Supramolecular Structure and Polymorphism of Alkali Metal Salts of

Aug 19, 2009 - Departments of Chemistry and Physics, The University of Texas at San Antonio, One UTSA Circle, San Antonio, Texas 78249. J. Phys. Chem...
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Supramolecular Structure and Polymorphism of Alkali Metal Salts of Guanosine 5′-Monophosphate: SEM and NMR Study Jason B. Hightower,† David R. Olmos,‡ and Judith A. Walmsley*,† Departments of Chemistry and Physics, The UniVersity of Texas at San Antonio, One UTSA Circle, San Antonio, Texas 78249 ReceiVed: May 11, 2009; ReVised Manuscript ReceiVed: July 24, 2009

Scanning electron microscopy images of the Na+, K+, and Rb+ salts of guanosine 5′-monophosphate (5′GMP) in the presence of the corresponding metal chloride have shown the formation of exceptionally large molecular aggregates. These are much larger than those previously reported in solution. Each cation system produced a solid with a different morphology. The SEM samples were prepared from concentrated aqueous (D2O) solutions containing various amounts of 5′-GMP and metal chloride, and they approached the limit of solubility for the 5′-GMP under these conditions. Straight or slightly curved “free standing” rods composed of bundles of parallel stacks of G-quartets were formed from solutions of 0.85-1.0 M Na2(5′-GMP) containing 0.25-0.50 M NaCl. The rods had varying lengths of 6000-40 000 nm and an average diameter of 2000 nm. Calculations estimate this diameter to correspond to approximately 650 parallel stacks of G-quartets. Alignment of the individual G-quartet stacks into bundles and rods occurred as a result of phosphate charge neutralization by the high concentration of Na+ ions. The SEM image of the K+ system showed the presence of two types of morphologies, a rodlike lattice formation interpreted to be formed of stacked G-quartets, and irregular twisting fibers of varying diameter. In conjunction with the 1H NMR data, the latter are proposed to be composed of continuous helices of doubly hydrogen-bonded guanines having the same H-bonding motif as the planar G-quartets. The Rb+ system had some similarities to both the Na+ system and the K+ system. 1H NMR spectra were different for each cation system, corresponding to the differences observed by SEM imaging of the solids. Polymorphism has been observed in telomeric sequences but has not been extensively explored in 5′-GMP. Introduction The self-association behavior of guanine ribonucleosides and ribonucleotides in the presence of certain monovalent cations was first observed in the 1960s1 and has been investigated extensively since that time.2-7 Elucidation of these systems has proven to be complex, with clarification of their structures of continuing interest. Early fiber diffraction investigations1,8-10 and other studies11,12 of the gels and solutions indicated the formation of stacked cyclic tetramers held together by Hoogsteen hydrogen bonds between the guanine bases (Chart 1A). In subsequent years, it was discovered that the same type of stacked hydrogen-bonded cyclic tetramers (G-quartets) could form in telomeric sequences at the ends of chromosomes in eukaryotic organisms.6,7,13-17 Numerous NMR spectral studies of telomeric sequences in solution, or models for them, have been performed.14,18-20 X-ray structural analysis of d(TGGGGT) has been reported in which it was found that Na+ ions were located in the central cavity of the cyclic tetramer or between planes of the stacked G-quartets, depending on the location of the G-quartet in the sequence.14,21-23 In d(G4T4G4), which has a folded structure, Na+ ions have reverse positions from those in the quadruplex sequence of d(TG4T),24 and the larger K+ ions were only observed between G-quartet planes but still within the central core of the structure.25 Crystal structures of a dimer and a trimer * To whom correspondence should be addressed. Phone: 210-458-5459. Fax: 210-458-7428. E-mail: [email protected]. † Department of Chemistry. ‡ Department of Physics.

of human telomeric DNA have been obtained on crystals grown from solutions containing K+ ions at physiological concentrations.26 Although both deoxynuclotide sequences and ribonucleotide sequences form G-quadruplexes, the arrangement of strands in RNA quadruplexes is different, with RNA found only with the parallel strand conformation27 while DNA quadruplexes can be either antiparallel or parallel.28 Cation-specific and sequence-specific polymorphism of the associated species has been observed in G-rich telomeric sequences, forming parallel or anitparallel structures, or mixtures of the two in some cases.29,30 The X-ray structure of partially disordered Na2(5′-GMP) crystal obtained by Lipanov et al. disclosed that the hexagonal unit cell was composed of three G-quartets stacked perpendicularly to the long axis with a 3.3 Å spacing and 30° rotation between the planes, although it was not possible to locate the Na+ ions.31 This was in agreement with earlier fiber diffraction results of guanosine 5′-monophosphate (5′-GMP) gels and solutions.1,8-10 More recently, a neutron diffraction study of the condensed hexagonal phase of K2(5′-GMP) was conducted to determine the number of K+ ions/G-quartets residing in the inner cavity of the helix and the number interacting with the outer edges.32 In spite of the time elapsed since the initial discovery of the unique behavior of guanine nucleosides and nucleotides, certain aspects of the solution structure of the monoribonucleosides and monoribonucleotides remain unclear. NMR spectroscopy of gels33,34 and solutions at neutral to slightly basic pH has been a highly informative technique for their investigation.35-40 Studies

10.1021/jp904383y CCC: $40.75  2009 American Chemical Society Published on Web 08/19/2009

Alkali Metal Salts of Guanosine 5′-Monophosphate

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CHART 1: (A) G-Quartet, R ) 5′-Ribophosphate; (B) Continuous Hydrogen-Bonded Helix of 5′-GMP

on 0.4-1.0 M aqueous solutions of 5′-GMP at low temperature showed each of the alkali metal cations produced a different 1 H spectrum.35,36 With each of the cations (Na+, K+, or Rb+), multiple species or multiple nucleotide environments were found, with stacked G-quartets believed to be present. Wu and Kwan have recently revisited the aqueous solution structure of Na2(5′-GMP) using DOSY and NOESY methods to clarify some aspects of the structure.41 They reported head-to-tail stacking of the G-quartets in the helical strand with alternating C2′-endo and C3′-endo sugar pucker, similar to that found in Z-DNA. It has been assumed that the G-quartet structure was formed in the presence of K+ ion, and there is evidence for that structure from a neutron diffraction study32 and for a derivatized guanosine in organic solvent,42 but another study of potassium 5′-GMP in aqueous solution indicated that a continuous helical hydrogen-bonded structure seemed more consistent with the NMR spectral data under the conditions employed (Chart 1B).43 As part of understanding the solution structure of guanine nucleotides, the number of G-quartets that could form a cylindrical stack has been of considerable interest. Dynamic light scattering coupled with NMR spectroscopy has been used to estimate this number under various conditions of nucleotide concentration, cation type, temperature, and pH.44-46 The reported number of G-quartets in the stacks ranges from 545 to 8744 for 5′-GMP and as many as 133 for 2′-deoxy-5′-GMP.47 The concentration dependence of the degree of stacking is illustrated by the work of Wu et al.44 in which they found that solutions containing 18-34 wt % Na2(5′-GMP) contained cylindrical stacks of ∼24-87 G-quartet units. They also reported stacking of monomers in the same solutions. A number of imaging studies of various types have been reported for the mononucleotides,48-50 as well as for oligonucleotides51,52 and telomeric sequences.53,54 Using optical microscopy, Spada et al. observed various phases (isotropic, cholesteric, and hexagonal) for the ammonium salts of 2′-, 3′-, and 5′-GMP in aqueous solutions and observed a phase concentration dependence.47 An AFM study of the self-assembly of 5′-GMP on mica revealed the formation of long, straight wires composed of G-quartets.55 In that study, it was found that the nature of the cation had no effect because of the large concentration of K+ ions on the mica surface. We report herein the results of a study in which scanning electron microscopy (SEM) was used to image fibers of the selfassociated Na+, K+, and Rb+ salts of 5′-GMP. The fibers were grown on graphite-coated Al studs from concentrated aqueous solutions of 5′-GMP containing 0.5-1.0 M chloride salt of the corresponding cation. It was observed that the morphology

varied with the nature of the cation. In particular, Na2(5′-GMP) had a more regular order than the K+ or Rb+ compounds, and it contained much larger structures than previously reported for self-assembled Na2(5′-GMP).44,47 1H NMR spectra of concentrated solutions, similar to those used for imaging, were also obtained and are consistent with the SEM data. Experimental Section Materials. Na2(5′-GMP) (21% H2O), H2(5′-GMP) · H2O, 12 M DCl, and D2O (99.8 atom %) were obtained from SigmaAldrich and used as received. Alkali metal chlorides and RbOH were of ultrapure grade from Alfa Products. Aluminum mounts (Ted Pella, size 12 mm diameter, 9 mm height) and colloidal carbon (graphite conductive adhesive 154 from Electron Microscopy Sciences) were used to prepare samples for SEM analysis. Preparation of K2(5′-GMP) and Rb2(5′-GMP). K2(5′-GMP) was prepared by dissolving H2(5′-GMP) in ∼40 mL of deionized H2O and titrating the solution to pH 7.8-8.2 with 0.20 M KOH. The solution was lyophilized to obtain the dry K2(5′-GMP) · xH2O, where x ) 2-3. It was transferred to a vial in a glovebag under N2 atmosphere because it is hygroscopic. Rb2(5′-GMP) was prepared in an analogous manner. Preparation of M2(5′-GMP)/MCl Solutions. All solutions were prepared in D2O in order to check the 1H NMR spectra to ascertain the nature of the self-association of the starting solutions for the SEM experiments. A sample of M2(5′-GMP) was weighed into a small vial, and 1.5 mL of D2O was added to form an approximately 1.1-1.2 M solution. The 5′-GMP dissolved readily, forming a slightly viscous solution. A measured amount of MCl solution in D2O was added to this to give the desired concentrations of each component. If there was any residual cloudiness in the solution after mixing, it was filtered through a 0.8 µm Milex-AA Millipore filter unit. The pH* was adjusted to 7.8-8.2 using 3 M DCl/D2O and a Fisher Accumet 925 pH meter. The 5′-GMP concentration in the final solution was determined from its UV absorption at 252 nm (ε ) 1.37 × 104 M-1 cm-1) on a Perkin-Elmer Lambda 19 spectrometer. Methods. Scanning Electron Microscopy (SEM). Samples were analyzed using a JEOL 840A SEM instrument attached to an Oxford INCA Electron Dispersion Spectroscopy (EDS) unit. Prior to sample application, aluminum studs were coated two times as evenly as possible with colloidal carbon in a 2-propanol carrier. These were allowed to dry for 3-4 h in a closed container, and those with cracks or ridges in the carbon surface were discarded. Using a Pasteur pipet, a small amount

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Figure 1. SEM image, solid from 0.85 M Na2(5′-GMP) + 0.52 M NaCl.

of freshly prepared nucleotide/salt solution was lightly applied to the carbon surface to produce a very thin layer, being careful not to scratch the carbon surface. The sample was allowed to air-dry in a closed container for 2-3 days, resulting in a thin, transparent film across the carbon surface. It was important that the sample be completely dry before analysis. The aluminum stud with the freshly dried sample was placed in a Pelco SC-7 Gold Auto Sputter Coater under argon gas for 30 s at 40 mA. The sample was then immediately analyzed. The presence of nucleotide, as indicated by the presence of phosphorus (in phosphate), was confirmed using EDS of various regions of the sample. Multiple analyses were done on each M2(5′-GMP)/MCl system. 1 H NMR Measurements. 1H NMR spectra were obtained in D2O on a Varian INOVA 500 MHz spectrometer with variable temperature capability. The spectral parameters were as follows: 128 acquisitions, 7990.8 Hz spectral width, 73.1° pulse width, acquisition time of 1 s, and delay of 2 s. Tetramethylammonium chloride was used as an internal reference (3.185 ppm). Spectra were taken at 25 and 3 °C. The spectra of all M2(5′GMP) compounds were obtained with and without added MCl. Results Scanning Electron Microscopy. A variety of different combinations of M2(5′-GMP) and MCl concentrations (where M ) Na+, K+, or Rb+) were prepared and analyzed. If the total cation concentration was large, precipitation or a milky sample resulted and was unusable. The point at which this occurred was dependent on the specific cation. For example, this occurred in samples of 1.0 M Na2(5′-GMP) with 1.0 M NaCl. The optimum molar ratio of M2(5′-GMP) to MCl was about 2:1, but the total cation concentration without precipitate formation for both the K+ and Rb+ systems was approximately one-half that for the Na+ system. At the higher relative concentrations of MCl, salt crystals were observed as well as 5′-GMP fibers. Na2(5′-GMP)/NaCl. Three separate sets of mounts were analyzed by SEM with reproducible results. These were 0.98 M Na2(5′-GMP)/0.50 M NaCl, 0.98 M Na2(5′-GMP)/0.25 M NaCl, and 0.85 M Na2(5′-GMP)/0.52 M NaCl. Large fibers (rods) were observed which were of varying lengths and generally straight or slightly curved (Figure 1). They are seen to have grown from the solution in a direction out of plane to the surface of the mount, so that they are “free standing”. Other clusters of parallel rods can be observed lying parallel to the surface of the mount.

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Figure 2. Increased magnification of a section of Figure 1.

Measurement of the “free standing” rods showed that they ranged in length from about 6000 to 40 000 nm, which is much longer than those observed in aqueous solution.44,45 The larger rods had a diameter of 1000-3000 nm. Increased magnification showed that the larger rods contained ridges running parallel with the long axis, and examination of the base of the rods indicated that they were a result of the aggregation of multiple smaller rods whose diameter was 250-500 nm (Figure 2). The ends of the rods were generally flat, which is consistent with vertical stacks of planar G-tetrads. Since the diameter of a single stack of G-quartets has been estimated to be 2.4-2.6 nm,32,46 both the larger and smaller rods contain a large number of parallel G-quartet stacks with the high NaCl concentrations providing the necessary cations to neutralize the accumulated negative charge of the stack and to hold the individual stacks together, mostly by coordination or electrostatic interactions of the Na+ ions with the phosphate groups on adjacent parallel G-quartet stacks. This is in addition to the Na+ ions that are known to occupy the channel within the center of the G-quartet stacks. K2(5′-GMP)/KCl. Multiple attempts were made to obtain SEM images corresponding in type and quality to those of the Na2(5′-GMP)/NaCl system. Solutions of K2(5′-GMP) having concentrations of 0.50-0.85 M and containing 0.25-0.56 M KCl were used. In only one case was fiber formation observed and the fibers were curved, of varying diameter along a fiber, and unaligned with each other (Figure 3). Some of the fibers were very long, up to 2.7 × 105 nm, approximately an order of magnitude longer than the longest rods of Na2(5′-GMP). They did not appear to be composed of discrete bundles, nor did they appear to have flat ends; they were very different in appearance from the rods observed in the Na2(5′-GMP)/NaCl system. At the higher KCl concentrations, substantial amounts of KCl crystals were observed on the mounts. In addition to the fibrous material observed for K2(5′-GMP), there were other regions on the same mount that displayed a lattice-like arrangement of thin fibers oriented ∼90° to each other and lying flat on the surface of the mount (Figure 4). They were encrusted with KCl crystals, making it difficult to image them clearly. This raises the question of whether or not there are two polymorphs of K2(5′-GMP) present. Additional attempts to obtain the fibers resulted in a mass of solid with some indication of rod formation with a large amount of KCl crystals on them. Reduction of the K2(5′-GMP) and KCl concentrations resulted in disks with scattered pockets of solid with undefined structures.

Alkali Metal Salts of Guanosine 5′-Monophosphate

Figure 3. SEM image of the fibrous region of a solid from 0.50 M K2(5′-GMP) + 0.25 M KCl.

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Figure 5. SEM image, solid from 0.57 M Rb2(5′-GMP) + 0.20 M RbCl.

Figure 4. SEM image of the lattice-like region of a solid from 0.50 M K2(5′-GMP) + 0.25 M KCl.

Figure 6. 1H NMR spectra of the H8 region of M2(5′-GMP) in D2O at neutral pH and 3 °C: (A) 0.98 M Na2(5′-GMP) + 0.25 M NaCl; (B) 0.57 M Rb2(5′-GMP) + 0.27 M RbCl.

Rb2(5′-GMP)/RbCl. As in the case of the K2(5′-GMP)/KCl system, multiple attempts were made to obtain good disks for imaging. Solutions of Rb2(5′-GMP) having concentrations of 0.30-0.57 M and containing 0.20-0.50 M RbCl were used. Lower concentrations were required because of the lesser solubility of Rb2(5′-GMP) in water. The disks from two different solutions were suitable, and gave the same results, both showing a mass of fibers (Figure 5). These were aligned along the long axis but also showed considerable curvature in places, similar to the K2(5-GMP)/KCl system. No fibers growing out of the plane of the disk were observed. The fibers appeared to be formed of bundles of smaller fibers, similar to the Na2(5′-GMP)/ NaCl system, but the striations indicated that the bundles had a twist to them. Measurement of what appeared to be discrete fibers/rods gave a diameter in the 1100-2100 nm range. 1 H NMR Spectroscopy. It has been known since 1978 when Pinnavaia et al. first obtained the 1H NMR of various 5′-GMP salts in solution that the nature of the self-associated species was cation specific35,36 and this area has been extensively investigated. The spectra of the H8 region of the Na+- and Rb+containing system are shown in Figure 6. In both, there is a considerable amount of line broadening as a result of the selfassociation of the 5′-GMP and some viscosity line broadening in the more concentrated solutions. This line broadening was observed in all regions of the spectrum.

The spectrum of the disodium salt is by far the simplest of the three that strongly self-associate (Na2(5′-GMP), K2(5′-GMP), and Rb2(5′-GMP)). The four resonances observed in the H8 region of the NMR spectrum (Figure 6A) and the resonances in the H1′ region are indicative of a specific orientation of G-quartets. Three of the four H8 resonances (HR, Hβ, and Hδ) are invariant in chemical shift with respect to changes in 5′GMP concentration, temperature, or added NaCl, and had been assigned to the signals resulting from a 30° rotation of the G-quartet about the helical axis.31 The fourth one (Hγ) has been interpreted as arising from stacked monomers. However, based on their DOSY and NOESY data, Wu and Kwan41 assigned the HR and Hδ to the G-quartet structure and the Hβ to a symmetrical hydrogen-bonded dimer. Dimeric structures are a possibility, but the invariant integrated intensity of the HR, Hβ, and Hδ under varied conditions needs to be explained if two different species are present. In contrast to Na2(5′-GMP), the large number of overlapping resonances in the 1H NMR spectra of Rb2(5′-GMP) and K2(5′GMP) (Figure 6B and 7, respectively), especially the latter, indicate a greater complexity for these solutions. This could be the result of tilting of the bases out of planarity due to the larger sizes of the K+ and Rb+ ions in order for their more effective coordination to the CdO of the 5′-GMP,56 or a more disordered

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Figure 8. Proposed 5′-GMP structures: (A) Na2(5′-GMP); (B) K2(5′GMP) G-quartet; (C) K2(5′-GMP) continuous H-bonded helix.

Figure 7. 1H NMR spectra of K2(5′-GMP) in D2O at pD 8.6 and 25 °C: (A) 0.49 M K2(5′-GMP); (B) 0.54 M K2(5′-GMP) + 0.54 M KCl (× denotes resonance postulated to arise from stacked G-quartets).

structure. However, in the 1H NMR spectrum of the H8 region of the 0.54 M K2(5′-GMP) containing 0.54 M KCl, four narrower resonances are observed rising out of a very broad base which spans approximately 1.0 ppm (Figure 7B), as we have reported previously.43 It was noted that increased concentrations of KCl enhanced the intensity of the narrower resonances.43 This suggests that two types of self-associated structures could be present in solution, similar to the SEM observations. Discussion The SEM images have shown that each cation system is different, in agreement with the solution data from NMR spectroscopy. At least part of the difference can be ascribed to the size difference between Na+, K+, and Rb+ and its effect on their bonding in the G-quartet structure. Of the three 5′-GMP systems imaged, the sodium salt formed the most regular structures and provided SEM results which were easily reproduced. In these systems, the cations have two different bonding or interaction environments: (1) the channel in the center of the G-quartet; (2) the outer perimeter of the helix or stack where the cation interacts electrostatically with the phosphate ions. Using an interhelix distance determined by Federiconi et al., it is possible to estimate the number of parallel stacks in a bundle or rod. They investigated the condensed hexagonal phase of K2(5′-GMP) containing various concentrations of polyethylene glycol (PEG) and KCl and determined that the interhelix distance (interaxial) ranged from 30.7 ( 0.1 to 32.1 ( 0.1 Å.32 A similar determination of the interhelix distance for the Na2(5′GMP) system was not reported, but if the smaller distance is used as an approximation for the smaller Na+ ion in our Na2(5GMP)/NaCl system, it can be estimated that a rod of 2000 nm diameter contained approximately 650 parallel stacks of Gquartets. A similar approximation for the smaller diameter fibers that were observed coming together to form the larger rods (Figure 2) yielded 119 G-quartet stacks per rod, using an average rod diameter of 375 nm. Not surprisingly, the rods contained many more individual G-tetrads than those reported in solution.44,45,47 The K2(5′-GMP)/KCl system is clearly very different from that of the Na+ system, in both its NMR spectra and SEM images. There is strong indication in both kinds of data that at

least two types of structures were present. Polymorphism has been demonstrated for d(T4G4)n in the presence of added KCl, where both parallel and antiparallel G-quadruplex structures exist simultaneously, and this is a possible explanation for our data.29,30 Polymorphism in the presence of NaCl has not been observed for d(T4G4)n unless the loop region is changed to T7.29 The region in the SEM of the K2(5′-GMP)/KCl system containing a lattice of thin rods oriented at ∼90° to each other could be interpreted as a polymorph formed by the stacking of G-quartets, as found in the Na2(5′-GMP)/NaCl system and in DNA telomeres. A hydrogen-bonded helical structure would be consistent with the observed curved fibrous material, and variation in the thickness along the length of the fibers could be explained by a nonregularity in the interhelical interactions. The aqueous solution 1H NMR spectra of Na+ and K+ 5′GMP systems provide further evidence for structural differences. The solution structure for Na2(5′-GMP) aggregates has been reported to consist of a mixture of G-quartets possessing either a C3′-endo or a C2′-endo sugar conformation with the glycosidic angle in the anti range for the former and in the high anti (syn) range for the latter; a head-face to tail-face stacking was suggested (Figure 8A).41 On the other hand, the K2(5′-GMP) conformation has been shown to be anti, consistent with a structure similar to parallel strands in DNA, or with a continuous helical structure43 (Chart 1B, Figure 8B and C). In light of the SEM and 1H NMR data reported herein, it is proposed that the four sharper NMR lines for 0.54 M K2(5′-GMP) with 0.54 M KCl43 (Figure 7B) represent the formation of the G-quartet structure (Figure 8B), the relative concentration of which increases with increased K+ concentration, and the very broad region under the sharper lines represents the formation of the continuous helical structure (Figure 8C). However, the chemical shifts of the sharp resonances are not the same as those of the Na+ system; therefore, a K+-induced G-quartet must be different form the Na+-induced G-quartet structure, consistent with two different types of stacking (Figure 8A and B). Support for the presence of a mixture of species is provided by the early X-ray diffraction studies of Zimmerman and others of fibers pulled from 5′-GMP gels and from neutral solution (Na+ cation).1,10 These experiments revealed the formation of two different types of molecular arrangements, the stacked G-quartet structure and the continuous hydrogen-bonded helix. Although X-ray crystal structures of model DNA telomeric sequences have shown only the presence of planar G-quartets and G-quadruplexes with K+ ions located between the planes,25,26 these systems are constrained by the phosphodiester backbone, while the monophosphates are not. Examination of the 1H NMR spectra of the Rb2(5′-GMP)/ RbCl system indicated the presence of an irregular structure or a mixture of structures but which appeared somewhat less complex than those of K2(5′-GMP). The SEM images had some characteristics of those of the Na2(5′-GMP)/NaCl system in that

Alkali Metal Salts of Guanosine 5′-Monophosphate there were bundles of small rods combining to produce larger formations. However, they differed from the Na2(5′-GMP)/NaCl system in that the fibers had a much greater amount of curvature, including twisting and looping in places. The diameter of an individual bundle appeared to be approximately uniform, which would be expected for parallel stacks of G-quartets. It can be postulated that the curvature of the rods was the result of the larger size of the Rb+ ion, which requires that it be located between the planes of adjacent G-quartets and would allow a considerable degree of tilt. Conclusions Although the alkali metal salts of 5′-GMP have been investigated since the 1960s, there remain a number of unanswered questions about the structures of the self-associated species. Self-association of each of the three cation-5′-GMP systems reported here leads to the formation of different structures in aqueous solution and in the solid state. Polymorphism in telomeres has been well studied but has not been seriously considered in 5′-GMP until now. Evidence for a mixture of structures was observed in the K2(5′-GMP)/KCl system and in the Rb2(5′-GMP)/RbCl system as well. Certainly, the size of the cation has a significant effect and the increased degrees of freedom of 5′-GMP relative to DNA and RNA has a large effect, as well. According to the SEM images shown above, the Na+ and Rb+ systems have some similarities, but the Na+ system is much more regular. High concentrations of K+ shift the equilibrium toward stacked G-quartet formation43 but which is different in structure from the antiparallel G-quartet structure of the Na+ system. Acknowledgment. The authors thank the NIH MBRS program (grant S06-08194) for partial support of this research. References and Notes (1) Gellert, M.; Lipsett, M. N.; Davies, D. R. Proc. Natl. Acad. Sci. U.S.A. 1962, 48, 2013–2018. (2) Chantot, J. F.; Sarocchi, M.-T.; Guschlbauer, W. Biochimie 1972, 53, 347–354. (3) Guschlbauer, W.; Chantot, J.-F.; Thiele, D. J. Biomol. Struct. Dyn. 1990, 8, 491–511. (4) Davis, J. T. Angew. Chem., Int. Ed. 2004, 43, 668–698. (5) Keniry, M. A. Biopolymers 2001, 56, 123–146. (6) Burge, S.; Parkinson, G. N.; Hazel, P.; Todd, A. K.; Neidle, S. Nucleic Acids Res. 2006, 34, 5402–5415. (7) Huppert, J. L. Chem. Soc. ReV. 2008, 37, 1375–1384. (8) Sasisekharan, V.; Zimmerman, S. B.; Davies, D. R. J. Mol. Biol. 1975, 92, 171–179. (9) Tougard, P.; Chantot, J.-F.; Guschlbauer, W. Biochim. Biophys. Acta 1973, 308, 9–16. (10) Zimmerman, S. B. J. Mol. Biol. 1976, 106, 663–672. (11) Howard, F. B.; Frazier, J.; Miles, H. T. Biopolymers 1975, 16, 791– 809. (12) Juskowiak, B. Curr. Anal. Chem. 2006, 2, 261–270. (13) Blackburn, E. H. Nature 1991, 350, 569–573. (14) Neidle, S., Balasubramanian, S., Eds. Quadruplex Nucleic Acids; Royal Society of Chemistry: London, 2006. (15) Ou, T.; Lu, Y.; Tan, J.; Huan, Z.; Wong, K.-Y.; Gu, L. Chem. Med. Chem. 2008, 3, 690–713. (16) Dai, J.; Carver, M.; Yang, D. Biochimie 2008, 90, 1172–1183. (17) Pieraccini, S.; Giorgi, T.; Gottarelli, G.; Masiero, S.; Spada, G. P. Mol. Cryst. Liq. Cryst. 2003, 398, 57–73.

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