Spiropyran in Situ Switching: A Real-Time Fluorescence Strategy for

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Spiropyran in situ Switching: A Real-Time Fluorescence Strategy for Tracking DNA G-Quadruplexes in Live Cells Jin Li, Xinchi Yin, Bin Li, Xiaokang Li, Yuanjiang Pan, Jian Li, and Yuan Guo Anal. Chem., Just Accepted Manuscript • Publication Date (Web): 21 Mar 2019 Downloaded from http://pubs.acs.org on March 21, 2019

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Analytical Chemistry

Spiropyran in situ Switching: A Real-Time Fluorescence Strategy for Tracking DNA G-Quadruplexes in Live Cells Jin Li,† Xinchi Yin,‡ Bin Li,† Xiaokang Li,§ Yuanjiang Pan,‡ Jian Li,§ and Yuan Guo*,† †Key

Laboratory of Synthetic and Natural Functional Molecule Chemistry of the Ministry of Education, National Demonstration Center for Experimental Chemistry Education, College of Chemistry and Materials Science, Key Laboratory of Resource Biology and Biotechnology in Western China of the Ministry of Education, Northwest University, Xi’an 710127, P. R. China ‡Department of Chemistry, Zhejiang University, Hangzhou 310058, P. R. China §State Key Laboratory of Bioreactor Engineering, Shanghai Key Laboratory of New Drug Design, School of Pharmacy, East China University of Science and Technology, Shanghai 200237, P. R. China ABSTRACT: DNA G-quadruplexes (G4s) in vivo have been linked to cancer and other diseases such as neurological disorders. Nondestructive fast detection of endogenous DNA G4s can provide specific real-time information, which is of particular interest for clinic accurate diagnosis. However, tools to probe live-cell endogenous DNA G4s in real time are very limited. Herein, we report the design and development of a fluorescent molecule QIN for the real-time detection of endogenous DNA G4s in live cells with the aid of a new spiropyran in situ switching (SIS) strategy. The lipophilic spiropyran-linked QIN differs from the other probes in that it can enter live cells readily within 15 s, and can be in situ induced by DNA G4s to adopt its charged open form, causing a large red shift in the fluorescent emission wavelength. Live-cell super-resolution fluorescent imaging suggests that the SIS-based probe has high photostability and can be applied for the accurate detection of DNA G4s in complex biosystems with very high sensitivity and selectivity.

S

ince the discovery of endogenous DNA G-quadruplexes (G4s) formed from DNA guanine-rich sequences in human cells,1 the non-canonical four-stranded structured DNA existing in cell nuclei has received significant attention over the past few years as an important biomarker.2-10 In particular, in 2016, writing in Nature Genetics, Balasubramanian et al.5 reported that G4 motifs are enriched in cancer-related genes, which further verified the existence of endogenous DNA G4s as specific intracellular anticancer targets. Nondestructive realtime sensing and clear imaging of endogenous DNA G4s in live cells is of particular interest for clinic accurate diagnosis. In this regard, high-performance, cell-permeable smallmolecule fluorescent probes are highly desirable in view of their advantage in real-time live-cell monitoring in situ.11,12 In recent years, a number of small-molecule fluorescent probes targeting DNA G4s have been developed.13-20 Most of them were designed to be fluorescence light-up probes with positive charges due to the negatively charged phosphate backbone and loops of DNA G4s. However, questions remain as uptake of these charged probes by live cells is not rapid because of the strongly lipophilic and hydrophobic nature of cell membranes’ phospholipid bilayers.21,22 Because of that, to date, none of the reported fluorescent probes have managed to achieve real-time imaging of endogenous DNA G4s in live cells (at least 20 min, Table S1). Furthermore, ratiometric fluorescence imaging can normalize the uncertainties related to single intensity fluorophores, thereby improving accuracy,23,24 and yet, to the best of our knowledge, the monitoring of DNA G4s in a ratiometric manner has not been reported either.

Lysosomes, as the main degradative compartment in eukaryotic cells in which aberrant pH values can cause cellular dysfunction25 have been shown to be excellent pharmacological targets for selectively destroying cancer cells, according to recent studies using existing drugs.26,27 These studies suggest that the lysosomes in cancer cells are different from those in normal cells: they are larger, more numerous and present higher levels of cellular signaling. The clear visualization of lysosomes and the detection of lysosomal pH changes in live cells is therefore an important diagnostic tool in lysosome-related diseases, especially cancer. In such cases, lysosomes as cytoplasmic organelles and the G4 DNA present in nuclei28-30 both play the role of intracellular anticancer targets. If these two targets can be rapidly detected and clearly imaged in live cells at the same time, the results will be mutually confirmed, thus greatly improving the accuracy of

Figure 1. Chemical structures of probes and their open forms.

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Figure 2. Structure-based development of QIN.

the diagnosis and even leading to more precisely targeted therapies. Generally, spiropyran dyes are considered as a class of organic photochromic molecules,31,32 which can be converted reversibly into the ring-opened merocyanine forms depending on UV and visible light irradiation induced pKa change.33 By focusing on the dynamic structural feature of G4 DNA, we noted that the structures of the open forms of spiropyran are similar with those of the reported probes targeting DNA G4s, such as Thioflavin T,13 thereby we propose that spiropyrans might be developed as a robust G4 DNA switchable probe. We hereby present the development of a spiropyran-linked fluorescent molecule QIN (Figure 1, Supporting Information) for real-time tracking of endogenous DNA G4s, based on an innovative spiropyran in situ switching (SIS) strategy. 6Nitro-1’,3’,3’-trimethylspiro[2H-1-benzo-pyran-2,2’-indoline] (1), a known non-fluorescent spiropyran derivative, was modified through structure-based screening and optimization via 2, 3 and 4 to obtain a lead probe QIN which exhibited superior detection performance for DNA G4s (Figures 2 and 4a-d, Figures S1-S5). Our lead, the lipophilic spiropyranlinked QIN, differs from charged probes in that it can sense live-cell intranuclear endogenous DNA G4s in real-time by switching between hydrophobicity and hydrophilicity as well as clearly image extranuclear lysosomes in live cells simultaneously (Figure 3). The pKa of QIN is 5.9, which means that it exists predominantly in spirocyclic form at physiological pH and emits blue fluorescence (λem = 458 nm). Interestingly, when QIN selectively binds to G4 DNA, the pKa value increases to 7.4, which forces the probe to switch to its open form (QIN-OPH+, Figure 1) at physiological pH and emit red fluorescence (λem = 610 nm). In live cell systems, the pKa value (5.9) allows QIN to respond to the changes in extranuclear lysosomal pH values, ensuring discernment of lysosomal subtle pH changes in stressed cells undergoing certain stimulation, while the increased pKa value (7.4) is suitable for imaging of intranuclear endogenous DNA G4s. Based on the DNA G4 induced pKa shift mechanism, QIN was successfully applied for the second-level real-time in situ detection of endogenous DNA G4s in live cells.

HepG2, SMMC-7721, PC-3, A375, MCF-7, HPASMC and MRC-5 cells, respectively. Subsequently, DNase and RNase digest experiments were conducted. The U2OS and HepG2 cells staining QIN (20 μM) were cultured with 1000 µL PBS (control group), 1000 µL RNase-free DNase I solution (100 units·mL-1), or 1000 µL protease-free RNase A solution (100 units·mL-1) for 2 h, respectively. Finally, real-time imaging of QIN was carried out. The total time was 70 s and the exposure time was set to 5 s for each raw data capture.

RESULTS AND DISCUSSION Optical Response of QIN to DNA G4s. QIN as the spiro form emits at 458 nm in K+ buffer [Tris-HCl (25 mM) buffer at pH 7.4 containing KCl (20 mM)]. Upon the addition of single-stranded DNA and double-stranded DNA respectively (Table S2), no significant changes in the fluorescence spectra of QIN were observed (Figure S5a). However, a new long wavelength emission band centered at 610 nm emerged when QIN was treated with G4 DNA (Table S2 and Figure S5a), which can be attributed to the formation of its open form QINOPH+ by DNA G4s induction. Upon switching forms from closed to open, the resulting π-extended conjugation system gave rise to a red shift in the fluorescent emission wavelength (458 nm to 610 nm) and an apparent fluorescence change from blue to red was observed with the naked eye under a UV-lamp (Figure 4a). Moreover, the enhanced rigidity of the open form after binding with G4 DNA triggered a significant increase in fluorescent intensity at 610 nm (Figure S5a inset). As shown

EXPERIMENTAL SECTION The details of the materials and general experimental methods are given in the Supporting Information. Briefly, confocal and super-resolution imaging of QIN in live cells were performed firstly. The live cells were U2OS,

Figure 3. Schematic representation of DNA G4s in nuclei and lysosomes in cytoplasm probed by QIN.


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Analytical Chemistry in Figure 4a, it is clear that QIN had high selectivity for DNA G4s over single-stranded DNA and double-stranded DNA. Especially, the probe showed the best response to c-MYC G4 DNA among these DNA G4s, with a 16-fold increase at 610 nm in the fluorescence titration assay (Figure 4b). As the inset of Figure 4b showed, the fluorescent intensity of QIN at 610 nm was linearly related to the concentration of c-MYC G4 DNA within 0-7 μM with a detection limit at 70 nM. The dissociation constant (Kd) calculated from the fluorescence spectra was found to be 3.53 μM (Figure S5b). Compared to lower values of Kd reported in the literature,1 this value means that the binding process between QIN and c-MYC G4 DNA and fluorescence signal changes thereof might be reversible. More importantly, a sharp increase was observed within 3 s upon the addition of c-MYC G4 DNA (Figure S5c). After that, the fluorescent intensity at 610 nm enhanced gradually, and the intensity at 458 nm decreased correspondingly until plateaus were reached, suggesting the ratiometric sensing of DNA G4s by QIN (Figure S5d). We applied the circular dichroism (CD) technique to explore the modification of the conformation of c-MYC G4 DNA as a result of interaction with QIN. In the absence of QIN, the G4 DNA displayed a positive peak at ~265 nm and a negative peak at ~245 nm, confirming the parallel type of G4 DNA (black line in Figure 4c). After titration with QIN, there were no peak shape changes in the CD spectra of c-MYC G4 DNA while the ellipticities were remarkably increased, indicating that QIN was able to retain the existing conformation for c-MYC G4 DNA and exhibited a good response to the DNA (Figure 4c). These properties of QIN are in accordance with those of an ideal G4 fluorescent probe, such as the ability to provide true information about endogenous DNA G4s in live cells by not disturbing the structure of G4 DNA. The interaction of QIN with c-MYC G4 DNA was studied using UV-Vis spectroscopic titrations. The absorption spectrum of the free QIN in K+ buffer showed two absorption peaks centered at 289 nm and 567 nm, respectively (Figure 4d). Addition of c-MYC G4 DNA gave rise to a hyperchromic effect at 289 nm and a significant hypochromic effect (27%) along with a red shift (10 nm) at 567 nm, implying a strong interaction between the QIN-OPH+ and c-MYC G4 DNA. pH-Switchable Properties of QIN. As a spiropyran derivative, its reversible isomerization can be also achieved by pH.37 Therefore, the fluorescence spectral changes of QIN were examined in phosphate buffer solution at different pH values. As expected, QIN underwent ring-opening in the presence of acid (pH 5.0) to form the red fluorescent open form QIN-OPH+ (λem = 608 nm) and then converted back to the blue fluorescent spirocyclic form QIN (λem = 458 nm) under physiological buffer (pH 7.4) or basic (pH > 7.4) conditions (Figure S5e). Furthermore, the pKa of the probe was determined to be 5.9 based on the S curve and the Henderson-Hasselbalch equation (Figure 4e). The results suggested that the sensitive response of QIN to pH matched well with the physiological pH range (pH 4.5-6.0)34,35 of lysosomes in live cells, making it a promising probe for targeting lysosomes in live cells. Binding Mechanism. As described, at the physiological pH of 7.4, QIN exists predominantly in spirocyclic form and emits blue fluorescence, while strong red fluorescence of its open form was observed upon the addition of G4 DNA. To explain this behavior, pH-dependent fluorescent intensity

Figure 4. (a) Fluorescent intensities at 610 nm of QIN (10 μM) with different DNA (10 μM) in 25 mM Tris-HCl buffer containing 20 mM KCl. Single-stranded DNA: ss-DNA1, ssDNA2, ss-DNA3 and ss-DNA4. Double-stranded DNA: dsDNA1, ds-DNA2 and ct-DNA. Promoter G4 DNA: c-MYC, BCL-2 and c-KIT1. Telomere G4 DNA: 22AG. Concentrations of QIN and DNA were 10 μM. I0 and I represent the fluorescent intensity without and with DNA, respectively. Inset: visual fluorescence color changes of QIN in the presence of various DNA from left to right; the photo was taken under illumination of a hand-held UV lamp. (b) Fluorescence spectra of QIN (10 μM) upon titration with c-MYC G4 DNA in 25 mM Tris-HCl buffer (20 mM KCl, pH 7.4). Inset: a linear relationship between fluorescent intensity of QIN (10 μM) and concentrations of cMYC G4 DNA. The error bars indicate S. D. (n = 3). (c) CD spectra of c-MYC G4 DNA (10 μM) upon titration with QIN in 25 mM Tris-HCl, 20 mM KCl, pH = 7.4. r = [QIN]/[c-MYC G4 DNA] (0, 2.5, 5, 7.5, 10). (d) UV-Vis spectra of QIN (10 μM) upon titration with c-MYC G4 DNA (1-50 μM) in 25 mM TrisHCl buffer (20 mM KCl, pH 7.4). (e) Fluorescent intensity changes of QIN (10 μM) with different pH monitored at 610 nm. (f) The pKa shift of QIN (10 μM) upon binding to c-MYC G4 DNA monitored through fluorescent intensity change upon pH variation for the free QIN and c-MYC G4 DNA/QIN complex. λex = 550 nm, slit widths: Wex = 10 nm, Wem = 5 nm.

changes of QIN with c-MYC G4 DNA were investigated. The pKa of the c-MYC G4 DNA/QIN complex was calculated to be 7.4, affording a large upward shift of ~1.5 units from the free probe value (5.9) (Figure 4f). Obviously, at pH 7.4, QIN was transformed by G4 DNA into its protonated open form QIN-OPH+ (Figure S6). Therefore, the G4-induced pKa shift can cause QIN to switch forms from closed to open, thereby achieving the ratiometric response of G4 DNA. Remarkably, the fluorescent intensity of the complex system in the red channel was about ten times that of free QIN-OPH+, although both existed predominantly in the red fluorescent open form. The significant increase in fluorescent intensity might be due to an enhanced rigidity of QIN-OPH+ after binding with cMYC G4 DNA.



Figure 5. (a) Schematic diagram of the combination of QIN and c-MYC G4 DNA. (b) The docking model of (R)-QIN with c-MYC G4 DNA (PDB ID: 2L7V). (c) The docking model of (S)-QIN with c-MYC G4 DNA. (d) Grid scores of (R)-QIN @ c-MYC G4 DNA, (S)QIN @ c-MYC G4 DNA and QIN-OPH+ @ c-MYC G4 DNA, respectively. (e) The docking model of QIN-OPH+ with c-MYC G4 DNA. Docking models were represented using PyMOL 1.8. (R)-QIN, (S)-QIN and QIN-OPH+ are shown by magenta sticks; c-MYC G4 DNA model is shown by smudge planes and orange ribbon; the binding interactions are represented as dashed lines inside pockets: π-π stackings are shown in orange, the hydrogen bond in green and π-cation interactions in yellow. (f-i) Mass spectra of QIN (f), QIN with cMYC G4 DNA (g), QIN-OPH+ (h) and QIN-OPH+ with c-MYC G4 DNA (i). (j-m) Energy resolved mass spectra of QIN (j), QIN with c-MYC G4 DNA (k), QIN-OPH+ (l) and QIN-OPH+ with c-MYC G4 DNA (m).

To gain insight into the detection mechanism of the probe, we further explored why the pKa shifted. To begin with, a molecular docking method was used to determine the interaction of c-MYC G4 DNA with QIN and QIN-OPH+, respectively. From the docking results, QIN with a spirocarbon center exists in the form of two enantiomers (R and S enantiomers) and there is only a π-π stacking interaction between the julolidine moiety in both enantiomers and c-MYC G4 DNA (Figure 5b,c and Table S3), resulting in binding energies of -41.6 kcal/mol (R enantiomer) and -45.3 kcal/mol (S enantiomer), respectively (Figure 5d and Table S4). In comparison, QIN-OPH+ fits into c-MYC G4 DNA more comfortably by three interactions involving aromatic ring stacking, electrostatic binding of N+ with π-electron and hydrogen bonding (Figure 5a,e and Table S3), leading to a binding energy of -64.8 kcal/mol (Figure 5d and Table S4). The lower binding energy shows that QIN-OPH+ binds cMYC G4 DNA with higher affinity than QIN does, which theoretically explains why the G4 DNA can induce a pKa shift. To clarify it through experiments, mass spectrometry and collision-induced dissociation (CID) were used to study the interaction of c-MYC G4 DNA with QIN and QIN-OPH+,

respectively. A molecular ion peak at m/z 373 was observed in all the mass spectra (Figure 5f-i). Subsequently, we exploited the CID technique to induce fragmentation of the molecular ion (m/z 373), which generated the signals of three fragment ions (m/z 358, 214 and 202) (Figure 5j-m). As can be seen from the energy resolved mass spectra, the spectrum of QINOPH+ (Figure 5l) was not the same as those of the other three, which could be due to the fact that the more stable QIN-OPH+ required a higher fragmentation energy than QIN. However, the spectrum of the c-MYC G4 DNA/QIN-OPH+ complex (Figure 5m) was similar to that of QIN (Figure 5j), meaning that there was almost no free QIN-OPH+ in the solution after its binding to c-MYC G4 DNA. Consequently, the signals in Figure 5m should be assigned to residual QIN. Figure 5i further proved that only 7.2% of free QIN was retained after the addition of c-MYC G4 DNA to the QIN-OPH+ solution, again indicating that QIN-OPH+ had higher binding affinity for c-MYC G4 DNA than QIN. Additionally, it was observed that the absolute intensity of the molecular ion peak of QIN decreased by 68.2% when treated with c-MYC G4 DNA (Figure 5g). This indicated that QIN was induced partially by c-MYC G4 DNA to adopt its open, strong-binding form QIN-


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Figure 6. (a) Confocal images of live cancer cells (U2OS, HepG2, SMMC-7721, PC-3, A375 and MCF-7) and normal cells (HPASMC and MRC-5) co-stained with QIN (20 μM) and LysoTracker Green DND-26 (75 nM). Column 1: images of QIN 458 nm (blue channel, λex = 405 nm, λem = 425-475 nm); Column 2: merged images of LysoTracker Green DND-26 (511 nm) and QIN 610 nm. Inset: the intensity scatter plots of LysoTracker Green DND-26 (511 nm) and QIN 610 nm; Column 3: images of QIN 610 nm (red channel, λex = 561 nm, λem = 570-620 nm); Column 4: zoom images of the areas indicated with white squares in the red channels. The yellow and pale yellow dotted lines in the zoom images show the boundary of the nuclei. (b) Schematic representation of U2OS cells enriched c-MYC G4 DNA (upper). Super-resolution images of live U2OS cells co-stained with QIN (20 μM, red channel, λex = 561 nm, λem = 570-640 nm) and Hoechst33342 (10 μg/mL, blue channel, λex = 405 nm, λem = 435-485 nm) (lower, left). Super-resolution images of fixed U2OS cells co-stained with QIN (20 μM, red channel) and DAPI (5 μg/mL, blue channel) (lower, right). Column 1: images of fixed cells stained with QIN; Column 2: loss of QIN staining after DNase I treatment; Column 3: maintenance of QIN staining after RNase A treatment. (c) Time-lapse images of live U2OS cells after treatment with QIN (20 μM) illustrating the rapid cell uptake process (left). Average fluorescent intensity of images from 5 s to 70 s (right), data are presented as the mean ± SD (n = 3). Scale bars, 10 μm (a, c) and 5 μm (b).

OPH+, further verifying why the G4 DNA induced a pKa shift experimentally. Visualization of Endogenous DNA G4s in Live Cells. Encouraged by the in vitro results, QIN was used to investigate the visualization of endogenous DNA G4s in different live cells including cancer cell lines and normal cell lines (Figure 6a). The intracellular imaging results showed that the blue fluorescent probe QIN was taken up by all the above live cell samples, whereas red fluorescent signals appeared only in cancer cell nuclei, suggesting that DNA G4s are cancer-related and can in situ activate QIN to its open form. Here, more significant responses were observed in U2OS and HepG2 cells enriched by c-MYC G4 DNA (Figure 6a),36-39 which accords with the experimental results of selectivity in vitro. Next, we used super-resolution microscopy, a powerful imaging tool that can not only offer better visualization but also extract additional details, to investigate the interaction between QIN and DNA G4s in U2OS and HepG2 cells. As shown in Figure 6b (lower left), Figure S7 and Videos S1 (U2OS) and S2 (HepG2), a red fluorescent response to certain regions of the nucleus was clearly observed. Interestingly, the stained red regions in the HepG2 cell nuclei appeared hollow and lotus-root-like nucleolus structures (Figure S7 and Video

S2), which indicated that the G4-forming sequences were located mainly in the nucleoli. To explore whether the probe binds to G4 DNA, digestion experiments by DNase I and RNase A were conducted in U2OS and HepG2 cells (Figure 6b lower right, Figure S8). As with the in vitro studies (Figure S5f), the G4 foci clearly diminished after DNase I treatment, but not after RNase A treatment. Furthermore, we used pyridostatin (PDS),1 a G4-stabilizing ligand, to trap intracellular DNA G4s. As shown in Figure S9, more intense emission in red channel was observed, demonstrating that PDS captures DNA G4s to increase the number of QIN targets available. This observation supports the targeting of DNA G4s in cancer cells by QIN. The capability of QIN for real-time monitoring of DNA G4s in U2OS cells was evaluated. As shown in Figure 6c, the red fluorescent response for DNA G4s in the nuclei appeared within 15 s. To the best of our knowledge, this is the first time that second-level detection of DNA G4s in live cells has been achieved. Comparatively, the known DNA G4 probes have longer response times, from tens of minutes to several hours (Table S1), because they were designed to be fluorescence light-up probes with positive charges based on the negatively charged phosphate backbone and loops of DNA G4s. Owing



Figure 7. (a) Schematic illustration of the in situ switching mechanism of HAN in lysosome (upper). Zoom images of the areas indicated the blue square in the image of U2OS cell stained by QIN and the green square in the image of U2OS cell stained by HAN (lower). (b) Co-localization studies of live cancer cells stained with LysoTracker Green DND-26 and HAN (upper). Column 1: images of HAN 615 nm (red channel, λex = 561 nm, λem = 570-620 nm); Column 2: images of LysoTracker Green DND-26 (green channel, λex = 488 nm, λem = 500-550 nm); Column 3: merged images of green and red channels; Column 4: the intensity scatter plots of LysoTracker Green DND-26 and HAN 615 nm and Pearson’s correlation coefficients on the upper right corner of the intensity scatter plots; Column 5: intensity profiles of LysoTracker Green DND-26 (arrow in green channel) and HAN (arrow in red channel) in the regions of interest (ROIs). A colocalization study of HepG2 cells stained with MitoTracker Green FM (200 nM, green channel, λex = 488 nm, λem = 500-550 nm) and HAN (lower). (c) Super-resolution images of A549 cells co-stained with LysoTracker Green DND-26 and HAN. (d) Confocal images of MRC-5 cells stained with HAN from blue channel (HAN 460 nm) and red channel (HAN 615 nm) stimulated with chloroquine (200 μM). Column 1-4: blue, red and merged images after chloroquine stimulation for 5, 10, 20 and 30 min; Column 5: merged image of bright field and blue channel in 30 min (upper); merged image of bright field and red channel in 30 min (middle); merged image of bright field, blue channel and red channel in 30 min (lower). Scale bars, 10 μm (a, b and d), 5 μm (c, left) and 0.5 μm (c, right).

to the strongly lipophilic and hydrophobic nature of cell membranes’ phospholipid bilayers, these charged probes are not taken up rapidly by live cells, whereas our lipophilic and uncharged SIS-based QIN can pass through the cell membrane and nuclear membrane readily, and can also be in situ activated by DNA G4s existing in the nuclei to its charged open form QIN-OPH+, thereby achieving real-time imaging of endogenous DNA G4s in live cells. Subsequently, co-localization experiments confirmed that QIN could efficiently target lysosomes located in the cytoplasm with high Pearson’s correlation coefficients using LysoTracker Green DND-26 as a reference in the cells studied (Figure 6a, Figure S10). From these fluorescent colocalization images, we also found that lysosomes in cancer cells are larger and more unevenly distributed than those in normal cells. Then QIN was employed to visualize chloroquine-stimulated intracellular pH changes in live U2OS

cells. Chloroquine as a drug of lysosomal deacidification can cause the leakage of protons out of lysosomes in the form of protonated bases, further increasing lysosomal pH. As shown in Figure S11, the fluorescent intensity in the red channel was obviously reduced outside the U2OS cell nuclei, which was ascribable to the accumulation of QIN in the lysosomes. However, the red fluorescence inside the cell nuclei showed only a slight fluctuation, which further proved the interaction between QIN and G4 DNA. Therefore, the SIS-based probe QIN, which can achieve accurate and real-time detection of endogenous DNA G4s and lysosomes in live cells, has the potential to be applied in early and precise cancer diagnosis. Control Compound HAN. Encouraged by QIN, a control compound HAN (Figure 1) for selectively sensing c-MYC G4 DNA was designed by DOCK 6.7 (Figure S12, Table S5 and S6) and synthesized (Supporting Information). Unsurprisingly, from the docking results and in vitro studies (Figure S13a-e),


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Analytical Chemistry HAN exhibited high binding affinity towards c-MYC G4 DNA. However, confocal images of all the cells treated with HAN showed no response to endogenous DNA G4s (Figure 7a,b). These results further verified the uniqueness and remarkable advantage of QIN. In spite of failure to detect endogenous DNA G4s in live cells, the pH sensitive HAN (pKa 5.0, Figure S13f) displayed a lysosome-specific blue to red fluorescent response (Figure 7b,c). The pH dependent ratiometric fluorescent patterns of HAN match pH windows of lysosomes (pH 4.5-6.0), suggesting that HAN can be induced in situ into its open form (HAN-OPH+, Figure 1) by H+ under the acidic lysosomal environment specifically. Subsequently, the localization of HAN in HepG2 cells was investigated by co-staining with MitoTracker Green FM or LysoTracker Green DND-26. As shown in Figure 7b, the merged image of the probe at red channel and MitoTracker Green FM exhibited a significantly different fluorescence and a low Pearson’s correlation coefficient of 0.44 demonstrated HAN did not target mitochondria. In contrast, the fluorescence of HAN almost completely overlaid with that of LysoTracker Green DND-26 and the Pearson’s correlation coefficient was as high as 0.96. Similar results were found in the other cancer cells studied, confirming that HAN had a high lysosome-targeting capability, as a result of a pH-modulated SIS system. Using the lysosomal probe, we also obtained clear super-resolution images of lysosomes in A549 cells (Figure 7c). Moreover, compared to the commercial LysoTracker Green DND-26, the SIS-based probe HAN presented a low background noise (Zoom images in Figure 7c), resulting in a marked improvement in sensitivity. To determine whether HAN also exhibited rapid cell uptake, real-time imaging of live cells was investigated (Video S3). As expected, HAN required only approximately 3 s to stain lysosomes in HepG2 cells, suggesting its fast targeting ability for lysosomes. Next, to prove that HAN could monitor dynamic pH changes of lysosomes in live stressed cells, chloroquine-treated MRC-5 cells were stained with HAN and imaged by confocal microscopy. As depicted in Figure 7d, the fluorescent intensity of MRC-5 cells exhibited a clear timedependent decrease in the red channel, while the blue fluorescent intensity showed a gradual time-dependent increase. Taken together, HAN, the SIS-based lysosometargeting fluorescent pH probe, could be used to successfully discern the subtle pH of lysosomes in chloroquine-treated cells.

high lysosome-targeting capability, and thus was successfully applied to monitor lysosomal pH changes in stressed cells undergoing chloroquine stimulation. Our SIS strategy therefore opens up new avenues for the rapid and accurate detection of cancer-related DNA structures in live cells and may be amenable to other biological targets.

ASSOCIATED CONTENT The Supporting Information is available free of charge on the ACS Publications website. Experimental details, spectroscopic data, molecular docking data, fluorescence imaging, X-ray crystallographic data summary, HRMS and NMR spectra of new compounds (PDF) Crystallographic data of QIN-OPH+ and HAN-OPH+ (CHECKCIF) Crystallographic data of QIN-OPH+ and HAN-OPH+ (CIF) Video S1 (AVI) Video S2 (AVI) Video S3 (AVI)

AUTHOR INFORMATION Corresponding Author *E-mail: [email protected]

ORCID Yuan Guo: 0000-0003-2081-7264

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT The authors acknowledge the National Natural Science Foundation of China (No. 21472148, 21072158 and 21532005), Open Funding Project of the State Key Laboratory of Bioreactor Engineering and Academic Backbone of Northwest University Outstanding Youth Support Program. The authors thank Prof. Guangfu Yang at Central China Narmal University, Prof. Minyong Li at Shandong University and Prof. Sanping Chen and Prof. Xi Chen at Northwest University for insightful discussions on this work. The authors thank Xiaoting Li and Peng Ji at the Demo Laboratory of ZEISS Microscopy Customer Centers in Shanghai for assistance with super-resolution imaging. The authors also thank Qingfeng Xiao and Zhifeng Cheng at Nikon Biological Imaging Center in Shanghai for assistance with SIM imaging.

REFERENCES

CONCLUSIONS In summary, we have used a structure-guided method to develop a lead probe QIN based on a spiropyran in situ switching (SIS) strategy, which is the first fluorescent probe for the second-level real-time imaging of endogenous DNA G4s in live cells. Our lead, the hydrophobic QIN, can pass through the cell membrane and nuclear membrane in a few seconds, and can be in situ induced by DNA G4s to adopt its charged open form QIN-OPH+. Upon switching forms from closed to open, the resulting π-extended conjugation system causes a red shift in the fluorescent emission wavelength (458 nm to 610 nm) and the ratiometric fluorescent response to DNA G4s leads to negligible background fluorescence in the red channel from the unbound free probe QIN. While also designed on the basis of the SIS strategy, HAN was originally used as a control compound compared to QIN because HAN was unable to image DNA G4s in cell nuclei although it had a good response in vitro. The SIS-based HAN, however, had a

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For Table of Contents Only

A spiropyran in situ switching strategy for real-time fluorescent sensing of certain important biomarkers in live cells is proposed. With the aid of the new strategy, our lead molecule QIN as the fluorescent probe for DNA G-quadruplex structures was developed and for the first time applied for the second-level real-time detection of endogenous DNA G4s in live cells.


Spiropyran in Situ Switching: A Real-Time Fluorescence Strategy for

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