Insights into Competition between K+ and Pb2+ Binding to G

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B: Biophysics; Physical Chemistry of Biological Systems and Biomolecules +

2+

Insights into Competition between K and Pb Binding to Gquadruplex and Discovery of a Novel K-Pb -quadruplex Intermediate +

2+

Ze Yu, Wei Zhou, Ge Ma, Yunchao Li, Louzhen Fan, Xiaohong Li, and Yi Lu J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.8b08161 • Publication Date (Web): 14 Sep 2018 Downloaded from http://pubs.acs.org on September 14, 2018

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The Journal of Physical Chemistry

Insights into Competition between K+ and Pb2+ Binding to G-quadruplex and Discovery of a Novel K+-Pb2+-quadruplex Intermediate Ze Yu,a Wei Zhou, a Ge Ma,a Yunchao Li,a Louzhen Fan,a Xiaohong Li,a,* and Yi Lub,* a

College of Chemistry, Beijing Normal University, Beijing, 100875, P.R.China

b

Department of Chemistry, Department of Materials Science and Engineering, University of

Illinois at Urbana and Champaign, Urbana, IL 61801, USA.

ACS Paragon Plus Environment

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ABSTRACT.

Numerous studies have reported cation-dependent stability and topological changes of Gquadruplexes (G4s), but competitions between cations at different concentrations for binding with G4s and their effects on polymorphism of G4 have been rarely investigated, which greatly limited the application of G4. Herein, with PW17 (a G4 forming DNA sequence) as a model system, the competition between K+ and Pb2+ binding to G4 was clarified. Using a combination of circular dichroism and electrospray ionization mass spectroscopy, it was found that 10 μM Pb2+ could replace K+ in K+-stabilized PW17 (2K+-PW17), but the substituting efficiency is highly dependent on K+ concentration (0.5 mM ~ 10 mM). In contrast, K+ at < 10 mM could partly replace Pb2+ in stable and antiparallel Pb2+-stabilized PW17 (Pb2+-PW17), and completely substitute Pb2+ at K+ ≥ 10 mM. In these competing processes, a novel intermediate consisting of a Pb2+ and a K+ in the same G4 (K+-Pb2+-PW17) was firstly discovered, which acted as a bridge to achieve the switches. Through measuring the energetics of different concentrations of K+ and Pb2+ inducing conformations, such a competitive binding was thermodynamically permitted.


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INTRODUCTION DNA could adopt a diverse range of structural conformations including duplexes, triplexes and quadruplexes (G4).1 Of particular interests is G4, which was found in cells from bacteria to human, and involved in cellular regulation.2 At the same time, G4-forming sequences had also been discovered through in vitro selection of DNA aptamers as unique structural elements in therapeutics,3 nanotechnology4 and conductive devices.5 G4-forming sequences could fold into multiple G4 conformations, such as parallel, antiparallel, or hybrid, and the exact conformation was influenced by DNA sequence, strand concentration and the presence of cations.6 Since the G4 conformations played a major role in determining their functions, a detailed understanding of factors and mechanisms in influencing G4 conformational transformation and stability was important for their applications. Among the factors that influenced the G4 conformation and stability, cations played a prominent role.7-8 While numerous studies had reported cation-dependent stability and topological changes of G4s, most of them focused on the effect of cation,9-16 but rarely on the effect of competition of metal ions under different concentrations.17-20 Actually, biological and most of environmental systems contained multiple cations and each of the cations had very different concentrations. Thus, investigation of the competition between different cations (at different concentrations) binding to G4s and their effects on polymorphism of G4 was critical, which could promote understanding the structures of G4s and further exploring their applications in biological and environmental systems. In order to deeply explore the issue, our attention focused on K+-stabilized G4 (K+-G4) and Pb2+-stabilized G4 (Pb2+-G4), two major cations (K+ and Pb2+) that were known to stabilize G4.7, 14, 21-22

Generally, it was believed that for a G4 forming DNA sequence, Pb2+-G4 was more stable


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than K+-G4 and Pb2+ could substitute K+ in K+-G4, regardless of concentrations of K+.9-11, 14, 21-30 On the other hand, though G4-based sensors had been reported for detecting Pb2+ 25-26, 30-44 or K+,4549

and the developed Pb2+ sensors exhibited a really high selectivity over K+. Moreover, the

stability of K+-G4 could gradually enhance with increasing K+ concentration.8, 49-50 Under this situation, particular issues of how K+ concentrations affected Pb2+-G4 stability and how K+ and Pb2+ could compete to bind with G4 were really elusive. Herein, with PW17 (a G4 forming DNA sequence) as a model system,21, 51 the competition between K+ and Pb2+ binding to G4 was clarified through circular dichroism and electrospray ionization mass spectrometry. It was found that (1) 10 μM Pb2+ could induce 4 µM PW17 transforming into stable and parallel Pb2+-stabilized PW17 (Pb2+-PW17); (2) 10 μM Pb2+ could completely and partially replace K+ (ranging from 0.5 mM to 10 mM) in K+-stabilized PW17 (2K+PW17) and transform into antiparallel Pb2+-PW17, corresponding to the gradually decreased substituting efficiency. In contrast, K+ at < 10 mM could partly substitute Pb2+ in stable Pb2+-PW17 and totally substitute Pb2+ at K+ ≥ 10 mM with antiparallel Pb2+-PW17 transforming into parallel 2K+-PW17. In the competing process, a novel intermediate consisting of K+ and Pb2+ in the same G4 (K+-Pb2+-PW17) was firstly discovered, which acted as a bridge to achieve the switches. The results were supported by measurement of the energetics of different concentrations of K+- and Pb2+-induced conformations. This study provided a novel insight into further investigating the competitions between two major cations that stabilize G4 and their effects on polymorphism of G4 structures.


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EXPERIMENTAL SECTION Materials. The purified oligonucleotide was obtained from Sangon Biotech (Shanghai) Co., Ltd. The applied DNA sequence was: PW17: 5’-GGGTAGGGCGGGTTGGG-3’. Tris (Tris(hydroxymethyl)aminomethane), TMAA (trimethylammonium acetate), HAc, KClO4, KCl, Pb(ClO4)2 ·3H2O (ACS reagent for above) and methanol (HPLC) were purchased from SigmaAldrich (St. Louis, MO) and used without further purification. The stock solution of 100 μM DNA was prepared in 10 mM Tris-HAc buffer (pH 8.0). The DNA solution was heated at 90 °C for 10 min to dissociate any intermolecular interaction, and gradually cooled to room temperature before use. Deionized water (18.2 MΩ cm resistivity) from a Millipore Milli-Q system was used throughout this work. Circular Dichroism. Circular Dichroism (CD) spectra of 4 μM PW17 in the 10 mM Tris-HAc buffer (pH 8.0) in the presence of different concentrations of K+ and Pb2+ were collected by a Chirascan CD Spectrometer (Applied Photophysics Ltd.) at room temperature. Different ions were added at 2 h intervals. The optical chamber (1 mm path length) was deoxygenated with dry purified nitrogen (99.99%) before use and the nitrogen atmosphere was kept during experiments. Two scans from 220 to 340 nm at 0.5 nm intervals and 60 nm/min were accumulated and averaged. The background of the buffer (10 mM Tris-HAc buffer, pH 8.0) solution was subtracted from the CD data. The thermal stabilities of PW17 in the presence of 10 μM Pb2+ or different concentrations of K+ were characterized in heating experiments by recording the CD at 310 nm (Pb 2+) or 265 nm (K+) as a function of temperature from 20 to 90 °C, by using a Chirascan CD Spectrometer (Applied Photophysics Ltd.). The heating rates were 1 °C per minute. And a sensitive temperature probe to record instant temperature in all tests. Experiments were performed with 1 mm path length quartz cuvettes.


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Electrospray Ionization Mass Spectroscopy (ESI-MS). All ESI mass spectra were obtained with a Thermo Scientific™ LCQ Fleet™ ion trap mass spectrometry (San Jose, CA). The electrospray source conditions were 2.5 kV spray voltage, 150 °C capillary temperature and in negative mode. Samples were prepared by mixing oligonucleotides at a 4 μM strand concentration with different ions and TMAA (100 mM).52 Methanol (20%) was added to acquire a good spray before the MS analysis.53 HPLC was used as an automated injector and flow rate is 2.50 μL/min. The obtained results were analyzed by ProMass software. The time-dependent MS was directly carried out on a Thermo Scientific™ LCQ Fleet™ ion trap mass spectrometry (San Jose, CA). One MS spectrum was accumulated every 12 s. ProMass software was used for analysis as well. RESULTS AND DISCUSSION Circular dichroism spectroscopic and mass spectrometric studies of Pb2+-stabilized PW17 It has been reported that Pb2+ could induce G-rich DNA sequences into Pb2+-stabilized G4 (called Pb2+-G4 hereafter) 21-23, 26, 42, 54-56, but the detailed transforming process was not explored. Herein, with PW17 (a G4 forming DNA sequence) as a model system,21, 51 circular dichroism (CD) spectroscopy was applied to monitor conformation changes of 4 μM PW17 in the presence of Pb2+. As shown in Figure 1a, PW17 exhibited a positive peak at 257 nm, a negative peak at 240 nm, and a shoulder peak at 295 nm.21 Upon adding Pb2+ from 0 to 20 μM, these characteristic peaks gradually disappeared. Meanwhile, a positive peak at 310 nm and a negative peak at 265 nm, typical of antiparallel G4,11, 21-22 were concurrently observed, and finally saturated at 10 μM Pb2+. These results indicate that PW17 gradually transforms into antiparallel G4 with increasing Pb2+ concentration22, 32, 57. To corroborate this conclusion, the transforming process was tracked with electrospray ionization mass spectroscopy (ESI-MS). As shown in Figure 1b, a peak corresponding


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to calculated molecular weight (MW) of PW17 was initially observed at m/z = 5404. Upon gradually adding Pb2+ from 2 μM to 20 μM, a new peak located at m/z = 5609 was detected and its peak height enhanced with increasing concentrations of Pb2+. At the same time, the peak at m/z = 5404 concurrently decreased and finally completely disappeared at 8 μM Pb2+. The MW difference of 205 (m/z = 5609 - 5404) corresponded to a single Pb2+, suggesting the presence of a monomeric Pb2+-stabilized PW17 (called Pb2+-PW17 hereafter). These results indicated that 10 μM Pb2+ was enough to induce 4 μM PW17 transforming into stable and antiparallel Pb2+-PW17.

Figure 1. (a) CD spectra of PW17 in the presence of Pb2+ from 0 (black), 1, 2, 3, 4, 6, 8, 10 (red) to 20 (blue) μM in Tris-HAc buffer (10 mM, pH = 8.0). (b) ESI-MS of PW17 in the presence of Pb2+ from 0 to 20 μM in Tris-HAc buffer (10 mM, pH = 8.0).

Pb2+ competing with K+ in K+-stabilized PW17 As reported previously, Pb2+-G4 was structurally more compact than K+-stabilized G4 (K+-G4), and Pb2+ could displace K+ in K+-G4 14. Based on these observations, both biosensors for Pb2+ and related logic gates have been developed, which all displayed a high selectivity over K+, in which K+ concentration ranging from 10 µM to 10 mM was used, respectively.10, 14, 21, 24-30, 35, 55. Actually,


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the stability of K+-G4 is strictly dependent on K+ concentration (0.1 mM ~ 10 mM).43 To offer deeper insight into Pb2+ competing with K+ in K+-G4 and further binding with G4, we collected the CD and ESI-MS spectra of K+-stabilized PW17 (2K+-PW17), Pb2+-PW17 and the addition of 10 μM Pb2+ to 2K+-PW17 (Figure 2).

Figure 2. CD spectra and corresponding ESI-MS spectra of PW17 in the presence of 0.5 mM ((a) and (b)); 2 mM ((c) and (d)); and 10 mM K+ ((e) and (f)) (Curve 1), in the presence of 10 μM Pb2+ (Curve 2), and adding 10 μM Pb2+ to K+-stabilized PW17 shown as Curve 1 (Curve 3).


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In the presence of 0.5 mM K+, K+ could induce 4 μM PW17 transforming into unstable K+PW17, displaying a negative peak at 245 nm and a positive peak at 265 nm (Figure 2a, Curve 1).49 Upon adding 10 μM Pb2+, these characteristic peaks disappeared and were replaced with a new CD spectrum with a negative peak at 264 nm and a positive peak at 310 nm (Figure 2a, Curve 3), which is almost identical to that of 4 μM PW17 in the presence of 10 μM Pb2+ (Figure 2a, Curve 2). These results were further corroborated by results from ESI-MS (Figure 2b): (1) in the presence of 0.5 mM K+, a strong mass peak at m/z = 5404 (PW17) and a weak one at m/z = 78 (2K) + 5404 (2K+-PW17) were observed, indicating that the formed 2K+-PW17 was unstable (Curve 1)

49, 58

;

(2) in the presence of 10 μM Pb2+, only one mass peak at m/z = 5609 was detected, corresponding to stable Pb2+-PW17 (Curve 2); (3) upon addition of 10 μM Pb2+ to the system containing 0.5 mM K+ and 4 μM PW17, the mass peaks corresponding to PW17 and 2K+-PW17 disappeared, and a new peak of Pb2+-PW17 was detected (Curve 3). These results show that 10 μM Pb2+ could completely replace K+ in unstable 2K+-PW17, and further transformed into stable and antiparallel Pb2+-PW17. Alternatively, in the presence of 2 mM K+, the intensity of positive peak at 265 nm and that of negative peak at 245 nm increased (Figure 2c, Curve 1), suggesting that the formed 2K+-PW17 was relatively stable as discussed before 49. When 10 μM Pb2+ was added, the two peaks decreased and a positive peak at 310 nm appeared (Figure 2c, Curve 3), which was different from those in the presence of 10 μM Pb2+ (Figure 2c, Curve 2) or of 2 mM K+. Meanwhile, from the ESI-MS spectra (Figure 2d), the characteristic mass peaks corresponding to 2K+-PW17 (Curve 1) and Pb2+PW17 (Curve 2) are concurrently detected (Curve 3). Interestingly, a low-abundant species was found. We assign this species as a K+-Pb2+-PW17 intermediate, which will be discussed later in


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this manuscript. These results show that 10 μM Pb2+ could partly replace K+ in relatively stable 2K+-PW17 and further transform into antiparallel Pb2+-PW17. However, when K+ was increased to 10 mM (Figure 2e, Curve 1), the intensity of positive peak at 265 nm and that of negative peak at 245 nm further increased, indicating that parallel 2K+-PW17 was more stable49. Upon adding 10 μM Pb2+, CD spectroscopy (Curve 3) is totally different from that of 10 μM Pb2+ (Curve 2), but almost identical to that of 10 mM K+ (Curve 1). The results from ESI-MS spectra (Figure 2f) are consistent with those from CD spectroscopic study, which showed that 10 μM Pb2+ could not replace K+ in stable and parallel 2K+-PW17. Based on the discussion above, upon increasing K+ concentration from 0.5 mM to 10 mM, the efficiency of Pb2+ replacing K+ in 2K+-PW17 gradually decreased until such a substitution was not observed at 10 mM K+. That was to say that Pb2+ substituting K+ in 2K+-PW17 was highly K+ concentration-dependent, which broadened the understanding of Pb2+ substituting K+ in K-G4. It could be expected that 0 ~ 10 μM Pb2+ cannot substitute K+ in stable 2K+-PW17.

K+ competing with Pb2+ in Pb2+-stabilized PW17 and discovery of a K+-Pb2+-PW17 intermediate Having found that Pb2+ could completely replace K+ in 2K+-PW17 at K+ < 1 mM, but not at K+ ≥ 10 mM, we wonder if K+ can replace Pb2+ in stable Pb2+-PW17, and Pb2+-PW17 can further transform into 2K+-PW17, and also if such a competing process depends on K+ concentration. As shown in Figure 1, 10 μM Pb2+ was enough to induce 4 μM PW17 transforming into stable Pb2+PW17. Upon further adding K+ ranging from 1 mM to 40 mM, CD spectroscopy was applied to


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track the conformational change caused by K+ as shown in Figure 3: the ellipticity at 310 nm and 245 nm (characteristic of antiparallel Pb2+-PW17) decreased, with concomitant increase of the ellipticity at 265 nm (characteristic of parallel G-quadruplex)21, 49. The final CD spectrum was similar to that of directly adding 10 mM K+ to 4 μM PW17 (Figure 1a, red curve).49 These results demonstrate that the conformational switch from antiparallel Pb2+-PW17 to parallel G4 is accomplished. According to the literatures,9, 11, 14, 22 such a conformational switch from Pb2+-G4 to K+-G4 upon addition of K+ was not explored. Expanding these literature precedence, our results suggest that it is possible for K+ to replace Pb2+ in Pb2+-PW17 and cause conformational switch from antiparallel to parallel G4.

Figure 3. (a) CD spectra of 4 µM PW17 in the presence of 10 μM Pb2+ with adding different K+ concentrations: 0 (black), 1, 2, 3, 5, 10, 20 and 40 mM (blue), and in the presence of only 10 mM K+ (red) in Tris-HAc buffer (10 mM, pH = 8.0). (b) CD intensity at 265 nm, 310 nm and 245 nm as a function of K+ concentration.

To further testify K+ replacing Pb2+ in Pb2+-PW17 and the conformational changes described above, ESI-MS was utilized to investigate the cation embedded in G4 upon adding K+ to the system containing 10 µM Pb2+ and 4 µM PW17. As shown in Figure 4a, in the presence of 10 μM Pb2+


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and 4 μM PW17, a MS peak located at m/z = 5609 was observed, which corresponded to stable Pb2+-PW17. On the other hand, in the presence of 10 mM or 20 mM K+, 4 μM PW17 displayed only one peak at m/z = 5482, which corresponded to 2K+-PW17. It means that 20 mM, even 10 mM K+ could induce 4 μM PW17 transforming into stable 2K+-PW17.49 When 1 mM K+ was added to the system containing 10 μM Pb2+ and 4 μM PW17, a new MS peak located at m/z = 5482 was detected firstly, indicating the formation of 2K+-PW17.49 Upon further increasing K+ ranged from 2 mM to 10 mM, the peak intensity at m/z = 5482 increased, while the peak intensity at m/z = 5609 concurrently decreased and totally disappeared at 20 mM K+. These results suggested that: (1) K+ could compete with Pb2+ in Pb2+-PW17 and further bind with PW17, (2) as a competing result, when K+ ≥ 10 mM, Pb2+ in Pb2+-PW17 was expelled out and Pb2+-PW17 transformed into 2K+-PW17, and (3) the generated switch from Pb2+-PW17 to 2K+-PW17 strictly depended on K+ concentration (1 ~ 10 mM). More importantly, when the concentration of K+ increased from 1 mM to 9 mM, a small peak at m/z = 5648 (39(K) + 205(Pb) + 5404), consistent with MW of a K+ and a Pb2+ co-existing in a PW17 (K+-Pb2+-PW17), was detected and finally disappeared at 10 mM K+. In the competing process, the detection of this K+-Pb2+-PW17 intermediate is exciting, because this intermediate possibly act as a bridge to achieve the switch from Pb2+-PW17 to 2K+-PW17. In order to verify K+-Pb2+-PW17 intermediate as a switching bridge, we collected time-resolved ESI-MS spectra of the system containing 10 µM Pb2+ and 4 µM PW17 with the addition of 1 mM K+ (Figure 4b). Upon adding 1 mM K+, the peak corresponding to MW of K+-Pb2+-PW17 appeared after 0.4 min, but no peak corresponding to 2K+-PW17 was observed. After 0.6 min (only 12 s interval), the peak of 2K+-PW17 was then detected, with a slight decrease of peak intensity of Pb2+-PW17. From 1 min to 10 min, the mass spectral peaks corresponding to 2K+-PW17, Pb2+-PW17 and K+-Pb2+-


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PW17 remained unchanged. Together with results from Figure 4a, it was concluded that K+-Pb2+PW17 was indeed a competing intermediate, bridging the transformation from antiparallel Pb 2+PW17 into parallel 2K+-PW17. Based on the results and the fact that the presence of K+ atmosphere was beneficial to the transformation of antiparallel Pb2+-PW17,50, 59 it was proposed as shown in Figure 4c: (1) a K+ close to surface of Pb2+-PW17 inserted into the vacancy of Pb2+-PW17, and further transformed into K+-Pb2+-PW17 intermediate; (2) the second K+ was further close to Pb2+ in K+-Pb2+-PW17 and inserted with Pb2+ being expelled out, promoting the formation of parallel 2K+-PW17. Additionally, it was also noticed that the intensity of K+-Pb2+-PW17 intermediate was fixed at a same level when the concentration of K+ ranged from 1 mM to 9 mM (Figure 4a). In our opinion, there might be an equilibrium between Pb2+-PW17 and 2K+-PW17 via K+-Pb2+-PW17 intermediate, exhibiting that higher K+ concentration (such as 5 mM or 9 mM) was used, more 2K+-PW17 was formed, and lower K+ concentration (such as 1 mM) was applied, less 2K+-PW17 was formed. The results expanded understanding of the competition between K+ and Pb2+ binding with G4.


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Figure 4. (a) ESI-MS of PW17 in the presence of 10 μM Pb2+ with the subsequent addition of 1, 2, 5, 9, 10 and 20 mM K+, and in the presence of only 10 mM K+ (from top to bottom). (b) Time resolved MS spectra of Pb2+-PW17 (10 μM Pb2+) upon adding 1 mM K+. (c) Representative image of K+ substituting Pb2+ in Pb2+-PW17. To provide deeper insight into this interestingly competing process, the thermodynamic parameters were measured by obtaining the temperature-dependent CD spectral changes in the presence of 4 µM PW17 and 10 μM Pb2+ or different concentrations of K+ (Figure 5). In the presence of 10 μM Pb2+, the ellipticity at 310 nm as a function of temperature was recorded. Based on the melting curve, a melting temperature(Tm) of about 60 °C was calculated.58 Similarly, in the presence of 1 mM, 10 mM, 20 mM and 30 mM K+, based on temperature-dependent changes of the ellipticity at 265 nm, the Tm of 28 °C, 61°C and 68 °C were obtained, respectively. When K+ was increased to 30 mM, the Tm reached to 72 °C. From these Tm values, the corresponding thermodynamic data were calculated through van’t Hoff analysis (Table 1).58 The ΔGo (298K) of PW17 transforming into Pb2+-PW17 (10 μM Pb2+) was ̶ 13.4 kJ/mol, which was much lower than ΔGo = ̶ 3.0 kJ/mol (2K+-PW17, 1 mM K+), higher than ΔGo = ̶ 15.9 kJ/mol (2K+-PW17, 10 mM K+), and even much higher than ΔGo = ̶ 21.0 kJ/mol (2K+-PW17, 20 mM K+) and ΔGo = ̶ 24.4 kJ/mol (2K+-PW17, 30 mM K+). Integrated with the results of Figure 2 and Figure 4, these stability differences provided thermodynamic reason that 10 μM Pb2+ could totally replace K+ in 2K+-PW17 (K+ at < 1 mM), and K+ at ≥ 10 mM could completely substitute Pb2+ in stable Pb2+-PW17.


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1.0 0.8

Fraction

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


0.6

b

a c d e

0.4 0.2 0.0 20

30

40

50

60

70

80

Temperature (OC)

Figure 5. Melting curves of PW17 in the presence of 10 μM Pb2+ (curve a), 1 mM K+ (curve b), 10 mM K+ (curve c), 20 mM K+ (curve d) and 30 mM K+ (curve e). Table 1. Thermodynamic data for PW17 incubated with K+ and Pb2+ Tm

ΔH

ΔS

ΔGo (298K)

(°C)

(kJ·mol-1)

(kJ·mol-1·K-1)

(kJ·mol-1)

10 μM Pb2+

60

-125.1

-0.375

-13.4

1 mM K+

28

-90.5

-0.301

-3.0

10 mM K+

61

-148.8

-0.443

-15.9

20 mM K+

68

-167.3

-0.491

-21.0

30 mM K+

72

-178.2

-0.516

-24.4

2+

+

C (Pb /K )

CONCLUSIONS In summary, with PW17 (a G4 forming DNA sequence) as a model system, Pb2+ and K+ competitively binding with PW17 was investigated. While 10 μM Pb2+ could displace K+ in 2K+PW17, the substituting efficiency was gradually decreased with increasing K+ from 0.5 mM to 10


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mM. In contrast, K+ (1 mM ~ 10 mM) could partly substitute Pb2+ in stable Pb2+-PW17 and totally substitute Pb2+ at K+ ≥ 10 mM with antiparallel Pb2+-PW17 transforming into parallel 2K+-PW17, which is totally different from the previous reports that Pb2+-G4 is more stable than K+-G4 and the presence of K+ cannot alter the stability of Pb2+-G4. Importantly, a K+-Pb2+-PW17 intermediate was firstly discovered in K+ concentration ranging from 1 mM to 9 mM, which acted as a bridge to achieve these switches between Pb2+-PW17 and 2K+-PW17. Based on the results, the switching process from antiparallel Pb2+-PW17 to parallel 2K+-PW17 was proposed. Moreover, Pb2+ and K+ competitively binding with PW17 was thermodynamically permitted. From the study, we provided a novel insight into further investigating the competitions between two major cations that stabilize G4 and their effects on polymorphism of G4 structures, and also a new clue for further exploring the potential function and the practical applications of G4 in biochemical and environmental fields.

AUTHOR INFORMATION Corresponding Author E-mail: [email protected] (Prof. X. Li) [email protected] (Prof. Y Lu) Notes The authors declare no competing financial interests. ACKNOWLEDGMENT


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This work was supported by the National Science Foundation of China (21673022, 21573024), the Major Research Plan of National Natural Science Foundation of China (21233003) and the Fundamental Research Funds for the Central Universities. REFERENCES 1.

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Graphic for Manuscript

Competition between K+ and Pb2+ binding to PW17 and accompanied conformation switches are clarified, which is strictly K+ concentration dependent and goes through a novel K+-Pb2+-quadruplex intermediate.


Insights into Competition between K+ and Pb2+ Binding to G

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