Kinetics and Mechanism of Conformational Changes in a G

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Kinetics and Mechanism of Conformational Changes in a G-Quadruplex of Thrombin-Binding Aptamer Induced by Pb2+ Wei Liu,†,‡,§,|| Yan Fu,^ Bin Zheng,†,‡,§ Sheng Cheng,‡,§,|| Wei Li,# Tai-Chu Lau,‡,§ and Haojun Liang*,†,‡,|| †

)

Hefei National Laboratory for Physics Sciences at the Microscale, University of Science and Technology of China, Hefei, Anhui 230026, P. R. China ‡ Advanced Laboratory of Environment Research and Technology (ALERT), Joint Advanced Research Center, USTC-CityU, Suzhou, Jiangsu 215124, P. R. China § Department of Biology and Chemistry, City University of Hong Kong, Tat Chee Avenue, Kowloon, Hong Kong, P. R. China CAS Key Laboratory of Soft Matter Chemistry, Department of Polymer Science and Engineering, University of Science and Technology of China, Hefei, Anhui 230026, P. R. China ^ Key Laboratory of Systems Bioengineering, Ministry of Education, School of Chemical Engineering & Technology, Tianjin University, Tianjin 300072, P. R. China # Key Laboratory for Green Chemical Technology, Ministry of Education, School of Chemical Engineering & Technology, Tianjin University, Tianjin 300072, P. R. China

bS Supporting Information ABSTRACT: It has been shown that guanine-rich DNA can fold into a G-quadruplex with certain metal cations. The spectral characteristics, thermostability, and kinetics for the formation of a Pb2+-driven G-quadruplex of thrombin-binding aptamer (TBA) were measured in the current work using a combination of ultraviolet (UV) and circular dichroism (CD) spectroscopy along with stopped-flow technique. CD spectra demonstrated that TBA could fold into a unique G-quadruplex with a strong positive peak at 312 nm. Analysis of the titration data reveals that the binding stoichiometry is 1:1 for the titration of TBA with Pb(NO3)2, which is in accordance with the localization of the Pb2+ ion between the adjacent G-quartets. Thermal denaturation profiles indicate that the Pb2+-induced intramolecular G-quadruplex is more stable than those driven by Na+ or K+ ions. Kinetic studies suggest that the Pb2+-induced folding G-quadruplex of TBA probably proceeds through the rapid formation of an intermediate Pb2 +
TBA complex, which then isomerizes to the fully folded structure. Conformational changes transpire after the addition of Pb(NO3)2 to the Na+- or K+-induced G-quadruplexes, which may be attributed to the replacement of Na+ or K+ ions by Pb2+ ions and the generation of a more compact structure of the Pb2+
TBA structure. The relaxation time, τ, of folding the G-quadruplex is reduced from 1.05 s in the presence of Pb2+ ions alone to 0.34 s under the cooperation of initially added Na+ ions, while τ is increased to 8.33 s under the competition of initially added K+ ions.

’ INTRODUCTION G-quadruplex nucleic acid structures, discovered by Davis et al. in 1962,1 consist of stacks of two or more square planar arrays of four Hoogsteen hydrogen-bonded guanines called a G-quartet, where each base is both a hydrogen bond donor and hydrogen bond acceptor. Interests in G-quadruplexes have been stimulated by their emerging roles in biological regulation, as well as the potential use of these structures to design anticancer drugs and drug targets.2
6 Quadruplex DNA is also an excellent module in designing devices for nanotechnology, and G-quartets are likely to form higher order structures, such as synapsable DNA or G-wire.7
9 On the other hand, functional molecules such as a thrombinbinding aptamer (TBA), a catalytic porphyrin metalation DNA r 2011 American Chemical Society

and an inhibitor of HIV replication, could form a G-quaduplex structure, where two G-quartets are interconnected through the lateral TT and TGT loops in antiparallel conformation; it also possesses a high affinity for thrombin.10
13 In addition to some chemical ligands or inorganic molecules,14
16 a variety of metal cations could drive TBA into G-quadruplexes and stabilize the structures.17,18 For monovalent cations, the stability follows the trend K+ > Rb+ > NH4+ > Na+ > Li+.19,20 Various measurements have revealed that K+ ions occupy the cavity between the adjacent G-quartets formed by eight carbonyl groups of guanines, whereas Received: August 4, 2011 Revised: September 23, 2011 Published: September 27, 2011 13051

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The Journal of Physical Chemistry B Na+ ions exhibit smaller ionic radii that allow them to be coordinated in the plane of each G-quartet.21 Divalent cations, such as strontium,22,23 barium,24 and manganese,25,26 have also been shown to coordinate with G-quadruplexes. Pb2+ is a poisonous heavy metal cation that can cause cognitive and motor impairment, with behavioral alterations.27
30 In particular, interaction of Pb2+ ions with genetic materials may contribute to its damage effects in humans, and its ability to stabilize folded DNA structures may give rise to lead’s genotoxicity.31 Pb2+ ions are found to be significantly more potent than K+ ions in inducing folded DNA structures.32
34 Compared with that of the K+induced complex, the positive peak of the Pb2+-induced complex has a red shift of almost 20 nm and is centered at 312 nm in the circular dichroism (CD) spectra. Shafer et al. identified that the Pb2+ ion binds symmetrically between the two guanine quartets in the Pb2+
TBA complex and have obtained quantitative distances of M
O and O
O bonds.34
36 Various methods have been applied to probe into the G-quadruplexes and the conformational changes of cation-induced folding of TBA.37 In addition to electrospray ionization mass spectrometry (ESI-MS),20,23 ultraviolet (UV) and CD spectroscopy were performed to monitor the formation of G-quadruplexes. Isothermal titration and differential scanning calorimetry were used to examine the energies in the folding process.38
40 X-ray crystallography and electron paramagnetic and nuclear magnetic resonance were performed to determine the tertiary structure of TBA as well as the number and location of the binding sites of the DNA G-quadruplex.25,41,42 Although several reports have focused on the cation-induced folding of TBA, little information concerning the kinetics and mechanism of the process is available.21 Kinetic studies can provide insight into the potential pathway of folding as well as the folding intermediates. Recently, Claires et al. investigated the kinetics and mechanism of K+- and Na+induced folding models of human telomeric DNA by UV stopped-flow technique.43,44 The oligonucleotide folding in KCl consists of a single exponential process, while the folding in NaCl consists of three exponential processes. Additionally, the kinetics of the structure transition from antiparallel to parallel of the tetra-quadruplex induced by Ca2+ ions have been investigated.45 In the present work, the detailed kinetic studies for the Pb2+induced folding process of TBA into the G-quadruplex structure are discussed. The conformational changes from random single strand to the packed G-quadruple and the cation binding stoichiometry were investigated using UV/vis and CD measurements. Equilibrium titrations obtained here show that a single Pb2+ ion is involved in the folding of TBA. Thermal denaturation profiles indicate that the Pb2+-induced G-quadruplex is more stable than those induced by Na+ or K+. The proposed mechanism of the Pb2+-induced folding quadruplex of TBA involves the rapid formation of an intermediate Pb2+
TBA complex which then isomerizes to the fully folded structure. The kinetics of the Pb2+-induced conformational switch in the Na+- or K+-induced G-quadruplexes of TBA have also been studied.

’ EXPERIMENTAL METHODS Chemicals and Reagents. The G-rich oligonucleotide (TBA: GGTTGGTGTGGTTGG), 2-(N-morpholino)ethanesulfonic acid (MES) and tris(hydroxymethyl)aminomethane (tris) were purchased from Shanghai Sangon Biological Engineering Technology & Services Co., Ltd. (China). All nitrate salts were of analytical grade and were used without further purification. The

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oligomer samples were dissolved in a buffer solution consisting of 10 mM MES/tris at pH 6.1. The oligomer solutions were then heated in a dry bath until the temperature reached 95 C (ABSON, U.S.), equilibrated for 15 min at this temperature, and then slowly cooled to room temperature. The concentrations of single-strand DNA were determined at 260 nm by UV/ vis spectrophotometer, using a molar extinction coefficient of 146.0 mM
1 cm
1.46 CD Spectra. CD spectra were measured on a JASCO J-810 spectropolarimeter at 25 C, maintained by a Julabo temperature controller. The final concentrations of the oligomer were in the range of 15
25 μM. Each measurement was recorded from 220 to 350 nm in a sealed 1 mm path-length quartz cuvette at a scanning rate of 100 nm min
1 with a response of 0.1 s and data interval of 0.2 nm. The final spectra were the average of three measurements. The scan of the buffer alone under the same conditions was used as the blank, which was subtracted from the average scan for each sample. The cell-holding chamber was flushed with a constant stream of dry nitrogen gas to avoid water condensation on the cell. Equilibrium Titration. The extent of TBA folding was evaluated by measuring UV absorbance changes at 303 nm as a function of the concentration of added Pb(NO3)2. These experiments are required prior to the kinetic experiments, not only for establishing the expected changes in absorption, but also for determining the minimum concentration of Pb(NO3)2 necessary to induce complete folding. With serial additions of concentrated solutions of Pb(NO3)2, the UV absorption spectra were measured at 1 nm intervals from 220 to 350 nm on a Shimadzu 1800 spectrophotometer (Shimadzu, Japan) equipped with a digital circulating water bath maintained at 25 C. Equal volumes of Pb(NO3)2 and oligomer in the same buffer were mixed together in a sealed tandem cuvette with a path length of 10 mm. The concentration of the oligomer before and after mixing was 5.0 and 2.5 μM, respectively. After each addition of Pb(NO3)2, the difference spectra ΔAλ = AλU
AλF were obtained by subtracting the absorption spectrum from the spectrum of the fully unfolded oligonucleotide. The resulting titration data (Δε) were then plotted as a function of the concentration of Pb(NO3)2. Thermal Denaturation Studies. Structures of nucleic acids are sensitive to temperature. Hence, UV melting profiles can be used to determine the melting temperature (Tm) of the cation
TBA complexes and to determine the temperature range in which G-quadruplexes could be stable. In a typical experiment, the melting curve was obtained by monitoring the UV absorbance, either at 295 nm (for K+ and Na+ systems) or at 303 nm (for Pb2+ system), as a function of temperature using a sealed 10 mm path-length quartz cell. The samples were first held at 15 C for 5 min and then heated to 95 C with a heating rate of 0.2 C nm
1. Tm of the complexes were calculated by fitting the experimental curves with a Sigma plot.47 Tm is the midpoint temperature of the order
disorder transition of the complex. Kinetic Experiments. The kinetics of the Pb2+-induced oligomer folding process were performed by monitoring the CD intensity changes on a JASCO J-810 spectropolarimeter equipped with SFM300 multimixing equipment (Biologic). The concentration of the oligomer before and after mixing was 50 and 25 μM, respectively. Before measurement, the DNA samples were thermally treated as described above and incubated at the desired temperature for several minutes. A circulating water bath was used to maintain a constant reaction temperature. The kinetic experiments were performed under pseudo-first-order 13052

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Figure 1. CD spectra of cation-induced G-quadruplex of TBA in the presence of various cations in the buffer (10 mM MES/tris, pH 6.1) at 25 C. Concentrations of oligonucleotide were 25 μM after mixing.

conditions with the concentrations of Pb(NO3)2 at least in 10fold excess of that of the oligomer. At least five successive mixing experiments were performed and averaged for analysis. Control experiments consisting of mixing oligomer with cation-free buffer were also performed. Kinetics of the conformational switches were conducted by adding Pb(NO3)2 to the solutions containing G-quadruplexes induced by Na+ or K+ ions.

’ RESULTS AND DISCUSSION CD Spectra. CD spectroscopy has been proven to be a potent technique for characterizing the formation of known structural motifs. Figure 1 shows the CD spectra of 25 μM TBA in buffer solution in the presence of different metal cations. The addition of metal cations results in significant changes in the CD spectra. The spectra of folded G-quadruplexes stabilized by K+ or Na+ ions exhibit a positive CD peak near 295 nm.48,49 The addition of 0.5 mM Na+ showed no effect on the structure of TBA, until the concentration of Na+ was increased up to 50 mM. On the other hand, the G-quadruplex could be formed in the presence of 0.5 mM K+, and there was only a slight increase of the peak intensity when [K+] was increased to 50 mM. In the presence of Pb(NO3)2, the wavelength for the maximum CD intensity was shifted by almost 20 to 312 nm, and the minimum peak is located at 268 nm. The spectrum did not vary with time or oligomer concentrations, indicating the formation of a stable, folded structure.33 In addition, the CD intensity of Pb2+-induced G-quadruplex was about 3 and 6 times larger than those of K+ and Na+, respectively. The intensity increase and location shift of the Pb2+-induced G-quadruplex may reveal a change in the dimension of the G8 cage, which is in accordance with the decrease in the cage size as observed in the X-ray crystal structure.32 The overall difference in the CD spectra of the G-quadruplex with various cations may be attributed to the difference in the coordination number of cations, partial formation of G-quadruplexes with some cations and/or the overall tightness of each G-quadruplex. Folding Equilibrium Titration. Previous studies have shown that the UV absorption spectrum of the Pb2+-driven folded G-quadruplex possesses an absorption maximum at ∼303 nm, which is distinctly different from that of the unfolded oligonucleotide.37,39 To monitor the expected absorbance change and cation binding number, TBA was titrated with Pb(NO3)2 at micromolar concentrations at 25 C. The absorbance change at 303 nm was monitored as a function of the mole ratio of cation to oligomer, and results are shown in Figure 2. Similar titrations with NaNO3 or KNO3 were also performed, and the results are

Figure 2. Spectrophotometric titration of TBA (2.5 μM) with Pb(NO3)2. (a) Spectral changes in the titration process. The arrow in the figure indicates decreasing absorbance changes upon successive additions of cations. The final cation concentrations after mixing are shown at the right of the panel. (b) Plots of absorbance changes at 303 nm as a function of the mole ratio of Pb(NO3)2 to TBA. The temperature was maintained at 25 C.

shown in Figure S1. The difference spectra Δε = ελU
ελF were obtained by subtracting the absorption spectrum from the spectrum of the fully unfolded oligomer. The spectral titration in Figure 2a shows that the Pb2+-induced folding of TBA is accompanied by relatively smaller absorbance changes between 235 and 285 nm but larger absorbance changes between 285 and 340 nm, with maxima and minimum at ∼245/∼275 and ∼303 nm, respectively. As shown in Figure 2a, the absorbance at 303 nm increases with increasing concentration of Pb(NO3)2, as indicated by the direction of the arrow. The titration curve derived from monitoring absorbance changes at 303 nm is shown in Figure 2b, which shows that a single Pb2+ ion is enough to stabilize the aptamer. On the other hand, in the titration of TBA with NaNO3 or KNO3, the absorbance continuously increases until the concentrations of cations exceed those of the oligomer a thousand-fold. Chaires et al. obtained similar results, and they analyzed the results using the Hill equation, which indicates that Na+ ions are within the central cavity of each quartet, while the K+ ion is coordinated between the two quartets.43 Our results suggest that the G-quadruplexes assembled by Na+ or K+ ions are not very stable, and there are free cations surrounding the complexes. The structural conformer of the G-quadruplex can be regulated by surrounding conditions such as the presence of identical or different cations. On the other hand, the Pb2+-driven G-quadruplex is much more stable, and is not affected even by the addition of a 20-fold excess of NaNO3 or KNO3. The Pb2+ ions are proposed to be coordinated in the same site as K+ ions. However, the Pb2+-stabilized G-quadruplex has shorter M
O and O
O bonds than those assembled by Na+ or K+ ions;32
34 such a compact structure contributes to the unusually high stability of the Pb2+-induced G-quadruplex. Thermal Denaturation Profiles. UV melting experiments were performed to examine the stability of cation-induced 13053

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Figure 3. Denaturaion profiles for the G-quadruplex of TBA (2.5 μM) induced by various cations in buffer solution (10 mM MES/tris, pH 6.1). The UV melting curve was measured at 303 nm for Pb(NO3)2 (0.5 mM), while experiments were performed at 295 nm for NaNO3 or KNO3 (50 mM).

G-quadruplexes. As shown in Figure 3, the unfolding profiles of the cation-induced G-quadruplexes were monitored at 303 or 295 nm because of large absorbance changes at these wavelengths. The UV melting analysis of TBA in the presence of 0.5 mM Pb(NO3)2 reveals a cooperative monophasic melting transition at 55 ( 2 C. Such a high melting temperature shows that the Pb2+-induced G-quadruplex of TBA is stable under physiological conditions. In the presence of 50 mM NaNO3 or KNO3, Tm of the G-quadruplexes of TBA were determined to be 24 and 48 C, respectively. The low melting temperature for the Na+-induced G-quadruplex indicates that the complex should be very unstable. The higher Tm for the G-quadruplex induced by Pb(NO3)2 than those of NaNO3 or KNO3 is consistent with higher binding affinity of the Pb2+ ions. In addition, the melting temperature (Tm) remains constant over a 10-fold increase in strand concentration, indicating that the G-quadruplex is formed intramolecularly. Folding Kinetics. Time-Tracing Curves of Pb2+-Induced Folding of G-Quadruplex. Given that the time scale of the folding of G-quadruplex is very short, the stopped-flow mixing technique was used to investigate the mechanism of conformational change in the G-quadruplex of TBA induced by Pb2+ ions. Time-tracing curves of the folding process were obtained by monitoring the CD intensity at 312 nm, the maximum signal of the spectrum of the Pb2+-induced G-quadruplex. In the presence of at least 10-fold excess of Pb(NO3)2, clean pseudo-first-order kinetics were observed for over three half-lives. The experimental and calculated profiles at 312 nm, as well as the residual plot, are shown in Figure 4. The observed pseudo-first-order rate constant, kobs, was obtained from the time-tracing curves, which were analyzed by a nonlinear least-squares fitting procedure. A set of five curves was analyzed, and the average kobs was determined to be 0.95 ( 0.04 s
1. Previous studies have shown that monovalent cation binding to the G-quadruplex occurs on a millisecond time scale.50,51 The rate constant for Pb2+ is slower, but is similar to that of the Mg2+-induced folding of the Tetrahymena ribozyme to form a stable tertiary intermediate structure, which has a rate constant of >2 s
1.52 This suggests that folding of oligonucleotide induced by divalent metal ions is slower than that by monovalent metal ions. Considering the unusually high binding efficiency of Pb2+ ions, the traces obtained in the present study can be regarded as the structural transition, rather than Pb2+ ion binding to TBA. Given that the Pb2+-induced G-quadruplex formed intramolecularly with respect to TBA, to avoid large signal-to-noise ratio, the kinetics were investigated over a limited

Figure 4. Representative experimental process curve of the folding of TBA (15 μM) induced by Pb(NO3)2 (0.2 mM) traced with CD intensity at 312 nm. All measurements were performed in a buffer containing 10 mM MES/tris (pH 6.1) at 25 C. The traces were fitted to a single exponential function. The residual plot indicates the deviation of the experimental and fitted absorbance changes.

oligomer concentration. Limited solubility of Pb(NO3)2 in the aqueous buffer thwarted the usage of cation concentrations of more than 1.0 mM. If the time-tracing curves included only one step under pseudo-first-order conditions, then the observed rate constant would linearly increase with [Pb(NO3)2]. However, this was not the case. The observed rate constant, kobs, is independent of the concentration of Pb(NO3)2, suggesting rate-saturation kinetics, which is in accordance with the high stability of the Pb2+-induced G-quadruplex of TBA. The simplest mechanism for the Pb2+-induced folding of a G-quadruplex of TBA is the initial rapid formation of an intermediate Pb2+
TBA complex, which then isomerizes to the fully folded structure, as shown in eq 1 below. ðTBAÞU þ Pb2þ h I f ðTBA
Pb2þ ÞF

ð1Þ

where (TBA)U and (TBA
Pb2+)F indicate the unfolded and folded structures of TBA, respectively. Conformational Switch from Na+/K+
TBA Complexes to Pb2+-Induced G-Quadruplex. K+ and Na+ are the major intracellular metal cations, which are also able to induce folding of the G-quadruplex. We find that Pb2+ ions are not only able to induce folding of the G-quadruplex from random-coiled DNA, but can also “switch” quadruplex conformations. To determine whether the effect of Pb2+ ions on the structural rearrangements is cooperative or competitive with Na+/K+ ions, we investigate conformational switch in the G-quadruplexes of TBA induced by NaNO3 or KNO3 in the presence of Pb(NO3)2. Figure 5 illustrates the time-tracing profiles for Na+/K+ f Pb2+ exchange using CD stopped-flow at 312 nm. A large increase in CD intensity was observed immediately after the addition of Pb(NO3)2, and the changes in the spectra are indicative of a gradual progression from the Na+- or K+-induced form to the Pb2+ coordinating complex. The time-tracing curves were fitted by a single exponential function, and the kobs values were determined to be 2.92 ( 0.14 s
1 and 0.12 ( 0.001 s
1 for adding Pb(NO3)2 to the Na+
TBA and K+
TBA complexes, respectively. Δθ increases from 9 to 12.5 mdeg for the exchange of Pb2+ for Na+ in TBA, which may be attributed to the tighter structure of the Pb2+-induced G-quadruplex. The kobs increases slightly from 13054

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Figure 5. Time-tracing curves of conformational changes by measuring CD intensity at 312 nm for (a) Na+/Pb2+ and (b) K+/Pb2+ exchanges. TBA (30 μM) was annealed in the buffer (10 mM MES/tris, pH 6.1) containing 100 mM Na+ or K+ ions and then mixed with Pb(NO3)2 (0.4 mM) at 25 C. Each profile was fitted to a single exponential function. The residual plots indicate the deviation of the experimental and fitted absorbance changes.

the presence of K+ ions, TBA was induced by K+ ions to a relatively stable G-quadruplex with the same binding site as Pb2+ ion, which probably makes exchange with Pb2+ ion more difficult. In short, the initially added Na+ ion has a cooperative effect with Pb2+ ion on the conformational transition of the TBA chain, while K+ ion has competitive effect on this transition.

Figure 6. Proposed mechanism of conformational switch in the quadruplex of TBA induced by Pb2+ ions. Guanine bases of TBA are shown as rectangles. τ = 1/k.

0.95 s
1 to 2.92 s
1, suggesting that the Na+ ion may have some cooperative effects on the structural change of the G-qudruplex induced by Pb2+ ions. The use of Pb2+ ions is proposed to probably be much easier in assembling the G-quadruplex after the formation of the complex of TBA induced by Na+. In the case of K+, Δθ increases from 9 to 16 mdeg for the exchange of Pb2+ for K+ ions. The larger Δθ may be interpreted as the stronger binding ability of Pb2+ to the two G-quartets of TBA, causing shrinking of the G8 cage. The kobs decreases from 0.95 s
1 to 0.12 s
1, suggesting that there is competition between K+ and Pb2+ ions for coordinating to the G-quadruplex of TBA. This phenomenon probably results from the same binding sites of K+ and Pb2+ions for assembling the G-quadruplex, which is consistent with the titration data. The equilibrium time of Pb2+/K+ exchange is remarkably longer than that of Pb2+/Na+, which is also consistent with the fact that K+ ions have higher affinity for TBA than Na+ ions, and therefore it is more difficult to be displaced by Pb2+ ions. From kinetic and equilibrium analyses, a mechanism of conformational switch in the G-quadruplex of TBA induced by Pb2+ ions is proposed as shown in Figure 6. In this mechanism, a single Pb2+ ion is sufficient to fully assemble TBA with the Pb2+ ion coordinating between the adjacent quartets. In the presence of Na+ ions, TBA folds to an incompact G-quadruplex with Na+ ions in the central cavity of each quartet, which may make it slightly easier for Pb2+ ions to fold a compact G-quadruplex. In

’ CONCLUSIONS In the present study, we have reported the kinetics and mechanism of the conformational switch in the G-quadruplex of TBA induced by Pb2+ ion. CD and UV spectroscopy illustrates the characteristic spectra of the G-quadruplex of TBA in the presence of various cations. Equilibrium titrations demonstrate that at micromolar concentrations, the binding is stoichiometric and a single Pb2+ ion is sufficient to fully assemble TBA with Pb2+ ion coordinating between the adjacent quartets. Thermal denaturation experiments show that the Tm of the TBA
Pb2+ complex is markedly higher than those of the Na+- or K+-induced ones, which is consistent with the fact that Pb2+ ions have higher binding affinity for TBA. The kinetic studies show that the Pb2+induced folding of TBA into a G-quadruplex probably proceeds through the rapid formation of an intermediate Pb2+
TBA complex that subsequently isomerizes to the fully folded structure. Pb2+ ion is a potent agent in assembling G-quadruplexes, and it can drive conformational change in the Na+ or K+-induced G-quadruplex, which may be ascribed to the exchange of Pb2+ for Na+ or K+ ions and the generation of a more compact structure of Pb2+-induced G-quadruplex. Na+ ions were cooperative, while K+ ions were competitive with Pb2+ ions in the structural transition. Analysis of folding kinetics of the chain indicates that initially added Na+ ion has a cooperative effect with Pb2+ ion on the conformational transition of the TBA chain, while K+ ion has competitive effect on this transition. We believe that the present findings provide significant novel physical insight into the mechanism of the conformational changes in G-quadruplex structures. ’ ASSOCIATED CONTENT

bS

Supporting Information. Figures showing the spectral changes in the titration of TBA with NaNO3 or KNO3 (Figure

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Pb2+ complex (Figure S2). This material is available free of charge via the Internet at http://pubs.acs.org.

’ AUTHOR INFORMATION Corresponding Author

*E-mail: [email protected].

’ ACKNOWLEDGMENT The work described in this paper was supported by the Program of the National Natural Science Foundation of China (Grant Nos. 20934004 and 20874094), NBRPC (Grant No. 2010CB934500), and the “Bairen” fund of CAS. We thank Dr. Y. L. Jiang for his suggestions and discussions. ’ REFERENCES (1) Gellert, M.; Lipsett, M. N.; Davies, D. R. Proc. Natl. Acad. Sci. U.S.A. 1962, 48, 2013–2018. (2) Olaussen, K. A.; Dubrana, K.; Dornont, J.; Spano, J. P.; Sabatier, L.; Soria, J. C. Crit. Rev. Oncol. Hematol. 2006, 57, 191–214. (3) Han, H. Y.; Hurley, L. H. Trends Pharmacol. Sci. 2000, 21, 136–142. (4) Neidle, S.; Parkinson, G. Nat. Rev. Drug Discovery 2002, 1, 383–393. (5) Muniyappa, K. Med. Chem. Res. 2010, 19, 28–29. (6) Kelland, L. R.; Ireson, C. R. Mol. Cancer Ther. 2006, 5, 2957–2962. (7) Marsh, T. C.; Vesenka, J.; Henderson, E. Nucleic Acids Res. 1995, 23, 696–700. (8) Neidle, S.; Parkinson, G. N.; Lee, M. P. H. Nature 2002, 417, 876–880. (9) Chen, F. M. Biophys. J. 1997, 73, 348–356. (10) Macaya, R. F.; Schultze, P.; Smith, F. W.; Roe, J. A.; Feigon, J. Proc. Natl. Acad. Sci. U.S.A. 1993, 90, 3745–3749. (11) Jing, N. J.; Hogan, M. E. J. Biol. Chem. 1998, 273, 34992–34999. (12) Sugimoto, N.; Toda, T.; Ohmichi, T. Chem. Commun. 1998, 1533–1534. (13) Forman, S. L.; Fettinger, J. C.; Pieraccini, S.; Gottareli, G.; Davis, J. T. J. Am. Chem. Soc. 2000, 122, 4060–4067. (14) Jia, G.; Feng, Z.; Wei, C.; Zhou, J.; Wang, X.; Li, C. J. Phys. Chem. B 2009, 113, 16237–16245. (15) Cheng, X. H.; Liu, X. J.; Bing, T.; Zhao, R.; Xiong, S. X.; Shangguan, D. H. Biopolymers 2009, 91, 874–883. (16) Fu, Y.; Wang, X.; Zhang, J. L.; Xiao, Y.; Li, W.; Wang, J. K. Biomacromolecules 2011, 12, 747–756. (17) Chowdhury, S.; Bansal, M. J. Phys. Chem. B 2001, 105, 7572–7578. (18) Engelhart, A. E.; Plavec, J.; Persil, O.; Hud, N. V. Metal Ion Interactions with G-Quadruplex Structures. In Nucleic Acid
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