from the European Regional Development Fund. Thecomputational resources were partially provided by MetaCen-trum, Czech Republic (grant LM2010005).

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