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J. Phys. Chem. B 2008, 112, 1130-1134
Tunable Thermoassociation of Binary Guanosine Gels Yuehua Yu, Darren Nakamura, Kevin DeBoyace, Adam W. Neisius, and Linda B. McGown* Department of Chemistry and Chemical Biology, 118 Cogswell Building, Rensselaer Polytechnic Institute, Troy, New York 12180 ReceiVed: October 1, 2007; In Final Form: NoVember 6, 2007
It is well-known that aqueous solutions of individual guanosine compounds can form gels through reversible self-assembly. Typically, gelation is favored at low temperature and acidic pH. We have discovered that binary mixtures of 5′-guanosine monophosphate (GMP) and guanosine (Guo) can form stable gels at neutral pH over a temperature range that can be tuned by varying the relative proportions of the hydrophobic Guo and the hydrophilic GMP in the mixture. Gelation was studied over the temperature range of 5-40 °C or 60 °C at pH 7.2 using visual detection, circular dichroism (CD) spectroscopy, and CD thermal melt experiments. Solutions with high GMP/Guo ratios behaved similar to solutions of GMP alone while solutions with low GMP/Guo formed firm gels across the entire temperature range. Most interesting were solutions between these two extremes, which were found to exhibit thermoassociative behavior; these solutions are liquid at refrigerator temperature and undergo sharp transitions to a gel only at higher temperatures. Increasing the GMP/Guo ratio and increasing the total concentration of guanosine compounds shifted the onset of gelation to higher temperatures (ranging from 20 to 40 °C), narrowed the temperature range of the gel phase, and sharpened the reversible phase transitions. The combination of self-assembly, reversibility, and tunability over biologically relevant temperature ranges and pH offers exciting possibilities for these simple and inexpensive materials in medical, biological, analytical, and nanotechnological applications.
Introduction It has long been known that some guanosine nucleosides and nucleotides can self-assemble to form reversible gels under certain experimental conditions.1-4 The building block of these structures is a guanosine tetrad or “G-quartet”, which is formed through Hoogsteen (G:G) hydrogen bonding between each of four guanines and their neighbors. Gel formation in solutions of individual guanosine compounds has been widely studied over the years using visual detection, bulk physical measurements, absorption and circular dichroism spectroscopies, X-ray diffraction, light scattering, neutron scattering, and NMR.5-12 On the basis of the results, models have been derived that describe supramolecular, columnar structures formed by selfassembly of G-quartets through π-π stacking6 and stabilized by centrally located metal cations that are coordinated to the eight oxygen atoms in the guanines.11 An alternative model has been proposed for 5′-guanosine monophosphate (GMP) in which the GMP monomers associate through Hoogsteen hydrogen bonding to form a continuous, helical network that is further stabilized by base stacking and cations.2 In either case, as the concentration of the guanosine compound continues to increase, the isotropic solutions of columnar or helical aggregates eventually organize to form higher-ordered, anisotropic liquid crystalline phases (gels) with cholesteric or hexagonal organization.9 Much of the interest in guanosine self-assembly arises from the biological significance of G-quartet structures formed by G-rich sequences of DNA and RNA,4,13-16 including the implications of guanosine self-assembly for the origin of life.17 Exploration of potential applications of guanosine gels has only recently begun to attract interest, with the major focus on * Corresponding author. E-mail:
[email protected].
columnar “G-wires” and layered thin films of guanosine “nanoribbons” as molecular wires for nanoelectronics.13,18-22 A second area of interest has been the enantiomeric selectivity exhibited by lipophilic derivatives of guanosine.23,24 Prior work in our group has focused on applications of guanosine gels for chemical, chiral, and biological separations.25,26 The potential of guanosine gels in the broader arenas of nanotechnology and biotechnology is worth exploring because of the reversibility, tunability, physical stability, aqueous solubility, biocompatibility, and chemical and chiral selectivity of the gels, as well as their potential for reversible encapsulation and reversible introduction of functionality. We recently discovered that guanosine gels formed by binary mixtures of the soluble 5′-guanosine monophosphate (GMP) and relatively insoluble guanosine (Guo) in aqueous solution exhibit unique thermoresponsiveness that can be controlled by adjusting the Guo/GMP ratio, cation content, and pH. At neutral pH and room temperature, GMP alone is too soluble in water to form firm gels, while Guo is too insoluble to form a stable gel even in the presence of high K+ concentrations. The present studies of GMP-Guo mixtures reveal, not surprisingly, that GMP helps to solubilize Guo while the insolubility of Guo promotes gelation at lower concentrations of GMP. We were particularly intrigued by the dramatic effect of the proportion of the two guanosine compounds on the apparent thermoresponsiveness of the gels. In some proportions, the gels are “thermodissociative”; that is, they gel with decreasing temperature, as is typical of solutions of individual guanosine compounds. However, in other proportions, the gels exhibit the opposite, “thermoassociative” behavior, that is, they are liquid solutions at low temperatures and organize into gels at higher temperatures. This behavior has not been previously reported for guanosine gels, which until now
10.1021/jp709613p CCC: $40.75 © 2008 American Chemical Society Published on Web 01/09/2008
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Figure 3. Classification of binary solutions as liquid (open circle), viscous (open triangle), or gel (solid square) across the temperature range or as thermoassociative (solid dash), as a function of GMP and Guo concentrations. Diagonal line corresponds to XGMP ) 0.83.
Figure 1. Classification of binary solutions as liquid (open circle), viscous (open triangle), or gel (solid square) as a function of GMP and Guo concentrations, at 5 °C (top), 25 °C (center), and 37 °C (bottom). Diagonal lines correspond to XGMP ) 0.83.
Figure 4. CD spectra at 5 °C (solid line) and 25 °C (dashed line) of solutions that remain liquid over the range 5-40 °C. (A) 0.10 M GMP, 0.02 M Guo, (XGMP ) 0.833); (B) 0.30 M GMP, 0.01 M Guo (XGMP ) 0.968); (C) 0.30 M GMP, 0.05 M Guo (XGMP ) 0.857).
Figure 2. Examples of binary solutions characterized as liquid (left), viscous (middle), and gel (right).
have been studied only in solutions of individual guanosine compounds and not in mixtures. In the present work, we describe systematic studies of the effects of the proportions of the two guanosine compounds on the thermoresponsiveness of the guanosine gels as determined by visual detection and circular dichroism (CD) techniques. Experimental Section Gel solutions were prepared in 25 mM Tris buffer, pH 7.2, 0.05 M KCl using 5′-guanosine monophosphate (GMP), guanosine (Guo, g98% purity), potassium chloride, and Trizma buffer, all from Sigma-Aldrich (St. Louis, MO). All stock buffer
and KCl solutions were prepared on a monthly basis and stored in the refrigerator when not in use. The constituents of the gel solutions were placed into a glass vial, heated in water bath at ∼70 °C, and stirred for 15 min. The vials were then stored in the refrigerator overnight. The following day, the solutions were observed visually and classified immediately as liquid, viscous, or gel at refrigerator temperature. They were then allowed to come to room temperature and the visual classification repeated, followed by heating to 37 °C and visual classification. Following these classifications, the solutions were immediately returned to the refrigerator for storage. Circular dichroism (CD) spectroscopy and thermal melt experiments were performed using a Jasco J-715 spectropolarimeter. A 20 µL aliquot of sample was placed in a 0.01 mm path length quartz cuvette that was then placed in the sample chamber at least 15 min prior to measurement to ensure thermal equilibration. Thermal melt experiments were performed over the temperature range of 5 °C to either 40 °C or 60 °C with a temperature ramp of 1 °C/min. Samples were equilibrated in
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Figure 7. Temperature of onset of gelation of binary solutions as a function of XGMP. For solutions marked as 5 °C, onset of gelation is e5 °C since these solutions were gels under refrigeration.
Figure 5. Examples of CD spectra of higher ordered phases at 25 °C. (A) 0.25 M GMP, 0.05 M Guo, (XGMP ) 0.833); (B) 0.05 M GMP, 0.06 M Guo (XGMP ) 0.455); (C) 0.20 M GMP, 0.06 M Guo (XGMP ) 0.769); (D) 0.25 M GMP, 0.06 M Guo (XGMP ) 0.806).
Figure 6. CD spectra of a refrigerated solution containing 0.15 M GMP/0.03 M Guo (XGMP ) 0.83) at 0, 60, 120, and 180 min immediately following placement of the sample in the CD sample compartment that was maintained at 25 °C.
the cuvette at low temperature for at least 15 min before measurement. For the three-dimensional plots of CD spectrum versus time, the circulating water bath used to control the temperature of the CD sample chamber was heated from approximately 11 to 32 °C. In these experiments, it was possible to monitor only the temperature of the water bath and not of the sample chamber, and so the spectra are shown as a function of time. Results Visual Detection of Thermal Response. On the basis of visual observations of solutions at refrigerator (5 °C), room (25 °C) and heated bath (37 °C) temperatures, we derived a “matrix” of the thermoresponsiveness of the binary gels as a function of GMP and Guo concentrations. Figure 1 shows the state of the gels at each temperature. Figure 2 shows examples of solutions that were classified as “liquid”, “viscous” or “gel”. The term “gel” is used here to describe only those solutions that were firm gels, maintaining their shape with no evidence
Figure 8. Examples of thermal melt curves from 5 to 60 °C for solutions containing 0.06 M Guo and varying GMP. (A) 0.10 M GMP (XGMP ) 0.625); (B) 0.25 M GMP (XGMP ) 0.806); (C) 0.30 M GMP (XGMP ) 0.833).
of flowing in the inverted sample container. The gels are further classified in Figure 3 as liquid, viscous, or gel over the entire temperature range, or as thermoassociative. The diagonal lines in Figures 1 and 3 correspond to a constant GMP mole fraction (XGMP) of 0.83 (5:1, GMP/Guo). The absence of gelation in the GMP solutions with little or no Guo even at high GMP concentrations and low temperature is due to the relatively high solution pH of 7.2. CD Spectra. On the basis of their CD spectra, the binary solutions generally fall into two groups, those that remain liquid over the temperature range studied and those that form higherordered viscous or gel phases. The CD spectra of the solutions that remain liquid (Figure 4) resemble spectra reported for assembled monomers in solutions of individual guanosine compounds,5,12,27,28 in which self-assembly is indicated by the exciton couplet in the main absorption band of the guanosine moiety.12 Although the solutions remain liquid, their spectra indicate some structural dependence on temperature and composition. The CD spectra of the binary solutions that form higherordered phases generally resemble spectra that have been reported for cholesteric phases formed in solutions of individual
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Figure 9. CD spectra of solutions vs time as they were heated from approximately 11 to 32 °C. (A) 0.25 M GMP, 0.05 M Guo (XGMP ) 0.833); (B) 0.25 M GMP, 0.06 M Guo (XGMP ) 0.806); (C) 0.30 M GMP, 0.06 M Guo (XGMP ) 0.833); (D) same solution as C with reverse temperature scan from high to low temperature.
guanosine compounds.12 Examples of spectra measured at 25 °C are shown in Figure 5. The spectrum in Figure 5A is for a viscous, thermoassociative solution (0.25 M GMP, 0.05 M Guo, XGMP ) 0.833) that is in transition between liquid and gel at 25 °C. This solution exhibits a positive CD spectrum that differs significantly from the liquid solutions. The spectra in Figure 5B-D are for solutions containing 0.06 M Guo and increasing amounts of GMP. The spectrum in Figure 5B is for a solution containing 0.05 M GMP (XGMP ) 0.455) that is a stable gel over the temperature range studied. Figure 5C,D shows spectra of thermoassociative solutions with 0.20 M GMP (XGMP ) 0.769) and 0.25 M GMP (XGMP ) 0.806), respectively. The spectra in Figure 5B-D are typical of guanosine gel phases. It is interesting that the CD magnitude increases dramatically with increasing GMP. During the course of the experiments we noticed that a few solutions at the lower total concentration range with XGMP near 0.83 showed unusual variability. We therefore performed a study of one such solution containing 0.15 M GMP/0.03 M Guo (XGMP ) 0.83). The solution was stored in the refrigerator and then placed directly in the CD sample compartment that was maintained at 25 °C. CD spectra were acquired immediately while the sample was still cold and then at regular time intervals over a period of 180 min. The results are shown in Figure 6. The solution began as a liquid at low temperature, formed a gel during the first hour in the 25 °C sample chamber, and then returned to liquid by 180 min. This indicates that the gel phase of this solution, which is stable at higher temperatures, is only metastable at 25 °C. Thermal Melt Experiments. The plot in Figure 7 shows the temperature of the onset of stable gelation for binary gels as a function of XGMP. At low XGMP, the onset of gelation is e5 oC. As XGMP approaches 0.8 (4:1, GMP/Guo) the thermoassociative behavior appears, and the onset of gelation moves to higher temperatures as XGMP increases. Figure 8 shows examples of thermal melt curves for a thermodissociative gel and two thermoassociative gels, all containing 0.06 M Guo with varying GMP. The curves show well-defined temperature ranges for the gel phases that become narrower and shift to higher temperatures as XGMP is increased. Figure 9 shows three-dimensional plots of CD spectra that were collected as a function of time for three thermoassociative gels as the circulating water bath of the CD instrument was
heated from approximately 11-32 °C, in order to further investigate the solution transitions. A solution containing 0.25 M GMP and 0.05 M Guo (XGMP ) 0.833) begins as a liquid and then transitions to a more organized phase with a positive CD spectrum (Figure 9A). This is consistent with the positive CD spectrum at 25 °C for this solution (Figure 5A), corresponding to the visibly viscous phase. A solution containing 0.25 M GMP and 0.06 M Guo (XGMP ) 0.806) also begins as a liquid, passes through the intermediate “viscous” phase with positive CD, and then becomes a gel with negative peaks (Figure 9B), which is consistent with the negative CD spectrum for this solution at 25 °C (Figure 5D). A solution containing 0.30 M GMP and 0.06 M Guo (XGMP ) 0.833) begins as a liquid and passes directly into a highly viscous phase with a strongly negative CD spectrum (Figure 9C). The reversibility of this transition is indicated by Figure 9D, which shows the reverse experiment in which the temperature of the circulating water bath was decreased from approximately 32 to 11 °C. Discussion This work demonstrates unique tunability and thermoresponsiveness of gels formed by binary mixtures of guanosine compounds. In certain proportions, the two compounds form gels that are thermoassociative, that is, change from liquid to gel upon heating, sometimes passing through an intermediate viscous phase. Thermoassociative behavior is well-known in polymeric hydrogels that undergo a discontinuous phase transition from solution to gel above a certain “lower critical solution temperature” (LCST).29-31 The change in phase is due to loss of water that is contained at lower temperatures through hydrophilic interactions but expelled above the LCST as the solution becomes dominated by hydrophobic interactions. Such “coil-to-globule” transitions are also exhibited by biopolymers such as proteins. Binary G gels differ from polymeric hydrogels in that they are formed by reversible self-association of monomers that is governed by solvent composition, pH, and monomer concentration, which is a temperature-dependent function of monomer solubility. In this respect, they are similar to lyotropic liquid crystalline phases formed by amphiphilic molecules but with the important difference that the monomeric guanosine compound first self-associates through hydrogen bonding to form
1134 J. Phys. Chem. B, Vol. 112, No. 4, 2008 G-quartets. The G-quartet is the basic building block of larger aggregates that are stabilized by π-π stacking, templated through complexation with metal cations, and driven toward higher-order aggregation by electrostatic, hydrophobic, and hydrophilic forces. Whereas gels formed by individual guanosine compounds exhibit “thermo-thinning” or thermodissociative behavior, the present work demonstrates that thermoassociative behavior can be achieved in binary mixtures containing a hydrophilic guanosine derivative and the hydrophobic guanosine. The unique thermoassociative behavior is attributed to incorporation of Guo into the GMP aggregates, thereby promoting solubilization of Guo and reducing repulsive forces among anionic GMPs, serving to stabilize higher order aggregates and promote formation of liquid crystalline gels. As the total concentration of guanosine compounds increases, the temperature required to form and maintain the stable, liquid crystalline gel phases increases. As XGMP increases, the onset of gelation shifts to higher temperatures, and the total temperature range of the gel phase decreases. Conclusions The unique tunability afforded by the thermal dependence of the gel phase on the composition of the mixture, combined with the molecular reversibility of gel formation, opens new possibilities for design of biocompatible, environmentally benign materials that are simple and economical to prepare. The region of greatest thermal tunability for the formation of stable gels at pH 7.2 occurs at GMP concentrations of 0.20 M GMP or above, in the approximate range of 0.75-0.85 XGMP. Studies of the effects of solution pH and cations that are currently underway should yield additional tunability. Future studies will focus on the molecular structure of the gels in order to elucidate the interactions leading to the unusual gelation properties of these binary mixtures. References and Notes (1) Davis, J. T. Angew. Chem., Int. Ed. 2004, 43, 668-698. (2) Gellert, M.; Lipsett, M. N.; Davies, D. R. Proc. Natl. Acad. Sci. U.S.A. 1962, 48, 2013-2018. (3) Guschlbauer, W.; Chantot, J. F.; Thiele, D. J. Biomol. Struct. Dyn. 1990, 8, 491-511.
Yu et al. (4) Simonsson, T. Biol. Chem. 2001, 382, 621-628. (5) Chantot, J. F.; Haertle, T.; Guschlbauer, W. Biochimie 1974, 56, 501-507. (6) Spindler, L.; Olenik, I. D.; Copic, M.; Romih, R.; Cerar, J.; Skerjanc, J.; Mariani, P. Eur. Phys. J. E 2002, 7, 95-102. (7) Sasisekharan, V.; Zimmerman, S.; Davies, D. R. J. Mol. Biol. 1975, 92, 171-179. (8) Proni, G.; Gottarelli, G.; Mariani, P.; Spada, G. P. Chem. Eur. J. 2000, 6, 3249-3253. (9) Mariani, P.; Ciuchi, F.; Saturni, L. Biophys. J. 1998, 74, 430435. (10) West, R. T.; Garza, L. A.; Winchester, W. R.; Walmsley, J. A. Nucleic Acids Res. 1994, 22, 5128-5134. (11) Walmsley, J. A.; Burnett, J. F. Biochemistry 1999, 38, 1406314068. (12) Pieraccini, S.; Giorgi, T.; Gottarelli, G.; Masiero, S.; Spada, G. P. Mol. Cryst. Liq. Cryst. 2003, 398, 57-73. (13) Marsh, T. C.; Vesenka, J.; Henderson, E. Nucleic Acids Res. 1995, 23, 696-700. (14) Keniry, M. A. Biopolymers 2001, 56, 123-146. (15) Lee, J. S. Nucleic Acids Res. 1990, 18, 6057-6060. (16) Aggerholm, T.; Nanita, S. C.; Koch, K. J.; Cooks, R. G. J. Mass. Spectrom. 2003, 38, 87-97. (17) Furuta, H.; Ogawa, Y.; Yanagawa, H. Nucleic Acids Res. Symp. Ser. 1988, 20, 87-88. (18) Calzolari, A.; Di Felice, R.; Molinari, E.; Garbesi, A. Appl. Phys. Lett. 2002, 80, 3331-3333. (19) Marsh, T. C.; Henderson, E. Biochemistry 1994, 33, 10718-10724. (20) Gottarelli, G.; Spada, G. P.; Garbesi, A. Comp. Supramolec. Chem. 1996, 9, 483-506. (21) Samori, P.; Pieraccini, S.; Masiero, S.; Spada, G. P.; Gottarelli, G.; Rabe, J. P. Colloids Surf., B 2002, 23, 283-288. (22) Rinaldi, R.; Branca, E.; Cingolani, R.; Di Felice, R.; Calzolari, A.; Molinari, E.; Masiero, S.; Spada, G. P.; Gottarelli, G.; Garbesi, A. Ann. N. Y. Acad. Sci. 2002, 960, 184-192. (23) Mezzina, E.; Mariani, P.; Itri, R.; Masiero, S.; Pieraccini, S.; Spada, G. P.; Spinozzi, F.; Davis, J. T. Chemistry 2001, 7, 388-395. (24) Andrisano, V.; Gottarelli, G.; Masiero, S.; Heijne, E. H.; Pieraccini, S.; Spada, G. P. Angew. Chem., Int. Ed. Engl. 1999, 38, 2386-2388. (25) Dowling, V. A.; Charles, J. A. M.; Nwakpuda, E.; McGown, L. B. Anal. Chem. 2004, 76, 4558-4563. (26) Case, W. S.; Glinert, K.; McGown, L. B. Electrophoresis 2007, 28, 3008-3016. (27) Chantot, J. F.; Sarocchi, M. T.; Guschlbauer, W. Biochimie 1971, 53, 347-354. (28) Guschlbauer, W.; Chantot, J. F.; Thiele, D. J. Biomol. Struct. Dyn. 1990, 8, 491-511. (29) Haraguchi, K.; Li, H. J. Angew. Chem., Int. Ed. 2005, 44, 65006504. (30) Khattak, S. F.; Bhatia, S. R.; Roberts, S. C. Tissue Eng. 2005, 11, 974-983. (31) Yoshioka, H.; Mori, Y.; Tsukikawa, S.; Kubota, S. Polym. AdV. Tech. 1998, 9, 155-158.
