An Off–On Two-Photon Carbazole-Based Fluorescent Probe: Highly

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An off-on Two-Photon Carbazole-based Fluorescent Probe: Highly Targeting and Super-Resolution Imaging of mtDNA Fengli Gao, Liuju Li, Jiangli Fan, Jianfang Cao, Yueqing Li, Liangyi Chen, and Xiaojun Peng Anal. Chem., Just Accepted Manuscript • Publication Date (Web): 06 Feb 2019 Downloaded from http://pubs.acs.org on February 6, 2019

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Analytical Chemistry

An off-on Two-Photon Carbazole-based Fluorescent Probe: Highly Targeting and Super-Resolution Imaging of mtDNA Fengli Gao,† Liuju Li,‡ Jiangli Fan,*†II Jianfang Cao,§ Yueqing Li,I Liangyi Chen*‡ and Xiaojun Peng†II †

Department State Key Laboratory of Fine Chemicals, Dalian University of Technology, 2 Linggong Road, 116024, Dalian, China ‡ Institute of Molecular Medicine, Peking University, 100871, Beijing, China § School of Chemical and Environmental Engineering, Liaoning University of Technology, 169 Shiying Road, 121001, Jinzhou, China I School of Pharmaceutical Science and Technology, Dalian University of Technology, 2 Linggong Road, 116024, Dalian, China II Research Institute of Dalian University of Technology in Shenzhen, Gaoxin South fourth Road, Nanshan District, Shenzhen 518057, China *

E-mail: [email protected], [email protected]

ABSTRACT: Many mitochondria related diseases are associated with the mutation of mitochondrial DNA (mtDNA). Therefore, visualizing its dynamics in live cells is essential for the understanding of the function of mtDNA transcription and translation. By employing carbazole as the framework and designing a module for DNA minor groove binding, here we have developed a novel fluorescent probe with large stokes shift (λab = 480 nm and λem = 620 nm), CNQ, for mtDNA detection and visualization. It is almost non-fluorescence in PBS buffer, and exhibits 182-fold enhancement in fluorescence within 20 s after the application of mtDNA in the solution, with the detection limit of 55.1 μg/L. Using dual-color Hessian structured illumination microscopy, we have demonstrated that CNQ labeled mtDNA structures distinct from those labeled by TFAM-EGFP. Finally, we have used two-photon confocal scanning microscopy (λex = 850 nm) to monitor the nondestructive doxorubicin-induced mtDNA damage in live cells.

KEYWORDS mtDNA • super-resolution imaging • two-photon fluorescent probe • carbazole derivative

Human mtDNA is consisted of 16569 base pairs (bp) and organized as a circular, covalently closed and double-stranded structure, which is the first significant part of human genome to be sequenced.1 Enzymes and proteins of the electron transport chain are encoded by mtDNA and nuclear DNA, which fulfill the cell respiratory function and provide energy for cells. 2-3 On the other hand, mtDNA exhibits more than 10 times higher mutation rates than that of nuclear DNA, which is due to the lack of the protection of histone, imperfection of repair function and high susceptibility to reactive oxygen species. 4-5 Since mutations in mtDNA are associated with the carcinogenic process and a number of hereditary human diseases, 6-8 real-time monitoring the damage of mtDNA is important. The commonly used methods for mtDNA detection include polymerase chain reaction (PCR)9-10 and fluorescence in situ hybridization (FISH),1112 while the low hybridization degree and poor reproducibility limited their further application. Therefore, to develop a new method for accurate localizing and imaging mtDNA will help to address the problem. With the development of various fluorescence probes, fluorescence microscopy has become powerful enough to provide spatiotemporal information regarding nearly all processes within live cells.13-15 However, the performance of these probes under super-resolution microscopy are seldom known. Furthermore, two-photon microscopy (TPM), which employs

two photons with long wavelength (>700 nm) as the excitation source, effectively avoid background interference owing to the auto-fluorescence of the biomatrix, has widely been used in imaging cells and tissues.16-17 Up to now, various fluorescent probes have been developed for nucleic acid in living cells, while the majority of them focused on nuclear DNA18-27 and very few of commerce available dyes are mtDNA specific. Furthermore, calf thymus DNA was used instead of mtDNA in buffer solution test, insufficient evidence was presented in cell experiment to prove the specificity of fluorescent probe on mtDNA.28-31 In this paper, we have designed a novel fluorescent probe CNQ based on carbazole derivative with large stokes shift (λex=480 nm, λem=620 nm) for specific labeling of mtDNA in living cells, can be imaged with Hessian-structured illumination microscopy (Hessian-SIM) and also two-photon microscopy. The molecular structure of CNQ is presented as below (Figure1(a)). We selected carbazole as the starting point due to its excellent optical properties and large maximum action two-photon absorption cross section (δΦ).32-33 The introduction of pyridine increased the electron delocalization together with methylated quinoline, as positively charged molecule is easier to be transported to mitochondria in living cells. The detailed synthetic procedures and characterizations of CNQ and 1

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intermediates were shown in ESI and characterized by 1H NMR, 13C NMR and HRMS spectra, respectively.

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Viscosity measurement. The viscosity experiment was performed by Brookfield viscometer at the temperature of 25.0 ± 0.1°C in a constant water bath. 20.0 mL of mtDNA (200 μg/L) buffer solution (PBS, 10.0 mM, pH=7.4) was tested to obtain the specific viscosity of mtDNA with (η) and without CNQ (η0). The classical DNA intercalator (EB) and groove-binding ligand (berenil) were also recorded as references. In order to eliminate the interference of organic solvent, the same volume of DMSO which dissolved the CNQ was added to the mtDNA solution and the viscosity measurement plots were measured. Thermal melting analysis. The temperature at which half of the dsDNA dissociated into ssDNA was called melting temperature (Tm). The mode of interaction between CNQ and mtDNA were further monitored by DNA thermal melting analysis. The thermal denaturation properties of mtDNA was obtained according to the absorbance variation at 260 nm in PBS buffer solution (10 mM, pH=7.4). The concentration of mtDNA was 10.0 μg/mL and the absorbance with the change of temperature ranging from 50-95°C were measured at the absence and presence of CNQ (5.0 μM). In order to eliminate the interference of organic solvent, the same volume of DMSO which dissolved the CNQ was added to the mtDNA solution and the thermal melting plots were measured as well. Cell incubation and staining MCF-7 (human breast adenocarcinoma) cells were cultured in DMEM medium supplemented with 10% fetal bovine serum (FBS) and penicillin/streptomycin from Gibco in CO2 incubator at 37°C. The cells were seeded into a glass bottom dish (35.0 mm dish with 20.0 mm bottom well) with 2.0 mL of culture medium in the incubator until the cell density was approximately 50%, then incubated with 2.0 μM of CNQ for 30 min and washed with PBS for three times before imaging. The excitation wavelength was 488 nm and the emission spectra were recorded in the range of 620 ± 20 nm. All the parameters were remained the same throughout the cell experiments. Cell maintenance and preparation for super-resolution imaging. MCF-7 cells were cultured in high-glucose DMEM supplemented with 10% FBS and 1% 100 mM sodium pyruvate solution in an incubator at 37°C with 5% CO2, detached using trypsin-EDTA, seeded onto poly-L-lysine-coated coverslips, and cultured for an additional 24 h before the transfection. MCF-7 cells were transfected with TFAM-EGFP using the LipofectamineTM 2000 reagent according to the manufacturer’s instructions. After transfection, the cells were cultured for an additional 20-28 h before the experiments. To label DNA, MCF-7 cells were incubated with 1.0 μg/L PicoGreen in highglucose DMEM at 37°C for 15 min before being washed and imaged in HBSS solution containing Ca2+, Mg2+ but no Phenol Red. The cells were tested for mycoplasma contamination before use.

Figure 1. (a) The molecular structure of two-photon fluorescent probe CNQ; (b) The minor-groove binding of CNQ and mtDNA.

EXPERIMENTAL SECTION Materials. All the solvents and reagents in this work were of analytical grade without further purification. PBS buffer solution (pH = 7.4, 10.0 mM) was used in the whole experiment. Flash column chromatography was performed using neutral silica gel (particle size: 40-63 μm, pore size 60 Å) and silica gel (200-300 mesh) purchased from Beijing Innochem Technology Co., ltd and Qingdao Ocean Chemicals, respectively. Doubly purified water used for all the experiments was taken from a Milli-Q system. All the interferences including amino acids and proteins were prepared with ultrapure water for the concentration of 5.0 mM. The mtDNA was extracted from MCF-7 cells and then amplified by techniques-polymerase chain reaction (PCR) to produce the final samples. Doxorubicin was purchased from energy chemical, PicoGreen dsDNA reagent and LipofectamineTM 2000 reagent were purchased from Life Technologies Co. (USA) and Thermo Fisher Scientific, respectively. Ultraviolet-visible and fluorescence spectroscopic studies. The ultraviolet-visible absorption and fluorescence emission spectra of CNQ in the absence and presence of mtDNA were performed using a 1.0 cm × 1.0 cm quartz cell and were recorded in the wavelength of 400-650 nm and 500-800 nm, respectively. CNQ was dissolved in dimethyl sulfoxide (DMSO) to prepare a concentration of 1.0 mM as the stock solution. Experiments were performed by fixed CNQ concentration (5.0 μM) and titrated with increasing mtDNA concentrations (0130.0 μg/L). The samples were gently stirred for 1.0 min before the spectrum was recorded. Each experiment was carried out for five times in replicate determinations (n=5). CD spectra determination. Circular dichroism (CD) is a powerful tool for the analysis of secondary structures and conformations adopted by nucleic acids and proteins. Many DNAbinding ligands are optically inactive and achiral, while upon interaction with DNA, an induced CD (ICD) signal could be acquired, so it can be assumed the interaction mode between ligand and DNA according to the different ICD signals. As the classical intercalation binding needed more space of adjacent base pairs to extend the DNA helix leading to a significant increase of the relative viscosity, when small molecules bind to DNA grooves via an electrostatic interaction or groove binding, a slight shortening in the axial length of DNA was obtained and therefore, the relative viscosity showed a slight change. To the solutions of mtDNA (0-0.3 mg/mL), a fixed concentration of CNQ (10.0 μM) was added and the spectrum of each sample was recorded in the range of 225-575 nm in PBS buffer solution (pH=7.4, 10 mM). Furthermore, the effect of different acidity solutions (pH=4.0-10.0) on CD spectra were recorded as well.

RESULTS AND DISCUSSION Spectral specificity of CNQ on mtDNA. The absorption and emission spectra of CNQ (5.0 μM) in the abscence and presence of mtDNA in PBS(10.0 mM, pH=7.4) were recorded without any adjunction of organic solvents. CNQ possessed an absorption spectrum peak at 480 nm (Figure S1a) and showed a very low intrinsic fluorescence (Figure S1b). Upon gradual addition of low concentrations of mtDNA (≤2.5 μg/mL), the corresponding fluorescent intensity showed only a small increment along with the emission wavelength almost located 2

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Analytical Chemistry different parts. Less orbital overlap of CNQ led to the low background fluorescence peaked at 682 nm which is close to the experimental emission peaked at 700 nm. Positively charged quinoline could be easily interacted with negatively charged phosphoryl group of mtDNA, and the vinylpyridine was in an unrestraint state. In the presence of low concentrations of mtDNA, twin peaks represented the emission of two different conjugated structures (long wavelength was the conjugation of whole molecule, the short wavelength was the conjugation of carbazole and quinoline). As shown in the molecular orbital diagram (Figure S3) and the potential energy curve (Figure S4), the molecule transited to excited state after absorbing the energy. The electron density redistribution from the quinoline heterocycle toward the carbazole group was blocked due to the easy rotation of 4. Then the overlap of electronic cloud distribution between the left part and right part of LUMO orbital was low. Therefore, the weak fluorescence emitted by the whole molecule corresponds to the long wavelength emission. Because of the bonds of 1 and 3 were easier to rotate, the conjugation between quinoline and carbazole presented low fluorescence emission in the low concentration of mtDNA. In the presence of high concentrations of mtDNA, the S1-S0 transition (LUMO→HOMO) is the electron density redistribution from the quinoline heterocycle toward the carbazole group and is of ICT character. Therefore, the right part of conjugated structure from quinoline ring and carbazole group expressed fluorescent. Environment-induced conformational changes lowered the HOMO and increased the LUMO. This leads to an increase in the HOMOLUMO gap, in agreement with the hypsochromic shift to 590 nm (experimental data is 620 nm) in the presence of high concentrations of mtDNA. On the other hand, there are several methane bonds in CNQ that may be involved in the rotations (Figure S3). As shown in Figure S4, the bonds of 1 and 3 with lower barriers in the S1 state were easier to rotate, which led to the nonradiative deactivation and low background fluorescence of CNQ. When this free rotation was restricted by binding with mtDNA, there was a large fluorescence enhancement. Characterization of the interaction between CNQ and mtDNA. The secondary structure changes of mtDNA after the interaction with CNQ were recorded by circular dichroism (CD) experiment. When mtDNA (0-0.3 mg/mL) was gradually added to the PBS buffer solution of CNQ (10.0 μM), the positive bands (279 nm) and negative bands (245 nm) which represent base stacking and the right-hand B helicity form34 of mtDNA became stronger. Meanwhile, an obvious and positive ICD signal appeared which was consistent with an expected groove binding mode (Figure 3a). Furthermore, during the course of binding titration, only the intensity of observed ICD changed, not the shape, indicating a single binding mode. And the titration curve of ICD intensity (at 480 nm) versus the logarithm of the mtDNA concentration (Figure 3b) was similar to that of berenil (a minor groove binder).35 With the pH of PBS buffer solution decreasing (from 7.4 to 4.0) or increasing (from 7.4 to 10.0), the ICD signal of CNQ at 480 nm appeared a downward trend (Figure S5). While the negative bands (245 nm) intensity were sharply decreased in the higher acidity solution, owing to the right-hand B helicity form of mtDNA was destroyed, and the tertiary amine was protonated as well. Furthermore, the viscosity of mtDNA increased significantly (Figure 3c) in the presence of crescent EB (intercalator), while the variation tendency of CNQ was closer to that of berenil (minor groove binder). In general, the Tm of DNA increases about 58°C due to the intercalation of small molecules while obviously

at 700 nm. Nevertheless, the emission wavelength had a large blue shift (approximately 80 nm) and there was a 182-fold fluorescent enhancement finally within the large concentrations of mtDNA (130.0 μg/mL) with the binding affinity of Ki=2.8×105 (M-1) (Figure 1a). Figure S2a showed the normalized two-photon fluorescence spectra of CNQ in the presence of different concentrations of mtDNA in PBS buffer solution. The two-photon absorption cross section of free CNQ was small (Figure S2b, black line), and it was increased up to 98.4 (GM) after the addition of mtDNA (130.0 μg/mL) at the excitation wavelength of 850 nm in PBS buffer solution (Figure S2b, red line). There was a good linear relationship between the fluorescence intensity of CNQ and mtDNA concentrations in the range of 0.42-2.50 μg/mL (Figure 2b) with the correlation coefficient of 0.993 and detection limit (3σ/k) of 55.1 μg/L. Time dependent (0-50 s) fluorescence responses (at 620 nm) of CNQ (5.0 μM) in the presence of mtDNA showed that the combination process could be completed within 20 s (Figure 2c). Indeed, the common amino acid (Ala, Ile, Lys, etc.), proteins (HSA and BSA), Yeast RNA, ssDNA (dA21and dT21) and dsDNA (Poly(A-T)9 and Poly(G-C)9) did not give obvious interaction at 100-fold excess concentrations (Figure 2d). The excellent selectivity for mtDNA over other analytes demonstrated that CNQ had potential applications for mtDNA detection in complicated biological environments.

Figure 2. a) Fluorescence spectra determination of CNQ (5.0 μM) with the titration of mtDNA (0-130.0 μg/mL) in PBS buffer solution (10 mM, pH=7.4); b) Linear relationship between the fluorescent intensity of CNQ and mtDNA (0.42-2.50 μg/mL), the embeddable graph represented the fluorescent spectra of CNQ in the same concentrations of mtDNA; c) Time course of fluorescence intensity of CNQ (5.0 μM) at 620 nm after addition of mtDNA (20.0 μg/mL), time range 0-50 s. d) Fluorescence responses of CNQ to mtDNA and other interferes in PBS buffer solution, 1-22: control, mtDNA, Ala, Ile, Lys, Trp, His, Tyr, Asn, Asp, Gly, GSH, Pro, Phe, Val, HSA, BSA, Yeast RNA, dA21, dT21, Poly(A-T)9 and Poly(G-C)9, λex=480 nm.

Gaussian calculation. To elucidate the mechanism of the fluorescence “off-on” process, the structure of CNQ was optimized, and the frontier molecular orbital (FMO) energies were calculated in water using Gaussian 09 (DFT/TDDFT in B3LYP/6-31G (d, p)). The calculated absorption of 460 nm is close to the experimental value of 480 nm, representing part of the conjugated molecule with the corresponding orbital transition of HOMO-1 to LUMO (Figure S3a). In the free state of CNQ, it is evident that the HOMO and LUMO are localized on 3

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no enhancement in case of groove binding. 36 As presented in Figure 3d, the Tm of mtDNA was not affected by DMSO and only increased a little bit with the addition of CNQ.

co-stain with CNQ in MCF-7 cells and RWPE-1 cells, respectively. Figure S9 and S10 indicated that the distribution of CNQ was almost the same as that of commercial mitochondria probe with the colocalization Pearson’s correlation factors (Rr) of 0.96 and 0.9. Meanwhile, other kinds of subcellular organelle commercial dyes like NBD C6-ceramide (commercial Golgi apparatus probe), LysoTraker® Green DND-26 (commercial lysosome probe), ER-TrackerTM Green (commercial endoplasmic reticulum probe) and Hoechst 33342 (commercial DNA probe) were also coincubated with CNQ in MCF-7 cells, respectively. As assumed, their Rrs were 0.56, 0.59, 0.57 and 0.29, respectively (Figure S11), suggesting that CNQ was selectively localized on mitrochondria in living cells. As presented in Figure S12, the permeability of nuclear membrane was destroyed after immobilized treatment of cells, therefore CNQ could be located in nucleus. As anticipated, the fluorescence of CNQ dramatically diminished with the treatment of DNase while remained almost the same after the RNase treatment and thus univocally affirmed the selective DNA targeting property of CNQ. All the results indicated that the two-photon fluorescent probe of CNQ was selectively stained mtDNA in living MCF-7 cells. MTT assay. The MTT assays revealed that the cell viabilities were not affected by incubation with 2.0 μM CNQ for 12 h, and the survival rate was higher than 83% when the incubation concentration increased to 15.0 μM, indicating the low cytotoxicity of CNQ (Figure S13). In addition, CNQ had a better photostabiliy than MitoTracker® Green FM, after irradiation under a 500 W iodine tungsten lamp at room temperature for 6 h, the absorption intensity of CNQ was as high as 95%, much higher than MitoTracker® Green FM with 63% (Figure S14). Super-resolution fluorescence imaging of CNQ. Encouraged by its good performance in aqueous system and living cells imaging for mtDNA, CNQ was used to superresolution fluorescence microscopy as it could offer new promises to study molecular processes in detail. 38-40 As a nucleiod component, mitochondrial transcription factor A(TFAM) is also a packaging factor of mtDNA.41-43 Therefore,

Figure 3. a) The CD spectrum of CNQ (10.0 μM) with the addition of mtDNA (0, 1×10-4, 5×10-3, 0.01, 0.03, 0.05, 0.08, 0.1, 0.2 and 0.3 mg/mL) in PBS buffer solution (10.0 mM, pH=7.4). b) The titration curve of ICD intensity (at 480 nm) versus the logarithm of the mtDNA concentration from the raw data; c) Effect of increasing concentrations of CNQ, EB (intercalator), beneril (minor groove binder) and DMSO on the viscosity of mtDNA at 25°C; d) Melting temperature profiles of mtDNA in the absence and presence of CNQ and DMSO in PBS buffer solution, the Tm of mtDNA was 88.1°C in PBS buffer solution.

Molecular docking calculation. The molecular docking methodology can further simulate the interaction of ligand on the active site of receptor and record the optimum mode according to the theoretical prediction. Here the simulate calculation was completed based on the module of DSLigandFit on the crystal structure of 5JGN. According to the automatically search, two binding sites with small groove combination model were screened (Figure S6a), while the site 2 (green) was matched with the molecular structure of CNQ very well. Therefore, the experimental results of circular dichroism, viscosity, thermal melting, and molecular docking calculation demonstrated the interaction between CNQ and mtDNA was in the manner of minor groove binding. Cell images of CNQ. Then MCF-7 cell line was used to investigate the intracellular localization of CNQ. Under two-photon excited wavelength of 850 nm, CNQ (2.0 μM) showed strong red fluorescence in the cytoplasm (Figure S7) and the fluorescent intensity was kept steady within a long time. Since mtDNA is dsDNA, then, CNQ (2.0 μM) was co-incubated with PicoGreen (1.0 μg/L), an ultrasensitive fluorescent doublestranded DNA (dsDNA) stain in MCF-7 cells. Results were presented in Figure S8, PicoGreen and CNQ had a good co-localization in cytoplasm (Figure S8d), therefore, CNQ was proved to selectively stain dsDNA in cytoplasm. The structure-activity relationship approach expresses physicochemical properties of probes numerically and relates these values to the staining or non-staining of mitochondria. Hydrophilicity-lipophilicity is modelled by the logarithm of the water/octanol partition coefficient (logP). The mitochondrion-specific probes are assigned numerically as 0