Structural Conversion of Intramolecular and ... - ACS Publications

ARTICLE pubs.acs.org/JPCB

Structural Conversion of Intramolecular and Intermolecular G-Quadruplexes of bcl2mid: The Effect of Potassium Concentration and Ion Exchange Chang-Ting Lin,†,‡ Ting-Yuan Tseng,†,‡ Zi-Fu Wang,†,§ and Ta-Chau Chang*,†,‡,§ †

Institute of Atomic and Molecular Sciences, Academia Sinica, P.O. Box 23-166, Taipei 10617, Taiwan, Republic of China Institute of Biophotonics, National Yang-Ming University, Taipei 11221, Taiwan, Republic of China § Department of Chemistry, National Taiwan University, Taipei 10617, Taiwan, Republic of China ‡

ABSTRACT: The gel assay, circular dichroism, and differential scanning calorimetry results all demonstrate that a major monomer component of bcl2mid exists at low [Kþ] and an additional dimer component appears at high [Kþ]. This implies that bcl2mid is a good candidate for elucidating the mechanisms of structural conversion between different G-quadruplexes. We further discovered that the conversion between the monomer and dimer forms of bcl2mid does not occur at room temperature but is detected when heated above the melting point. In addition, the use of the lithium cation to keep the same ionic strength in a Kþ solution favors the formation of the bcl2mid dimer. We also found that the bcl2mid dimer is more stable than the monomer. However, after the bcl2mid monomer is formed in a Kþ solution, there is no appreciable structural conversion from the monomer to the dimer detected with addition of Liþ at room temperature. Furthermore, the spectral changes of bcl2mid when transitioning from sodium form to potassium form take place upon Kþ titration. The absence of the dimer form for bcl2mid after the direct addition of 150 mM [Kþ] at room temperature suggests that the spectral changes are not due to rapid unfolding and refolding. In addition, this work reveals the conditions that would be useful for NMR studies of G-quadruplexes.

’ INTRODUCTION A large number of potential G-quadruplex-forming sequences are found in the human genome.1-3 Among them, the 30 overhang G-rich single strand of human telomeres can form G-quadruplex (G4) structures under physiological conditions both in vitro4,5 and In Vivo in metaphase chromosomes.6,7 The folding of telomeric DNA into G4 structures is important in inhibiting the activity of telomerase;8,9 thus, such a structure might be a potential target for the therapeutic intervention of cancer.10-12 In this case, it would be important to know telomeric G4 structure for drug design. However, the G-rich sequences can adopt various G4 structures and can possibly coexist in mixtures. For example, NMR analysis showed that the human telomeric sequence, AG3(T2AG3)3 (HT22), forms an antiparallel basket G4 structure in a Naþ solution,4 while X-ray crystallography showed that HT22 forms a parallel propeller G4 structure in the presence of Kþ.5 On the other hand, the precise G4 structures of HT22 and (T2AG3)4 (HT24) in Kþ solution still remain undetermined,13-16 which is due to the coexistence of two different intramolecular G4 structures that cause problems for structural analysis.17,18 In addition, a spectral conversion of HT24 from the “sodium form” to the “potassium form” has been observed upon Kþ titration.18-23 Two possible mechanisms involving structural changes between different types of structures via unfolding18-20 and structural changes within a single conformational state without unfolding21-23 were proposed for this r 2011 American Chemical Society

spectral conversion. Investigation of structural diversity among various G4 structures and verification of the proposed structural conversion are essential for developing new anticancer drugs and for exploring their potential biological roles.24,25 We propose the G-rich sequence with various G4 structures that can be easily separated and identified may be useful in verifying the proposed mechanisms for structural conversion. Since the intramolecular and intermolecular conformations can be easily distinguished by gel assays, a G-rich sequence that can adopt intra- and intermolecular G4 structures under different conditions is a good candidate to study when investigating the mechanisms of structural conversion. Gabelica et al.26 found that the formation of intermolecular G4 assemblies is favored by short loops in the Kþ solution. In addition to human telomeric G-quadruplexes, a number of nontelomeric G4 structures have been identified in human gene promoters such as the c-myc oncogene,27-30 the bcl-2 gene,31-33 the VEGF gene,34 the KRAS gene,35 and the c-kit oncogene.36 Such promoters that can form G4 structures may play a critical role in gene transcription and regulation.37 Hurley et al.28 have examined the G-quadruplexes of AG3TG4AG3TG4 (c-myc18) and TG4AG3TG4AG3TG4AAG2 (c-myc27) in the c-myc gene Received: August 12, 2010 Revised: January 13, 2011 Published: February 22, 2011 2360

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The Journal of Physical Chemistry B promoter region, and the intermolecular conformation of c-myc27 in Kþ solution has been previously described.27,28 In addition to c-myc gene promoter sequences, the middle four consecutive G-tracts of the P1 promoter in the human bcl-2 gene, G3CGCG3AGGAAG5CG3 (bcl2mid), are also likely to form the major G4 structure in the bcl-2 promoter region.32,33 NMR analysis has suggested that there are at least two conformations of bcl2mid that coexist in a Kþ solution. However, the modified sequences of G3CGCG3AGGAATTG3CG3 (bcl2midM)33 for bcl2mid and T2G3(T2AG3)3A (HT24-M) for HT24 are predominantly a hybrid type of G4 structure in a Kþ solution.38 Is it possible to verify the two major components of bcl2mid in Kþ solution? In addition, it is of interest to examine whether the spectral conversion of bcl2mid from the “sodium” form to the “potassium” form can be detected upon Kþ titration. Can the study of bcl2mid in comparison with HT24 allow us to determine the structural conversion mechanism of G-quadruplexes? We have combined gel mobility, circular dichroism (CD), and thermal melting to examine the possible structural diversity of these G-rich sequences including human telomeres and two gene promoter sequences (for the bcl2 and c-myc genes) when in the presence of varying [Kþ]. The effect of salt concentration on G4 intramolecular structure has been documented by a number of groups,39-42 and it was found that increasing the salt concentration would increase the melting temperature of the G4 structure. However, the formation of intramolecular and intermolecular G4 structures as a function of salt concentration has not been documented. The studies of the gel assays together with differential scanning calorimetry (DSC) results of bcl2mid show that a major intramolecular conformation occurs at a low [Kþ] while an additional intermolecular conformation occurs at a high [Kþ]. Although the formation of intermolecular G4 structures is unlikely in the promoter region, the distinct intra- and intermolecular G4 structures of bcl2mid allow one to examine the possible unfolding and refolding of G-quadruplexes with the addition of Kþ. In addition, the intramolecular component of bcl2mid predominates at a low [Kþ] and the addition of further Kþ can stabilize the initial G-quadruplex structure without structural conversion. One may determine the intramolecular structure of bcl2mid that is formed at a low [Kþ] using NMR.

’ EXPERIMENTAL SECTION DNA Samples. All oligonucleotides were purchased from Bio Basic Inc. and used without further purification. The molar concentration of DNA was determined by monitoring the optical absorbance at 260 nm using a Hitachi U3200 UV-visible spectrometer. Table 1 lists the DNA sequences studied in this work and the calculated molar extinction coefficients from each sequence using the nearest-neighbor method.43 A stock solution consisting of 10 mM Tris-HCl at pH 7.5 and various [KCl] was mixed with each DNA sample and then sonicated for 5 min, heated to 95 °C for ∼5 min, cooled slowly to room temperature, and then stored for 48 h at 4 °C before use. PAGE. PAGE was conducted using 20% polyacrylamide gels. Electrophoresis gels were run at 250 V/cm for 4 h at 4 °C. After photographing with UV shadowing, gels were poststained with 20 μM BMVC-2 for 1 min at room temperature, rinsed with distilled water, and then photographed under UV light (254 nm) by a digital camera. In addition, the fluorescence images of the gels were recorded on a FluoChem HD2 (Alpha Innotech, USA).

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Table 1. G-Rich Sequences Studied in This Work sequence

abbreviation

ε260 (M-1 cm-1)

50 -TTAG3TTAG3TTAG3TTAG3

HT24

50 -TTG3TTAG3TTAG3TTAG3A

HT24-M

244 300

50 -G3CGCG3AGGAAG5CG3

bcl2mid

231 300

50 -G3CGCG3AGGAATTG3CG3

bcl2mid-M

227 300

50 - AG3TG4AG3TG4

c-myc18

186 900

50 -TG4AG3TG4AG3TG4AAG2

c-myc27

279 900

244 600

The relative quantities of the major components in each line were then measured using the Alphaview program. Circular Dichroism (CD). The CD spectra had an average of 10 scans on a J-715 spectropolarimeter (Jasco, Japan) with a 2 nm bandwidth at a 50 nm/min scan speed and a 0.2 nm step resolution. The CD spectra were measured under N2 between 210 and 350 nm to monitor the G4 structures. Thermal melting and annealing curves were obtained by monitoring the CD intensity at either 295 or 265 nm at a rate of 1.0 °C/ min. Three independent scans were recorded for each sample. The melting temperature (Tm) was measured from the first differentiation of the melting curve. The enthalpy (ΔH) can be obtained from a van’t Hoff analysis of the CD melting curve using the equation ΔH vH ¼ - R½d ln K=dð1=TÞ where K is the equilibrium constant obtained from the ratio of the folded to unfolded fraction near Tm.44 This analysis is valid for two-state transition, and in addition, one can determine the differential binding of counterions, Δn(Kþ), between the quadruplex and coil states by the following equation:45,46 ΔnðK þ Þ ¼ ½d ln K=d T m ½d T m =d lnðK þ Þ ¼ 1:11½ΔH=RT m 2 ½d T m =d lnðK þ Þ Differential Scanning Calorimetry (DSC). DSC thermograms were measured using an N-DSC III calorimeter (New Castle, DE). The data acquisition and analysis were carried out through the built-in software (NDSC Run version 3.6 and NanoAnalyze version 2.0). Each calorimetric experiment of 200 μM was performed by scanning from the sample from 20 to 105 °C at 1.0 °C/min. The corresponding baseline (bufferbuffer) scans were subtracted from the buffer-sample scans prior to their normalization and analysis. Standard thermodynamic parameters including ΔH, entropy (ΔS), and free energy change (ΔG) can be obtained from DSC data using the following relationship Z ΔCP dT, ΔScal ¼ ΔH cal =T m , and ΔG ΔH cal ¼

¼ ΔHð1 - T=T m Þ where the heat capacity of ΔCP is assumed to be independent of temperature. The ΔHvH/ΔHcal ratio shows the nature of the transition; for a two-state transition, ΔHvH = ΔHcal, whereas, for a non-two-state transition, ΔHvH 6¼ ΔHcal.45

’ RESULTS Gel Electrophoresis. The gel mobility was applied to evaluate the possible existence of intermolecular G-quadruplexes. 2361

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The Journal of Physical Chemistry B Figure 1A and B shows the UV shadowing and poststained gels of telomeric sequences of HT24 and HT24-M (lanes 1-2) and gene promoter sequences of bcl2mid, bcl2mid-M, c-myc18, and c-myc27 (lanes 3-6) in the presence of 150 mM Kþ and stained with 3,6-bis(1-methyl-2-vinylpyridinium) carbazole diiodide (BMVC-2), respectively. We found that BMVC-2 is a better gel staining dye for G4 structures than BMVC (paper in preparation). The gel assays show a major intramolecular component in HT24 and HT24-M. However, more than one major component can be found in the gel assays of bcl2mid, bcl2mid-M, c-myc18, and c-myc27. Moreover, the intramolecular component in c-myc18 is much less than its intermolecular component. These results are consistent with the findings documented by Gabelica et al.26 They found that G-rich sequences with long loops tend to form intramolecular

Figure 1. UV shadowing of 40 μM telomeric sequences of HT24 and HT24-M (lanes 1-2) and 80 μM gene promoter sequences of bcl2mid, bcl2mid-M, c-myc18, and c-myc27 (lanes 3-6) in the presence of 150 mM Kþ (A) and 5 mM Kþ (C); the gels were stained with 20 μM BMVC-2 (B, D).

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G-quadruplexes while sequences with short loops favor the formation of intermolecular G-quadruplexes. Figure 1C and D shows the UV shadowing and poststained gels of the same G-rich sequences in the presence of 5 mM Kþ. These gel assays show no appreciable difference in telomeric sequences but a considerable difference in the gene promoter sequences upon changing the [Kþ]. The high order component of bcl2mid detected at a high [Kþ] is almost negligible at low [Kþ]. On the other hand, the intramolecular component of c-myc18 that is rarely detected in the presence of 150 mM Kþ is clearly observed in the presence of 5 mM Kþ. It appears that the [Kþ] plays an important role in determining their structures. To further examine the relative changes of each component of bcl2mid and c-myc27 at different [Kþ], gel assays of c-myc27 and bcl2mid in the presence of 150, 70, 20, 5, and 1 mM Kþ were performed, as shown in Figure 2A and B. Considering that the same amount of DNA is used in each lane, one can compare the relative intensity of the intramolecular component in each lane. The gels were then put in a FluoChem HD2 (Alpha Innotech, USA), and the fluorescence images were recorded. The fluorescence intensity of each component of bcl2mid and c-myc27 as a function of [Kþ] was measured using Alphaview, and the normalized quantities of intramolecular components were plotted in Figure 2C. The results indicate that as the [Kþ] increases there are fewer intramolecular components formed in both cases. Considering some random Kþ ions in solution that might diffuse from one lane to another in the gel assays especially at different Kþ concentrations, it is better to examine whether the diffuse Kþ ions can destroy the original G4 structure. No appreciable difference from Figure 2B was found when adding extra 20 mM Kþ in the buffer of the gel assays for the bcl2mid in 150, 70, 20, 5, and 1 mM Kþ solutions. To further examine whether a possible salt gradient induced by the 150 mM Kþ (lane 1) to the 1 mM Kþ (lanes 2-7) in the gel assays can affect the original G4 structure, Figure 2D shows no discernible

Figure 2. Gel assays of 80 μM c-myc27 (A) and bcl2mid (B) in the presence of 150, 70, 20, 5, and 1 mM Kþ stained with 20 μM BMVC-2. The HT24, HT36, and HT54 in the first lane of part A were used as gel markers. The relative quantities of the intramolecular components of c-myc27 and bcl2mid were plotted as a function of [Kþ] (C). The poststained gels of bcl2mid in the presence of 150 mM (lane 1) and 1 mM (lanes 2-7) Kþ (D). The plots of the relative quantity of intramolecular component in each lane normalized by the quantity in lane 7 (E). 2362


The Journal of Physical Chemistry B

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Figure 3. CD spectra of 20 μM G-rich sequences in 5 and 150 mM Kþ solutions.

difference in lanes 2-7 of bcl2mid in the poststained gels. Further measurement of each intramolecular component in lanes 2-7 by using Alphaview confirms this finding, as shown in Figure 2E. These results suggest that the effect of the diffuse Kþ ions to destroy the original G4 structure in the gel assays is negligible in our work. Circular Dichroism. The CD spectra have been routinely applied to characterize the G4 structures.47-49 Although CD cannot give precise structures of these G-quadruplexes, it can be used to distinguish parallel and antiparallel G4 structures based on their strand orientation.50 Figure 3 shows the CD spectra of these G-rich sequences in 5 and 150 mM Kþ solutions. Two positive CD bands occur at ∼265 and ∼295 nm for HT24, HT24-M, bcl2mid, and bcl2mid-M, while a positive CD band occurs at ∼265 nm for c-myc27 and c-myc18 in both [Kþ]. It is known that the mixed type G4 structure is the major component in HT24-M18 and bcl2mid-M,33 while the propeller type G4 structure is predominant in c-myc27 and c-myc18.29,30 With reference to the gel assays, the increase in intensity of the 265 nm CD band of these gene promoter G-rich sequences in a 150 mM Kþ solution is probably due to increased intermolecular G4 structures at a high [Kþ]. Moreover, the intramolecular component of c-myc18 is minor in the presence of 150 mM Kþ; this implies that the parallel type G4 structures are the major conformation for the intermolecular components of c-myc18. These results suggest that the parallel type of the G4 structures is the major intermolecular component of these gene promoter sequences in Kþ solution. Melting Temperature. It is known that the potassium cation can not only induce G4 structures but also stabilize G4 structures.26,39-41,50 Figure 4A-D shows the CD signals at 295 nm for HT24 and HT24-M and at 265 nm for bcl2mid and c-myc18 as a function of temperature and different [K þ], respectively. Figure 4E shows the plot of Tm of HT24, HT24-M, bcl2mid, bcl2mid-M, c-myc27, and c-myc18 as a function of ln[Kþ] with the linear regression fits. The Tm increases as the [Kþ] increases; this suggests that as the [Kþ] increases the G4 structures become more stable. The important finding is that a linear plot of Tm vs ln[Kþ] with a similar slope of ∼6.8 was obtained for these G-rich sequences. Although the Tm value of c-myc18 is higher than that of c-myc27 and bcl2mid by ∼10 °C (and much higher than that of HT24 by ∼30 °C in the same [Kþ]), similar slopes of Tm vs ln[Kþ] suggest that the Tm increases as a function of the [Kþ]

and independent of the G4 conformations. Note that a linear plot of Tm vs ln[Kþ] with a similar slope (∼7.0) was obtained from fluorescence melting data of the T(G3A)3G3T40 and TG3TG3T4G3TG3T41 as a function of Kþ. We consider that the Tm increases noted as the [Kþ] increases are mainly due to the electrostatic interaction between the cations and the DNA scaffold; this can reduce electrostatic repulsion of the DNA phosphate groups and result in a more stable structure. On the other hand, a distinctly different Tm for these G-rich sequences in the same [Kþ] is likely due to different G4 structures. For example, the Tm of the hybrid type G4 structure of HT24-M is higher than that of the antiparallel G4 structures of HT24. Further studies on the Tm of bcl2mid as a function of the DNA concentration in 5 mM Kþ solution show no appreciable difference, as shown in Figure 4F. Note that the high order components of bcl2mid are absent from the gel assays; this is shown in the inset of Figure 4F. The results suggest that the Tm of bcl2mid is independent of DNA concentration up to 200 μM but does indeed depend on the [Kþ]. Marky et al.45 found that the Tm of intramolecular complexes of HT22 and c-myc27 are independent of strand concentration. Vorlíckova et al.22 also reported similar CD results up to 40 mM HT22 using an extremely thin cell. Differential Scanning Calorimetry (DSC). Sheardy et al.42 used DSC and found two melting temperatures of 51 and 66 °C for HT24 in 150 mM Kþ solution. Here, Figure 5A shows the DSC melting curves of bcl2mid as a function of [Kþ]. The DSC shows a monophasic transition at low [Kþ] but a biphasic transition at high [Kþ]. These DSC results are consistent with the gel assays, as different structures may be verified by their distinct melting characteristics. Figure 5B shows the 265 and 295 nm CD melting curves obtained from the CD spectra of bcl2mid in 5 and 150 mM Kþ solutions as a function of temperature. The plots show similar melting curves in 5 mM Kþ solution but different melting curves in 150 mM Kþ solution. Further study of the Tm values of transition one measured from DSC melting curves shows very good agreement with those obtained from 295 nm CD melting curves, as shown in Figure 5C. These findings confirm that an intramolecular hybrid G4 structure exists in 5 mM Kþ solution, while the intramolecular hybrid G4 structure and high order intermolecular parallel G4 structures coexist in 150 mM Kþ solution. In addition, the DSC melting curves together with the gel assays suggest that the Tm of bcl2mid is higher in intermolecular than intramolecular G4 structures. 2363



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Figure 4. CD signals at 295 nm for HT24 (A) and HT24-M (B) and at 265 nm for bcl2mid (C) and c-myc18 (D) as a function of temperature and different [Kþ]. The plots of Tm of 20 μM HT24, HT24-M, bcl2mid, bcl2mid-M, c-myc27, and c-myc18 as measured from CD as a function of ln[Kþ] with linear regression fits (E). The CD melting curves at 265 nm for 20, 40, 80, and 200 μM of bcl2mid in 5 mM Kþ solution (F). The inset shows the corresponding gel assay.

Moreover, the DSC thermogram allows one to measure the enthalpy (ΔHCal) for the unfolded-folded transition. Using a two-state transition, one can calculate the entropy (ΔSCal) at melting temperature simply by using the equation ΔSCal = ΔHCal/Tm and the free energy change (ΔG°) at temperature T by the equation ΔG° = ΔHCal - TΔSCal. Marky et al.44 suggested that the simple two-state model is valid when the ΔHCal measured from DSC is equal to the ΔHvH obtained from the CD melting curve. For simplicity, we have placed our attention on bcl2mid at a low [Kþ]. The ratios of ΔHCal/ΔHvH were found to be 1.02, 0.99, and 0.92 at 20, 5, and 1 mM Kþ solutions, respectively. This suggests that the first transition of bcl2mid takes place in a two-state manner at a low [Kþ]. Table 2

lists the thermodynamic information obtained from the melting results at a low [Kþ]. Folding and Unfolding of G4 Structures. In the gel assays of bcl2mid, a single intramolecular component is detected at a low [Kþ] while additional intermolecular components are found at a higher [Kþ]. The decrease of the ∼295 nm band and the increase of the ∼265 nm band in the CD spectra of bcl2mid upon changing the [Kþ] from 5 to 150 mM allows one to monitor the changes that take place between the intra- and intermolecular components. The combination of these two methods may be useful in elucidating the possible structural conversion via unfolding and refolding between intra- and intermolecular G4 structures. 2364



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Figure 5. DSC melting thermograms of 200 μM bcl2mid as a function of [Kþ] in 150, 70, 20, 5, and 1 mM Kþ solution (A). The plots of CD intensity at 265 and 295 nm obtained simultaneously from the CD spectra of bcl2mid in 5 and 150 mM Kþ solution as a function of temperature (B). The plots of the Tm values obtained from 295 nm CD melting curves and DSC melting curves as a function of ln[Kþ] with linear regression fits (C).

Table 2. Thermodynamic Information for the Formation of G-Quadruplxes [Kþ]

Tm (CD295nm)

Tm (DSC)

ΔHvH

ΔHCAL

(mM)

(°C)

(°C)

(kcal mol-1)

(kcal mol-1)

1

50.6

50.4

-35.1

-32.2

0.98

-0.10

-1.3

-0.92

5

58.7

59.9

-33.4

-33.2

1.01

-0.10

-2.3

-0.90

20

69.7

69.5

-40.3

-41.0

1.09

-0.12

-3.9

-1.05

ΔHvH/ΔHCAL

ΔSCAL

ΔG(37°C)

ΔnKþ

(kcal K-1 mol-1)

(kcal mol-1)

(mol-1)

Transition 1

Figure 6A shows the gel assays of bcl2mid in 5 mM Kþ solution overnight and then mixed with an equal amount of the 150 mM Kþ solution for 2 h (and vice versa) at room temperature. The relative quantities of the major components of bcl2mid in each line are measured. Surprisingly, no appreciable change is found for these components. Furthermore, Figure 6B shows no discernible change in the corresponding CD spectra. These results suggest that the structural change between intraand intermolecular G4 structures is not detected after altering [Kþ] at room temperature for 2 h. In addition, the existence of the high order component of bcl2mid is unlikely due to the aggregation of two intramolecular G4 structures. Since increasing [Kþ] can reduce the electrostatic repulsion experienced by the DNA phosphate anions and thus result in more stable structure, one would expect that the Tm of the bcl2mid should change as the [Kþ] changes. Indeed, Figure 6C shows the change in the corresponding melting curves. Moreover, the increase in the Tm when mixing 150 mM Kþ solution with the original bcl2mid in 5 mM Kþ solution occurs much faster than the decrease in the Tm when mixing 5 mM Kþ solution with the original bcl2mid in 150 mM Kþ solution. This indicates that the reaction rate of Kþ-G4 association is much faster than that of Kþ-G4 dissociation. Considering the relatively high melting temperature of ∼62 °C for bcl2mid in 5 mM Kþ solution, this is likely the reason why no discernible structural conversion of bcl2mid is detected after altering the [Kþ] at room temperature. We next evaluate the thermal annealing effect after altering [Kþ]; the same stock solutions of bcl2mid in the presence of 5 mM Kþ were mixed with equal amounts of the 150 mM Kþ solution at room temperature for 2 h and then annealed at different temperatures for 1 h. After these samples had slowly cooled to room temperature and were stored in 4 °C for 24 h, Figure 6D shows the gel assays carried out at 4 °C. We found no appreciable

structural conversion at 55 °C, while some structural conversion was noticed at 65 °C, and even more at 75 °C. In addition, the corresponding CD spectra show an increase at 265 nm and a decrease at 295 nm as a function of annealing temperature, as shown in Figure 6E. It is likely that the change in [Kþ] can affect the stability of the G4 structure, but it is not easy to unfold the initial G4 structure and form different types of G4 structures below the melting temperature. On the other hand, structural change occurs when the sample is annealed above the melting temperature. This further suggests that there is an energy barrier present for structural conversion. Ionic Strength Effect. We now examine the ionic strength effect by adding the lithium cation (Liþ) to keep the 150 mM ionic strength constant. Figure 7A and B shows the gel assays of 40 μM HT24 and HT24-M and 80 μM bcl2mid, bcl2mid-M, c-myc18, and c-myc27 in a 5 mM Kþ/145 mM Liþ mixed solution and a late addition of 145 mM Liþ in 5 mM Kþ solution. We found that a late addition of Liþ shows no appreciable change in the gel assays. However, the presence of Liþ in mixed solution shows little effect for HT24, HT24-M, c-myc18, and c-myc27 but significant effect for bcl2mid and bcl2mid-M. In addition, the presence of Liþ in a mixed solution slightly increased the Tm for HT24 but significantly increased the Tm of bcl2mid, as shown in Figure 7C and D. In addition, Figure 7E (lanes 2-5) shows the gel assays of bcl2mid as a function of the Kþ and Liþ mixed solutions. Surprisingly, the monomer conformation of bcl2mid dominated in 5 mM Kþ solution (Figure 2B) was only barely detected in the mixed solution of 5 mM Kþ and 145 mM Liþ. In addition, we found that, as the [Liþ] increased in the Kþ and Liþ mixed solution, more of the dimer conformation was evident in the gel assays. Figure 7F shows the DSC results of bcl2mid in the Kþ and Liþ mixed solutions. Considering the major component of bcl2mid dimer in the gel assays and the DSC band in the melting curve in the 5 mM Kþ and 145 mM Liþ mixed solution, 2365



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Figure 6. Gel assays of 40 μM bcl2mid in 150 mM Kþ solution overnight (lane 1) and mixed with an equal amount of 5 mM Kþ solution for 2 h (lane 2) and vice versa (lanes 3 and 4) at room temperature (A) along with their corresponding CD spectra (B) and Tm measurements (C). The gel assays of 80 μM bcl2mid in 5 mM Kþ solution overnight (lane 1), mixed with an equal amount of 150 mM Kþ solution for 2 h (lane 2) and then heated up to 55, 65, and 75 °C (lanes 3-5) for 1 h (D). Their corresponding CD spectra (E).

it appears that the ∼78 °C Tm measured in both CD melting and DSC melting in the 5 mM Kþ and 145 mM Liþ mixed solution is mainly due to the dimer conformation. Similarly, the ∼99 °C Tm measured in DSC melting in a 70 mM Kþ and 80 mM Liþ mixed solution is also due to the dimer conformation. We conclude that the presence of Liþ favors the dimer formation of bcl2mid in the Kþ and Liþ mixed solutions. Since the dimer component of bcl2mid is not observed in a 150 mM Liþ solution, it is not clear why the bcl2mid can be converted significantly from the monomer in a 5 mM Kþ solution to the dimer in a 145 mM Liþ and 5 mM Kþ solution. Nevertheless, it is of interest to examine whether the initial monomer structure of bcl2mid in a 5 mM Kþ solution can be converted to the dimer with a late addition of 145 mM Liþ. Figure 7E (lanes 6-9) shows no appreciable change from the monomer to the dimer, indicating that the structural conversion induced upon late addition of Liþ is negligible. In addition, the CD spectra show no discernible difference at 295 nm with a late addition of Liþ; however, there is an appreciable decrease at 295 nm in a 145 mM Liþ and 5 mM Kþ mixed solution, as shown in Figure 7G. The CD results are consistent with the gel assays; this suggests that the possible unfolding and refolding from monomer to dimer that is induced by late addition of Liþ is negligible at room temperature. In addition, Figure 7H shows the plots of Tm for HT24 and bcl2mid as a function of ln[Kþ] in the presence of Liþ to keep the ionic strength constant at150 mM. Again, a linear plot with a slope of ∼6.4 was obtained for HT24, while a slope ∼8.0 was obtained for bcl2mid. Note that the ∼6.4 slope obtained in the presence of Liþ is slightly different from the ∼6.8 slope measured in the absence of Liþ for HT24 as a function of ln[Kþ]. However,

the ∼8.0 slope obtained in the presence of Liþ is likely due to the dimer of bcl2mid, while the ∼6.8 slope measured in the absence of Liþ is likely due to the monomer of bcl2mid. The major effect of the Liþ is to convert the bcl2mid monomer to the dimer form in mixed solutions of Liþ and Kþ. Structural Change of bcl2mid Induced by Naþ/Kþ Ion Exchange. To further examine whether Naþ/Kþ ion exchange could induce structural conversion of bcl2mid, Figure 8A shows CD spectra of 20 μM bcl2mid in a 150 mM Naþ solution with Kþ titration; each CD spectrum was recorded immediately after Kþ titration with the exception of the dashed line, which was taken after adding 90 mM Kþ and waiting 24 h. Before Kþ titration, the CD band at 295 nm was stronger than that at 265 nm in Naþ solution. Upon Kþ titration, the spectra show a slight decrease in the 295 nm CD band and an appreciable increase in the 265 nm CD band. Since the CD band at 265 nm is stronger than that at 295 nm in a 5 mM Kþ solution, as shown in Figure 3, the spectral change from the sodium form to the potassium form indeed occurs upon Kþ titration. Similar spectral changes of HT24 induced by Naþ/Kþ ion exchange were previously reported.21 In this case, if the spectral change of bcl2mid is due to the unfolding and refolding of G4 structures as the ions change, one would expect to observe the intermolecular structures upon adding 150 mM Kþ. To verify whether the intermolecular (specifically bimolecular) components of bcl2mid are formed after adding 150 mM Kþ, Figure 8B shows gel assays of 40 μM bcl2mid in 150 mM Naþ solution (lane 1), 150 mM Kþ solution (lane 2), and 150 mM Naþ solution with the addition of 150 mM Kþ cation before and after thermal annealing (lanes 3 and 4). A major intramolecular component of bcl2mid is found in the 150 mM Naþ solution. 2366



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Figure 7. Gel assays of 40 μM HT24 and HT24-M (lanes 1-2) and 80 μM bcl2mid, bcl2mid-M, c-myc18, and c-myc27 (lanes 3-6) in a 5 mM Kþ/145 mM Liþ mixed solution (A) and a late addition of 145 mM Liþ in 5 mM Kþ solution (B). The CD signals at 295 nm for HT24 (C) and 265 nm for bcl2mid (D) as a function of temperature in different mixed solutions of Kþ and Liþ under the same ionic strength ([Kþ] þ [Liþ] = 150 mM). The gel assays of bcl2mid (E) in 150/0, 70/80, 20/130, 5/145, and 1/149 mM Kþ/mM Liþ mixed solution (lanes 1-5) and those with a late addition of 80, 130, 145, and 149 mM Liþ to the bcl2mid in 70, 20, 5, and 1 mM Kþ solution (lanes 6-9), respectively. Finally, bcl2mid in 150 Liþ solution (lane 10). The DSC melting thermograms of bcl2mid (F) in 5 mM Kþ/145 mM Liþ and 70 mM Kþ/80 mM Liþ two different mixed solutions. The CD spectra of bcl2mid in different solutions (G). The plots of Tm of HT24 and bcl2mid as a function of ln[Kþ] in different mixed solutions with linear regression fits (H).

After adding 150 mM Kþ, there is no bimolecular component observed before annealing; however, the bimolecular component

appears after annealing. The appearance of the bimolecular component after annealing indicates that unfolding the intramolecular 2367



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Figure 8. CD spectra of 20 μM bcl2mid in 150 mM Naþ solution upon Kþ titration (A). The gel assays of 40 μM bcl2mid in 150 mM Naþ solution (lane 1), 150 mM Kþ solution (lane 2), and 150 mM Naþ solution overnight with addition of 150 mM Kþ cation before and after annealing (lanes 3-4) (B). The CD spectra of bcl2mid in a 150 mM Naþ solution, with the addition of 150 mM Kþ at 10 °C, heating to 25 and 95 °C, and annealing at 25 and 10 °C (C).

G4 structures of bcl2mid and then refolding the bimolecular G4 structures can be achieved by thermal annealing. However, the absence of the bimolecular component before annealing suggests that the unfolding of the G4 structures is not very likely to be induced simply by Naþ/Kþ exchange even though the spectral conversion is observed upon Kþ titration. Vorlíckova et al.22 illustrated that extensive changes in the CD pattern are not necessarily due to a change in quadruplex topology. Figure 8C shows the CD spectra of bcl2mid in a 150 mM Naþ solution, with the addition of 150 mM Kþ at 10 °C, heating to 25 and 95 °C, and annealing at 25 and 10 °C. After addition of 150 mM Kþ, spectral conversion is apparent (the decrease of the 295 nm CD band is associated with the increase of the 265 nm CD band). During heating at 95 °C, the 295 nm CD band is almost negligible, which implies that the unfolding of the antiparallel G4 structure has low melting temperature, while the presence of the 265 nm CD band indicates the existence of the parallel G4 structure, which has high melting temperature. After annealing, the significant increase of the 265 nm CD band is consistent with the appearance of the bimolecular component in the gel assays. This is attributed to the formation of bimolecular G4 structures. Accordingly, we believe that there is an energy barrier against the unfolding of the G4 structure.

’ DISCUSSION Potassium Concentration Effect. Although the studies of gel assays and CD spectra are not sufficient for determining the precise G4 structures, the gel assays allow us to differentiate between intramolecular and high order components and the CD spectra can be used to distinguish parallel and antiparallel G4 structures. The combination of these two methods clearly indicates that the [Kþ] plays a critical role in the formation of G4 structures. At a low [Kþ], intramolecular G4 structures are the major conformation of these G-rich sequences. At a high [Kþ], the intermolecular components appear in the gene promoter sequences but are not detected in human telomeric sequences. In addition, the gene promoter sequences have more intermolecular components at high [Kþ]. Although the intramolecular component of these sequences can adopt various types of G4 structures, the parallel form of the G4 structure dominates over the intermolecular G-quadruplexes. The importance of Kþ cations to the G-rich sequences is not only to induce the G-quadruplex but also to stabilize the G4 structure. The obtained negative Δn(Kþ) values (Table 2)

indicate that quadruplex formation favors an uptake of Kþ to stabilize the G4 structure.41,44 Two types of Kþ interactions are found in the G4 structure: (1) tightly residing inside the G4 channel and (2) loosely coordinating to phosphate groups. The ΔG values listed in Table 2 correspond to the formation energies of G-quadruplexes under different [Kþ] which are mainly due to the stacking of G-quartets and additional contributions such as hydrogen bonding, base-base interaction, loop interaction, and solvent effect. ΔG is the energy difference between the folded and unfolded states. The difference between the ΔG values obtained from two different [Kþ] is likely due to the additional salt effect such as loosely coordinating to phosphate groups of DNA. Furthermore, similar slopes of Tm vs ln[Kþ] demonstrate that the increase in Tm as a function of [Kþ] is quite independent of G4 conformations. Considering no discernible conformation of intermolecular structure existed in HT24 but major components of intermolecular structures existed in c-myc18, the similar ratios of Tm/ln[Kþ] suggest that the additional [Kþ] can reduce the electrostatic repulsion of the DNA phosphate groups and result in more stable G4 structure. G-Quadruplex Structures. NMR analysis showed that two types of G4 structures coexisted in HT24 and bcl2mid when in Kþ solutions, while the hybrid type of the G4 structure dominated in the modified sequences of HT24-M and bcl2mid-M in Kþ solution.18,33 Sheardy et al.42 used DSC to verify the presence of two types of G4 structures of HT24 by finding two melting temperatures of 51 and 66 °C when in a 150 mM Kþ solution. The DSC results of bcl2mid (Figure 5A) show a monophasic transition at low [Kþ] and a biphasic transition at high [Kþ]. For example, the Tm of the monophasic transition is 70 °C in 20 mM Kþ solution; it is 80 °C for the first transition and it is >95 °C for the second transition in a 150 mM Kþ solution. In addition, the gel assays revealed that the unimolecular component is the major component at low [Kþ], while a bimolecular component clearly appears at high [Kþ]. It is probable that this bimolecular component detected in bcl2mid at high [Kþ] makes NMR structural analysis of bcl2mid in Kþ solution difficult. Separating each conformation of HT24 and bcl2mid is a prerequisite for determining their individual G4 structures. The two different intramolecular conformations of HT24 cannot be distinguished in the gel assays; however, the intra- and intermolecular conformations of bcl2mid are clearly identified in the gel assays. Particularly, the gel assays revealed that the intramolecular component is the major component at a low [Kþ]. Moreover, the increase of [Kþ] can further increase the Tm of 2368


The Journal of Physical Chemistry B the initial G4 structure. Thus, it is possible to determine the intramolecular structure of bcl2mid when it is formed at a low [Kþ] using NMR. Structural Conversion. A major intramolecular component at a low [Kþ] with an additional intermolecular component at a high [Kþ] of bcl2mid can be used to verify the possible structural conversion of bcl2mid upon Kþ titration. The addition of Kþ indeed stabilizes the G4 structure as revealed by an increase of Tm. However, the CD spectra and the gel assays of bcl2mid show no appreciable change before and after the change of [Kþ] at room temperature; this implies that the structural conversion between intra- and intermolecular structures does not occur within 2 h. It is likely that the initial [Kþ] plays a critical role in determining the structural topology and the change of [Kþ] can disrupt the stability of the G4 structures at room temperature. However, the structural conversion upon addition of Kþ occurs through thermal heating above the melting temperature. It is rational to suggest that heating a compound above its melting temperature will cause it to unfold its intramolecular G4 structure. Thus, the structural conversion of bcl2mid with changing [Kþ] is unlikely to occur below the melting temperature. To further study the specific effect of [Kþ] under the same ionic strength, we have added the proper amount of neutral monovalent Liþ to keep the ionic strength constant at 150 mM. Of particular interest is that the presence of Liþ can dramatically convert the bcl2mid monomer (which dominated in the 5 mM Kþ solution) into the bcl2mid dimer in a 5 mM Kþ and 145 mM Liþ mixed solution. It appears that the presence of Liþ in the mixed solution favors the formation of the bcl2mid dimer. Moreover, the Tm of the bcl2mid dimer is higher than that of the bcl2mid monomer by more than 10 °C; this implies that the dimer is more stable than the monomer. However, no appreciable change from the monomer to the dimer for bcl2mid takes place in a 5 mM Kþ solution with a late addition of 145 mM Liþ; this indicates that the initial intramolecular G4 structure of bcl2mid formed in a 5 mM Kþ solution is not easily unfolded with the late addition of Liþ at room temperature. The gel assays show a major monomer component with several very weak high order components of bcl2mid in a 150 mM Naþ solution. However, there is no discernible dimer component. If the intramolecular G4 structure of “sodium forms” can be rapidly unfolded after the addition of a high [Kþ], the unfolded bcl2mid is able to form both intra- and intermolecular structures. Thus, it is possible to examine whether the structural conversion of bcl2mid occurs from the monomer conformation in Naþ solution to the dimer conformation upon Kþ titration. Careful comparison of the CD spectra of bcl2mid at low [Kþ] (Figure 3) and the spectral change from the “sodium form” to the “potassium form” are observed upon Kþ titration (Figure 8A). Moreover, with the direct addition of 150 mM Kþ, the results show that the dimer is not detected at room temperature but appears after thermal annealing (Figure 8B). This implies that there is an energy barrier present that impedes the unfolding of an intramolecular G4 structure. We believe that the spectral changes of bcl2mid induced by Naþ/Kþ ion exchange from the “sodium form” to the “potassium form” upon Kþ titration are unlikely due to the proposed mechanism of unfolding and refolding.18-20 Instead, structural changes are perhaps occurring within a single conformational state at room temperature.21-23 In summary, a major intramolecular component of bcl2mid is present at a low [Kþ] and an additional intermolecular

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component is present at a high [Kþ]; this is revealed by the DSC results and gel assays which provide a good model system for determining the spectral conversion of bcl2mid from the “sodium form” to the “potassium form” upon Kþ titration. The major findings of this work are summarized as follows: (1) The importance of [Kþ] is not only to stabilize the G4 structure but also to determine the formation of various G4 structures. For example, as the [Kþ] increases, an increasing amount of the high-order components are detected in the gel assays of bcl2 and c-myc G-rich sequences. (2) The melting temperature of these G-rich sequences increases as a function of Kþ concentration. Similar ratios of Tm/ln[Kþ] from these G-rich sequences suggest that the additional Kþ are loosely coordinated to phosphate groups of DNA and can further stabilize the G4 structure. (3) The conversion between intra- and intermolecular topologies of bcl2mid upon altering [Kþ] is not appreciable at room temperature but discernible when heated above the melting temperature. Considering that as [Kþ] increases so does the melting temperature, it is of interest to note that the unfolding of bcl2mid is also dependent on [Kþ]. (4) The changes in melting temperature of bcl2mid upon altering [Kþ] suggest that the Kþ-quadruplex association is faster than the Kþ-quadruplex dissociation. (5) It is surprising that the presence of Liþ in the mixed solution of Kþ and Liþ favors the formation of the bcl2mid dimer. Moreover, the increase of Tm from the monomer to the dimer suggests that the dimer is more stable than the monomer. However, no structural conversion from the monomer to the dimer of bcl2mid takes place in a 5 mM Kþ solution with a late addition of 145 mM Liþ; this supports the notion that the unfolding of intramolecular G4 structures of bcl2mid is not appreciable at room temperature. (6) The spectral changes of bcl2mid from the “sodium form” to the “potassium form” indeed occur upon Kþ titration. The absence of the dimer conformation after direct addition of 150 mM Kþ at room temperature suggests that the spectral changes are unlikely due to a rapid unfolding and refolding; instead, the structural changes are likely occurring within a single conformational state. (7) Moreover, it is possible to determine the intramolecular G4 structure of bcl2mid when it is formed at a low [Kþ] using NMR. This is because the intramolecular component of bcl2mid dominates at a low [Kþ]. In addition, increased [Kþ] can further stabilize the initial G4 structure without structural conversion at room temperature.

’ AUTHOR INFORMATION Corresponding Author

*E-mail: [email protected].

’ ACKNOWLEDGMENT This work was supported by Academia Sinica (AS-98-TPA04) and the National Science Council of the Republic of China (Grant NSC-98-2113-M001-025). We thank Dr. Shu-Chuan Jao (Biophysics Core Facility in Institute of Biological Chemistry, Academia Sinica) for kindly providing the DSC measurements. 2369



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