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Molecular Rotor-Based Fluorescent Probe for Selective Recognition of Hybrid G‑Quadruplex and as a K+ Sensor Lingling Liu,† Yong Shao,*,† Jian Peng,† Chaobiao Huang,‡ Hua Liu,† and Lihua Zhang† †

Institute of Physical Chemistry, Zhejiang Normal University, Jinhua 321004, Zhejiang, People’s Republic of China Department of Chemistry, College of Chemistry and Life Sciences, Zhejiang Normal University, Jinhua 321004, People’s Republic of China

‡

S Supporting Information *

ABSTRACT: This work demonstrates the significant fluorescence enhancement of thioflavin T (ThT) when binding to G-quadruplexes possessing hybrid structures by using UV−vis absorption spectra, fluorescence spectra, and Tm experiments to confirm the binding events. ThT binding does not disturb native G-quadruplex structures preformed in Na+ and K+ solutions. The fluorescence enhancement is caused by the rotation restriction of benzothiazole (BZT) and dimethylaminobenzene (DMAB) rings in the ThT excited state upon its Gquadruplex binding. This molecular rotor mechanism as a means of fluorescence enhancement is confirmed using a nonrotor analogue of ThT. Hydroxylation and electrolyte experiments demonstrate that ThT stacks on the tetrad of the hybrid G-quadruplexes, whereas electrostatic forces contribute more to ThT binding for other G-quadruplex structures. By stacking on the tetrad, the ThT binding favors selective identification of DNA hybrid G-quadruplex structures with enhanced fluorescence and can serve as a conformation probe to monitor G-quadruplex structure conversion between hybrid and other structures. Using these properties, we developed a selective and label-free fluorescent K+ sensor with a detection limit of 1 mM for K+ in the presence of 100 mM Na+. The coexistence of other metal ions produces a fluorescence response comparable to K+ alone. We believe that ThT can potentially provide structure identification of hybrid G-quadruplexes and aid in the construction of Gquadruplex-based sensors.

T

ligands that can recognize one of the structures with high selectivity, while causing minimal disturbance to the native Gquadruplex structures themselves. Fluorescent techniques provide a powerful and visual means of studying the folding and structure of G-quadruplexes.11−13 Due to the strong distance dependency of energy transfer between donor and acceptor, fluorescence resonance energy transfer (FRET)14,15 is a powerful tool for monitoring Gquadruplex conformation with labeled fluorophores as probes. Alternatively, a fluorescent nucleotide analogue (for example, 2aminopurine16) has been inserted into G-quadruplexes to follow external stimuli-induced conformational changes. Recently, it was found that you can follow the folding of a Gquadruplex structure using its intrinsic fluorescence.17 However, the majority of research efforts have focused on developing G-quadruplex-specific fluorescent ligands with a molecular size comparable to G-tetrad (e.g., porphyrin and its complex,18−22 Zn2+-phthalocyanine,23−25 Pt2+-dipyridophenazine,26 Ru2+-phenazine,27,28 bisquinolinium/thiazole orange conjugate,29 triphenylmethane dye,30−32 cresyl violet,33 berber-

he selective recognition of DNA structures by small molecules is a promising development in therapeutic drug screening and biosensor construction. G-quadruplex is a fourstranded structure derived from a guanine (G)-rich DNA sequence,1,2 with a stacked Hoogsteen hydrogen-bonded Gtetrad as the basic structural motif. Since stable G-quadruplex structures exhibit inhibitory effects on the catalytic activity of telomerase,3,4 the G-quadruplex frequently found at the 3′ end of vertebrate telomeric DNA is considered to be an evolutionarily conserved structural element, essential for chromosome end protection.5 Additionally, the G-quadruplexes in some gene promoter regions such as oncogene c-myc6,7 and c-kit8,9 are believed to have the potential to regulate gene expression.10 Human G-quadruplexes are found to have at least five typical structures (Scheme 1), depending on the G-tetrad core arrangement and the direction of the intervening variablelength loops linking the tetrads: propeller-type parallel, baskettype antiparallel (one diagonal and two lateral loops), chairtype antiparallel (three lateral loops), (3 + 1) hybrid-1 (one propeller and two lateral loops in succession from the 5′ end), and (3 + 1) hybrid-2 (two lateral and one propeller loops in succession from the 5′ end). Additionally, the G-quadruplex structures always coexist with abundant double-stranded duplex DNA in cells. Thus, there is a great demand to develop novel © 2014 American Chemical Society

Received: October 15, 2013 Accepted: January 10, 2014 Published: January 10, 2014 1622

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Scheme 1. ThT and the Typical G-Quadruplex Structures: (a) Hybrid-1, (b) Hybrid-2, (c) Basket Antiparallel, (d) Chair Antiparallel, and (e) Parallela

a

The hybrid structures are more efficient in lighting up ThT fluorescence for a given G-quadruplex sequence.

Table 1. Oligonucleotides Used in This Work, and the Identified Structures along with Their Measured Binding Constants name 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

Tel22 Tel26 wtTel26 TAG3 TG3 TAG3T TTAG3 mPu22 1XAV PS2.M T30695 2O3M T3TT T3TT3 T3T3T3 FM

sequence

structure in Na+

5′-AG3(TTAG3)3-3′ 5′-A3G3(TTAG3)3AA-3′ 5′-TTAG3(TTAG3)3TT-3′ 5′-TAG3(TTAG3)3-3′ 5′-TG3(TTAG3)3-3′ 5′-TAG3(TTAG3)3TT-3′ 5′- TTAG3(TTAG3)3-3′ 5′-TGAG3TG4AG3TG4AA-3′ 5′-TGAG3TG3TAG3TG3TAA-3′ 5′-GTG3TAG3CG3TTGG-3′ 5′-(G3T)4-3′ 5′-(AG3)2CGCTG3AGGAG3-3′ 5′-G3TTTG3TG3TG3-3′ 5′-G3TTTG3TG3TTTG3-3′ 5′-G3TTTG3TTTG3TTTG3-3′ 5′-GAGGTGTGAGTGTGAGTGTGAG-3′ 3′-CTCCACACTCACACTCACACTC-5′

54,55

basket basket54 basket54 basket54 basket54 basket54 basket54,65 hybrid-124,56 partial parallel68 chair21,69 chair72 partial hybrid75 parallel31 antiparallel31 antiparallel31

ine,34 and Hoechst 3325835), in order to achieve a strong binding. Although these hydrophobic macrocyclic fluorophores benefit from potential cell permeability, it is at the expense of recognition selectivity among various G-quadruplex structures, and even between G-quadruplex and duplex DNAs. More seriously, sometimes these G-tetrad-sized macrocyclic fluorophores can themselves induce DNA folding toward Gquadruplex, or convert preformed G-quadruplex from one conformation to another.33 Additionally, most of these fluorophores18−26 are efficient only for G-quadruplexes possessing a parallel structure. Developing probes for other frequently observed G-quadruplex structures (e.g., the K+stabilized hybrid structure that usually exists in human telomeric G-quadruplex) and for adaptive recognition without disturbing the native conformation is still a challenge.31,33 On the other hand, in order to achieve a selective therapy by targeting the G-quadruplex, it is necessary to find ligands that can not only differentiate G-quadruplex from duplex DNA but also target a specific type of G-quadruplex.

structure in K+ 56,57

hybrid-1 hybrid-157−59 hybrid-258,59 hybrid-1 or -257−62 hybrid-158,63 hybrid-1 or -258−64 hybrid-157,65 parallel24,66,67 parallel58,66 chair70,71 chair73,74 parallel58,76 parallel31 parallel31 hybrid-131

binding constant KNa/KK/105 M−1 0.5 0.9 0.5 0.6 0.5 0.4 0.8 15.7 2.1 2.0 1.3 4.2 3.8 0.8 0.4 0.06

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.2/2.8 0.2/3.9 0.2/5.1 0.2/3.8 0.3/3.1 0.2/8.6 0.2/6.8 0.3/2.4 0.4/2.0 0.2/2.4 0.2/1.8 0.5/1.9 1.1/2.9 0.3/1.7 0.1/4.4 0.02

± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.3 0.9 0.6 0.4 1.1 1.6 1.3 0.7 0.4 0.5 0.3 0.4 1.6 0.4 0.3

In this work, we use thioflavin T (ThT) as a novel fluorophore to target G-quadruplex. ThT has long been chosen as an extrinsic fluorescent probe to identify amyloid fibrils, particularly those with a β sheet-rich structure.36−39 In addition to these misfolded protein fibril aggregates, acetylcholinesterase40 and serum albumins41 in their folded native states can also significantly enhance ThT fluorescence. Although ThT has been used as a fluorescent marker for amyloid fibrils for more than 50 years,42 it is only recently that researchers have confirmed43−47 that the ThT nonradiative process is controlled by a molecular rotor mechanism. Scheme 1 shows that, upon excitation, rotation of the benzothiazole (BZT) and dimethylaminobenzene (DMAB) rings around the single C−C bond between the rings quenches ThT radiative transition in favor of a nonradiative twisted internal charge-transfer (TICT) state; with the dihedral angle at 90°, this effect is especially noticeable. Thus, any interaction restricting this rotation and enforcing planarization of the dye results in an enormous increase in fluorescence emission. However, even with this new understanding of ThT fluorescence mechanisms and its fruitful 1623

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application in the identification of β sheet-rich amyloid fibrillation, the DNA structure-dependent switching of ThT fluorescence still requires investigation.48,49 Recently, we found that ThT can be fluorescent when binding to duplex DNAs possessing a cavity structure.50 We now investigate the Gquadruplex structure dependence of ThT fluorescence lighting up, finding that the ThT binding model and fluorescence performance are strongly dependent on the G-quadruplex structure. The hybrid structure of the G-quadruplex is very efficient in lighting up ThT fluorescence, and we demonstrate a molecular rotor mechanism to tune the emission property via a binding-induced change in ThT conformation. This Gquadruplex structure-dependent switching of ThT fluorescence has the potential to identify the hybrid structure for a given Gquadruplex sequence. The G-quadruplex structure-selective binding of ThT, and subsequently the turn-on fluorescence response as a result of the binding-induced ThT conformation change, provide a new way to construct DNA-based sensors. To prove this type of application, we successfully use the switched ThT fluorescence as the readout for a K+ sensor with high selectivity and low fluorescence background.

ThT was titrated with G-quadruplex for measurement of the binding constants, with the fluorescence intensity at 487 nm plotted as a function of DNA concentration. The data were fitted by KaleidaGraph (Synergy Software, PA) according to a 1:1 binding model.52 The ThT concentration used in the analysis was dependent on the DNA type in order to get an obvious fluorescence signal, where the DNA concentration for each titration was always higher than that of ThT so that interaction occurred mainly at the strong DNA binding site for the formation of a 1:1 complex.53 For FM with multiple ThT binding sites, the binding constant was only roughly estimated, and we used an even larger excess concentration of DNA to make the reaction mainly occur at the strongest DNA binding site for an approximate 1:1 association model. The hydroxylation of ThT (0.5 μM) in the presence of Gquadruplexes (15 μM) was conducted in corresponding sodium and potassium phosphate buffers (pH 8.4). Excess Gquadruplex was used to keep the ThT in a bound state. The solutions were incubated at 35 °C, and the hydroxylation kinetics measured using the fluorescence of ThT as a function of incubation time. To confirm the electrostatic contribution to the ThT binding, we measured the dependence of 1 μM ThT fluorescence at 487 nm in the presence of 1 μM G-quadruplex on the added Tris-HCl (pH 7.5) concentrations. The solutions always contained either 0.1 M KCl or NaCl, and the Tris-HCl (pH 7.5) concentration varied between 25 and 100 mM. UV−vis Absorption Spectra and Difference Spectra. UV−vis absorption spectra were determined with a UV2550 spectrophotometer (Shimadzu Corp., Kyoto, Japan) at room temperature using a quartz cell with a path length of 1 cm. In order to investigate DNA conformation changes upon ThT binding, absorption difference spectra (ADS) between K+- and Na+-defined DNA structures were measured in the presence and absence of ThT, respectively. The ADS spectra were obtained by subtracting the corresponding NaCl spectra from the KCl spectra. Thus, the effect of ThT on the DNA absorbance was mostly eliminated in ADS, with the resulting ADS for the DNA mainly reflecting the structural changes between the varied base stacking in Na+ and K+. Due to the DNA binding-induced shift in absorbance wavelength, the ADS in the ThT absorbance region only indicates the difference in binding capacity of ThT to the Na+- and K+-defined quadruplex structures. DNA Melting Temperature (Tm) Measurements. The melting temperatures (Tm) of the DNA in the presence and absence of ThT were determined using a UV2550 spectrophotometer (Shimadzu Corp., Kyoto, Japan), equipped with a TMSPC-8 Tm analysis system, which can simultaneously control the chamber temperature and detect up to eight samples with a micro multicell. The absorbance of the Gquadruplex at 295 nm as a function of solution temperature was collected in 0.5 °C increments, with a 30 s equilibration time applied after each temperature increment. In order to avoid the partial ThT hydroxylation at neutral pH that would occur at the high solution temperature required for the Tm measurements, the Tm experiments were carried out in a phosphate buffer (25 mM) at pH 5.3 containing 0.1 M Na+ or K+. Note that only the partial melting curve was obtained for sample T30695 due to the high Tm value in the K+ solution (more than 90 °C), and a sigmoidal fitting method was used to estimate the actual Tm value.

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EXPERIMENTAL SECTION Materials and Reagents. DNA species (Table 1) were synthesized by TaKaRa Biotechnology Company, Ltd. (Dalian, China) and purified by HPLC. The DNA concentrations were measured by first dissolving DNA in pure water and detecting the UV absorbance at 260 nm using extinction coefficients calculated by nearest neighbor analysis. Thioflavin T (ThT, ultrapure grade) was obtained from AAT Bioquest, Inc. (California) and used as received. 2-(4′-Methylaminophenyl)benzothiazole (BTA-1) was purchased from Sigma Chemical Company (St. Louis). The ThT solution was freshly prepared before each experiment in order to avoid storage-induced oxidation.51 Milli-Q water (18.2 mΩ; Millipore Company, Billerica, MA) was used in all experiments. All other chemicals were analytical-reagent grade (Sigma Chemical Company, St. Louis, MO) and used without further purification. Fluorescence Measurements. Fluorescence spectra were acquired with a FLSP920 spectrofluorometer (Edinburgh Instruments Ltd., Livingston, U.K.) at 18 ± 1 °C, which was equipped with a temperature-controlled circulator (Julabo Labortechnik GmbH, Seelbach, Germany). Fluorescence was measured in a quartz cell with a path length of 1 cm. To prepare the G-quadruplex solution, the strand was annealed in a thermocycler (first at 92 °C, then slowly cooled to room temperature) in 25 mM Tris-HCl buffer (pH 7.5), containing 100 mM NaCl or KCl. To prepare the DNA duplex solutions (FM, Table 1), the two strands were mixed in equimolar amounts and annealed using a similar procedure as for the Gquadruplex. ThT at the specified concentration was added into the DNA solutions, and the resulting solutions were allowed to incubate for 15 min before fluorescence measurements were taken. For the experiments to identify K+ selectivity over other metal ions, 10 mM ethylenediaminetetraacetic acid disodium salt (EDTA) was further added to avoid hydrolization that occurs to some heavy metal ions in the detection solution (pH 7.5). ThT was added to the DNA solution to an appropriate molar ratio in 25 mM Tris-HCl buffer (pH 7.5) containing the desired metal ions. After mixing, the solution was incubated for 15 min with gentle stirring. The resulting solution was examined at room temperature within 2 h. 1624

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Figure 1. (A) Excitation and emission spectra of ThT (2 μM) in 0.1 M KCl and NaCl solutions in the presence of Tel22 and FM (1 μM). (B) Absorption spectra of ThT (3 μM) in 0.1 M KCl and NaCl in the absence and presence of Tel22 (3 μM). Inset: photographs of these solutions under UV illumination (from left to right: ThT in K+, ThT-Te22 in K+, ThT in Na+, and ThT-Te22 in Na+).

Application. Tap water and commercially bottled mineral water were used as samples to confirm the feasibility of applying this method as a K+ sensor. KCl (1 mM) was added into the samples to test the recovery. To 100 μL of water samples were added 1 μM Tel22 and 1 μM ThT in a 25 mM Tris-HCl buffer (pH 7.5) containing 100 mM NaCl with and without the standard 1 mM K+. The added K+ was measured by the primarily established work curve.

achieved with the naked eye under UV illumination (Inset of Figure 1B). Previously, a 1:1 binding of ThT to human telomeric Gquadruplexes was obtained by fluorescence titration of ThT with DNA.48,53 Such fluorescence titration of ThT by DNA was also used here to evaluate the binding constant. The typical titration curves are provided in Figure S1 of the Supporting Information, and the results were listed in Table 1. The formation of 1:1 ThT complexes is confirmed by the good fitting to the data with a 1:1 binding model (Table 1). Accordingly, the binding constant for ThT binding to the K+defined Tel22 hybrid structure is (2.8 ± 0.3) × 105 M−1, 5.6 times higher than that of its binding to the Na+-defined basket antiparallel structure [(0.5 ± 0.2) × 105 M−1]. In comparison to G-quadruplexes, the ThT binding strength to FM was even weaker [(0.06 ± 0.02) × 105 M−1], obtained by an approximation to the 1:1 binding model for the strongest FM binding site that should mainly occur based on the excess concentration of FM used for the analysis. Other human telomeric sequences in the K+ solution also form a hybrid-type intramolecular G-quadruplex structure having a (3 + 1) strand arrangement in a fashion similar to Tel22.54,64 In order to verify the selective ThT fluorescence lighting up in response to the hybrid structure binding, and investigate the effects of DNA sequence on hybrid-selective emission, the fluorescence ratios (FK/FNa) of ThT in K+ and Na+ solutions were also measured for the other human telomeric sequences, including Tel26, wtTel26, TAG3 , TAG3T, TG3, and TTAG3. These DNAs contain the same 5′-G3(TTAG3)3-3′ quadruplex core as Tel22 but have different 5′ and 3′ flanking nucleotides (Table 1), and their conformations have been well-documented. In a Na+ solution, the folded topology for these quadruplexes is the basket antiparallel structure.54−65 As shown in Figure 2, ThT exhibits an enhanced fluorescence response on binding to the K+defined hybrid structure, with a FK/FNa ratio between 6 and 26, depending on the flanking nucleotides. It is widely accepted that the folding manner of G-quadruplexes is dependent on the 5′ and 3′ flanking nucleotides.79−82 For the basket-type antiparallel structure usually adopted by human telomeric Gquadruplexes in Na+, a diagonal TTA loop and two lateral TTA loops position over the top and bottom tetrads separately, and the extended 5′ and 3′ flanking ends appear in the same direction along the quadruplexes (the top tetrad, Scheme 1). These factors offer steric obstacles to ThT binding. On the other hand, the hybrid-type folding has a propeller TTA loop and only one lateral TTA loop positions over each of the top and bottom tetrads. Additionally, the extended 5′ and 3′

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RESULTS AND DISCUSSION ThT Targeted G-Quadruplex Hybrid Structures with a Restricted Molecule Rotating-Induced Fluorescence Enhancement. In order to investigate the selective binding of ThT to a G-quadruplex structure, we first compared the achieved fluorescence of Tel22 to a fully matched DNA duplex (FM, Table 1). Tel22 is one of the most frequently investigated human telomeric G-quadruplex sequences. Note that one of the strands in FM has an identical base composition to Tel22, but the base context arranges in such a way that there is no expectation of a G-quadruplex structure. As shown in Figure 1A, ThT is still very weakly emissive in the presence of FM. However, in the K+ solution, Tel22 induces 60 times the fluorescence intensity compared with FM. The excitation and emission bands are located at 440 and 487 nm. Additionally, the emission in the Na+ solution is 21 times weaker than the emission in the K+ solution for Tel22. These results illustrate that ThT can fluorescently target the Tel22 structure defined by K+. It is well-known that Tel22 mainly adopts a basket antiparallel structure in Na+ solutions54,55 and a (3 + 1) hybrid1 structure in K+ solutions.56,57 However, only a parallel Gquadruplex structure has been observed by X-ray for solid crystal Tel22 in the presence of K+.77 Our results suggest that it is possible for ThT to target hybrid G-quadruplex structures with an accompanying significant fluorescence enhancement. The specific interaction of ThT with the hybrid quadruplex structure was further verified by absorption spectra. As shown in Figure 1B, Tel22 induces a clear red shift in ThT absorption in the presence of K+, a characteristic that indicates formation of a ThT emissive state39 with a restricted rotation of BZT and DMAB rings that thus results in a greater degree of planarizing of the molecule.43−47 The observed red shift in the ThT absorption spectra also implies that the bound ThT ground state, and subsequently the Franck−Condon excited state, are far from contact with the water solvent that is more polarity than DNA environment.78 Furthermore, the difference in fluorescence resulting from the hybrid structure and its basket antiparallel counterpart means that recognition can even be 1625

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ruplexes exhibit distinct FK/FNa fluorescence dependencies that are significantly different from the telomeric fluorescence dependencies. To further verify the selective fluorescing of ThT in response to the hybrid structure binding, we assessed the G-quadruplexes formed by T3TT, T3TT3, and T3T3T3 sequences (Table 1) without the 5′ and 3′ flanking nucleotides. These sequences are representatives of the oxytricha telomeres. Among these sequences, only T3T3T3 has the potential to form the hybrid1 structure in a K+ solution, while the parallel and antiparallel structures dominate for the other sequences under both K+ and Na+ conditions.31 As shown in Figure 2, T3T3T3 indeed experiences a higher response in FK/FNa than T3TT and T3TT3, although the fluorescence enhancement factor for T3T3T3 is still lower than most of the human telomeric G-quadruplexes. Accordingly, only T3T3T3 exhibits the most difference in binding constants in K+ and Na+ solutions (Table 1). PS2.M (Table 1) folds to the chair antiparallel structure in both K+ and Na+ solutions21,69−71 and is of particular interest as complexing with PS2.M enhances the peroxidase activity of hemin.83 T30695 (Table 1) is an HIV integrase inhibitor. In K+, it also adopts a chair antiparallel structure,73,74 while its Na+defined structure displays a CD spectrum similar to its spectrum in K+,72 also suggesting a primarily chair folding structure in Na+. These control DNAs were used as the chair G-quadruplex structure representatives. However, we observed rather weak and comparable fluorescence responses in K+ and Na+ solutions for ThT binding to these quadruplexes (Figure 2, panels A and B), and the binding strengths are similar in Na+ and K+ for each DNA (Table 1). These results indicate that ThT is inefficient for fluorescently targeting the G-quadruplex chair structure. DNA melting (Tm) experiments were also used to investigate the interactions of ThT with these G-quadruplexes. Positively charged ThT can be hydroxylated to a neutral state, thus forming a more hydrophobic species. The high temperature required for Tm experiments speeds up this process, which mainly occurs at neutral and alkaline pH.84 As shown in Figure S2 of the Supporting Information, there is a significant change in absorbance at 295 nm after hydroxylation, in addition to the absorbance change at 412 nm. However, G-quadruplex melting as a function of solution temperature is also usually monitored at 295 nm. In order to avoid this drawback in this work, the Tm measurements were carried out at a pH of 5.3 for the corresponding sodium and potassium phosphates, as ThT hydroxylates much less at this pH.84 Furthermore, the ThT concentration was 8 times higher than the DNA concentration. As shown in Figure 3, the K+-defined structures exhibit a higher thermal stability than the corresponding Na+-defined structures. Among these G-quadruplexes, both of the mPu22 and 1XAV cmyc quadruplexes have higher melting temperatures than the telomeric ones in 0.1 M K+, in agreement with the previous work.2 The addition of ThT induces a Tm change of between −1.3 and 6.3 °C, indicating ThT binding to the Gquadruplexes. However, in comparison with the fluorescence results, not all bindings significantly enhance ThT emission, showing the quadruplex structure-dependent molecular rotation inhibition of ThT. Note that an 11 °C increase in Tm has been reported for Tel22 in a K+ solution at concentration conditions similar to those used in this work, but that experiment was only carried out at a pH of 7.2,48 suggesting the effect of ThT hydroxylation on Tm in that work. Additionally, only small Tm changes were observed at the

Figure 2. (A) Dependence of ThT (2 μM) fluorescence intensity ratios (FK/FNa) in 0.1 M K+ and Na+ solutions on G-quadruplex sequences (1 μM). Inset: dependence of FNa/FK on the same sequences. From 1 to 15 for the quadruplexes: Tel22 (1), Tel26 (2), wtTel26 (3), TAG3 (4), TG3 (5), TAG3T (6), TTAG3 (7), mPu22 (8), 1XAV (9), PS2.M (10), T30695 (11), 2O3M (12), T3TT (13), T3TT3 (14), and T3T3T3 (15). (B) Photographs of these solutions under UV illumination (the same sequence from left to right as in A). The pink-filled circles refer to the hybrid quadruplexes.

flanking ends are pointing into different tetrads in opposite directions (Scheme 1). Thus, in comparison to the counterpart basket structure, ThT is most likely to bind favorably to the hybrid structure because of less steric interference. This is confirmed by the 4.3−21.5 times higher binding constants for ThT binding to the hybrid structures of these DNAs than the binding constants of the corresponding basket structures (Table 1). The oncogene c-myc and c-kit G-quadruplexes have a form that is different from the human telomeric G-quadruplexes and can form parallel structures in K+ solutions.6−10 These sequences provide a useful platform for carrying out control experiments with human telomeric structures. Here we used mPu22, 1XAV (from c-myc), and 2O3M (from c-kit, Table 1) as G-quadruplex models. As shown in Figure 2A, these oncogene G-quadruplexes indeed exhibit much weaker FK/ FNa responses than the telomeric sequences, whereas the FNa/ FK ratios, especially for mPu22 and 2O3M, are higher than those of the telomeric sequences (inset of Figure 2A). This comparison thus predicts a somewhat hybrid folding structure in Na+ for these oncogene DNAs. For example, mPu22 in K+ exhibits a typical parallel structure,24,66,67 while its structure24 in Na+ shows a circular dichroism (CD) spectroscopy profile similar to that of Tel22 in K+,56 suggesting the formation of a hybrid structure. This structure characteristic is confirmed by the 6.5 times higher binding constant in Na+ than in K+ (Table 1). 2O3M also adopts a parallel structure in a K+ solution.58,76 Even though its Na+-defined structure can not be accurately resolved, even by NMR,76 we believe it also contains a partial hybrid folding,75 as confirmed by the higher ThT binding constant in Na+ compared to K+ (Table 1). 1XAV in Na+ and K+ exhibits almost similar CD spectra, suggesting the formation of a main parallel structure in both solutions58,66,68 that is reflected by its comparable binding constants in K+ and Na+ solutions (Table 1). Apparently, these oncogene G-quad1626

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Figure 4. Absorption difference spectra of DNA (1 μM). The difference spectra were obtained by subtracting the NaCl spectra from the corresponding KCl spectra. Black curves: no ThT; red curves: 5 μM ThT. From 1 to 15 for the quadruplexes: Tel22 (1), Tel26 (2), wtTel26 (3), TAG3 (4), TG3 (5), TAG3T (6), TTAG3 (7), mPu22 (8), 1XAV (9), PS2.M (10), T30695 (11), 2O3M (12), T3TT (13), T3TT3 (14), and T3T3T3 (15).

Figure 3. Dependence of DNA melting temperature (Tm) on sequence. The melting temperatures were obtained in a phosphate buffer (25 mM, pH 5.3) containing 0.1 M Na+ or K+ with 2 μM DNA and 16 μM ThT. From 1 to 15 for the quadruplexes: Tel22 (1), Tel26 (2), wtTel26 (3), TAG3 (4), TG3 (5), TAG3T (6), TTAG3 (7), mPu22 (8), 1XAV (9), PS2.M (10), T30695 (11), 2O3M (12), T3TT (13), T3TT3 (14), and T3T3T3 (15).

ThT binding is very different from the results of some previous reports using G-tetrad-size-alike macrocyclic fluorophores as probes. For example, cresyl violet33 can convert G-quadruplex from an antiparallel structure to a hybrid structure, while a derived porphyrin analogue86 (although not a fluorescent probe) can convert a c-myc parallel quadruplex to an antiparallel one. On the other hand, it is the G-quadruplex structures that determine the ADS responses of the bound ThT between 350−500 nm (Figure 4). As observed in Figure 1B, we see that DNA with a hybrid structure only in K+ will produce a positive ADS peak at longer wavelengths and a negative ADS peak at relatively shorter wavelengths in the region of 350−500 nm, while DNA with a hybrid structure only in Na+ will produce inverse ADS peaks at the corresponding wavelengths. Indeed, all of the human telomeric DNAs (Tel22, Tel26, wtTel26, TAG3, TG3, TAG3T, TTAG3) and oxytricha telomeric T3T3T3 induce a 450 nm positive peak and a 400 nm negative peak, indicating that ThT is favorable to binding to the K+-determined (hybrid) structures (Table 1) with a restricted-molecule-rotating conformation. On the other hand, mPu22 and 2O3M induce a 450 nm negative peak, showing ThT is favorable to binding to their Na+-determined structures (also hybrid24,75). These results are in a good agreement with the fluorescence experiments illustrated in Figure 2. Previous quantum-chemical calculations indicate that the presence of the methyl group bound to nitrogen N5 of the BZT ring prevents the formation of a strictly planar conformation for free ThT molecules in an aqueous solution.78 In accordance with our results, the interaction of ThT with G-quadruplexes, particularly those with a hybrid folding, overcomes this coplanar energy barrier between the BZT and DMAB rings and results in the observed fluorescence enhancement. This molecular rotor mechanism had already been previously recognized as the mechanism responsible for ThT enhanced fluorescence response upon binding to amyloid fibrils.43−47 In this work, we used the commercially available BTA-1 (Figure 5A) to confirm that it is this same molecular rotor mechanism responsible for the enhanced G-quadruplex-switched ThT emissions. Compared to ThT, eliminating the methyl group at nitrogen N5 of the BZT ring in BTA-1 recovers the coplanar conformation.78 Note that the difference in the mono- and dimethyl group substitutions in the aniline moieties of ThT and BTA-1 has no effect on the molecular conformation. The strong emission of BTA-1 alone in an aqueous solution verifies

concentration ratios similar to those used in the fluorescence experiments ([ThT]:[DNA] = 2:1, Figure S3 of the Supporting Information), indicating that ThT binding exerts only a weak disturbance on the native quadruplex structures. By contrast, others have reported that G-tetrad-size-alike macrocyclic fluorophores26−29 can induce a 14−25 °C increase in Tm at ligand-to-DNA concentration ratios varying from 1:1 to 5:1. CD is used as a sensitive tool to identify distinct Gquadruplex structures, while induced CD (ICD) is usually observed upon binding of a fluorophore to a chiral DNA environment.85 ThT also has a strong absorption band between 200−300 nm that overlaps the DNA absorption region (Figure S2 of the Supporting Information), and a strong ICD response occurs when ThT interacts with DNA.48 Thus, it is not easy to accurately identify G-quadruplex structures by CD spectra as a result of ThT binding, since the intrinsic CD signal of the Gquadruplex is overlaid with the significant ICD signal between 200−300 nm of the bound ThT. In order to overcome this drawback, in this work the absorption difference spectra (ADS) of the DNA is used to measure ThT binding and as a tool to identify whether the G-quadruplex structures change upon ThT binding. The spectra were obtained by subtracting the NaCl spectra from the corresponding KCl spectra (i.e., AK − ANa). As shown in Figure 4, all of the telomeric G-quadruplexes of Tel22, Tel26, wtTel26, TAG3, TG3, TAG3T, and TTAG3 exhibit a 275 nm positive ADS peak and a 297 nm negative ADS peak in the absence of ThT (black curves), showing the different foldings in K+ and Na+. However, the other DNA samples exhibit distinct structures that are different from the telomeric G-quadruplexes in either K+ or Na+. This is confirmed by, for example, the 262 nm positive ADS peak for mPu22 and 2O3M. Thus, the ADS peaks are DNA structure-dependent and can be used to identify any alterations in G-quadruplex conformation induced by ThT binding. Interestingly, the presence of ThT (red curves) gives an almost similar ADS response for a given DNA between 230 and 300 nm as exhibited by each DNA in the absence of ThT, indicating that ThT binding only slightly disturbs the native G-quadruplex structure, in agreement with the Tm experiments. The fact that no new peak appeared also confirms that the effect of ThT itself on DNA absorbance between 230−300 nm is mostly eliminated using ADS. The DNA structure stability upon 1627

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binding sites in the G-quadruplex: the groove, the top/bottom tetrad, and in between the tetrads (for intercalation reactions). Molecular modeling and quantum chemical calculations indicate that the DNA binding pattern of ThT is strongly dependent on the quadruplex structures. The calculated docking results show that ThT only binds to the parallel quadruplex structure in the DNA groove region, but that stacking on the top tetrad, as depicted in Scheme 1, occurs for the other quadruplex structures.48 Furthermore, a 1:1 complex with ThT stacking on the tetrad is the main contributor to the enhanced fluorescence signal.53 Therefore, based on our results, we expect that only the hybrid G-quadruplex can have ThT stacking on the tetrad and so induce a restriction in ThT rotation, while the other binding modes such as the groove binding that occurs for parallel G-quadruplexes48 have less potential to achieve a rotation-restricted ThT state favorable for fluorescence emission. Both the ThT binding strength and the binding-favored ThT molecular rotation restriction contribute to increased fluorescence. The BZT ring in ThT hydroxylates at alkaline pH and at elevated temperatures to produce a nonfluorescent species.84 We predict that if ThT binds to hybrid G-quadruplexes with the BZT ring lying at the tetrad according to the stacking model, the bound ThT should be well-protected from hydroxylation. To further confirm the binding pattern of ThT at the G-quadruplexes, a ThT solution in the presence of excess DNA was incubated in potassium and sodium phosphate buffers at pH 8.4 and 35 °C. The hydroxylation kinetics was then measured along with ThT fluorescence as a function of incubation time (Figure 6A). Excess DNA was used to keep most of the ThT in a DNA-bound state. The hydroxylation conditions of pH 8.4 and 35 °C did not disturb the Gquadruplex formation, as confirmed by the unchanged ThT fluorescence dependence on K+/Na+. As shown in Figure 6A using Tel26 and mPu22 as the typical examples, the hybrid structures of Tel26 (in K+) and mPu22 (in Na+) indeed exhibit lower hydroxylation kinetics than their corresponding counterpart structures (basket antiparallel and parallel, Scheme 1). The hybrid structure of mPu22 in Na+ seems to provide an even better protection for ThT than the K+-defined Tel26 hybrid structure. Thus, ThT stacking on the tetrad of hybrid structures makes it less exposed to solvent, at least for the BZT ring, than its binding to other G-quadruplex structures. Since the fluorescence originates from cooperation between the BZT and DMAB rings according to the molecular rotor mechanism,

Figure 5. (A) Structure of BTA-1. The rings of BZT and MAB are almost coplanar. (B) Fluorescence spectra of BTA-1 in the absence and presence of Tel22. Inset: the corresponding absorption spectra. (C) Dependence of BTA-1 (2 μM) fluorescence intensity ratios in 0.1 M K+ and Na+ (FK/FNa) solutions on the G-quadruplex sequences (1 μM). From 1 to 15 for the quadruplexes: Tel22 (1), Tel26 (2), wtTel26 (3), TAG3 (4), TG3 (5), TAG3T (6), TTAG3 (7), mPu22 (8), 1XAV (9), PS2.M (10), T30695 (11), 2O3M (12), T3TT (13), T3TT3 (14), and T3T3T3 (15).

(Figure 5B) that it is not an efficient molecular rotor, although the excitation and emission wavelengths are different from those of ThT. This result is also strengthened by the fact that G-quadruplexes result in comparable BTA-1 fluorescence responses independent of the hybrid, parallel, and antiparallel structures (Figure 5C). The notion that BTA-1 emission is controlled by a mechanism other than molecular rotor was also confirmed by the unchanged absorption spectra in the presence of G-quadruplex structures (inset of Figure 5B). Analysis of the G-Quadruplex Structure-Dependent Binding Mode of ThT. Although we found that the binding constants varied among the G-quadruplex sequences, the role of the G-quadruplex structures on ThT binding can be identified for a given G-quadruplex sequence: DNA with a hybrid structure exhibits a higher binding constant than its counterpart structure, whereas DNA with a structure other than hybrid exhibits a somewhat comparable binding constant in Na+ and K+ solutions (Table 1). There are three possible ThT

Figure 6. (A) Dependence of emission intensity of 0.5 μM ThT at 487 nm on hydroxylation time. The solutions were incubated in 0.1 M potassium and sodium phosphate buffers (pH 8.4) at 35 °C in the presence of 15 μM mPu22 (circle) and Tel26 (triangle). F0 and F are the fluorescence intensities at the initial and extended incubation times at 35 °C, respectively. (B) Dependence of 1 μM ThT fluorescence at 487 nm in the presence of 1 μM mPu22 (circle) and Tel26 (triangle) on Tris-HCl (pH 7.5) concentrations. The solutions contain either 0.1 M KCl or NaCl at room temperature. F0 and F are the fluorescence intensities at 25 mM and increased Tris-HCl concentrations, respectively. The solid lines are the fitted curves according to the first-order exponential decay. 1628

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after K+ introduction, since no time evolution was observed in fluorescence, which indicates fast kinetics in the folding.87 Thus, ThT can serve as a useful probe to follow G-quadruplex structure conversion. The results presented in Figure 7 also point toward a potential sensor platform for K+ detection. It is well-known that K+ plays an important role in the human body by maintaining extracellular osmolarity, balancing pH, and ensuring normal neuromuscular function.88 A deficiency or excess of K+ can also cause hyperkalemia and hypokalemia. As such, a highly sensitive and specific system for K+ detection would be extremely useful. However, one must consider the selectivity of the proposed method for K+ identification, owing to the presence of sodium and other cations in normal physiological conditions, and so K+ detection with high selectivity and low fluorescence background remains a challenge.15,21,24,30,31,89 To test the specificity of this new sensor, metal ion selective assays were conducted by measuring the fluorescent response of the ThT/Tel22 complex toward 2 mM solutions of metal ions (Na+, Mg2+, Fe3+, Fe2+, Al3+, Pb2+, Zn2+, Ni2+, Mn2+, Cu2+, Co2+, and Cd2+). Note that some of the heavy metal ions were subject to hydrolization in the detection solution (pH 7.5), so they were pretreated with sufficient EDTA before analysis (10 mM for this work). This EDTA treatment was also useful for avoiding the possible effects of these multivalent metal ions upon their backbone binding-induced DNA conformation changes90 and specific ion-favored G-quadruplex structures (for example, Pb2+91). As shown in Figure 8A, a dramatic enhancement in ThT fluorescence only appears for K+, suggesting that the probe indeed has a high selectivity for the K+ assay. Additionally, the coexistence of the other metal ions with K+ created a response similar to the florescence achieved with K+ alone (Figure 8B). Therefore, the method proposed here is highly selective for K+ over other metal ions (see Table S1 of the Supporting Information for a comparison with the other reported methods). In particular, ThT itself seems to have no direct interaction with these metal ions in the presence of EDTA, thus assuring a high selectivity. The feasibility of this detection method was further verified by recovery experiments. We added 1 mM K+ to each of three water sample solutions, then measured the added K+ contents with recoveries in the range of 97−108% (Table S2 of the Supporting Information). These results show the potential for using the proposed G-quadruplex/ThT platform to develop a selective K+ sensor.

the DMAB ring should also simultaneously stack on the tetrad of hybrid G-quadruplex structures (Scheme 1). Additionally, since ThT is a positively charged molecule, its binding to G-quadruplexes in the presence of 0.1 M K+ and Na+ with increasing Tris-HCl concentrations was also investigated to show the possible contribution of the electrostatic force in G-quadruplex binding. As shown in Figure 6B, the ThT fluorescence exponentially decays for Tel26 in Na+ and mPu22 in K+ with increasing Tris-HCl concentrations, whereas Tel26 in K+ and mPu22 in Na+ (both are hybrid structures) exhibit almost stable emissions. Subsequently, we can conclude that ThT binding to hybrid G-quadruplexes and its stacking on the tetrad experience much less contribution from electrostatic force than binding to the other structures. Thus, the theoretically predicted groove binding of ThT to parallel Gquadruplex48 is believed to have a significant component resulting from electrostatic interaction. However, according to the 1:1 binding stoichiometry in G-quadruplex−ThT complexes obtained by fluorescence titration, and the remaining fluorescence at high electrolyte concentration, there should also be other interaction forces besides the electrostatic force simultaneously tuning the ThT binding to G-quadruplexes possessing structures other than hybrid. In this case, it is possible that only the DMAB ring would stack on the tetrad, while the BZT ring is likely to be located at the DNA site that is more exposed to the solvent (Scheme 1), as confirmed by the hydroxylation and electrolyte experiments. Monitoring Structure Conversion of G-Quadruplex with ThT as the Probe and a K+ Sensor. ThT’s selective fluorescence lighting up upon binding to the hybrid structure creates a new way to easily monitor structure conversion of the G-quadruplex. As shown in Figure 7 (using Tel22 as a typical

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Figure 7. Fluorescence spectra of 1 μM ThT and 1 μM Tel22 with increasing K+ concentration in 25 mM Tris-HCl buffer (pH 7.5) containing 100 mM Na+. Inset: Dependence of fluorescence intensity (F) at 487 nm on the added K+ concentration relative to F0 in the absence of K+.

CONCLUSIONS In this work, we have demonstrated that ThT serves as a novel fluorescent probe that can selectively target G-quadruplexes with a hybrid structure through a turn-on response. UV−vis absorption spectra, fluorescence spectra, and Tm experiments were used to confirm the binding specificity. Moreover, the ThT binding showed no significant disturbance on the native G-quadruplex structures in both Na+ and K+ solutions. The fluorescence enhancement is believed to be caused by rotation restriction of BZT and DMAB rings in the ThT excited state upon its G-quadruplex binding through a molecular rotor mechanism. This mechanism was confirmed by comparing the florescence using a ThT analogue of BZT-1 that is not a molecular rotor. Hydroxylation and electrolyte experiments demonstrate that the ThT binding model is strongly dependent on the native G-quadruplex structure. Being stacked on the tetrad occurs for the hybrid structure, whereas electrostatic

example), the fluorescence intensity at 487 nm gradually increases as the K+ concentration increases from 0.1 to 50 mM, even in the presence of 100 mM Na+, indicating that the Na+defined structure converted to a K+-defined hybrid structure. Introducing K+ into a Na+ solution containing ThT/Tel22 results in an emission that is comparable to the emission that occurs when introducing ThT into a K+/Na+ solution containing Tel22, meaning that the initial presence of ThT has no effect on conversion. A good linear relationship was found for K+ concentrations, varying from 0.1 to 20 mM (inset of Figure 7), with a detection limit of 1 mM in coexistence with 100 mM Na+. The conversion obviously finished immediately 1629

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Figure 8. Selective responses of ThT/Tel22 to K+. (A) The presence of individual metal ion (2 mM, F0 and F stand for the fluorescent intensities in the absence and presence of the metal ions, respectively). (B) The coexistence of the other metal ions (2 mM) with K+ (2 mM) (F0 and F stand for the fluorescent intensities at K+ alone and K+, respectively, in coexistence with the other metal ions). Experimental conditions: 25 mM Tris-HCl (pH 7.5) containing 1 μM Tel22, 1 μM ThT, and 10 mM EDTA. (9) Shirude, P. S.; Okumus, B.; Ying, L.; Ha, T.; Balasubramanian, S. J. Am. Chem. Soc. 2007, 129 (24), 7484−7485. (10) Balasubramanian, S.; Hurley, L. H.; Neidle, S. Nat. Rev. 2011, 10 (4), 261−275. (11) Vummidi, B. R.; Alzeer, J.; Luedtke, N. W. ChemBioChem 2013, 14 (5), 540−558. (12) Ma, D. L.; He, H. Z.; Leung, K. H.; Zhong, H. J.; Chan, D. S. H.; Leung, C. H. Chem. Soc. Rev. 2013, 42 (8), 3427−3440. (13) He, H. Z.; Chan, D. S. H.; Leung, C. H.; Ma, D. L. Nucleic Acids Res. 2013, 41 (8), 4345−4359. (14) Lee, J. Y.; Kim, D. S. Nucleic Acids Res. 2009, 37 (11), 3625− 3634. (15) Kim, B.; Jung, I. H.; Kang, M.; Shim, H. K.; Woo, H. Y. J. Am. Chem. Soc. 2012, 134 (6), 3133−3138. (16) Gray, R. D.; Petraccone, L.; Trent, J. O.; Chaires, J. B. Biochemistry 2010, 49 (1), 179−194. (17) Dao, N. T.; Haselsberger, R.; Michel-Beyerle, M. E.; Phan, A. T. FEBS Lett. 2011, 585 (24), 3969−3977. (18) Arthanari, H.; Basu, S.; Kawano, T. L.; Bolton, P. H. Nucleic Acids Res. 1998, 26 (16), 3724−3728. (19) Wei, C.; Wang, J.; Zhang, M. Biophys. Chem. 2010, 148 (1−3), 51−55. (20) Zhang, Z. X.; Sharon, E.; Freeman, R.; Liu, X. Q.; Willner, I. Anal. Chem. 2012, 84 (11), 4789−4797. (21) Li, T.; Wang, E.; Dong, S. Anal. Chem. 2010, 82 (18), 7576− 7580. (22) Qin, Y.; Hurley, L. H. Biochimie 2008, 90 (8), 1149−1171. (23) Alzeer, J.; Vummidi, B. R.; Roth, P. J. C.; Luedtke, N. W. Angew. Chem., Int. Ed. 2009, 48 (49), 9362−9365. (24) Qin, H. X.; Ren, J. T.; Wang, J. H.; Luedtke, N. W.; Wang, E. K. Anal. Chem. 2010, 82 (19), 8356−8360. (25) Alzeer, J.; Luedtke, N. W. Biochemistry 2010, 49 (20), 4339− 4348. (26) Ma, D. L.; Che, C. M.; Yan, S. C. J. Am. Chem. Soc. 2009, 131 (5), 1835−1846. (27) Yao, J. L.; Gao, X.; Sun, W. L.; Fan, X. Z.; Shi, S.; Yao, T. M. Inorg. Chem. 2012, 51 (23), 12591−12593. (28) Yao, J. L.; Gao, X.; Sun, W. L.; Fan, X. Z.; Shi, S.; Yao, T. M. Dalton Trans. 2013, 42 (16), 5661−5672. (29) Yang, P.; De Cian, A.; Teulade-Fichou, M. P.; Mergny, J. L.; Monchaud, D. Angew. Chem., Int. Ed. 2009, 48 (12), 2188−2191. (30) Kong, D. M.; Guo, J. H.; Yang, W.; Ma, Y. E.; Shen, H. X. Biosens. Bioelectron. 2009, 25 (1), 88−93. (31) Kong, D. M.; Guo, J. H.; Yang, W.; Ma, Y. E.; Shen, H. X. Anal. Chem. 2009, 81 (7), 2678−2684. (32) Bhasikuttan, A. C.; Mohanty, J.; Pal, H. Angew. Chem., Int. Ed. 2007, 46 (48), 9305−9307. (33) Verma, S. D.; Pal, N.; Singh, M. K.; Shweta, H.; Khan, M. F.; Sen, S. Anal. Chem. 2012, 84 (16), 7218−7226.

force contributes more of the binding force for the other Gquadruplex structures. As a G-quadruplex conformation probe, ThT can be used to monitor G-quadruplex structure conversion between the hybrid structure and other conformations, and a G-quadruplex/ThT platform has potential as a label-free fluorescent K+ sensor. In conclusion, we believe that ThT has potential application for structure identification of Gquadruplexes and for the construction of G-quadruplex-based sensors.

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ASSOCIATED CONTENT

S Supporting Information *

Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Fax: 86 579 82282595. 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 (Grant 21075112), the Zhejiang Provincial Natural Science Foundation of China for Distinguished Young Scholars (Grant LR12B05001), and the Foundation of State Key Laboratory of Electroanalytical Chemistry, Changchun Institute of Applied Chemistry (Grant SKLEAC2010001). C.H. thanks the support of the National Natural Science Foundation of China (Grant 21175117).

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