Investigation of Na+ and K+ Competitively Binding with a G

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Article Cite This: J. Phys. Chem. B 2019, 123, 5405−5411

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Investigation of Na+ and K+ Competitively Binding with a G‑Quadruplex and Discovery of a Stable K+−Na+-Quadruplex Ge Ma, Ze Yu, Wei Zhou, Yunchao Li, Louzhen Fan, and Xiaohong Li* Key Laboratory of Theoretical and Computational Photochemistry, Ministry of Education, College of Chemistry, Beijing Normal University, Beijing 100875, China

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ABSTRACT: DNA G-quadruplex (G4) could adopt multiple conformations, and the exact conformation is related to the presence of cations. However, the fact that cations with various concentrations could competitively bind with G4 is rarely investigated, which greatly limits the potential applications of G4based sensors. Here, with PW17 (a G4-forming DNA sequence) as an example, Na+ and K+ with different concentrations competitively binding with PW17 are clarified by circular dichroism spectroscopy and electrospray ionization mass spectroscopy. Although Na+ could induce PW17 switching to unstable and antiparallel Na+-stabilized PW17 (2Na+-PW17) (CNa+ = 5−70 mM) and further to stable and hybrid 2Na+-PW17 (CNa+ = 70−800 mM), K+ (CK+ = 0.1−10 mM) could replace Na+ in 2Na+-PW17 with 2Na+-PW17 transforming into K+-stabilized PW17 (2K+-PW17). Moreover, the replacing ability strictly relied on CK+ and CNa+. In the switching process, a stable intermediate including a K+ and an Na+ in one G4 (K+− Na+-PW17) is firstly detected. Importantly, the stable K+−Na+-PW17 is detected at 0.5 mM K+ and 140 mM Na+. Based on the facts, the interferences of Na+ with the performance of PW17-based K+ sensors are investigated. With the stable K+−Na+-PW17 as a sensing probe and protoporphyrin IX (PPIX) as a G4 fluorescent read-out probe, a linear relationship between CK+ (500 nM−10 mM) and PPIX fluorescence is observed, which provides a fluorescence assay for detecting K+ with the co-existing 140 mM Na+. This study exhibits clear evidence of Na+ and K+ competitively binding with G4 and finds a novel and stable K+−Na+PW17, which provides a clue to further explore G4 functions in Na+-contained samples.

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enzymes,37,38 and small molecules.39,40 According to the literature, many studies focus on the structure of G4s induced by a single cation, such as Na+, Pb2+, K+, etc.41,42 However, in most cases, these cations are co-existing. For example, Na+ and K+ are abundantly present in physiological systems.21 Thus, Na+ and K+ could competitively bind with G4. Generally, it was believed that K+ can substitute Na+ in Na+-stabilized G4 (Na+-G4) with Na+-G4 transforming into K+-stabilized G4 (K+-G4).21,43−48 Yang et al.27 found that the stability of K+-G4 was better than Na+-G4, even in case of containing 100 mM Na+. Braunlin et al.30 reported competitive binding experiments and confirmed that the binding affinity of K+ was stronger than that of Na+. Hud et al. reported that the selectivity for K+ versus Na+ in G4 was determined by relative free energies of hydration,44 which was theoretically verified by Gu et al.45 and Guerra et al.49 Such a substitution was experimentally and theoretically explored, and was believed: Na+ in Na+-G4 could be substituted by K+, followed by a structural rearrangement to produce the G-triplex intermediate (a partly folded structure with three tandem guanine repeats bound together by Hoogsteen hydrogens50) and further

INTRODUCTION DNA could exist in different conformations, such as duplexes, triplexes, quadruplexes (G4s), and i-motifs.1−5 Among them, G4s are not only detected in cells but also found in regulating cellular functions.6,7 G4-forming DNA sequences (GDSs, such as DNA aptamers) have been screened by the systematic evolution of ligands by the exponential enrichment (SELEX) approach.8−10 In addition, GDSs also include some G-rich DNA sequences due to the ability to transform into G4. As unique structural elements, the selected GDSs have been applied in therapeutics,11 nanotechnology,12 and conductive devices.13−15 It has been reported that G4 could adopt parallel, antiparallel, or hybrid G4 conformations,16,17 and the concrete conformation is related to the G-rich DNA sequence and its concentration, even the co-existing cations.18 Actually, the cation concentration also seriously affects the G4 conformation.19−22 However, cation-concentration-dependent G4 multiple conformations are rarely investigated.23 Thus, a detailed understanding of cation-concentration-dependent G4 structural transformation is really important for further exploring their applications in biochemical and biomedical fields. Metal cations (such as Pb2+,24−26 K+,27−29 Na+,19,30 and 2+21,31 ) are widely applied to induce GDSs transforming into Sr G4s. Based on the facts, G4s have been used to construct various sensing platforms for metal ions,32−34 nucleic acids,35,36 © 2019 American Chemical Society

Received: March 26, 2019 Revised: May 30, 2019 Published: June 4, 2019 5405

DOI: 10.1021/acs.jpcb.9b02823 J. Phys. Chem. B 2019, 123, 5405−5411

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The Journal of Physical Chemistry B transform into K+-G4.21,51,52 Recently, Tang’s group proposed that K+-G4 and Na+-G4 were co-existing, and the relative amounts of Na+-G4 and K+-G4 are dependent on the Na+/K+ ratio.46 However, Na+/K+ concentration-dependent substituting process is really elusive.48 Clarifying this issue is beneficial to fully understand the allosteric mechanism and provides a clue for further investigation of cation-concentration-dependent competitively binding with G4 and their impacts on G4based sensing platforms. In this paper, with PW17 (a GDS) as an example,42,53,54 the Na+/K+ concentration-dependent transformation of Na+stabilized PW17 (2Na+-PW17) into K+-stabilized PW17 (2K+-PW17) was investigated by circular dichroism (CD) spectroscopy and electrospray ionization mass spectrometry (ESI-MS). The results demonstrated: (1) Na+ (0−800 mM) could induce PW17 transforming into 2Na+-PW17 and undergo a structural switch from PW17 to unstable and antiparallel 2Na+-PW17 and to stable and hybrid 2Na+-PW17, which are strictly dependent on CNa+; (2) K+ could substitute Na+ in 2Na+-PW17 with 2Na+-PW17 transforming into parallel 2K+-PW17 through a K+−Na+-PW17 intermediate. In this switching process, the stable K+−Na+-PW17 was detected at 140 mM Na+ and 0.5 mM K+. According to these findings, the effect of CNa+ on the performance of PW17-based K+ sensors was investigated. With the stable K+−Na+-PW17 as a sensing probe and protoporphyrin IX (PPIX) as a G4 fluorescent readout probe, K+ detection with the co-existing 140 mM Na+ could be achieved with CK+ ranging from 500 nM to 10 mM. This study demonstrates a clear evidence of Na+ and K+ competitively binding with G4 and their effects on G4-based K+ sensors, which provides enlightenment for the design of G4-based sensing platforms in live organisms.

Tris−HAc buffer (pH 7.4), fully mixed, and incubated for 2 h. Subsequently, K+ (0.01−10 mM) was added and incubated for another 2 h, respectively. Before MS analysis, TMAA (50 mM) was further added.55 Negative ion ESI mass spectra of the samples were recorded by a Thermo Scientific LCQ Fleet ion trap mass spectrometry (San Jose, CA/Sangon Biotech Co. Beijing).56 The obtained MS spectra were analyzed by ProMass software (an automated biomolecular deconvolution software),57,58 and the deconvolution result was singly charged in positive mode. Fluorescence Spectroscopy. A fluorescence spectrometer (Model FS5, Edinburgh Instruments, U.K.) was used to detect the fluorescence emission spectra of PPIX. Spectra were collected from 580 to 665 nm, with an excitation wavelength of 410 nm. A quartz cuvette with a 3 mm × 10 mm path length was applied for obtaining the spectra at 3 nm excitation and emission slit widths. For this, 1 μM PW17 was incubated with Na+ (0, 20, 140, and 800 mM) for 2 h and K+ (10 nM−10 mM) for another 2 h, respectively. Finally, 1 μM PPIX was added and kept for 40 min in the dark. The emission fluorescence intensity of PPIX reached a maximum at λmax = 632 nm, which was used by linear regression analysis. Error bars are the standard deviations from three measurements.

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RESULTS AND DISCUSSION Formation of Na+-Stabilized PW17. Although Na+ could induce G-rich DNA sequences (GDSs) transforming into a Gquadruplex (G4), the detailed process is not clear.30,59,60 Herein, with PW17 (a GDS) as a model system,42,53,54 the detailed transforming process is explored. First, in the presence of Na+, the conformational switch of 4 μM PW17 is tracked with circular dichroism (CD) spectroscopy (Figure 1). Figure

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EXPERIMENTAL SECTION Materials. Purified PW17 was synthesized and provided by Sangon Biotech Co. (Shanghai, China), and the base sequence was 5′-GGGTAGGGCGGGTTGGG-3′. KAc, NaAc, HAc, Tris (Tris-(hydroxymethyl)aminomethane), protoporphyrin IX (PPIX), dimethyl sulfoxide (DMSO), and trimethylammonium acetate (TMAA) were purchased from Sigma-Aldrich (St. Louis, MO) and used without purification. Deionized water (18.2 MΩ cm resistivity) from a Millipore Milli-Q system was used, and 100 μM DNA stock solutions were prepared by dissolving PW17 in 10 mM Tris−HAc buffer (pH 7.4), annealed (first at 90 °C for 10 min and slowly cooled to room temperature), and diluted before use. KAc and NaAc with different concentrations were prepared with 10 mM Tris− HAc buffer; 1 M TMAA was prepared using ultrapure water, and 200 μM PPIX was dissolved in DMSO and stored in the dark at −20 °C. Circular Dichroism Spectroscopy. Circular Dichroism (CD) spectra were collected by a Chirascan CD Spectrometer (Applied Photophysics Ltd.) at room temperature from 225 to 340 nm, two scans at 60 nm·min−1 with 0.5 nm intervals. The optical chamber (3 mm × 10 mm path length) was deoxygenated with nitrogen (99.99%) before use, and a nitrogen atmosphere was maintained. In 10 mM Tris−HAc solutions, the samples contained 4 μM PW17 and different Na+ concentrations and various K+ concentrations at 2 h intervals. The background of the buffer solution was subtracted from CD data. Electrospray Ionization Mass Spectroscopy (ESI-MS). Na+ (10−800 mM) was added to 4 μM PW17 in 10 mM

Figure 1. CD spectra of 4 μM PW17 in the presence of Na+ in 10 mM Tris−HAc (pH = 7.4): (a) 0 mM (black line), 10 mM, 20 mM, 40 mM, 50 mM, and 70 mM (blue line); (b) 70 mM (blue line), 100 mM, 140 mM, 300 mM, 500 mM, 600 mM, and 800 mM (wine red line). (c) Deconvoluted spectra (from ESI-MS spectra via ProMass software) of PW17 with different concentrations of Na+.

1a shows that 4 μM PW17 in 10 mM Tris−HAc (pH = 7.4) exhibited a negative CD peak at 240 nm, a positive CD peak at 257 nm with a shoulder peak at 295 nm, indicating that PW17 was randomly dispersed.24,42,61 When 5−70 mM Na+ was added, respectively, the negative peak at 240 nm gradually decreased and switched to a positive one at 250 nm, and also 5406

DOI: 10.1021/acs.jpcb.9b02823 J. Phys. Chem. B 2019, 123, 5405−5411

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The Journal of Physical Chemistry B the positive peak at 257 nm decreased and changed to a negative one at 264 nm. Moreover, the shoulder peak at 295 nm further increased. These characteristic peaks (positive peaks at 250 nm and at 295 nm and a negative peak at 264 nm) indicated that Na+ induced 4 μM PW17 switching to antiparallel G4.21 As a continuation, upon further addition of 70−800 mM Na+ (Figure 1b), the positive peak at 250 nm switched to a negative one at 243 nm and the negative peak at 264 switched to a positive one, accompanied by an almost declined positive peak at 295 nm. These switches indicated that antiparallel G4 possibly transformed into a hybrid G4.62−64 Electrospray ionization mass spectrometry (ESI-MS) was further applied to corroborate the forming process.55,56,65 The full-scale spectra are shown in Figure S1, and the deconvoluted spectra are shown in Figure 1c. For 4 μM PW17, a distinct mass peak at m/z = 5404 (5403 + 1) was detected, corresponding to a protonated PW17 ([PW17 + H]+).24,42 Upon gradually adding Na+ from 10 to 50 mM, the second peak at m/z = 5426 was detected. The molecular weight (MW) difference of m/z = 23 (5426−5403) is an Na+, demonstrating the formation of an Na+-stabilized PW17 (Na+PW17). It was suggested that the formed Na+-stabilized PW17 was loose and unstable at CNa+ ≤ 50 mM.60 Subsequently, when Na+ increased to 70 mM, a third new peak at m/z = 5449 appeared. The MW difference of m/z = 46 (5449 − 5403) indicated the formation of 2Na+-PW17. The result showed that unstable Na+-PW17 started to become compact and stable.42 While Na+ was further increased to 800 mM, the mass peak intensity (MPI) at m/z = 5404 gradually reduced and finally disappeared at CNa+ = 700 mM. Meanwhile, the MPI at 5449 was increased, demonstrating that the 2Na+PW17 formed was gradually stable. Meanwhile, it was noticed that the MPI at m/z = 5426 was slightly increased at CNa+ = 70−600 mM, further decreased at CNa+ = 700 mM, and finally disappeared at CNa+ = 800 mM. Integrated with the CD results, it was concluded that PW17 transformed into unstable and antiparallel 2Na+-PW17 at CNa+ ≤ 70 mM and further gradually switched to stable and hybrid 2Na+-PW17 at 70 mM < CNa+ ≤ 700 mM. Finally, the stable and hybrid 2Na+-PW17 formed at CNa+ = 800 mM. The results demonstrated that Na+ could induce PW17 undergoing a conformational transformation from random coil to antiparallel G4 and further to hybrid G4, which was strictly Na+ concentration-dependent. K+ Competitively Substituting Na+ in Na+-Stabilized PW17. G4-based K+ sensors had been applied to detect K+ with the co-existing Na+ from 10 to 600 mM, even 800 mM.33,66−68 The sensing mechanism probably is K+ competitively binding with Na+ in Na+-G4.34,69 As discussed above, 800 mM Na+ induced 4 μM PW17 switching to a stable and hybrid 2Na+-PW17. With the stable and hybrid 2Na+-PW17 as a starting G4, the substituting process is first investigated. When K+ (0−10 mM) was added into the system containing 800 mM Na+ and 4 μM PW17, the conformational changes were recorded by CD spectra as shown in Figure 2a. The characteristic peaks of 2Na+-PW17 at 243 nm and at 264 nm further enhanced, and the shoulder peak at 295 nm decreased and almost disappeared at CK+ = 10 mM, indicating that K+ could induce hybrid 2Na+-PW17 transformation into parallel G4.21 At the same time, ESI-MS was applied to track the transforming process. The full-scale spectra are shown in Figure S2, and the detailed deconvoluted spectra are shown in Figure 2b. When 0.5 mM K+ was added, although G4

Figure 2. (a) CD spectra of PW17 in 10 mM Tris−HAc (pH = 7.4) containing 800 mM Na+ and various amounts of K+: from bottom to top: 0 mM (red line), 0.2 mM, 0.5 mM, 1 mM, 2 mM, 5 mM, and 10 mM (blue line). (b) Deconvoluted spectra (from ESI-MS spectra via ProMass software) of 4 μM PW17 in the solution of 800 mM Na+ and various amounts of K+ (from top to bottom: 0, 0.5, 0.6, 0.8, 1, 2, 5, 6, 8, and 10 mM).

conformation slightly changed, the detected MW was still located at m/z = 5449 (corresponding to [2Na+-PW17]+), indicating that 0.5 mM K+ could not substitute Na+ in 2Na+PW17. When 0.6−6 mM K+ was added, the MPI at m/z = 5449 was gradually decreased and finally disappeared at CK+ = 6 mM. Interestingly, in the case of 0.6 mM K+, a new peak at m/z = 5465 appeared. The difference of m/z = 62 (23 + 39 = 5465 − 5403), matching with the sum of an Na+ and a K+, indicated that a [K+−Na+-PW17]+ intermediate formed. Such a novel intermediate was detected for the first time in the transforming process from Na+-G4 to K+-G4. Upon increasing the K+ concentration from 0.6 to 2 mM, the MPI at m/z = 5465 gradually increased to the maximum. Meanwhile, at 2 mM K+, another new peak at m/z = 5481 (39 + 39 + 5403) appeared and further increased, showing the appearance of [2K+-PW17]+. These results suggested that (1) K+ first substituted a Na+ in 2Na+-PW17 with 2Na+-PW17 transforming into the [K+−Na+-PW17]+ intermediate and (2) the [K+−Na+-PW17]+ intermediate further transformed into [2K+PW17]+. To testify the illustration, continually adding 2−10 mM K+, the MPI of the [K+−Na+-PW17]+ intermediate gradually reduced and faded away at 10 mM K+, accompanied by the further increased MPI of [2K+-PW17]+. Integrated with the results of CD spectra, it was concluded that (1) 0.5 mM K+ induced a conformational change in stable 2Na+-PW17 but did not substitute Na+; (2) K+ (CK+ > 0.5 mM) could cause hybrid 2Na+-PW17 to transform into parallel G4 and also substitute Na+ in 2Na+-PW17 with 2Na+-PW17 switching to 2K+-PW17 through the K+−Na+-PW17 intermediate. It means that the stable 2Na+-PW17 is not sensitive to K+ at CK+ ≤ 0.5 mM. Alternatively, for the system containing 140 mM Na+ and 4 μM PW17 (unstable 2Na+-PW17), upon adding 0.01−10 mM 5407

DOI: 10.1021/acs.jpcb.9b02823 J. Phys. Chem. B 2019, 123, 5405−5411

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The Journal of Physical Chemistry B K+, the CD spectra showed that the peak at 264 nm switched to a positive one and the positive peak at 295 nm decreased and finally disappeared (Figure 3a). Meanwhile, a new peak at

and parallel 2K+-PW17. This means that 2Na+-PW17 (140 mM Na+) is not sensitive to K+ at CK+ < 0.1 mM. Similar results were also observed for the system containing 70 mM Na+ and 4 μM PW17 (Figures S4 and S5). For the case of 20 mM Na+ and 4 μM PW17 (unstable 2Na+-PW17), Figure 4a shows that the CD spectra, upon

Figure 3. (a) CD spectra of PW17 in 10 mM Tris−HAc (pH = 7.4) containing 140 mM Na+ and various amounts of K+: from bottom to top: 0 mM (red line), 0.2 mM, 0.5 mM, 1 mM, 2 mM, 5 mM, and 10 mM (blue line). (b) Deconvoluted peaks generated from ESI-MS spectra of PW17 in the solution of 140 mM Na+ and various amounts of K+ (from top to bottom: 0, 0.01, 0.1, 0.2, 0.3, 0.4, 0.5, 1, 2, 3, 5, and 10 mM) via ProMass software.

Figure 4. (a) CD spectra of PW17 in 10 mM Tris−HAc buffer (pH = 7.4) containing 20 mM Na+ and various amounts of K+: from bottom to top: 0 mM (red line), 0.2 mM, 0.5 mM, 1 mM, 2 mM, 5 mM, and 10 mM (blue line). (b) Deconvoluted spectra (from ESI-MS spectra via ProMass software) of PW17 in the solution of 20 mM Na+ and various amounts of K+ (from top to bottom: 0, 0.1, 0.2, 0.3, 0.4, 0.5, 1, 2, 3, 5, and 10 mM).

243 nm was detected and gradually enhanced. The results showed that K+ could also induce unstable 2Na+-PW17, transforming into a parallel G4.21 The switching process was also tracked by ESI-MS (Figure S3), and the deconvoluted spectra are shown in Figure 3b. The mass peaks located at m/z = 5404, 5426, and 5449 were detected, respectively, corresponding to [PW17 + H]+, [Na+-PW17]+, and [2Na+PW17]+ , indicating that the 2Na+ -PW17 formed was unstable.60 When 0.01 mM K+ was added, the detected MW remained unchanged. Continually enhancing K+ to 0.1 mM, the MPI of [2Na+-PW17]+ was slightly decreased, and a new peak at m/z = 5465 appeared, demonstrating the formation of the [K+−Na+-PW17]+ intermediate. When K+ increased to 0.2 or 0.3 mM, the mass peak of [2Na+-PW17]+ disappeared and the MPI of [K+−Na+-PW17]+ further increased. At 0.4 mM K+, the mass peak of [Na+-PW17]+ finally disappeared. As a continuation, the mass peak of [PW17 + H]+ disappeared at 0.5 mM. Excitedly, at this point (0.5 mM K+ and 140 mM Na+), only the mass peak of [K+−Na+-PW17]+ remained, indicating that K+−Na+-PW17 was relatively stable.42 Upon further increasing the K+ concentration from 1 to 10 mM, the MPI of [K+−Na+-PW17]+ was gradually reduced, and the mass peak of [2K+-PW17]+ at m/z = 5481 appeared at 1 mM K+ and further enhanced. The results clearly showed that (1) K+ first competed with an Na+ in unstable 2Na+-PW17 with 2Na+PW17 transforming into a stable K+−Na+-PW17 intermediate; (2) another K+ further competed with Na+ in the K+−Na+PW17 intermediate with K+−Na+-PW17 switching to stable

addition of 0.1−10 mM K+, are similar to that shown in Figure 3a. Such a switching process was also tracked by ESI-MS (Figure S6), and the deconvoluted spectra are shown in Figure 4b. The mass peaks located at m/z = 5404 ([PW17 + H]+) and 5426 ([Na+-PW17]+) were detected, demonstrating that the formed 2Na+-PW17 was less stable.55 When 0.2 mM K+ was added, the mass peak of [Na+-PW17]+ decreased and a really weak mass peak at m/z = 5465 appeared ([K+−Na+-PW17]+ intermediate), indicating that K+ first substituted an Na+ in unstable 2Na+-PW17. Further increasing the K+ concentration to 0.5 mM, the [K+−Na+-PW17]+ MPI slightly increased, indicating that the formed K+−Na+-PW17 was unstable. Two new peaks at m/z = 5442 ([K+-PW17]+) and 5481 ([2K+PW17]+) concurrently appeared, showing the formation of unstable 2K+-PW17.42,70 When K+ increased to 1 mM, [Na+PW17]+ disappeared, followed by the disappearance of [K+PW17]+ at CK+ = 2 mM and that of [K+−Na+-PW17]+ at CK+ = 3 mM. Meanwhile, the MPI of [2K+-PW17]+ further increased until CK+ = 10 mM. The transforming process was similar to that of K+, inducing PW17 switching to 2K+-PW17.42 The results showed that 20 mM Na+ could hardly interfere with the formation of 2K+-PW17. Influence of Na+ on the Performance of PW17-Based + K Sensors. On the basis of the discussions above, Na+ could interfere with the forming process of K+-G4, which was 5408

DOI: 10.1021/acs.jpcb.9b02823 J. Phys. Chem. B 2019, 123, 5405−5411

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The Journal of Physical Chemistry B dependent on the applied Na+ concentration. Herein, the influence of Na+ on the performance of the PW17-based K+ sensor was explored with PW17 as a sensing probe and protoporphyrin IX (PPIX) as a G4 fluorescent read-out probe. In the presence of 20 mM Na+, Figure 5a shows an outline of

concentration was not observed (Figure 5c). These results indicated that (1) Na+ affected the performance of the PW17based K+ sensor, which was dependent on Na+ concentration; (2) the higher the Na+ concentration was applied, the more seriously the K+ detection was interfered. Interestingly, when the stable K+−Na+-PW17 was applied as a sensing probe and PPIX as a G4 fluorescent read-out probe, Figure 6 shows a

Figure 6. Relationship of PPIX fluorescence emission intensity change (F − F0) at 632 nm vs applied 500 nM−10 mM K+ (10 mM Tris−HAc buffer, pH = 7.4). F represents PPIX emission fluorescence in the presence of K+ and F0 represents that in the absence of K+. Error Bars denote the standard deviations from three separate measurements.

linear relationship between K+ concentration (500 nM−10 mM) and PPIX fluorescence change with a regression equation of F − F0 = 24 944.53 lg[K+] + 161 565.19 (R2 = 0.9984). The measured detection limit was 500 nM.

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CONCLUSIONS In this paper, with PW17 as an example, Na+ and K+ with various concentrations competitively binding with PW17 was explored with circular dichroism (CD) spectroscopy and electrospray ionization mass spectroscopy (ESI-MS). Even if Na+ could induce PW17 switching to unstable and antiparallel 2Na+-PW17 (CNa+ ≤ 70 mM) and further to stable and hybrid 2Na+-PW17 (70 mM < CNa+ ≤ 800 mM), 0.1−10 mM K+ could replace Na+ in 2Na+-PW17 with 2Na+-PW17 finally transforming into 2K+-PW17. Such a replacing efficiency was strictly dependent on CNa+ and CK+. Importantly, a K+−Na+PW17 intermediate was first found in the switching process and then stable K+−Na+-PW17 was discovered at 0.5 mM K+ and 140 mM Na+. Based on the above facts, the interference of Na+ with PW17-based K+ sensors was investigated. It was found that 70−140 mM Na+ affected the performance of the PW17-based K+ sensor until the sensor did not work at CNa+ = 800 mM. With the stable K+−Na+-PW17 as a sensing probe and PPIX as a G4 fluorescent read-out probe, the fluorescent detection of K+ (500 nM−10 mM) was achieved in the presence of high CNa+ (140 mM) and the measured detection limit was 500 nM. This study provided (1) clear evidence of Na+ and K+ competitively binding with PW17, which was beneficial for deeply understanding G4 functions in Na+contained samples; (2) a positive motivation for clarifying the structure of K+−Na+-G4; and (3) a clue to further generalize such competitively binding behavior for other G-rich DNA sequences.

Figure 5. Relationship of PPIX fluorescence emission intensity change (F − F0) at 632 nm vs applied K+ (10 nM−10 mM) with (a) 20 mM, (b) 140 mM, and (c) 800 mM Na+ (10 mM Tris−HAc buffer, pH = 7.4). F represents PPIX emission fluorescence in the presence of K+ and F0 represents that in the absence of K+. Error Bars denote the standard deviations from three separate measurements.

the relationship between K + concentration and PPIX fluorescence change (F represents PPIX emission fluorescence in the presence of K+, and F0 represents that in the absence of K+). Two linear fluorescence ranges (at 10 nM−0.5 mM K+, the linear regression equation is F − F0 = 5784.35 lg[K+] + 57 683.16 (R2 = 0.9900), and at 0.5−10 mM K+, the linear regression equation is F − F0 = 193 664.41 lg[K+] + 680 860.90 (R2 = 0.9981)) were observed, which were consistent with that of the absence of Na+ (Figure S7).42 The result shows that 20 mM Na+ did not affect the sensor performance. Alternatively, when Na+ increased to 140 mM, the formed 2Na+-PW17 became more stable than that of 20 mM Na+. In this case, further addition of 10 nM−10 mM K+, Figure 5b shows the relationship between K+ concentration and PPIX fluorescence change. In the range of 10 nM−0.5 mM K+, the linear relationship was destroyed. Within the scope of 0.5−10 mM K+, the linear relationship was observed and the regression equation is F − F0 = 201 560.53 lg[K+] + 684 803.00 with R2 = 0.9957, which was corresponding to the process of K+ substituting Na+ in stable K+−Na+-PW17 to transform into 2K+-PW17. Further increasing Na+ to 800 mM, the stable 2Na+-PW17 was detected (Figure 1c). Although K+ could substitute Na+ in 2Na+-PW17, and finally 2Na+-PW17 transformed into 2K+-PW17 at 10 mM K+, the linear relationship between PPIX fluorescence change and K+

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The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jpcb.9b02823. 5409

DOI: 10.1021/acs.jpcb.9b02823 J. Phys. Chem. B 2019, 123, 5405−5411

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Deconvolution parameters, all RAW mass spectra, and additional CD, ESI-MS, and fluorescence spectroscopy data (PDF)

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel.: +86-10-58802741. ORCID

Yunchao Li: 0000-0002-5554-7252 Louzhen Fan: 0000-0003-0958-7015 Xiaohong Li: 0000-0003-2043-1206 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the National Natural Science Foundation of China (21673022) and the Fundamental Research Funds for the Central Universities.

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