Mitochondria-Directed Fluorescent Probe for the Detection of

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A mitochondria-directed fluorescent probe for the detection of hydrogen peroxide near mitochondrial DNA Ying Wen, Keyin Liu, Huiran Yang, Yi Liu, Liming Chen, Zhongkuan Liu, Chunhui Huang, and Tao Yi Anal. Chem., Just Accepted Manuscript • Publication Date (Web): 24 Sep 2015 Downloaded from http://pubs.acs.org on September 29, 2015

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Analytical Chemistry

A mitochondria-directed fluorescent probe for the detection of hydrogen peroxide near mitochondrial DNA Ying Wen, Keyin Liu,# Huiran Yang,$ Yi Liu, Liming Chen, Zhongkuan Liu, Chunhui Huang, and Tao Yi* Department of Chemistry and Concerted Innovation Center of Chemistry for Energy Materials, Fudan University, Shanghai 200433, China. Email: [email protected]; Fax: (+86) 21-55664621 ABSTRACT: It is important to detect hydrogen peroxide (H2O2) near mitochondrial DNA (mtDNA) because mtDNA is more prone to oxidative attack than nuclear DNA (nDNA). In this study, a mitochondria-targeted fluorescence probe, pep3-NP1, has been designed and synthesized. The probe contains a DNA-binding peptide, a H2O2 fluorescence reporter and a positively-charged red emissive styryl dye to facilitate accumulation in mitochondria. Due to groove binding of the peptide with DNA, the styryl dye of pep3-NP1 intercalated into the bases of DNA, leading to an increase in red fluorescence intensity (centered at 646 nm) and quantum yield. In this case, pep3-NP1 was a turn-on probe for labeling DNA. Subcellular locations of pep3-NP1 and MitoTracker suggested that pep3-NP1 mostly accumulated in the mitochondria of live cells. Namely, as an intracellular DNA marker, pep3NP1 bound to mtDNA. In the presence of H2O2, pep3-NP1 emitted green fluorescence (centered at 555 nm). Thus, the ratio of green with red fluorescence of pep3-NP1 was suitable to reflect the change of the H2O2 level near mtDNA in living cells. The detecting limit for H2O2 was estimated at 2.9 and 5.0 µM in vitro and in cultured cells, respectively. The development of pep3-NP1 could help in studies to protect mtDNA from oxidative stress.

Hydrogen peroxide (H2O2), as the most important marker for reactive oxygen species (ROS),1 can influence growth, development and fitness of living organisms. As a second messenger for intracellular signal transduction,2 H2O2 can activate many signaling pathways to stimulate cellular proliferation, differentiation and migration. Therefore, the level of H2O2 in normal cells is precisely regulated. However, under stress or stimulation by exogenous chemicals, aberrantly production of H2O2 may result in oxidative stress. The overproduced H2O2 can attack cellular structures or biomolecules, such as proteins,3 liposomes4 and DNA.5 Oxidative modifications of DNA bases, including the oxidation of purines and pyrimidines, the generation of alkali labile sites and strand breaks,6 may induce mutations and even cancer, if not repaired in a timely manner. In terms of DNA oxidative damage, mitochondrial DNA (mtDNA), which encodes 13 polypeptides involved in oxidative phosphorylation, is a more sensitive target than nuclear DNA (nDNA), due to a combination of the following factors: 1) unlike nDNA, mtDNA lacks protection from histones; 2) mtDNA is close to the site of oxidant production and has a relatively low DNA repair capacity.7 Indeed, the levels of oxidized bases in mtDNA have been reported to be 10- to 20- fold higher than those in nDNA.8-10 Once mtDNA is damaged, the encoding of critical proteins for the respiratory chain becomes deficient, amplifying ROS production and mitochondrial dysfunction.11-14 Oxidative damage of mtDNA, therefore, deserves more extensive attention. To reduce mtDNA oxidative damage, it is critical to monitor the oxidant level near mtDNA. Fluorescence labeling is a powerful technique for this purpose because of its nondisruptive and real-time features. Chang’s group15-17 and other

researchers18-22 have developed several H2O2 fluorescent probes, some of which could be targeted to important subcellular organelles, such as the nucleus,23-25 endoplasmic reticulum25 and mitochondria.20, 25-27 However, currently available H2O2 fluorescent probes directed to mitochondria have not been reported to have the capability of binding to DNA, so these probes cannot be used to precisely monitor the level of H2O2 near mtDNA.

Scheme 1. (a) Chemical structure of pep3-NP1 and (b) the schematic diagram of its binding modes toward DNA and H2O2.

We recently reported a versatile H2O2 fluorescence reporter, NP1, based on 1,8-naphthalimide.28 It was a highly sensitive ratiometric probe carrying an azide group, which rendered it easy to be linked to other molecules via the click reaction. To further develop fluorescence probes for monitoring the levels of H2O2 near mtDNA, herein, pep3-NP1 was designed and synthesized based on NP1, by introducing a DNA-binding peptide tagged with a positively charged red-emitting styryl fluorophore (Scheme 1a). This lipophilic cation may facilitate

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accumulation of the probe in mitochondria.29–31 An additional advantage to use the styryl fluorophore was that its red fluorescence was sensitive to the polarity of the microenvironment due to its internal charge transfer property,32 which made pep3-NP1 to be used as a detection signal for DNA binding. The DNA-binding peptide, containing two TrpThr-Lys tripeptide units with each being attached via the Cterminus to a central lysine spacer,33 could bind to the minor grooves of DNA. Once the probe bound to DNA, the red fluorescence intensity of the styryl fluorophore should be greatly enhanced due to the low polarity of DNA grooves. On the other hand, the probe would emit green fluorescence in the presence of H2O2. Thus, pep3-NP1 could be used as both an intracellular DNA marker and a H2O2 sensor. Particularly, pep3-NP1 was suitable to reflect the change of the H2O2 level near mtDNA by using the ratio of green with red fluorescence (Scheme 1b). Compared with NP1, pep3-NP1 has two advantages: 1) the probe can accumulate in mitochondria and target DNA, making it especially suitable to detect H2O2 in the mtDNA microenvironment; and 2) the ratiometric output signal when detecting H2O2 changes from blue/green to green/red fluorescence by laser excitation at a wavelength in the visible range (455 nm), due to incorporation of the red-emissive styryl dye. The red-shift of the excitation wavelength and the output fluorescent signal is a more biocompatible system. With this precise design, we have developed a mitochondria-directed ratiometric fluorescent probe for the detection of hydrogen peroxide near mitochondrial DNA.

EXPERIMENTAL SECTION Materials. Calf thymus DNA (CT DNA), H2O2, tertbutylhydroperoxide (tBHP), hypochlorite (NaOCl), 3(aminopropyl)-1-hydroxy-3-isopropyl-2-oxo-1-triazene (NOC5), xanthine oxidase, xanthine sodium salt, catalase, potassium superoxide, ferrous perchlorate, tris[(1-benzyl-1H1,2,3-triazol-4-yl)methyl]-amine (TBTA), proline (Pro), cysteine (Cys), arginine (Arg), sucrose (Suc), glucose (Glu), glutathione (GSH) and bovine serum albumin (BSA) were purchased from Sigma-Aldrich (St. Louis, MO, U.S.). All organic solvents were supplied by Shanghai Sinopharm Chemical Reagent Co., Ltd. MitoTracker green and LysoTracker green were supplied by Shanghai Beyotime Co., Ltd. The peptide was synthesized on a solid-phase synthesizer by Bankpeptide biological technology Co., Ltd. NP1 was synthesized as described previously.28 Instruments. 1H NMR spectra were recorded with Bruker DRX 500 spectrometer at 400 MHz. Proton chemical shifts were reported in parts per million downfield from tetramethylsilane (TMS). Electrospray ionization mass (ESI-MS) spectra were measured on a Bruker Micro TOF II 10257 instrument. UV visible spectra were measured on a Shimadzu UV-2550 spectrometer. Steady-state emission spectra were recorded on an Edinburgh FLS-920 spectrometer with a Xe lamp as the excitation source. An Edinburgh FLS5 spectrophotometer with integrating sphere was used to measure absolute fluorescent quantum yield. Confocal imaging was performed on an Olympus FV1000 confocal fluorescence microscope with a 60 × oil-immersion objective lens. HPLC separation was carried out on a Waters Alliance 2695 HPLC system. The detector was a Waters 2996 diode array detector with the wavelength range from 210 to 400 nm. Fractions were collect-

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ed manually and were lyophilized on a Christ Alpha 1-4/LD-2 freeze-dryer. The MTT assay was performed on a Tecan Infinite M200 monochromatic-based multifunction microplate reader. Cell culture. The HeLa, MCF-7 and HepG2 cells were provided by the Institute of Biochemistry and Cell Biology, China and were grown in RPMI 1640 media supplemented with 10% fetal bovine serum at 37ºC in a humidified atmosphere containing 5% CO2. CLSM imaging. Cells were plated on 14 mm glass coverslips and were incubated overnight. The cells were washed with RPMI 1640 media and then incubated with 5 µM pep3NP1 in dimethyl sulfoxide (DMSO)/RPMI 1640 (0.5%, v/v) for 3 h at 37ºC. After washing three times, the cells were subjected to CLSM imaging with the excitation wavelength at 458 nm. The green and red channels were set at 535 ± 25 and 635 ± 25 nm, respectively. For the co-location experiments, the cells loaded with pep3-NP1 were incubated with MitoTracker (500 nM) or LysoTracker (1 µM) for another 30 min. These cells were excited at 488 nm, and the emission was collected at 530 ± 20 nm. Cell viability. Cytotoxicity was assessed by performing 3(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay on the HeLa cells. Cells were seeded into a 96well plate at 2 × 103/well and were cultured at 37ºC and 5% CO2 for 24 h. Different concentrations of pep3-NP1 (0, 1, 2.5, 5, 7.5 and 10 µM) were then added to the wells. After incubation for 6 or 12 h, MTT (0.5 mg/mL) was added to each well and the plate was incubated for 4 h. The optical densities at 490 nm were measured.

RESULTS AND DISCUSSION Synthesis and characterization of pep3-NP1. The synthetic details for pep3-NP1 are provided in the Electronic Supplementary Information (Scheme S1 in the Supporting Information). Compound 1 was synthesized as reported previously.34 A mixture of 1, potassium iodide and 3bromopropanoic acid was stirred under reflux to produce a red solid (2) in 60% yield. The peptide was custom-synthesized by a commercial supplier. One of the two N-terminuses of the peptide was functionalized with 2, and the other was modified with 5-hexynoic acid to produce pep3. Click reaction between pep3 and NP1 provided pep3-NP1, which was purified by HPLC (ESI-MS: calcd. for C91H113N19O15B+ [M]+, 1722.8757; found, 1722.8799; calcd. for C91H113N19O15B2+ [M+H]2+, 861.9418; found, 861.9452; calcd. for C91H113N19O15B3+ [M+2H]3+, 574.9638; found, 574.9659).


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Figure 1 Absorption (50 µM, solid lines) and fluorescence emission (5 µM, λex = 352 nm, dashed lines) spectra of pep3NP1 (red), 2 (blue) and NP1 (black) in Tris-HCl (pH 7.2, 1% DMSO).

The basic spectroscopic properties of pep3-NP1 were assessed in vitro (Tris-HCl, pH 7.2, 1% DMSO). The probe (50 µM) showed two absorption bands (Figure 1, the red solid line) at 352 nm (ε = 9 020 M-1 cm-1) and 455 nm (ε = 10 700 M-1 cm-1), which were attributed to the 1,8-naphthalimide and styryl fluorophores, respectively. A weak blue fluorescence at 390 nm (Φ = 0.94%) from the 1,8-naphthalimide fluorophore and a red fluorescence at 646 nm from the styryl fluorophore (Φ = 2.8%, λex = 352 nm, Figure 1, the red dashed line) were observed. The much lower quantum yield for the blue fluorescence of pep3-NP1 compared with that of NP1 (6.1%) should be due to the fluorescence resonance energy transfer (FRET) from the 1,8-naphthalimide fluorophore to the styryl one, due to the spectral overlap between the absorption band of the styryl fluorophore 2 (Figure 1, the blue solid line) and the emission one of 1,8-naphthalimide (Figure 1, the black dashed line).28 With 455 nm excitation, the pep3-NP1 solution showed only weak red fluorescence of styryl fluorophore (Φ = 3.3%). Ability of pep3-NP1 to bind DNA. Due to the presence of DNA-binding peptide, pep3-NP1 could bind to DNA as expected. Binding of pep3-NP1 to DNA was studied by the absorption and fluorescence changes in vitro on calf thymus DNA (CT DNA) (Figure 2). In the presence of 200 µM CT DNA, the absorption of pep3-NP1 (50 µM) at 455 nm (ascribed to the styryl fluorophore) decreased, followed by a red-shift to 465 nm, and that at 352 nm showed slight hyperchromism (Figure 2a). According to previous reports, the observed large bathochromic shift of ~ 10 nm (455-465 nm) for pep3-NP1 in the presence of DNA should be due to strong interactions between the dye and DNA base pairs.32, 35 Indeed, the styryl group of pep3-NP1, having extended planarity and conjugation, is able to establish π-stacking interactions with DNA base pairs. However, it was interesting to note that no changes in the absorption spectrum or the fluorescence quantum yield of 2 were observed upon the addition of CT DNA, indicating that the styryl fluorophore itself had no ability to interact with DNA (Figure S1 in the Supporting Information). Thus, the change of the absorption spectrum of the styryl moiety in pep3-NP1 after the addition of DNA should be attributed to the proximity of the styryl fluorophore to the base pairs caused by binding of the peptide moiety in pep3-NP1 to DNA. The binding of the peptide to DNA groove may facilitate the chromophore to be intercalated into the stacks of base pairs as reported in our previous report.33 More binding evidence came from fluorometric titration experiments with increasing DNA concentrations. Addition of CT DNA (0-50 µM) to the solution of pep3-NP1 (2.5 µM) caused a significant enhancement of the red fluorescence intensity at 646 nm with a slight red-shift (5 nm, λex = 455 nm) (Figure 2b). The intercalation between the dye and the DNA bases hindered bond rotation and thereby decreased possible non-radiative processes. This led to increases in the fluorescence intensity and the absolute quantum yield. Indeed, the quantum yield of the red fluorescence of pep3-NP1 (2.5 µM) increased from 3.3% to 16% after binding with 50 µM of

DNA. A Job’s plot was made by using the change of fluorescence intensities at 646 nm versus the fractions of DNA with a total concentration of pep3-NP1 and DNA of 10 µM (Figure 2c). The maximum emission intensity at 646 nm appeared at 0.64, indicating that the stoichiometric binding ratio of pep3NP1 with CT DNA was 1:2. It should be pointed out that pep3-NP1 showed high selectivity in binding to DNA even in the presence of other biologically relevant macromolecules (Figure 2d). To verify the selectivity of pep3-NP1 for DNA, the change of the fluorescence intensities of pep3-NP1 at 646 nm were recorded upon the addition of proline (Pro), cysteine (Cys), arginine (Arg), sucrose (Suc), glucose (Glu), glutathione (GSH) and bovine serum albumin (BSA). These biologically relevant macromolecules did not induce any changes in the emission intensities of pep3-NP1, in contrast to a 15.5-fold increase after the addition of the same amounts of DNA, suggesting that these species had no interactions with pep3-NP1 and did not interfere with the response of pep3-NP1 to DNA. In a word, pep3-NP1 could be used as a turn-on probe for labeling DNA.

Figure 2 (a) Absorption spectra of pep3-NP1 (50 µM) without or with 200 µM CT DNA. (b) Fluorescent spectral changes of pep3-NP1 (2.5 µM) response to CT DNA with the concentrations at 0, 0.5, 1, 2, 5, 10, 15, 20, 30, 40 and 50 µM. (c) Normalized Job’s plot of CT DNA and pep3-NP1. The total concentration of CT DNA and pep3-NP1 was kept constant at 10 µM. Fluorescence emission intensity was measured at 646 nm. Square deviations of the fitted lines were 0.99169 and 0.99173. (d) Fluorescence changes of 2.5 µM pep3-NP1 responding to various biologically relevant macromolecules, including protein, sugars and amino acids. Bars represent the final integrated fluorescence intensity (Ff, 646 nm) over the initial integrated emission (Fi, 646 nm). Black bars represent the addition of an excess of the appropriate macromolecule (25 µg/mL BSA and 50 µM of Pro, Cys, Arg, Suc, Glu, GSH) to a pep3-NP1 solution. Red bars represent the subsequent addition of 50 µM CT DNA to the solution. Spectra were acquired in Tris-HCl (pH 7.2, 1% DMSO), λex = 455 nm.

Ability of pep3-NP1 to accumulate in mitochondria. With these in vitro data in hand, we studied whether pep3NP1 could be used as intracellular DNA marker. First, the subcellular location of pep3-NP1 was examined. Cervical cancer HeLa cells incubated with 5 µM pep3-NP1 at 37 °C for



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3 h showed strong red fluorescence (635 ± 25 nm) in cytoplasm as observed by using a confocal laser scanning microscopy (CLSM) (Figure 3a,f), suggesting that the probe bound to intracellular DNA. To verify the precise distribution of pep3-NP1 in live cells, the pep3-NP1-loaded cells were incubated with 500 nM Mito Tracker Green, a commercially available mitochondrial indicator (Figure 3b) or 1 µM LysoTracker Green, a lysosomal indicator (Figure 3g). The merged images and the correlation mapping of the fluorescent intensities showed a good colocalization of pep3-NP1 and MitoTracker, indicating that pep3-NP1 was predominantly accumulated in the mitochondria. Namely, pep3-NP1 bound to mtDNA. The colocalization experiments were also carried out in other types of cells, such as breast cancer MCF-7 and hepatocellular carcinoma HepG2 cells, which showed a similar intracellular distribution of pep3-NP1 (Figure S2).

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cence at 555 nm was ascribed to the replacement of the boronate group of pep3-NP1 by a hydroxyl group after reaction with H2O2.28 Kinetic measurements of the H2O2-mediated boronate deprotection were performed under pseudo-firstorder conditions (1 µM pep3-NP1, 1 mM H2O2), giving an observed rate constant of 4.87 × 10-4 s-1 (Figure S4). The selectivity of pep3-NP1 towards H2O2 was also verified. Fluorescence intensities of pep3-NP1 were determined at 0, 15, 30, 45, 60, 90 and 120 min upon the addition of H2O2 and other ROS/RNS (200 µM of nitric oxide (NO), superoxide (O2-), tert-butoxy radical (·OtBu), hypochlorite (OCl-), superoxide radical (O2-·), tert-butylhydroperoxide (tBHP), hydroxyl radical (HO·) and singlet oxygen (1O2), Figure 4c). The fluorescence intensity change ratio at 555 nm (R555) was calculated for all species and time points. At 2 h, H2O2 induced an obvious response with the R555 being 11.90, which was markedly greater than that obtained with other ROS/RNS (0.08-2.21). The result showed a high selectivity of pep3-NP1 toward H 2O 2.

Figure 3 CLSM images of HeLa cells co-labeled with (a-d) pep3-NP1 (5 µM)/MitoTracker (500 nM), and (f-i) pep3NP1 (5 µM)/LysoTracker (1 µM) at 37 °C. (a, f) Red channel: 635 ± 25 nm for pep3-NP1, λex = 458 nm; (b, g) green channel: 530 ± 20 nm for MitoTracker or LysoTracker, λex = 488 nm; (c, h) the brightfield image; and (d, i) the overlay images of red and green channels. (e, j) The correlation of Mito/LysoTracker and pep3-NP1 intensities. Scale bar = 20 µm.

Ability of pep3-NP1 to detect H2O2 in the presence of DNA. The ability of pep3-NP1 to detect H2O2 was investigated in vitro (Figure 4). After the addition of 1 mM H2O2, the absorbance of pep3-NP1 at 352 nm decreased, and that at 455 nm increased with a blue-shift over time (Figure S3a). At 2 h, the two absorption bands changed to one absorption band centered at 442 nm (Figure 4a). NP1 was reported to display one absorption band centered at 446 nm in the presence of H2O2 due to its chemospecific boronate switch.28 On the contrary, the absorption of 2, which exhibited a maximum at 413 nm, did not change after the addition of H2O2 (Figure S3b), indicating that the styryl dye itself did not react with H2O2. Consequently, the absorption band of pep3-NP1 at 442 nm could be attributed to the overlap of the absorption of the 1,8naphthalimide (after reaction with H2O2) and styryl fluorophores. The ability of pep3-NP1 to detect H2O2 was further studied by fluorescent spectral change. Upon treatment of 5 µM pep3NP1 with 200 µM H2O2, with 455 nm excitation, where the absorption of 1,8-naphthalimide after reaction with H2O2 overlapped with that of the styryl fluorophore, the green fluorescence intensity at 555 nm (Φ = 5.8%) drastically increased, whereas the red emission at 646 nm was almost unchanged. This behavior was consistent with that of NP1.28 The fluores-

Figure 4 (a) Absorption spectra of pep3-NP1 (25 µM) without or with 1 mM H2O2 at 2 h. (b) Fluorescent spectral changes of pep3-NP1 (5 µM) with time after the addition of H2O2 (200 µM) at 37°C. (c) The fluorescence intensity change ratio at 555 nm of pep3-NP1 (5 µM) in response to various ROS/RNS (200 µM) at 0 to 120 min. A to I represents H2O2, HO· , O2-, NO, O2-· , OCl-, · OtBu, tBHP, 1O2, respectively. (d) The fluorescent spectral changes of pep3NP1 (5 µM) in the presence of 1 µM CT DNA upon addition of H2O2 (200 µM) at 37°C. Spectra were acquired in TrisHCl (pH 7.2, 1% DMSO), λex = 455 nm.

To eliminate the possibility that DNA-binding interfered with the detection of H2O2, the fluorescent spectral changes of pep3-NP1 in the presence of DNA upon addition of H2O2 were studied. Addition of CT DNA (1 µM) made the fluorescence intensity of pep3-NP1 at 646 nm (F646) a rapid increase (twice as much within 10 s), and then remained almost constant with time (Figure 4d and Figure 5a). Meanwhile, with the addition of H2O2 (200 µM) in the presence of DNA (1 µM) at 37 °C, the fluorescence intensity of pep3-NP1 at 555 nm (F555) increased with time (Figure 4d and Figure 5a). Thus, ratio of the fluorescence intensities (F555/F646) could be used as a detection signal for production of H2O2 in the presence of DNA. The ratiometric probe can enable more accurate and quantitative analysis compared with a simple “turn on” probe, espe-


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cially in a cellular environment.36 Figure 5a showed that the ratio (F555/F646) increased from 0.08 to 0.71 with the increased reaction time with H2O2 (from 0 to 150 min) in the presence of CT DNA. In addition, a linear correlation between the ratio of the emission intensities (F555/F646) and the H2O2 concentration was observed with R2 being 0.9908 (Figure 5b). The detection limit of H2O2 in the presence of DNA was calculated with 3σ/k; where σ is the standard deviation of blank measurement; k is the slop in Figure 5b. The detection limit was estimated as 2.90 µM, which was comparable to those of the established H2O2 probes.37, 38

Figure 5 (a) Fluorescence intensity change of pep3-NP1 (5 µM) at 646 nm (black squares), and intensity ratio of F555/F646 (blue circles) with time after addition of H2O2 (200 µM) in the presence of CT DNA (1 µM) at 37°C. (b) Plot of the ratio (F555/F646) for pep3-NP1 (5 µM) vs [H2O2] in the range of 10-50 µM in the presence of CT DNA (1 µM) (each data was acquired 2 h after the addition of H2O2 at 37°C). Spectra were acquired in Tris-HCl (pH 7.2, 1% DMSO), λex = 455 nm.

Ability of pep3-NP1 to detect exogenous and endogenous H2O2 ex vivo. To assess the capability of pep3NP1 to detect H2O2 in cells, HeLa cells loaded with pep3-NP1 (5 µM) were treated with 200 µM H2O2 for 30 min. The fluorescence intensities from the green channel (λem = 535 ± 25 nm) clearly increased after H2O2 stimulation (Figure 6, a1 and b1). The ratio (RG/R) images were established by fluorescence detection at 535 ± 25 nm (green channel) and 635 ± 25 nm (red channel) using Carestream software. The red fluorescence showed the binding between pep3-NP1 and mtDNA (Figure 6, a2 and b2); the green fluorescence resulted from the monitoring of H2O2. Thus, the ratio of green with red fluorescence could be used to reflect the change of the H2O2 level near mtDNA. The control group was found to give a weak RG/R signal (< 0.80), while the RG/R values clearly increased in the group treated with H2O2 (> 1.15, Figure 6, a4 and b4). To further determine the detection limit of H2O2 in cultured cells, CLSM images of Hela cells in the presence of H2O2 of 0, 1, 5, 10, 50 and 100 µM were measured (Figure S5). We found that pep3-NP1 was capable of visualize H2O2 as low as 5 µM in living HeLa cells, which was comparable to the detection limit in vitro. Next, we assessed whether pep3-NP1 could be used to detect endogenous H2O2 in the mitochondria of living cells. To this end, we treated HeLa cells with paraquat, a model compound for endogenously produced ROS by intracellularly redox cycling in the mitochondria.39-46 HeLa cells were treated with 1 mM paraquat for 6 h before loading with pep3-NP1 (5 µM) (Figure 6, c and d). The control group gave low RG/R values (< 0.45, Figure 6, c4), whereas the cell treated with paraquat resulted in a marked increase in RG/R (> 1.15, Figure 6,

d4). These results indicated that pep3-NP1 is sensitive enough to detect local mitochondrial H2O2 production.

Figure 6 CLSM images of 5 µM pep3-NP1-loaded HeLa cells incubated (a1-a4 and c1-c4) without and (b1-b4 and d1d4) with H2O2 (200 µM)/ paraquat (1 mM). The ratio (RG/R) images were generated by measuring fluorescence at a range from green channel to red channel. Green channel: 535 ± 25 nm; Red channel: 635 ± 25 nm. λex = 458 nm; scale bar = 20 µm.

Moreover, pep3-NP1 was non-toxic to HeLa cells. The viabilities were estimated to be > 90% at 12 h in the presence of 1-10 µM pep3-NP1, as determined by the MTT assay (Figure 7). Thus, pep3-NP1 had the potential to be used in biological applications. Taken together, these data demonstrate that pep3-NP1 could be used to examine cellular H2O2 change near mtDNA.

Figure 7 Cell viability values (%) estimated by MTT assay in HeLa cells, which were cultured in the presence of 0-10 µM pep3-NP1 for 6 and 12 h.

CONCLUSIONS



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In summary, we have designed and developed a mitochondria-targeted fluorescence probe, pep3-NP1, which is capable of lighting up DNA and monitoring H2O2. The probe contains a DNA groove-binding peptide, a H2O2 fluorescence reporter (NP1) and a styryl dye with a positive charge. The positive charged styryl moiety rendered the probe to accumulate in mitochondria and the DNA-binding peptide made the probe bind to mtDNA. Compared with the mitochondria-directed H2O2 fluorescent probes reported previously, pep3-NP1 had an additional ability to label DNA, making it be able to be used as a probe to detect the level of H2O2 near mtDNA. This application of pep3-NP1 could help in studies to protect mitochondrial DNA from oxidative stress. The development of the fluorescence probes that are capable of binding to a variety of cellular structures (including, but not limited to, proteins, liposomes, and DNA) will help provide better understandings of the complicated regulation mechanisms inside cells and more accurately predict diseases.

ASSOCIATED CONTENT Supporting Information The synthetic details, additional spectra and additional CLSM cell images. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author * E-mail: [email protected]. Fax: (+86) 21-55664621. # Present address for Keyin Liu: University of Jinan, Institute of Fluorescent Probes for Biological Imaging, School of Chemistry and Chemical Engineering, School of Biological Science and Technology, Jinan 250022, China $ Present address for Huiran Yang: Nanjing University of Posts and Telecommunications (NJUPT), Institute of Advanced Materials (IAM), Nanjing 210046, China

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT The authors thank for the financial support of the National Basic Research Program of China (2013CB733700), the China National Funds for Distinguished Young Scientists (21125104), National Natural Science Foundation of China (51373039) and Specialized Research Fund for the Doctoral Program of Higher Education (20120071130008).

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Porteous, C. M.; Smith, R. A.; Hartley, R. C.; Partridge, L.; Murphy, M. P. Nat. Protoc. 2012, 7, 946-958.

(46) Dickinson, B. C.; Lin, V. S.; Chang, C. J. Nat. Protoc. 2013, 8, 1249-1259.

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