Mitochondrial G-quadruplex targeting probe with near-infrared fluorescence emission

Accepted Manuscript Title: Mitochondrial G-quadruplex targeting probe with near-infrared fluorescence emission Authors: Ling-Ling Li, Hao-Ran Xu, Kun Li, Qi Yang, Sheng-Lin Pan, Xiao-Qi Yu PII: DOI: Reference:

S0925-4005(19)30202-3 https://doi.org/10.1016/j.snb.2019.01.169 SNB 26095

To appear in:

Sensors and Actuators B

Received date: Revised date: Accepted date:

17 November 2018 9 January 2019 31 January 2019

Please cite this article as: Li L-Ling, Xu H-Ran, Li K, Yang Q, Pan SLin, Yu X-Qi, Mitochondrial G-quadruplex targeting probe with near-infrared fluorescence emission, Sensors and amp; Actuators: B. Chemical (2019), https://doi.org/10.1016/j.snb.2019.01.169 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Mitochondrial G-quadruplex targeting probe with near-infrared fluorescence emission

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Ling-Ling Li, Hao-Ran Xu, Kun Li*, Qi Yang, Sheng-Lin Pan and Xiao-Qi Yu

Key Laboratory of Green Chemistry and Technology, Ministry of Education, College of Chemistry, Sichuan University, Chengdu, 610064, China

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Table of contents:

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Highlights 

Highly selective ‘turn-on’ probe with near-infrared fluorescence emission for G-quadruplex is

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designed and synthesized.

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Probe NCT brings little disturbance to the configuration of G4.

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NCT can serve as a NIR dye in selectively imaging G4 in polyacrylamide gel electrophoresis.

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The imaging of mitochondrial G4 was achieved by NCT with high resolution optical microscope.

Abstract: A novel thiazole orange based fluorescent probe NCT has been reported with promising

G-quadruplex targeting ability. Interacting with G-quadruplexes will enhance its fluorescence intensity, but cause no obvious change in the CD spectrum. NCT exhibits high sensitivity and selectivity towards G4 over single strand DNA and double strand DNA. The viscosity-dependent fluorescence spectra and the excited-state lifetime experiment demonstrate that the restriction of the single bond

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between vinyl and quinolone group may be responsible for the ‘turn-on” of the fluorescence. Moreover, NCT is used as a special dye for imaging G4 selectively under both UV irradiation and

NIR irradiation in electrophoresis gel experiment, while commercially available dye Gel-Red can only stain DNA under UV irradiation. The mitochondria co-localization experiment, the high-resolution cellular imaging and the DNase digestion experiment have jointly proved that NCT is a potential

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probe for imaging G4 in mitochondria of living cells with excellent cell membrane permeability.

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Key words：G-quadruplexes, mitochondrial imaging, near-infrared fluorescence

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1. Introduction

G-quadruplexes (G4) are non-canonical nucleic acid structures that have attracted many research

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interests for several decades [1-3]. G4 can be classified into parallel, antiparallel or hybrid according

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to the orientation of nucleic acid chain [4]. Under physiological conditions, G4, which is formed by guanine-rich single-stranded DNA or RNA, is as stable as double helix structure [5, 6]. Literature has

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shown that G4 forming elements can be found in up to 43% of human genes [7]. Large amount of DNA G4 sequences have been identified in the promoter region of genes [8, 9], which play crucial

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roles in the regulation of genomic functions [10, 11]. Studies have shown that G-quartet stabilizers can bind to the ends of chromosomes and inhibit telomerase [12, 13], which can be used as potential

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anticancer drugs [14]. Nevertheless, the function of G-quadruplex in mammalian cells remains largely unknown. Therefore, developing tools to further our understanding of the roles played by G-quadruplex structures in biology is a demanding challenge [7, 15]. Fluorescent dyes including cyanines [16, 17], carbazoles [18, 19], quinolone [20], triphenylmethane [21], tetraphenylethene [22] and others [23-27] are widely explored for the design of G4 ligands due

to their good selectivity and optical properties [28, 29]. The interaction between these ligands and G4 will restrict their intramolecular rotation and reduce non-radiation energy dissipation [30, 31]. Consequently, the fluorescent signal of these dyes will be changed. Based on similar design strategy, thiazole orange (TO) has also been widely used for nucleic acid sensing because of its good binding

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abilities [3]. Many TO derivatives have been reported as G4 probes, however, their emission wavelengths are generally below 600 nm. NIR fluorescence (> 650 nm) is more desirable for

noninvasive biological imaging because of the little interference of cellular autofluorescence and less

light damage [32]. Till now, quite limited near-infrared probes have been developed for G4 detection, and only two of them have been used for cell imaging [33, 34]. On the other hand, even though the

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existence of G4 structures in cytoplasm remains controversial, most of the previously published G4

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probes with cell imaging abilities show fluorescence in cytoplasm [23,24,25,34]. Whether or not does mitochondrial DNA capable of adopting G4 configuration is a puzzle waiting to be solved.

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Developing mitochondria targetable G4 probes might help in solving this tricky problem. Moreover,

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commercially available nucleic acid dyes, such as Gel-Red, usually have no specific affinity to

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non-canonical structures of DNA. Developing new kind of G4 dyes for electrophoresis gel experiment with high selectivity is in urgent need. To meet these demands, we have designed a novel TO-based

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near-infrared emission mitochondria-targeted probe for G4. Crescent-shaped scaffold is proved to have better affinity and selectivity towards G4 [18, 35]. To

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construct such a crescent-shaped π-conjugated planar core, TO moiety is conjugated to coumarin via

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vinyl bond. The conjugation will not only endow the probe with improved selectivity, but also red-shift the maximum emission wavelength to the near-infrared region. Meanwhile, in order to prove

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the design concept and mechanism, probe CT, which is deprived of the thiazole moiety (Scheme 1) is also synthesized. All the probes and intermediates are characterized by 1H NMR, 13C NMR and HRMS. 2. Materials and methods 2.1. General methods, instrumentation, and measurements

Mass spectrometer (ESI-MS) and High-Resolution Mass Spectrometer (HRMS) data were recorded on a Finnigan LCQDECA and a Bruker Daltonics Bio TOF mass spectrometer, respectively. The 1H NMR and 13C NMR spectra measured on a Bruker AM400 NMR spectrometer and the δ scale in ppm referenced to residual solvent peaks or internal tetramethylsilane (TMS). Fluorescence emission

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spectra were obtained using FluoroMax-4 Spectrofluoro-photometer (HORIBA Jobin Yvon) at 298 K. Fluorescence lifetime measurements were carried out using a time-correlated single photon counting spectrometer (HORIBA TEMPRO-01). Circular dichroism studies were carried out on a JASCO

J-1500 Circular Dichroism Spectrometer. The cells were analyzed for membrane permeability by

acquiring the fluorescence using flow cytometry (BD Accuri C6). Cytotoxicity assay was carried out

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on BIO-RAD iMarkTM Microplate Reader. Gel electrophoresis results were photographed by Azure

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Biosystems, c600 bioanalytical imaging system and Molecular Imager® Gel Doc™ XR+ System.

and Carl Zeiss-I scan (resolution of 140 nm).

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2.2 Materials

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Fluorescence confocal laser scanning images were recorded with a LEICA TCS SP8，ZEISS LSM 780

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All oligonucleotides used in this study were purchased from Sangon (China). DNase was bought from

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Sigma-Aldrich (D4527). Unless otherwise indicated, all syntheses and manipulations were carried out under N2 atmosphere. All the solvents were dried according to the standard methods prior to use. All

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the solvents were either HPLC or spectroscopic grade in the optical spectroscopic studies.

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All the oligonucleotides and ctDNA were dissolved in relevant buffer. Their concentrations were determined from the absorbance at 260 nm based on respective molar extinction coefficients,

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respectively. The oligonucleotides were engaged in G4 formation, as determined by circular dichroism (CD) measurements. Stock solutions of G4s (100 nM) were dissolved in relevant buffer and stored at 4 °C. 2.3 Details for Fluorescence Measurements Stock solutions of NCT (1 mM) were prepared in DMSO for each measurement. All the oligonucleotides (100 nM) were dissolved in 10 mM Tris-HCl buffer containing 50 mM KCl at pH 7.4.

The fluorescence studies performed in 10 mM Tris-HCl buffer containing 50 mM KCl at pH 7.4. Each time a 3 mL of sensor solution (1 μM) was filled in a quartz cell (3.5 mL) of 1 cm of optical path length and the stock solution of G-quadruplex was added into the quartz cell using a microsyringe. The excitation and emission slits of fluorescence spectra were set at 5.0 nm if not specified. After each

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addition of sensors, the solution was stirred and allowed to equilibrate for at least 5 min. The fluorescence lifetime was measured by the method of time-correlated single-photon counting using a

picosecond spectrofluorometer. The excitation wavelength was 450 nm. The typical response time of this laser head was <1 ns. 2.4 Determination of the fluorescence quantum yield

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The fluorescence quantum yield (Q) is defined as the number of photons emitted per photon absorbed by

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the system. The general equation for calculating the fluorescence quantum yield is listed below.

In this equation, I (a) is the relative intensity of the exciting light at wavelength A, n is the average

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refractive index of the solution to the luminescence, D is the integrated area under the corrected

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emission spectrum, and A(a) is the absorbance of the solution at the exciting wavelength a. Subscripts x and r refer to the sample and reference solutions, respectively [36].

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In this paper, we used the same wavelength to excite the sample and the reference solution. Both measured samples are dissolved in water, therefore, the middle two formulas of the equations are 1. That

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means we only needed to determine the absorbance and integration of the emission spectra of peak area. Rhodamine B was used as reference in this experiment, Qr = 0.69.

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2.5 Calculations for detection limit The detection limit was calculated based on the fluorescence titration using the following equation [37]: DL =

3σ k

Here, σ was the standard deviation of blank measurements, k was the slope between emission intensity and G4 concentration. The fluorescence emission of NCT were measured 10 times to obtain the

standard deviation of blank measurements. 2.6 CD Studies The CD studies were performed in 10 mM Tris-HCl buffer containing 50 mM KCl at pH 7.4. A quartz cuvette with a 10 mm path length was used for the recording of spectra over a wavelength range of

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230-350 nm with a 2 nm bandwidth, 0.5 nm step size and time of 100 nm/min. The recorded spectra represented an average of three scans, zero-corrected at 320 nm and normalized (Molar ellipticity θ is

quoted in 105 deg cm2 dmol−1). The buffer baseline was collected in the same cuvette and was subtracted from the sample spectra. Origin 8.5 was used for final analysis of the data. 2.7 Gel Electrophoresis Studies

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Gel electrophoresis studies were carried out with 20% polyacrylamide-bisacrylamide (29:1) gel.

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Samples were prepared in 20 μL (final volume) of 1×TBE (tris-borate-ethylenediamine tetraacetic acid) buffer + 50 mM KCl + 2 μL 1× Loading buffer, with different concentrations of DNA. The

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electrophoretic migration was performed in 1 × TBE for 50 min at room temperature (150 V). After

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the migration, gels were analyzed after a post-staining step (20 μM NCT, 30 min; 3× Gel-Red, 30 min)

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with an Azure c600 bioanalytical imaging system (UV365 mode and IR700 mode). 2.8 Flow cytometry study on cell membrane permeability

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Exponentially growing Hela cells were seeded in a 60 mm petridish (1 × 106 cells/ petridish) and allowed to grow in DMEM complete media for 24 h. Then the cells were treated with various

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concentrations of NCT (0, 1.0, 3.0 and 5.0 μL) and DMSO (0.1% as control) in duplicate for 12 h.

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Then the cells were trypsinized, centrifuged and washed twice with 1 × PBS. The cell pellets were then resuspended in 2 mL of 70% ethanol and were stored at 4C for overnight. The overnight fixed

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samples were centrifuged and washed twice with 1× PBS. The cells were analyzed for cell membrane permeability by acquiring the fluorescence of NCT using flow cytometry. 2.9 Cytotoxicity experiment Toxicity toward Hela cells were determined by Cell Counting Kit-8 (CCK-8; Dojindo Laboratories, Kumamoto, Japan) following literature procedures [38]. Briefly, about 9000 cells per well were seeded in 96-well plates and cultured for 24 h. After removing the old medium, Hela cells were incubated with

0.5 μM, 1 μM, 2 μM, 4 μM and 8 μM NCT for another 24 h. The mediumwas replaced by 100 mL fresh medium containing 10 mL CCK-8 and the plates were incubated at 37 C for another 2 h. Then, the absorbance of each sample was measured using an ELISA plate reader (BioRad, imark) at a wavelength of 450 nm. The cell ability (%) was obtained according to the manufacturer's instruction.

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2.10 Imaging of living cells

Hela cells were cultured in Dulbecco's modified Eagle medium (DMEM) containing 10% fetal bovine serum and 1% antibiotic–antimycotic at 37 C in a 5% CO2 /95% air incubator. For fluorescence

imaging, cells (4×103 per well) were passed on confocal dishes and incubated for 24 h. Immediately

before the staining experiment, cells were washed twice with PBS (10 mM). Dish 1 was incubated with

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1 μM NCT for 15 min at 37 C, and then washed with PBS (10 mM) for 3 times. Dish 2 was incubated

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with 1 μM NCT for 15 min at 37 C, followed by incubation with 1μM Mito-Tracker Green for

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another 15 min at 37 C after washed with PBS (10 mM) for 3 times. These two dishes were analyzed

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with a fluorescent inverted microscope using an excitation wavelength of 488 nm. 2.11 Cell imaging without and with DNase treatment

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Hela cells (4×103 per well) were passed on confocal dishes and incubated for 24 h. Immediately before

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the staining experiment, cells were fixed by precooled 99% methanol (-4 °C) for 1 min and were then washed twice with PBS (10 mM). To permeabilize the cell membrane, the cells were cultured with 1%

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Triton X-100 (1 mL) for 2 minutes, then washed with PBS (1 mL * twice). The pretreated Hela cells were then stained with 1 μM NCT for 15 min at 25 ℃. After washed with PBS for 3 times, one of the

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dishes was treated with only PBS to make control group, the other dish was treated with 70 ug/mL DNase (37 ℃, 2 h). The imaging experiments were performed on a ZEISS LSM 780 confocal laser

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scanning microscope (CLSM). Equal parameters and exposure time were used for the control group and DNase group. 3. Results and discussions 3.1. Photophysical properties Following the synthesis and characterization, we systematically explored the photophysical properties of NCT. As shown in Figure S1, NCT displayed extremely week emission in Tris-buffer.

When 6 equiv. CM22 (prefolded G4) was added, a significant enhancement centered at 650 nm was observed (250 folds). Meanwhile, the fluorescence quantum yield of NCT increased from 0.10% to 3.12% after interacting with CM22 (Rhodamine B as the reference [39], Qr = 0.69). The results demonstrated that CM22 could significantly enhance the fluorescence intensity of NCT. Moreover,

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compared with the maximum fluorescence emission wavelength of TO in G4 (550 nm) [40], the incorporation of the diethylamino coumarin unit turned NCT into a near-infrared emission probe

(λem= 650 nm). Furthermore, the kinetic study (Figure S3) indicated the interaction of NCT with G4 could be completed within 30 min and the probe exhibited high photostability. 3.2. Sensitivity and selectivity of the probe towards G4

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To study the sensitivity and selectivity of the probe towards G4, the interaction between various

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types of nucleic acid and NCT were investigated. As can be seen in Figure 1A, NCT showed different

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fluorescence responses to different kinds of G4. The 110-250 equivalent emission enhancements

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indicated the ligand was sensitive towards all kinds of G4. To be specific, the fluorescence intensity of NCT showed most dramatic enhancement when interacting with CM22 (250-fold). In addition, 22AG,

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G4TTA, G3T3, HRAS, Ckit and Ckit* increased the fluorescence by177-fold, 169-fold, 133-fold,

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119-fold, 133-fold and 110-fold, respectively. Above results suggested that varied configurations of G4 could make a great difference on the fluorescence intensity of NCT. The exact mechanism behind

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such variations was yet to be studied. We then calculated the detection limits of NCT towards different G4 to be 3.1-8.7 nM (Table S1). As expected, CM22 exhibited the lowest detection limit (3.1 nM),

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indicating that NCT was most sensitive to CM22. Moreover, the detection limits were comparable to previously published results, that was to say, NCT might be applicable in detecting G4 in living cells.

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As another important feature of sensors, selectivity of NCT over other biologically relevant species including single-stranded DNA, double-stranded DNA, proteins and enzymes was subsequently studied. Single and double strand DNA (6 μM) caused only slight enhancement (10-25 folds, Figure 1A, 1B, Figure S2 and Figure S13); all the tested proteins (BSA and EEA) and enzymes (GO, amylase and others) led to negligible fluorescence changes. The high selectivity of the probe towards G4 was therefore further proved.

3.3. The experiment of Job’s plot Job’s plots revealed the stoichiometric coefficients of NCT differed with different G4. A majority of G4 showed a 1:1 bonding mode with NCT, including CM22, Ckit*, G3T3 and G4TTA. Other tested G4 showed disparate combination ratio such as 4:1, 2:1 and 1:2 with HRAS, Ckit, and 22AG,

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respectively (Figure S4). The results indicated the existence of different bonding mode between G4 and NCT, which might be attributed to the specific conformation of each kind of G4. 3.4. Fluorescence intercalator displacement (FID) assay

Fluorescence intercalator displacement (FID) assay was performed to evaluate the binding affinity of NCT with DNA (Figure S5). As mentioned above, TO could bind with double-strand DNA or G4

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accompanied by large fluorescence enhancement in water solution. Thus, the affinity of the probe

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towards G4 or ds-DNA could be determined through its ability of displacing TO from the target DNA.

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The fluorescence intensity of TO interacting with DNA was much stronger than that of NCT, so that the affinity of NCT for G4 DNA could be monitored via the decrease of emission intensity of TO.

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DC50, a value that was defined as the required concentration of the ligand to displace 50% TO from

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target DNA, could directly illustrate the affinity of probe binding to DNA. In agreement with fluorescence titration, NCT showed high binding affinity for G4 that all the DC50 values were below

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2.50 μM ([TO]=1 μM, Table S2). It was noteworthy that the DC50 values of NCT when binding to

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22AG, G3T3, CM22 and Ckit were even lower than half of the concentration of TO, which meant that NCT had stronger affinity for these G4 than TO. At the same time, the concentration of NCT needed

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to replace 50% TO from double stranded DNA was higher than 2.5 uM (Figure S6). These results indicated the excellent binding capacity and selectivity of NCT for G4 over duplex DNA.

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3.5. Gel electrophoresis experiments The high sensitivity, selectivity and binding ability of NCT towards G4 rendered us to further

explore the potential of NCT to serve as a fluorescent G4 dye in polyacrylamide gel electrophoresis (PAGE). Both prefolded G4 and duplex DNA were tested in this experiment. After electrophoretic migration, the polyacrylamide gels were immersed into a solution of NCT or Gel-Red for 30 min. As can be seen in Figure S7, under UV365 irradiation, NCT selectively showed the bands of three kinds

of G4 (G4TTA, 22AG and HRAS), whereas the bands for other type of G4 and duplex DNA were barely visualizable. Interestingly, when the gel was imaged in IR700 (λex/em = 660/700) mode, no trace of ds 26 were detected and all the G4 showed clear red bands with CM22 being the strongest. This result correlated well with the fact that CM22 could maximize the fluorescence intensity of NCT. In

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comparison, Gel-Red selectively stained HRAS, Ckit* and ds26, relatively weak signal was also detected for Ckit and CM22. However, no obvious bands were shown when the Gel-Red stained gels were imaged in IR700 mode. The reason for this difference might be attributed to the redshifted

wavelength of NCT compared with Gel-Red. Encouraged by this phenomenon, concentration related

PAGE experiments of HRAS, Ckit*, Ckit, G4TTA, G3T3, 22AG and CM22 were carried out (Figure

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S8). The NCT impregnated gel showed clearer bands with the increased concentration of G4TTA,

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22AG and HRAS under UV365 mode and all the G4 under IR700 mode. At the same time, duplex DNA showed distinct bands after stained by Gel-Red while no band could be observed after the gel

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was stained with NCT (Figure S9). The selective and NIR imaging capabilities of NCT endowed the

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3.6. Viscosity response of NCT

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probe with high potential in serving as a G4 staining agent in gel electrophoresis.

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To better understand the binding mechanism, the viscosity response of NCT was also investigated. We measured the fluorescence emission spectra of NCT in glycerol–Tris-HCl buffer mixtures of

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varied viscosity (Figure S10). The emission intensity at 650 nm in pure glycerol was 75-fold higher than that in pure Tris-HCl buffer. While little emission enhancement (6 folds) was observed in

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methanol and Tris-HCl buffer (Figure S11). The results implied that the viscosity rather than the polarity was the key factor for the change of fluorescence. Thus, we inferred that the rotational

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limitation caused by the interaction between G4 and NCT could be the main reason for fluorescence enhancement. 3.7. The interaction between CT and DNA In order to make it clear that whether benzothiazole moiety was restricted after interacting with G4, another promising probe CT was synthesized. When fluorometric titration experiment of CT was

carried out with increasing concentration of G4 (Ckit), much like NCT, the fluorescence intensity was enhanced dramatically at the emission wavelength of 657nm (Figure S12). This result proved that the restriction of the benzothiazole group was not the only reason for the increased fluorescence of NCT. We deduced that the restriction of the single bond between vinyl and quinoline group might also be

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responsible for the fluorescence change. Additionally, the fluorescence of CT could be turned on by both G4 and ds26 (Figure S13A). Figure S13B clearly show that the selectivity of CT to G4 was not as good as NCT. In other words, the existence of benzothiazole group affected little on the optical

property of NCT, but greatly on the selectivity of G4. This experiment verified our original design strategy that crescent-shaped molecules could bind to G4 with high selectivity.

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3.8. Fluorescence lifetime experiments

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The excited-state lifetime of NCT was used to corroborate the relationship between the emission

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spectrum change and the binding mode. The time-resolved fluorescence spectra were obtained in

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Tris-HCl buffer containing K+ (Figure S14). Due to a feasible torsional relaxation channel in excited state, NCT exhibited fast decay with the lifetime of 0.4 ns in the absence of DNA. Upon binding to

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different G4, the lifetime dramatically increased to more than 1 ns (Table S3), suggesting that G4

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restricted the intramolecular rotational diffusion of NCT and more energy was dissipated in the form of fluorescence emission. Likewise, the excited-state lifetime of NCT with single- and double-

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stranded DNA were also investigated. The slower time constant (0.8-0.9 ns) suggested the weaker binding affinity of NCT to single- and double- stranded DNA. Consistent with the previously obtained

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experimental results, the lifetime experiments also showed the better selectivity of NCT to G4. 3.9. Circular dichroism experiments

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It is important for fluorescent probes to cause as little interference as possible on the original state of

the targeted analyte. We therefore investigated the effects of NCT on the conformation of G4 by measuring the CD spectra of different G4 in the absence and presence of NCT (Figure 2 and Figure S15). In the absence of NCT, the CD spectra of parallel G4 CM22 and Ckit exhibited a characteristic positive peak at 265nm and a negative peak at 240 nm. Antiparallel G4 HRAS showed a characteristic

positive peak at 295nm and a negative peak at 260 nm. Hybrid-type G4 22AG, G3T3, G4TTA and Ckit* showed a positive peak at 290 nm and a characteristic shoulder peak at 265 nm. The addition of NCT to G4 solutions hardly affected their characteristic CD peaks in K+ buffer, indicating that the interaction between NCT and G4 did not cause significant conformational transition. The little

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disturbance of NCT to the topological stability of G4 made it a promising G4 probe for further biological studies. 3.10. Cytotoxicity experiments

The excellent performance of NCT led us to the study of its biocompatibility. A long-term cell

viability assay was performed to determine the cellular toxicity of NCT (Figure S16). We adopted

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CCK8 assay to determine the mitochondrial activity which was an indicator of cell viability. Varying

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concentrations of NCT (0, 0.5, 1, 2, 4, 8μM) were incubated with Hela cells for 24 h. The cell survival

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rate was above 50% at the concentration of 8 μM, suggesting NCT had relatively high cytotoxicity.

concentration.

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3.11. Flow cytometric analysis

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Since 1 μM NCT caused no detectable cell death, cell imaging experiments were carried out at this

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The cell membrane permeability was another factor needed to be studied. Three different concentrations of NCT were used to stain Hela cells and flow cytometric analysis was applied for

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counting stained cells. As shown in Figure 3 and Figure S17, 1μM NCT could stain 100% living cells. Simultaneously, the peak graph (Figure S18) showed a positive correlation between the fluorescence

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emission in cells and the concentration of NCT. This analysis demonstrated the excellent membrane permeability of NCT, and showed its potential in quantitative detection of G4 in living cells.

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3.12. Mitochondria co-localization experiment Encouraged by above findings, we used NCT as a turn-on probe to image G4 in human cervical

cancer cells using confocal laser scanning microscopy. Figure 4B showed NCT mainly distributed in the cytoplasm of Hela cells but almost none in the nucleus, the reason of which may be attributed to the poor nuclear membrane permeability of the probe. Co-staining experiments revealed that the

staining area by NCT (Figure 4B, red channel) was highly overlapped with the staining area of MitoTracker green (Figure 4A, green channel). The Pearson’s co-localization coefficient, which described the correlation of the intensity distribution between two channels, was calculated to be 0.82, implying NCT mainly targeted mitochondria of Hela cells. This special organelle targeting was

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probably due to the positive charge carried by NCT. According to the literature reported, there were many repeats of G-rich motifs in mitochondrial DNA (NCBI Reference Sequence: NC 001807.4). 3.13. High resolution cellular imaging

To have a better look of the image of mitochondrial G4, the cellular imaging experiment was further carried out with Carl Zeiss-I scan (resolution of 140nm). As could be seen in Figure 5, no hollow

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structure was found in the zoomed image of mitochondria stained by NCT, indicating the probe didn’t

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locate to the membrane, but probably in mitochondrial matrix. Based on the above results, we

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preliminarily concluded that NCT interacted with mitochondrial G4 in matrix which lead to the

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enhancement of the fluorescence intensity. The specific mitochondrial G4 targeted fluorescence probe NCT showed great potential for life process studying in the future.

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3.14. Cell imaging without and with DNase treatment To further prove the above conclusion, deoxyribouclease (DNase) digestion experiments were

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conducted. The treatment of DNase significantly quenched the fluorescence of NCT (Figure 6). Figure

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S19 vividly showed that about 7-fold fluorescence decrement were caused by the digestion of DNase. Thus far, the mitochondria co-localization experiment, the high-resolution cellular imaging and the

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DNase digestion experiment have jointly proved that mitochondria G4 was the switch that turned on the fluorescence of NCT intracellularly.

The specific mitochondrial G4 targeted fluorescent probe

NCT have demonstrated great potential for imaging G4 in living systems.

Conclusion

In conclusion, we have designed and synthesized a crescent-shaped probe NCT for G4 detection. NCT shows excellent selectivity to G4 with dramatic fluorescence enhancement, while ss- and ds-DNA will not have such effects. CD spectrum proves that binding to NCT does not affect the topological stability of G4. Additionally, NCT demonstrates excellent capability in serving as a NIR

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dye in selectively imaging G4 in polyacrylamide gel electrophoresis. The selective imaging of G4 in mitochondria make it a promising tool for studying genetic information outside the nucleus. Acknowledgement

This work was financially supported by the National ScienceFoundation of China (21572147 and

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Univetsity with CD measurement was greatly appreciated.

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21877082). The kind assitance of Prof. Peng Wu from the Analytical & Testing Center at Sichuan

Electronic Supplementary Information (ESI) available: [Materials and methods, DNA samples,

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synthesis and characterization, fluorescence titration, detection limits, fluorescence kinetics, Job’s

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plot, displacement assay, gel electrophoresis, viscosity and polarity responses, fluorescence decay

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Figure 1. (A) The fluorescence intensity enhancement of 1μM NCT at 650 nm against the ratio of

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[Sample] / [NCT]; (B) The histogram of the fluorescence intensity enhancement of 1μM NCT at 650

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nm against CM22 and biologically relevant interferants in 10 mM Tris-HCl buffer, 50 mM KCl, pH =

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7.4. λex = 466 nm. From left: CM22 (6 μM), enzymes and protein (100 μg/mL): Albumin from bovine

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serum (BSA), Albumin from ex egg (EEA), Lipase from Mucor javanicus (MJL), Lipase from Porcine pancreas (PPL), Acylase I from Aspergillus melleus (AMA), Gluten from wheat (WG), Albumin from

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milk (MA), Amylase, Catalase, Cellulase, Peroxidase from horseradish (HP), Glucose Oxidase (GO),

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Pepsin, Trypsin.

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Figure 2. CD spectra of 3μM G-quadruplex-forming oligonucleotides CM 22, Ckit, 22 AG and Ckit*

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in 10 mM Tris-HCl buffer, 50 mM KCl, pH 7.4, with different concentrations of NCT (0 μM, 0.5 μM,

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1 μM, 2 μM and 5 μM).

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Figure 3. Flow cytometric analysis of the fluorescence change in Hela cells incubated with different concentration of NCT. Scatterplot A) 0 μM and B) 1μM NCT treated cells. Q2-LL: percentage of

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unstained cells; Q2-LR: percentage of stained cells.

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Figure 4. Confocal fluorescence images of Hela cells stained with 1μM NCT for 15min and 1μM Mito

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Tracker green for 15min. (A) Mito Tracker channel (green); (B) NCT channel (red); (C) merged

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images of (A) and (B); (D) bright field; (E) merged images of (C) and (D); (F) co-localization images.

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Ex@488 nm for both the red channel (630-670nm), and the green channel (500-540 nm). Bars: 5 μM.

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Figure 5. (A), (D) and (G): Merged images of fluorescence image and bright field of Hela cells stained with 1μM NCT for 15min. (B), (E) and (H): Ex@488 nm excitation for the red channel (620-680nm). (C), (F) and (I): Partial enlarged details

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Figure 6. Fluorescence images of Hela cells stained with 1 μM NCT for 15 min without (A, B, C) and

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with (D, E, F) DNase treatment (λex = 488 nm, λem =630-670 nm).

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Scheme 1 The structures of the probes.

Table 1. DNA samples used in the present study. Sequence

Structure in K+ solution

CM22

5’-d(TGAGGGTGGGTAGGGTGGGTAA)-3’

Parallel G4

Ckit

5’-d(AGGGAGGGCGCTGGGAGGAGGG)-3’

HRAS

5’-d(TCGGGTTGCGGGCGCAGGGCACGGGCG)-3’

22AG

5’-d(AGGGTTAGGGTTAGGGTTAGGG)-3’

G3T3

5’-d(GGGTTTGGGTTTGGGTTTGGG)-3’

G4TTA

5’-d(TTAGGGTTAGGGTTAGGGTTAGGG)-3’

Ckit*

5’-d(GGCGAGGAGGGGCGTGGCCGGC)-3’

s15a

CGC GCG TTT CGC GCG

s15b

CGC GCG AAA CGC GCG

Single-Strand

ds15

s15a/s15b

Duplex

ds26

5’-d(CAATCGGATCGAATTCGATCCGATTG)-3’

Parallel G4

Antiparallel G4

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Hybrid-Type G4

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Name

Hybrid-Type G4 Hybrid-Type G4 Hybrid-Type G4 Single-Strand

Duplex