Quantum Dots–Ligand Complex as Ratiometric Fluorescent

Oct 1, 2016 - By complexing a nonionic G-quadruplex ligand with hybrid dual-emission quantum dots (QDs), a ratiometric fluorescent nanoprobe is develo...
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Quantum Dots—Ligand Complex as Ratiometric Fluorescent Nanoprobe for Visual and Specific Detection of G-quadruplex Haojun Jin, Yuqian Liu, Tianshu Xu, Xiaojun Qu, Feika Bian, and Qingjiang Sun Anal. Chem., Just Accepted Manuscript • Publication Date (Web): 01 Oct 2016 Downloaded from http://pubs.acs.org on October 2, 2016

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Quantum Dots—Ligand Complex as Ratiometric Fluorescent Nanoprobe for Visual and Specific Detection of G-quadruplex Haojun Jin, Yuqian Liu, Tianshu Xu, Xiaojun Qu, Feika Bian and Qingjiang Sun* State Key Laboratory of Bioelectronics, School of Biological Science & Medical Engineering, Southeast University, Nanjing 210096, China *Fax: (86) 25-83792349. Email: [email protected]

ABSTRACT: By complexing a non-ionic G-quadruplex ligand with hybrid dual-emission quantum dots (QDs), a ratiometric fluorescent nanoprobe is developed for G-quadruplex detection in a sensitive and specific manner. The QDs nanohybrid comprised of a green-emission QD (gQD) and multiple red-emission QDs (rQDs) inside and outside of a silica shell, respectively, is utilized as the signal displaying unit. Only the presence of G-quadruplex can displace the ligand from QDs, breaking up the QDs—ligand complexation, and inducing the restoration of the rQDs fluorescence. Since the fluorescence of embedded gQD stays constant, variations of the dual-emission intensity ratios display continuous color changes from green to bright orange, which can be clearly observed by naked eyes. Furthermore, by utilizing competitive binding of a cationic ligand versus the non-ionic ligand toward G-quadruplex, the nanoprobe is demonstrated to be applicable for assessing the affinity of a G-quadruplex-targeted anti-cancer drug candidate, exhibiting ratiometric fluorescence signals (reverse to that for G-quadruplex detection). By making use of the specificity of the ligand binding with G-quadruplex against double helix, this nanoprobe is also demonstrated to be capable of sensitive detection of one-base mutation, exhibiting sequence-specific ratiometric fluorescence signals. By functionalizing with a nuclear localization peptide, the nanoprobe can be used for visualization of G-quadruplex in the nucleus of human cells.

INTRODUCTION G-quadruplexes are defined by layers of stacked G-tetrads, each of which contains four guanine residues that interact 1 through Hoogsteen hydrogen bonding interactions. G-quadruplexes can form in vitro, and their topologies depend on the sequences and the experimental conditions 2 (e.g. coexisting metal ions, and degree of molecular 3 crowding). In vivo, G-quadruplexes have been evoked in controlling the expression of certain oncogenes as well as in 4 the perturbation of telomeric organization. Fluorescent probes have gained tremendous interest recently, providing a powerful and visual means of studying G-quadruplexes and 5-7 their biological functions. The probes reported to date are fluorescent G-quadruplex ligands, which structurally feature with an extended π-surface, together with net cationic 8 charges. By tuning their shape/size, these molecular probes have exhibited high affinity as well as improved specificity to recognize G-quadruplex against duplex (which is abundant 8-13 in nuclei). Despite this notable progress, these molecular probes still face significant challenges for the utilization as 14-16 valuable in vitro and cellular probes. The molecular probes suffer from guanine-mediated quenching through photo-induced electron transfer upon stacking on the 17 G-tetrad surface. More seriously, nonspecific electrostatic interaction of molecular probes with G-quadruplex and duplex DNA (both are anionic polyelectrolytes) always remains, which may result in false positives of fluorescence 18 signal. In this regard, non-ionic ligands are preferred, 19 whereas they have less solubility. Therefore, there is a high

demand for the development of new fluorescent probes with further improved specificity and sensitivity for recognizing G-quadruplexes. Ratiometric fluorescent assays can achieve improved sensitivity and accuracy by providing built-in correction for environmental conditions, as well as improved visualization 20 capability by displaying continuous color changes. The QDs are advantageous over organic fluorophores in terms of broad absorption and narrow, size-tunable emission, enabling different-band emissions by a single-wavelength 21,22 excitation, and thus are superior for constructing 23-25 multi-colored fluorescent systems. In the past few years, ratiometric fluorescent nanoprobes based on dual-color QDs systems have been successfully developed for visual and 26,27 28 29 sensitive detection of metal ions, TNT, amino acids, 30 and duplex DNA. In this work, we report on the development of a non-ionic G-quadruplex ligand, dipyrido-[3,2-a:2’,3’-c] phenazine-imidazolone (DI), complexed with a dual-emission QDs nanohybrid, as a nanoprobe for G-quadruplex detection. DI was synthesized via a Schiff base condensation reaction, with the π-surface size closely matching with that of G-tetrad. The QDs nanohybrid was constructed by embedding one CdSe/ZnS gQD in silica and assembling multiple InP/ZnS rQDs onto the silica surface, respectively (Scheme 1A). By a facile phase transfer process, DI dissolved in 1-butanol, goes to the surface of rQDs in aqueous phase, forming the complex (referred to as QDs-DI).

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DI quenches the rQDs fluorescence through charge transfer (CT). Importantly, displacement of DI can occur in the presence of even trace concentrations of G-quadruplex, breaking up the QDs—DI complexation (Scheme 1B). Consequently, the CT process is eliminated and the rQDs fluorescence is restored. Since the gQDs fluorescence stays constant, sensitive G-quadruplex detection is realized via ratiometric fluorescence signals, while duplex DNA/RNA cannot produce the similar result. Advances of the QDs-DI complex as a fluorescent G-quadruplex nanoprobe include: (1) since the DI ligand is non-ionic, this nanoprobe is advantageous in specificity and sensitivity over most of molecular probes (cationic ligands) for recognizing G-quadruplex, avoiding the interference from nonspecific electrostatic association; (2) this G-quadruplex assay is completely resistant to guanine-mediated quenching, since the fluorescence of DI is not utilized; (3) this nanoprobe is well suitable for quantitative visualization of G-quadruplex by displaying distinct emission-color changes. Interestingly, by utilizing a reverse displacement of the DI ligand (i.e., from G-quadruplex to QDs), applications of the nanoprobe for assessing the affinity of an anti-cancer drug candidate and recognizing one-base mutation can be demonstrated.

Scheme 1. Illustration of (A) the preparation of QDs-DI, and (B) principles for specifically detecting G-quadruplex and one-base mutation.

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TCI. Single-stranded DNA (ssDNA), 22AG, Oxy-4, molecular beacon (MB), and other forms of DNA as well as messenger RNA (mRNA) were purchased from Invitrogen (Shanghai, China), and their sequences are listed in Table S1 in the Supporting Information. The pure water (ρ> 18.2 MΩ cm) was obtained from a Pall Cascade AN synthesis system. Synthesis of DI, DPPZ and [Ru(bpy)2DI]·(PF6)2. The DI ligand was synthesized via a Schiff base condensation 31 reaction. Typically, 0.105 g of 1,10-phenanthroline-5,6-dione and 0.082 g of 5,6-diaminobenzimidazolone-2 were mixed and refluxed for 20 minutes in ethanol. Yellow needle-like crystals were collected and purified by recrystallization. 0.165 1 g of DI was obtained in a yield of 97.6%. H NMR [CF3COOD, 400 MHz]: δ 10.15 (1H, d), 9.44 (1H, d), 8.55 (1H, s), 8.46 (1H, q). MS: calcd For C19H10N6O 338.3; found 338. DPPZ and [Ru(bpy)2DI]·(PF6)2 were synthesized according to the 31,32 literatures, respectively. Preparation of the QD nanohybrid. The synthesis of oil soluble gQDs (CdSe/ZnS, EM: 508 nm) and water soluble mercaptopropionic acid-capped rQDs (InP/ZnS, EM: 585 nm) 33,34 were described previously. The gQD@SiO2 were prepared 27 by a reverse microemulsion approach. The concentrations of gQD@SiO2 and rQDs were estimated according to the 35 Dual-emission gQD@SiO2@rQD established method. nanohybrids were prepared by dropwise adding certain amounts of rQDs into 0.085 μM of gQD@SiO2 with stirring for 20 minutes. Preparation of the nanoprobes. The QDs-DI was prepared via a phase transfer process. Typically, Tris-HCl buffer (10 mM, pH 7.4) solution of gQD@SiO2@rQD and 1-butanol solution of DI, equal volume of each, were mixed and shaked for 2 min. After a 10-minute standing, the two solutions in the cuvette were stratified. The upper 1-butanol solution was abandoned and the aqueous solution was stored. The QDs-DPPZ was prepared following the similar procedure of QDs-DI except that DPPZ was used. The 2+ QDs-[Ru(bpy)2DI] was prepared by directly mixing 4 μM of 2+ the [Ru(bpy)2DI] solution with equal volume of the gQD@SiO2@rQD solution. To prepare the QDs-MB nanoprobe, gQD@SiO2@rQD was dispersed in borate-boric acid buffer (10 mM, pH 7.4), followed by the addition of 10 μM MB and 500 μM EDC. The mixture was allowed to react for 2 h at room temperature, followed by an ultra-filtration for 3 times to remove excess of EDC and MB. All the above nanoprobes were re-dispersed in 1% FBS buffer for further use.

EXPERIMENTAL SECTION Reagents and chemicals. Ethanol (99%), 1-butanol (99%), and o-phenylenediamine (98%) were supplied by Alfa Aesar. 5,6-dinitrobenzimidazolone-2 and 1,10-phenanthroline-5,6-dione (97%) were purchased from Aladdin. Calf thymus DNA (referred to as dsDNA), poly(AT)n, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC, 99%) and fetal bovine serum (FBS) were purchased from Sigma-Aldrich. Hemin was purchased from

Fluorescence measurements. All the fluorescence measurements were conducted with a 0.2 cm-thick quartz cuvette, an excitation wavelength at 405 nm and a 5 nm slit. Stability of QDs-DI was investigated by challenging it with + Tris-HCl buffer containing varying K concentrations (0-1000 mM) or Tris-HCl buffer at simulated physiological condition + + 2+ (100 mM for K , 140 mM for Na , 2.5 mM for Ca , 1 mM for 2+ 2+ 3+ 2+ Mg , 20 μM for Fe , 2.5 μM for Fe , 6 μM for Zn , 1 μM for 3+ 2+ 2+ 2+ 2+ Cr and Mn , 0.5 μM for Ni , Co and Cu , 1 μM for Cl , 22CO3 , HCO3 , H2PO4 and HPO4 , 4 μM for Cys, His and BSA, and 1 μM for Glucose). Biocompatibility of QDs-DI as well as gQD@SiO2@rQD was evaluated by the MTT assay. Typically, G-quadruplex assays were performed in 1% FBS + buffer containing 100 mM K . Prior to fluorescent detection,

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22AG or Oxy-4 was induced into G-quadruplex by heating at 90 °C for 5 min, cooling to room temperature, and incubating at 4 °C overnight. G-quadruplex detection was accomplished by equally mixing solutions of QDs-DI with different concentrations of 22AG or Oxy-4 G-quadruplex for fluorescence measurements. For specificity experiments, 2+ solutions of QDs-DI, QDs-DPPZ or QDs-[Ru(bpy)2DI] were equally mixed with solutions of 1.6 μM of G-quadruplex, poly(AT)n, ssDNA and mRNA, and 11.2 mg/L of dsDNA, respectively. For competition experiments, solutions of 40 nM of G-quadruplex co-existed with 0, 1, 5, 10, 20 and 50 equiv. of either dsDNA, poly(AT)n, ssDNA or mRNA were prepared and equally mixed with solutions of QDs-DI, respectively. For assessing the affinity of hemin with G-quadruplex, solutions of QDs-DI (in the presence of 4 μM 22AG G-quadruplex) were equally mixed with solutions of different concentrations of hemin, respectively. For one-base mutation detection, solutions of QDs-DI (in the presence of 1.6 μM G-quadruplex) or QDs-MB were equally mixed with solutions containing 1.6 μM of different DNA sequences and incubated for 1 h at room temperature, respectively. Cellular imaging. TAT-QDs-DI was prepared by 36 conjugating TAT peptides with gQD@SiO2, and subsequently assembling rQDs onto TAT-gQD@SiO2 and transferring DI onto rQDs. L02 cells and MCF-7 cells were seeded in a Lab-Tek 8 well chambered coverglass (Nunc), respectively. TAT-QDs-DI (0.5 μM for rQDs and 4 μM for DI) was added to the cell medium. After incubation for 2, 6, 10 and 24 h, the cells were washed with PBS, fixed with paraformaldehyde, stained with Hoechst and observed under a confocal laser-scanning microscope (For Hoechst, excitation wavelength was 405 nm, and emission in 450-470 nm was collected; for TAT-gQD@SiO2 and rQDs, excitation wavelength was 488 nm, and emission in 498-518 nm and 575-595 nm was collected, respectively). The fluorescence for single cell was analyzed by ImageJ. Calculation of the binding constant of DI with G-quadruplex. The binding constant was calculated with 37,38 the following equations: (1)

I =1− k × CDI I0  2  − (KnCG-quadruplex− KCDI +1) + (KnCG-quadruplex− KCDI +1) + 4KCDI   I'   =1 − k ×  I0 2 K   

1 2

      

(2) where in Eq. 1, I0 and I are the red-emission intensity of QDs in the absence/presence of DI, respectively; CDI is the concentration of DI transferred onto rQDs; k is the slope of the linear regression curve representing the relationship between I/I0 and CDI; in Eq. 2, I’ is the red-emission intensity of QDs-DI in the presence of G-quadruplex, and I0 is the same as in Eq. 1; K is the apparent binding constant of DI with G-quadruplex; n is the number of binding sites on a G-quadruplex; CG-quadruplex is the concentration of G-quadruplex. Instruments. Fluorescence spectra were collected with a fluorescence spectrometer (Hitachi F-7000, Japan). UV/Vis absorption spectra were acquired on a Hitachi U-4100

1

spectrophotometer. The H NMR spectrum was recorded on a Bruker DRX-400 NMR spectrometer. Mass spectrum was recorded on Micromass GC-TOF (70 eV). Fourier transform infrared (FT-IR) spectra were obtained on a Thermo Scientific Nicolet-5700 spectrometer with detector at 0.09 -1 cm resolution and 64 scans per sample. X-ray photoelectron spectroscopy (XPS) was acquired from a PHI 5000 VersaProbe spectrometer (ULVAC-PHI, Inc.) equipped with a monochromatic Al Kα source at room temperature. Zeta potential measurements were performed on a Malvern Zetasizer Nano-ZS particle analyzer. Transmission electron microscopy (TEM) images were taken on a JEOL JEM-2100 microscope. Confocal fluorescence images were taken on a confocal laser-scanning microscope (TCS SP8, Leica, Germany).

RESULTS AND DISCUSSION

Figure 1. (A) TEM image of gQD@SiO2@rQD. (B) Zeta potentials of NH2-gQD@SiO2 (a), rQD (b), gQD@SiO2@rQD (c) and QDs-DI (d). (C) XPS spectra of rQD (a), rQD-DI (b) and rQD-DI in the presence of 2 μM G-quadruplex (c). (D) Evolution of fluorescence spectra of gQD@SiO2@rQD as a function of DI concentration (5-70 μM) in 1-butanol. The right inset shows evolution of relative red-to-green fluorescence intensity ratio vs. the amount of DI transferred onto the QDs. The upper inset shows photographs of QDs-DI preparation via phase transfer. Formation of the QDs—Ligand complex. The formation process of QDs-DI complex was investigated in detail. Dual-emission QDs nanohybrid was first prepared by electrostatically assembling rQDs onto the surface of gQD@SiO2. The TEM image shows that the formed dual-emission QDs nanohybrid has a “sesame ball”-like structure, in which one gQD and multiple rQDs are located inside and outside of a ~11 nm-thick silica shell, respectively (Figure 1A). The thickness of silica shell well exceeds the 39,40 Förster radius (~5-7 nm), blocking fluorescence resonance energy transfer between the dual-emission QDs. As a result, the rQDs fluorescence is linearly increased with increased rQD-to-gQD@SiO2 ratio, while the gQDs fluorescence stays constant (Figure S1 in the Supporting Information). At an rQD-to-gQD@SiO2 ratio of 12:1, the QDs nanohybrid exhibits comparable dual emissions, along with

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the solution displaying bright orange color, which was selected as the scaffold to form QDs-DI complex. At this rQD-to-gQD@SiO2 ratio, zeta potential is dramatically changed from 37.6 mV for NH2-gQD@SiO2 to -42.4 mV for gQD@SiO2@rQD (Figure 1B). By phase transfer, QDs-DI complexes are formed, which remain mono-dispersed as the QDs nanohybrid (Figure S2 in the Supporting Information). The QDs—DI complexation was confirmed by FT-IR and XPS measurements. Characteristic vibration bands from DI -1 -1 -1 (3259.1 cm and 1737.5 cm for amide group; 1471.4 cm and -1 736.6 cm for benzene ring) appear in the FT-IR spectrum of QDs-DI (Figure S3 in the Supporting Information). The band peaked at 397.6 eV for N 1s signal and the band peaked at 1020.5 eV for Zn 2p3/2 signal, corresponding to Zn-N binding, appear in the XPS spectrum of rQD-DI (Figure 1C), indicating that the complexation is through the coordination of surface Zn atom of InP/ZnS rQDs with two nitrogen atoms 41 of the phenanthroline moiety of DI. Due to the covalent 42,43 bonding character of N-Zn-N coordination, the rQD—DI complexation has relatively high affinity, allowing the phase transfer accomplished within 2 min. With respect to dual strong emissions of the QDs nanohybrid, QDs-DI show quenched rQDs fluorescence, which is dependent on the amount of DI used during the phase transfer (Figure 1D, and Figure S4 in the Supporting Information). By recording the absorption changes of DI in 1-butanol before/after phase transfer, it is found that the amount of DI molecules transferred onto rQDs is proportional to the DI concentration at a constant volume, or the volume of 1-butanol solution at a fixed DI concentration (Figure S5 in the Supporting Information). Furthermore, by comparing the amount of DI transferred with that before phase transfer, the transfer efficiency of DI is calculated to be 20% within a wide concentration/volume range, i.e., one fifth of DI molecules in 1-butanol can be transferred onto rQDs in aqueous phase. Notably, the transfer of DI is saturated when large amounts of DI were used. For instance, by equally mixing solutions of 70 μM DI with gQD@SiO2@rQD (with the rQDs concentration of 1 μM), the transfer of DI starts to be saturated, indicating that at most 14 DI molecules can be transferred onto one rQD of the QDs nanohybrid. The quenching effect of rQDs fluorescence by complexed DI molecules is further analyzed by the Stern-Volmer equation (Figure 1D, the right inset). It can be seen that 1-8 DI molecules per rQD can quench the rQDs fluorescence with 5 -1 the high quenching constant of 1.7 × 10 M , whereas the quenching becomes less efficient at higher DI/rQD ratios. Thus, during the phase transfer, the DI/rQD ratio of 8 was controlled for constructing the nanoprobe. At this DI/rQD ratio, the fluorescence of rQDs is quenched to be ~18% of the original, along with emission color of the solution changed from bright orange to green (Figure 1D, the upper inset).

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Figure 2. (A) Fluorescence spectra of QDs-DI in the presence of various amounts of 22AG G-quadruplex. The inset: photographs of buffer solutions of rQD-DI and QDs-DI in the presence of different amounts of 22AG G-quadruplex. (B) Evolution of relative red-to-green fluorescence intensity ratio of QDs-DI vs. 22AG or Oxy-4 G-quadruplex concentration. The inset shows G-quadruplex structures of 22AG and Oxy-4. Visual detection of G-quadruplex by the QDs—ligand nanoprobe. Prior to G-quadruplex detection, fluorescence stability of the QDs-DI nanoprobe was evaluated under different experimental conditions (Figure S6 in the Supporting Information). It is found that the fluorescence of QDs-DI is insensitive to ionic strength (0-1000 mM KCl). It also is tolerant of different types of ions such as abundant + 2+ 2+ cellular cations (Na , Ca , Mg ), trace cations in organism 2+ 2+ 2+ 2+ 2+ 2+ 3+ 3+ (Fe , Zn , Cu , Ni , Co , Mn , Cr , Fe ), and anions (Cl , 22CO3 , HCO3 , H2PO4 , HPO4 ). Importantly, the fluorescence of QDs-DI is stable in the presence of physiologically important biomolecules such as Cys, His, BSA, and Glucose, indicating that DI complexed with rQDs cannot be displaced by these thio/amino/hydroxyl-containing species. As such, the QDs-DI was used to detect 22AG human telomeric DNA + + in 1% FBS buffer containing 100 mM K . At this K concentration, 22AG formed into G-quadruplex, which 44 consists of three layers of stacked G-tetrads. In all the assays, the final concentration of QDs-DI was kept as 0.5 μM for rQDs and 4 μM for DI. The response time was defined as 10 minutes (Figure S7 in the Supporting Information). As shown in Figure 2A, the QDs-DI exhibits a ratiometric fluorescent response to a dose of 22AG G-quadruplex. The red-to-green fluorescence intensity ratio is increased with increased G-quadruplex concentration. At a G-quadruplex concentration of 2 μM, the increment of red-to-green fluorescence intensity ratio reaches the maximum (4.3-fold), which indicates that the stoichiometry of DI/G-quadruplex is 2:1. Due to the well-resolved dual emissions and the large range of red-to-green fluorescence intensity ratios, the solutions display continuous color changes from green to bright orange, which can be clearly distinguished by naked eyes under a UV lamp (the inset of Figure 2A). Unlike the ratiometric fluorescence, the color changes of single-emission rQD-DI solution upon exposure of G-quadruplex are hard to observe. As shown in Figure 2B, the linear dynamic range is defined as 20 nM to 800 nM, which can be used for quantitative determination of

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G-quadruplex with a correlation coefficient of 0.995. Following the 3σ IUPAC criteria, the limit of detection is estimated to be 7 nM.

Figure 3. The confocal microscopy images of L02 cells and MCF-7 cells treating with TAT-QDs-DI for 24 h. From left to right: blue fluorescence images of the nucleus stained with Hoechst, green fluorescence images of TAT-gQD@SiO2, red fluorescence images of rQD and the overlay of bright-field and fluorescence images. Scale bars: 10 μm. Visualization of G-quadruplex in the nucleus of human cells. Since both silica-encapsulated CdSe/ZnS gQDs 45 and InP/ZnS rQDs are considered to be low toxicity, the nanoprobe exhibits good biocompatibility with living cells (Figure S8 in the Supporting Information). This allows us to visualize G-quadruplex structures in human cells by the nanoprobe. To facilitate nuclear internalization, the nanoprobe was functionalized with TAT peptides by conjugating the peptides onto the outer surface of silica 36 shell. The cellular uptake and subsequent localization of the nanoprobe (TAT-QDs-DI) for 2, 6 and 10 h incubations are shown in Figure S9 in the Supporting Information. After incubation for 10 h, the nanoprobe is populated in the cytoplasm and accumulated in the nuclei of L02 cells, as demonstrated by the green fluorescence from TAT-gQD@SiO2 and the blue fluorescence from Hoechst lighting up the nuclei. Figure 3 shows the confocal microscopy images for L02 cells and MCF-7 cells treating with TAT-QDs-DI for 24 h. The rQDs fluorescence can be clearly seen in the nuclei of these cells, and in contrast it is negligible in the cytoplasm, revealing the abundance of G-quadruplex in the nuclei and its deficiency in the cytoplasm. By the ImageJ analysis, the red-to-green fluorescence intensity ratios reach 0.74:1 for L02 cells and 0.65:1 for MCF-7 cells, respectively. These results suggest that by functionalization the QDs—ligand complex can be used as a reliable cellular probe for visualization of G-quadruplex in cells. High-affinity binding of DI with G-quadruplex. As proposed in our protocol, fluorescent detection by the nanoprobe relies on high-affinity binding of DI with G-quadruplex. Typically, there are three binding modes for ligand/G-quadruplex interactions: intercalation, end-stacking and electrostatic association. Since DI is non-ionic, electrostatic interaction is excluded. The preferential binding mode of DI with G-quadruplex was further investigated by treating the nanoprobe with different G-quadruplex structures. Oxytricha telomeric DNA (Oxy-4) induced G-quadruplex was used, which consists of four layers of stacked G-tetrads (the inset of Figure 2B). The fluorescence of QDs-DI also responds to Oxy-4 G-quadruplex (Figure S10 in the Supporting Information).

Interestingly, the nanoprobe exhibits the ratiometric fluorescence titration curve toward Oxy-4 G-quadruplex completely identical to 22AG G-quadruplex as shown in Figure 2B. The stoichiometry of DI/Oxy-4 G-quadruplex is also defined as 2:1, despite that Oxy-4 G-quadruplex has one more G-tetrad layer than 22AG G-quadruplex and consequently one more inner hydrophobic space between two adjacent G-tetrad layers for ligand intercalation. This result suggests that the preferential binding mode of DI with G-quadruplex is end-stacking (i.e., two DI molecules interact with G-tetrad layers at the two ends of one G-quadruplex by π-π stacking, irrespective of the number of inner G-tetrad layer). By nonlinear fit analysis of the fluorescence titration 5 data in Figure 2B with Eq.2, where k is deduced as 1.1 × 10 from Eq.1 and n is the stoichiometry, the apparent binding constant of DI toward G-quadruplex is calculated to be 2.7 × 6 -1 10 M . That the high-affinity binding toward G-quadruplex displaces DI from the rQDs was confirmed by the FT-IR and XPS measurements. By the presence of 2 μM of G-quadruplex, all characteristic vibration bands of DI disappear in the FT-IR spectrum of QDs-DI (Figure S3 in the Supporting Information), and the XPS spectrum of rQD-DI recovers to that of free rQDs (Figure 1C).

Figure 4. (A) Histogram on ratiometric fluorescence 2+ signals of QDs-DI, QDs-DPPZ and QDs-[Ru(bpy)2DI] in the presence of G-quadruplex, different forms of DNA or mRNA. (B) Histogram on ratiometric fluorescence signals of QDs-DI in the presence of 20 nM G-quadruplex coexisted with 1, 5, 10, 20, 50 equiv. of either different forms of DNA or mRNA. Specificity of the QDs—ligand nanoprobe for detecting G-quadruplex. Specificity toward G-quadruplex against other DNA forms and mRNA was evaluated by observing ratiometric fluorescent response of the nanoprobe (Figure 4A, and Figure S11 in the Supporting Information). In contrast to the remarkable response toward G-quadruplex, the fluorescence of QDs-DI does not respond to dsDNA, ssDNA or mRNA. By replacing DI with DPPZ, however, the fluorescence of QDs-DPPZ responds to G-quadruplex, dsDNA and mRNA. The distinct difference in specificity is ascribed to π-surface sizes of DI and DPPZ as well as their binding modes with G-quadruplex versus DNA/RNA double helix (Figure S12 in the Supporting Information). DPPZ, having a smaller π-surface size than DI, also binds with G-quadruplex via end-stacking. Therefore, the fluorescence of both QDs-DI and QDs-DPPZ responds to G-quadruplex. Differently, the binding mode of DPPZ and its analogues with dsDNA is intercalation from the minor groove, which is highly sensitive to their π-surface sizes of in-plane long

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axis. DPPZ has been established as the biggest ligand that 48 intercalate into dsDNA. DI, having the bigger size of in-plane long axis than DPPZ, cannot effectively intercalate into dsDNA. This is further emphasized by the finding that the fluorescence of QDs-DI also does not respond to poly(AT)n, which presents higher ratio of minor groove in the double helix than dsDNA (calf thymus DNA). The incapability of DI intercalation into double helix was confirmed by FT-IR measurements (Figure S13 in the Supporting Information). In the presence of excessive dsDNA or poly(AT)n, characteristic vibration bands of DI remain in the spectrum of QDs-DI, while those bands of DPPZ disappear from the spectrum of QDs-DPPZ, indicating that unlike DPPZ, DI cannot be displaced from the rQDs surface by either dsDNA or poly(AT)n. The mRNA has a secondary structure, with a loop and an 8-base pair stem, which provides double helix for DPPZ intercalation whilst not G-tetrad layer for DI stacking. Therefore, the presence of mRNA leads to the fluorescent response of QDs-DPPZ rather than QDs-DI. Both of G-tetrad layer and double helix are absent in ssDNA. Accordingly, the fluorescence of neither QDs-DI nor QDs-DPPZ responds to the ssDNA. Importantly, not only π-surface size but also electrical neutrality of DI contributes to its highly specific binding with G-quadruplex and consequently plays a vital role in the specific detection by the nanoprobe. As shown in Figure 4A, by replacing 2+ non-ionic DI with cationic [Ru(bpy)2DI] , the fluorescence 2+ of QDs-[Ru(bpy)2DI] responds to all DNA forms and mRNA, suffering from the interference from nonspecific electrostatic interaction between the cationic ligand and anionic polyelectrolytes. The high specificity of QDs-DI for recognizing G-quadruplex was further characterized by competition experiments. Up to a 50-fold excess of different forms of DNA or mRNA, respectively, were mixed with 20 nM of G-quadruplex, for fluorescent detection. The bare G-quadruplex solution was used as the control. As shown in Figure 4B, in all cases the nanoprobe exhibits identical fluorescent response, indicating that the DNA/RNA competitors cannot interfere the specific binding of DI with G-quadruplex. This means that 20 nM of G-quadruplex can be selectively detected by the nanoprobe from a mixed sample, in which 1 μM of other forms of DNA/RNA is coexisted.

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Figure 5. Scheme of assessing the affinity of hemin with G-quadruplex by QDs-DI. (A) Evolution of fluorescence spectra of QDs-DI (in the presence of 2 μM G-quadruplex) vs. hemin concentration (0-16 μM). Inset: photographs of buffer solutions of QDs-DI with 2 μM G-quadruplex in the presence of different amounts of hemin under a UV lamp. (B) Evolution of relative red-to-green fluorescence intensity ratio of QDs-DI (in the presence of 2 μM G-quadruplex) with respect to time upon exposure of different amounts of hemin. Assessing the affinity of hemin with G-quadruplex by the nanoprobe. Analogous to the established G-quadruplex 49 fluorescent “intercalator” displacement method, the QDs-DI can be used to assess the affinity of a G-quadruplex–targeted drug candidate such as hemin by observing the ratiometric fluorescent change of QDs. The assay was accomplished by sequentially exposing 2 μM of G-quadruplex and various concentrations of hemin to the nanoprobe. As shown in Figure 5A, the fluorescence of rQDs restored by the presence of G-quadruplex is gradually quenched with an increased hemin concentration. As a result, the nanoprobe exhibits a ratiometric fluorescent response to hemin (reverse to that for G-quadruplex detection), along with the solution color changed from orange to green (the inset of Figure 5A). Hemin does not affect the QDs fluorescence (Figure S14 in the Supporting Information), but 50 it binds with G-quadruplex via end-stacking. Thus, it is rationalized that hemin competitively displaces DI from the G-tetrad surface, and DI returns to the rQDs surface driven by metal affinity. This was confirmed by the re-appearance of characteristic vibration bands of DI in the FT-IR spectrum (Figure S3 in the Supporting Information). In this competitive assay, it is found that excess of hemin is necessitated to displace DI bound with G-quadruplex. By a 1:1 competitive binding of hemin versus DI with G-quadruplex, the presence of 4 μM of hemin only induces 15% quenching of the rQDs fluorescence. The presence of 4-fold excess of hemin can displace all DI molecules from G-quadruplex, resulting in the fluorescent “dark” state of QDs-DI. Moreover, the response time for this competitive assay is elongated with respect to that for G-quadruplex detection. As shown in Figure 5B, the response time of QDs-DI can be defined as ~20 min, independent of the hemin concentration. For G-quadruplex detection, the response time is predominantly determined by high affinity of DI/G-quadruplex interaction, while for the competitive assay, it is determined by the difference in binding affinity of hemin versus DI toward G-quadruplex. The elongated response time together with the necessitated high hemin concentrations indicate that the binding affinity of hemin with G-quadruplex is only slightly higher than that of DI/G-quadruplex interaction.

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Analytical Chemistry μM. The blank only contained the target sequence. As shown in Figure 6B, the ratiometric fluorescence signal of QDs-DI is gradually increased with the increased 1BM ratio in the mixture. The limit of detection (3σ) is defined to be 10:1 of target/1BM ratio. In other words, with the coexistence of 10 target sequences, 1 one-base mutant can be selectively detected by the nanoprobe. Together these results support that along with the typical QDs-MB nanoprobe, the QDs—ligand nanoprobe is capable of detecting one-base mutation with good sensitivity and specificity, by the presence of G-quadruplex.

CONCLUSIONS

Figure 6. (A) Histogram on ratiometric fluorescence signals of QDs-DI (in the presence of 0.8 μM G-quadruplex) and QDs-MB upon hybridization with different targets. (B) Histogram on ratiometric fluorescence signals of QDs-DI (in the presence of 0.8 μM G-quadruplex) upon hybridization with the target sequence mixed with varying ratios of 1BM. Detection of one-base mutation by the nanoprobe. Additionally, we applied the QDs-DI nanoprobe for hybridization-based one-base mutation detection, by making use of the specificity of DI binding with G-quadruplex against dsDNA (Scheme 1B). This was accomplished by pre-incubating QDs-DI with G-quadruplex, exposing to different DNA sequences, and suffering from hybridizations, respectively. All the DNA sequences are 30-base long, with varying base mutations (Table S1 in the Supporting Information). A QDs-MB nanoprobe also was prepared for comparison. As shown in Figure 6A, the two types of nanoprobes both exhibit distinctly discriminative ratiometric fluorescent responses toward different DNA sequences. The fluorescent response of QDs-DI is regarded to be induced by the displacement of DI: upon G-quadruplex hybridizing with a target and forming double helix, DI is forced to displace from the G-tetrad surface, complexing with the QDs again. This was confirmed by the re-appearance of characteristic vibration bands of DI in the FT-IR spectrum (Figure S3 in the Supporting Information). The complementarity of G-quadruplex/target pair governs the number of DI molecules displaced from G-quadruplex. The more is the DI molecules displaced, the stronger the quenching of rQDs is. As a result, the ratiometric fluorescence signal of QDs-DI is increased with the increased number of mutated base in the DNA sequence. In contrast, the QDs-MB nanoprobe exhibits the decreased signals with the increased mutation level. Despite this, both nanoprobes exhibit good specificity toward recognizing the DNA mutation. For one-base mutation, the relative red-to-green fluorescence intensity ratios between the one-base mutant (1BM) and the target (complementary sequence) are defined as 1.54: 1 by QDs-DI, and 1: 1.48 by QDs-MB, respectively (Figure 6A, and Figure S15 in the Supporting Information). When 9 bases (~1/3 of the total base number) are mutated (9BM), for both nanoprobes, the ratiometric fluorescent signals approach to those of completely noncomplementary sequence (NC). To further evaluate the sensitivity of the QDs-DI nanoprobe, we mixed the target and 1BM sequences at different molar ratios of 40:1, 20:1, 10:1, 5:1 and 3:1 with a total concentration of 0.8

In conclusion, a complex comprised of dual-emission QDs nanohybrid and non-ionic G-quadruplex ligand has been developed for homogenous fluorescent assay of G-quadruplex. On benefits of photophysical properties of QDs and molecular properties of the ligand, the complex is demonstrated to be suitable as a valuable nanoprobe for visual detection of G-quadruplex, exhibiting specific targeting, as well as distinct ratiometric fluorescent change. Furthermore, in the presence of G-quadruplex, this nanoprobe also has been demonstrated to be highly useful for assessing the affinity of an anti-cancer drug candidate, as well as sensitively recognizing one-base mutation. This prototype nanoprobe can be developed as a cellular probe for visualizing G-quadruplex structures in human cells by additionally but facilely introducing appropriate nuclear-targeting molecules within the nanosystem.

ASSOCIATED CONTENT Supporting Information DNA sequences, molecular structures of DI, DPPZ, 2+ [Ru(bpy)2DI] , G-tetrad, different forms of DNA, and mRNA, DLS, FT-IR, stability and biocompatibility of QD-DI, fluorescence spectra of QDs-DI, QDs-DPPZ, 2+ QDs-[Ru(bpy)2DI] and QDs-MB under different experimental conditions, confocal microscopy images of L02 cells treating with TAT-QDs-DI for different times. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author *Fax: (86) 25-83792349. Email: [email protected]

ACKNOWLEDGEMENT This work is financially supported by NSFC and Jiangsu Province (Grants: 21375015, 21545006, BK20141334).

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