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A Reliable FRET Probe Based on Structure-Switching DNA for Ratiometric Sensing of Telomerase in Living Cells Xue-Jiao Yang, Kai Zhang, Tingting Zhang, Jing-Juan Xu, and Hong-Yuan Chen Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b00267 • Publication Date (Web): 16 Mar 2017 Downloaded from http://pubs.acs.org on March 16, 2017

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Analytical Chemistry

A Reliable FRET Probe Based on Structure-Switching DNA for Ratiometric Sensing of Telomerase in Living Cells Xue-Jiao Yang, Kai Zhang, Ting-Ting Zhang, Jing-Juan Xu,* Hong-Yuan Chen

State Key Laboratory of Analytical Chemistry for Life Science and Collaborative Innovation Center of Chemistry for Life Sciences, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing 210023, China * Corresponding author. Tel/Fax: +86-25-89687294; E-mail: [email protected]

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ABSTRACT In situ detection and monitoring of telomerase is of great importance as it is a relatively specific cancer target. However, the complexity of biological system makes it difficult for the nanoprobe to keep absolutely stable and low background in living cells. This study designs a probe termed FRET nanoflares to achieve ratiometric fluorescent detection of intracellular telomerase with higher specificity, which can effectively resist the disturbance from DNase I and GSH, etc. The probe is composed of a gold nanoparticle (AuNP) which is functioned with telomerase primer sequences (TS) and flares fluorescently labeled donors and acceptors at two terminals. In the presence of telomerase, flares are displaced from the primer sequences and form hairpin structures, so that the donors and acceptors are brought into close proximity, resulting in high FRET efficiency. The probe exhibits good performance for efficiently distinguishing tumor cells from normal cells and monitoring the change of intracellular telomerase activity during treatment with telomerase-related drugs, showing great potential for cancer diagnosis and estimating therapeutic effect.

KEYWORDS: Telomerase, Ratiometric imaging, FRET, Biomarker, Nanoflares

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INTRODUCTION Human telomerase is a ribonucleoprotein enzyme that can maintain the protective structure at the ends of chromosomes by adding repetitive sequences (TTAGGG) to the 3’ end of telomeres.1-5 In most normal somatic cells, telomerase activity is repressed, and telomeres shorten progressively with cell division. In contrast, almost all tumor cells overexpress telomerase, which can elongate telomeres and subsequently ensure unlimited cellular proliferation.6 Therefore, as a unique and credible cancer target, telomerase has gained more and more interest for its potential in cancer early diagnosis and therapy.2,7,8 Over the past decades, a variety of strategies has been developed to probe telomerase activity, which could mainly be divided into two types.9 The most commonly used method is polymerase chain reaction (PCR)-based telomere repeat amplification protocol (TRAP) and its modified assays.9-11 Although the strategy performs satisfactory sensitivity and specificity, shortcomings are also obvious, such as, complicated manipulations and helplessness during in situ detection. Alternatively, several kinds of direct analysis strategies have been developed recently, including optical detection, array and biosensor chip, electrochemical strategies, micro- and nano-materials-assisted detection.9-21 Among them, fluorescence methods gain much attention because of its sensitivity and simplicity. Nevertheless, the single-intensity-based detection is susceptible to thermodynamic fluctuations, drifts of light sources, surroundings such as glutathione(GSH) and nuclease degradation, which might generate false positive signals.22 To avoid these weaknesses, a new and upgraded two-fluorophore-labeled probe has been well designed for imaging telomerase in living cells (Scheme 1). The probe consists of a gold nanoparticle assembled with duplexes of the telomerase primer sequence (TS) and the reporting sequence (Flare), which allows the good dispersity in aqueous system, low toxicity and wonderful membrane penetrability. Compared to the conventional single-dye probe, the FRET nanoflares possess higher resistance to the interference from intracellular surroundings, such as DNase I and GSH, etc. So the ratio-dependent probe could preferably avoid false positive signals and is expected to perform good specificity, which is critically important for imaging and detecting telomeres activity in living cells. In order to visualize the telomerase, the flares are fluorescently labeled with donors (FITC) and acceptors (TAMRA) at the 5’ and 3’ ends, respectively. In the absence of telomerase, flares hybridize with the primer sequences, separating of the donor and acceptor, and inducing low FRET efficiency. Moreover, due to the quench effect of gold nanoparticles23,24, only the fluorescence of donors can be 3



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detected. Whereas, in the presence of telomerase, the primer sequences can be elongated from its 3’ end to produce multiple TTAGGG repeats that is complementary to the corresponding stem at the 5’ end.10,25 Subsequently, flares displace from the primer sequences and form hairpin structures, resulting in high FRET efficiency between the donors and acceptors. Then the fluorescence of acceptors will enhance markedly and the fluorescence intensity of donors will decrease. Thus, the fluorescence emission ratio of acceptor to donor (FA/FD) can be used as a signal for imaging and semi-quantitation of telomerase.22 The sensitive and credible method achieves the analysis of telomerase in cell lysate with the LOD down to ~33 HeLa cells, meanwhile, in situ imaging and monitoring of intracellular telomerase is also satisfactory. EXPERIMENTAL SECTION Chemicals

and

Materials.

Trisodium citrate, chloroauric acid (HAuCl4·4H2O) and

(-)-epigallocatechin gallate (EGCG) were purchased from Sigma-Aldrich Inc. (St. Louis, USA). Tris (2-carboxyethyl) phosphine hydrochloride (TCEP), RNase inhibitor, deoxynucleotide triphosphates (dNTPs) solution mixture and DEPC-treated water were obtained from Sangon Biotechnology Co. Ltd. (Shanghai, China; DEPC = diethyl pyrocarbonate). Human telomerase (TE) ELISA kit was from Qiaodu

Biotechnology

Co.

Ltd.

(Shanghai,

China).

DNase

I

endonuclease,

3-(4,5-dimethylthiazol-2-yl)-2-diphenyltetrazolium bromide (MTT) and all the cell lines were obtained from KeyGen Biotech. Co. Ltd. (Nanjing, China). The 1× CHAPS lysis buffer was purchased from Millipore (Bedford, MA). All the chemicals were of analytical grade and were used without additional purification. All the oligonucleotides were synthesized and purified by Sangon Biotechnology Co. Ltd. (Shanghai, China). All the water used in the work was purified by a Millipore filtration system and was RNase-free by adding DEPC. Preparation of Gold Nanoparticles (AuNPs). The AuNPs were prepared according to the classical sodium citrate reduction method.26 Firstly, 100 mL of 0.01% HAuCl4 was heated to boil with stirring. When the solution began to reflux, 3.5 mL 38.8 mM sodium citrate was quickly added. Then the color changed from pale yellow to colorless and then deep red. The system was refluxed with stirring for another 15 min, and then cooled down to room temperature. The sizes of the AuNPs were verified by TEM. The concentration was calculated based on Beer-Lambert Law.27,28 Modification of AuNPs with Flare/Primer Duplexes. The thiolated primer sequences were reduced by TCEP at a concentration of 10 mM. After 30 min, primer sequences and flares were dissolved in PBS and then the solution was heated to 95 ºC and maintained 5~10 min, followed by 4


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cooling down to room temperature as slowly as possible for complete hybridization. Secondly, the above solution was added to the colloid gold and the mixture was shaken overnight. After 15 h, 2 M sodium chloride solution was added to the above solution gradually to achieve a concentration of 0.2 M.26,29,30 Finally, the mixture was centrifuged (12000 rpm, 30 min) and washed three times with PBS, the probes were dispersed in PBS buffer containing 0.2 M NaCl. Cell Culture and Telomerase Extraction. HeLa cells, HepG2 cells, L-O2 cells were cultured in DMEM medium (KeyGen Biotech, Nanjing, China) supplemented with 10% fetal calf serum (Gibco, Grand Island, NY), penicillin (80 U/mL), streptomycin (0.08 mg/mL) at 37 ºC in an atmosphere containing 5% CO2. A549 cells and MCF-7 cells were cultured in RPMI-1640 (KeyGen Biotech, Nanjing, China) supplemented with 10% fetal calf serum (Gibco, Grand Island, NY), penicillin (80 U/mL), streptomycin (0.08 mg/mL) in the same incubator as the above cell lines. All kinds of cells were collected during the exponential phase of growth and washed twice with ice-cold PBS buffer (0.01 M, pH 7.4). Then the cells were suspended in 100 µL 1× CHAPS lysis buffer (Millipore) to make the concentration 10000 cells/µL and incubated on ice for 30 min. After that, the mixture was centrifuged at 12000 rpm for 20 min at 4 ºC and the supernatant was collected for analysis or frozen at -80 ºC.15,18 All the tubes and tips were RNase-free. Detection of Telomerase Activity in Cell Extracts. The cell extracts were first diluted to a series of different concentrations. The fluorescence spectrum was measured after incubating the solution which contained RNase inhibitor, 10 µL extracts, 10 µL dNTPs (10mM) and 500 µL probe at 37 ºC for 1 h, while in the controlled experiment cell extracts was heat-treated at 95 ºC for 20 min before use. In Situ Imaging of Telomerase Activity. 1 mL HeLa cells (or MCF-7, A549, HepG2, L-O2 cells) were seeded in a confocal dish overnight. On the second day, the medium was removed, and then 100 µL fresh medium containing 30 µL probe was added to the confocal dish. After incubated at 37 ºC for 5 h, the cellular images were collected with a laser scanning confocal microscopy (LSCM). In order to verify the specificity of our probe, cells were pre-incubated with a telomerase-inhibiting drug EGCG for 24 h before the probe was added. After another 5 h, the cells were examined by LSCM. All the cell images were obtained with a Leica TCS SP5 laser scanning confocal microscopy under 488 nm excitation. The FITC and TAMRA channels were collected from 505 nm to 545 nm, and 560 nm to 600 nm, respectively. Normalized FRET imaging data were collected and processed by Image J software. Detection of Telomerase Activity by Flow Cytometry. flow cytometry analysis was performed on a CytomicsTM FC 500 cytometer (Beckman Coulter) by counting 10000 events. After incubated 5



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with the probe for 5 h in 35 mm dishes, the cells were collected and washed twice, and then dispersed in fresh PBS buffer for latter flow cytometry analysis. In the control group, the cells were only pretreated with the corresponding medium, not the probe. RESULTS AND DISCUSSION Characterization of the Probe. To detect and image telomerase in vitro and in living cells, a ratio-dependent fluorescent probe based on structure switch was designed. Firstly, gel electrophoresis was performed to track the reaction process and test the mechanism (Figure 1d). When equally mixing reporting sequences (Flare, lane 2) and telomerase primer sequences (TS, lane 3) together, most of them hybridized with each other to form a larger molecular weight DNA complex (Lane 4). After telomerase extraction was added to the reaction system and incubated at 37 ºC for 1 h, the TS was extended and the Flare was displaced from the duplex (Lane 5). In contrast, no difference was observed (Lane 6) while the extraction was heated beforehand. AuNPs were used for the nanocarriers, since they might possess many interesting properties, including distance-dependent optical features, protecting oligonucleotides from degradation, and the ability of entering cells without another transfection reagent.25,29,31 The TEM images of the synthetic AuNPs showed an average diameter of 15 nm with a deviation of ± 1.1 nm (80 particles, counted by Image J, Figure 1a). After the AuNPs were functioned with oligonucleotides, the maximum absorption in the UV-vis absorption spectra was red-shifted from 519 nm to 524 nm, and the characteristic peak of DNA at 260 nm appeared (Figure 1b). In addition, the dynamic light scattering (DLS) experiments showed that the average hydrodynamic size increased from 18.2 nm to 34.2 nm (see Figure S1 in the Supporting Information). Zeta-potential analysis indicated that the probe had a more negative zeta potential compared to the AuNPs (Figure 1c), further confirming that the AuNPs were successfully assembled with the TS/Flare duplexes. Then the amount of TS/Flare duplexes loaded on each gold nanoparticle was estimated to be ~ 79 according to fluorescent quantitative analysis (Figure S2). In Vitro Analysis of the Probe. In order to evaluate the response of the probe to telomerase, the fluorescence spectra of the reaction system containing the probe, dNTPs and excessive cell extracts in PBS buffer (0.01M) was recorded at different incubation times (Figure S3a). At the beginning, only the fluorescence of donors could be detected. When the incubation time was prolonged, the fluorescent intensity of donors decreased and the fluorescence of acceptors enhanced gradually, which reached equilibrium after ~ 60 min. In contrast, no change in the spectra and the value of fluorescence 6


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ratio (FA/FD) was observed in the absence of cell extracts, demonstrating the telomerase-triggered increase of the FA/FD value and the stability of the probe in PBS (Figure S3b). Moreover, the practicability of the probe in complex media such as DMEM and RPMI-1640 was investigated by time-dependent fluorescence change (Figure S3). The results showed that the probe could remain stable in cell culture medium, which offered the possibility of subsequent intracellular applications. Furthermore, to test the feasibility of the designed probe for telomerase analysis, extracts from human cervical cancer cells (HeLa) and breast cancer cells (MCF-7) were used as the source of telomerase. The probe was incubated with the cell extracts of different concentration at 37 ºC for 60 min, and then the fluorescence spectra were recorded. As can be seen from Figure 2 and Figure S4, when the number of HeLa and MCF-7 cells was raised from 0 to 40000 (or 45000) in the reaction system, the fluorescent intensity of donors and acceptors showed an obviously opposite trend. These results demonstrated the positive correlation between the acceptor-to-donor fluorescence ratio (FA/FD) and the number of cells, which wonderfully verified our scheme. On the basis of 3σ/k (σ: the standard deviation of the blank sample; k: the slope of the standard curve), the limit of detection (LOD) was calculated to be 33 HeLa cells or 41 MCF-7 cells. The inserts in Figure 2b and Figure S4b showed the linear detection ranges from 50 to 1000 HeLa and MCF-7 cells. Combining with the ELISA Kit analysis (Figure S5), the telomerase activities in each HeLa cell or MCF-7 cell were 3.92×10-9 IU and 3.17×10-9 IU, respectively. Nuclease Stability of the FRET Signals. Before performing the probe for imaging and detection of intracellular telomerase, the nuclease stability assay was carried out. DNase I endonuclease was used as the model, which could cleave dsDNA and ssDNA. Two groups of the FRET probe were used, and then 1µL of DNase I was added to one of them to achieve a final concentration of 20 U/L. The other untreated group was served as control. The prepared two groups of solution were monitored for a period of 120 min.27 Compared with the telomerase-induced increase of the ratio value (FA/FD), the mixture of FRET probe and DNase I in PBS (0.01 M) exhibited negligible change in fluorescence spectra (see Figure 3a). As a control, the single-dye probe in which the sequence Control-1 and TAMRA-labeled Control-2 (Table S1) were employed to modify AuNPs was prepared and used in this experiment. Figure 3 compared the responses of the FRET probe and TAMRA-labeled probe to DNase I. The results showed that the nuclease stability of FRET signal mainly owed to two aspects. On the one hand, the gold nanoparticles protected oligonucleotides from enzymatic hydrolysis and only 5.6% of Control-1/Control-2 were cleaved (see the insert in Figure 3b). On the other hand, the fluorescence 7



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ratio FA/FD could remain unchanged although the duplexes of TS and Flare were degraded, because acceptors still kept away from donors and no FRET effect could happen. However, the increased background signal of the TAMRA-labeled probe caused by DNase I could not be distinguished from a true target-induced fluorescence response. Therefore, compared with the conventional single-dye probe, the dual-labeled FRET probe could preferably avoid false positive signals in the enzyme environment, which was of great importance for intracellular imaging. Evaluation of Cytotoxicity of the Probe. The cytotoxicity of the nanoprobe was determined by MTT assay with human cervical cancer cell line (HeLa). The absorbance of MTT at 550 nm is dependent on the cell viability. As shown in Figure S6 in the Supporting Information, all the HeLa cells maintained more than 90% of cell viability after incubated with our probe for different times. The results demonstrated the satisfactory low cytotoxicity of our probe, indicating the probe was potential for noninvasive monitoring and detection of intracellular telomerase activity. Intracellular Imaging and Detection of Telomerase Activity. Evaluating relative telomerase activity in living cells is of great importance for cancer early diagnosis and treatment. For in situ imaging and monitoring of the intracellular telomerase activity with our low-cytotoxicity probe, HeLa (human cervical cancer cell line), MCF-7 (human breast cancer cell line), HepG2 (human liver hepatocellular carcinoma cell line) and L-O2 (normal human hepatocyte cell line) were selected as the model cell lines. Cells were seeded in 35-mm confocal dishes respectively for 24 h, followed by addition of 200 µL fresh culture medium containing 30 µL probe. According to the fluorescence imaging pre-experiments (not shown), the FRET signals (red color, TAMRA) in HeLa cells were gradually increased with the incubation time until 5 h, while the FRET signals in L-O2 cells remained negligible. Therefore, the optimized incubation time was determined to be 5 h, which is appropriate for use in the following experiments. Additionally, Z-stack analysis and counterstaining experiment of HeLa cells were performed to confirm the location of probes in cells. As shown in Figure 4 and Video S1, from the bottom to the top of the HeLa cell, the merged fluorescence intensity of FITC and TAMRA increased first and then decreased. The features indicated that the probes were inside the cell, rather than just being absorbed on the membrane. Then counterstaining analysis of the probe and Hoechst 33342, a nuclei dye, demonstrated the FRET signal mainly blinked in the cytoplasm (Figure S7). The ratio-type fluorescent probe could be used to semi-quantify the telomerase activity in situ. FRET imaging allowed the simultaneous recording of two emission intensity in different wavelength ranges in the presence or absence of telomerase, avoiding some false positive signals by chemical 8


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interferences and thermodynamic fluctuations. The feasibility of the FRET probe for semi-quantify the intracellular telomerase activity was investigated using several kinds of cell lines, such as HeLa, MCF-7, HepG2, L-O2 cells. As shown in Figure 5, cancer cells showed much higher telomerase activity than the normal L-O2 cells. Then the normalized FRET ratios of confocal images were processed by Image J software (Figure 5b). The values showed that the telomerase activity in different tumor cells differed to some extent, which might reflect the ability of cancer cells to proliferate. In particular, the relative telomerase activity in single HeLa cell and MCF-7 cell agreed with the experimental results in enzyme-linked immunosorbent assay (ELISA) within permissible error range. Furthermore, flow cytometry analysis was performed to verify the ability of the probe to detect the telomerase activity in massive cells. As shown in Figure 5c, the results were consistent with the confocal imaging analysis. All the fluorescence signals of TAMRA were very low in control groups and in L-O2 cells pretreated with the probe, while the experimental groups exhibited high fluorescence signals. Assessment of the Specificity of the FRET Signals. A control probe functioned with the duplexes of Control-3/Control-4 (see Table S1) which were lack of the telomerase primer sequences was used for the intracellular imaging experiments to investigate the specificity of the FRET signals (Figure S8). After 5 hour of incubation, only negligible fluorescence of TAMRA was observed in the control group, while the HeLa cells exhibited strong FRET signals in the experimental group. The results confirmed that the enhanced FRET signals should be attributed to the telomerase-triggered switch in DNA structure, not exonuclease or other enzymes in the cells. Then the designed probe was applied to dynamically monitoring the variation of intracellular telomerase activity after treated with a typical telomerase-inhibiting drug, epigallocatechin gallate (EGCG). In brief, HeLa cells were cultured with different amounts of EGCG (0, 50, 100, 150, 200 µg/mL) in medium for 24 h before the probe was added to each dish, and then the confocal imaging was performed. As the amount of EGCG increased, the fluorescence ratio of acceptors to donors (FA/FD) in the drug-treated cells became smaller and smaller (Figure 6), showing the dose-dependent inhibition of EGCG toward telomerase activity. Moreover, the confocal imaging analysis exhibited that the FRET efficiency between donors and acceptors in MCF-7 cells also decreased seriously when cells were pre-cultured with 200 µg/mL EGCG for 24 h (Figure S9). The results demonstrated the ratio-dependent fluorescent signals of the probe were correlated very well with the levels of the intracellular telomerase activity, and further verified the perfect specificity of the probe in 9



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ratiometric imaging of intracellular telomerase. In conclusion, the proposed probe was capable of monitoring changes of intracellular telomerase and distinguishing different kinds of cells, especially cancer cells from normal cells. CONCLUSION In summary, we have developed a reliable FRET fluorescent probe based on the structure change in DNA, which is effective for ratiometric imaging and monitoring telomerase activity in living cells. The probe is easy to prepare and possesses many advantages, such as high stability, good biocompatibility, satisfactory specificity. Furthermore, it is the fluorescence ratio of acceptors to donors, not single fluorescence intensity that indicates the telomerase activity, which avoids some false positive signals. Firstly, several in vitro experiments verified the feasibility of the proposed method and exhibited the LOD down to 33 HeLa cells with a wide linear range. Secondly, dynamically monitoring and semi-quantification of the intracellular telomerase activity in different kinds of cells were achieved, demonstrating the ability of the probe to distinguish cancer cells from normal cells. We anticipated that the proposed probe will help understanding the role of telomerase in biological processes and provide a potential tool for clinical diagnosis based on telomerase activity. ASSOCIATED CONTENT Supporting Information Detailed description of the sequences, the characterizations of the probe, and some additional figures. The video about Z-stack analysis. AUTHOR INFORMATION Corresponding Author *E-mail: [email protected] Tel: +86-25-89687294 Notes The authors declare no competing financial interest. 10


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ACKNOWLEDGMENTS This work was supported by the financial support by Ministry of Science and Technology of China (No. 2016YFA0201200), the National Natural Science Foundation of China (Grant No. 21327902 and 21535003). This work was also supported by the Priority Academic Program Development of Jiangsu Higher Education Institutions. REFERENCES (1) Morin, G. B. Cell 1989, 59, 521-529. (2) Hahn, W. C.; Stewart, S. A.; Brooks, M. W.; York, S. G.; Eaton, E.; Kurachi, A.; Beijersbergen, R. L.; Knoll, J. H.; Meyerson, M.; Weinberg, R. A. Nat Med 1999, 5, 1164-1170. (3) Bodnar, A. G.; Ouellette, M.; Frolkis, M.; Holt, S. E.; Chiu, C.-P.; Morin, G. B.; Harley, C. B.; Shay, J. W.; Lichtsteiner, S.; Wright, W. E. Science 1998, 279, 349-352. (4) von Figura, G.; Hartmann, D.; Song, Z.; Rudolph, K. L. J Mol Med (Berl) 2009, 87, 1165-1171. (5) Blasco, M. A. Nat Chem Biol 2007, 3, 640-649. (6) Shay, J. W.; Bacchetti, S. Eur J Cancer 1997, 33, 787-791. (7) Harley, C. B. Nat Rev Cancer 2008, 8, 167-179. (8) Shay, J. W.; Keith, W. N. Br J Cancer 2008, 98, 677-683. (9) Zhou, X.; Xing, D. Chem Soc Rev 2012, 41, 4643-4656. (10) Xiao, Y.; Dane, K. Y.; Uzawa, T.; Csordas, A.; Qian, J. R.; Soh, H. T.; Daugherty, P. S.; Lagally, E. T.; Heeger, A. J.; Plaxco, K. W. J Am Chem Soc 2010, 132, 15299-15307. (11) Qian, R.; Ding, L.; Ju, H. J Am Chem Soc 2013, 135, 13282-13285. (12) Zuo, X.; Xia, F.; Patterson, A.; Soh, H. T.; Xiao, Y.; Plaxco, K. W. Chembiochem 2011, 12, 2745-2747. (13) Qian, R.; Ding, L.; Yan, L.; Lin, M.; Ju, H. J Am Chem Soc 2014, 136, 8205-8208. (14) Liu, X.; Wei, M.; Liu, Y.; Lv, B.; Wei, W.; Zhang, Y.; Liu, S. Anal Chem 2016.

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Scheme 1. Schematic Illustration of the FRET Probe for Ratiometric Imaging of Intracellular Telomerase

The picture is not drawn in accordance with the actual proportion.

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Figure 1. (a) TEM image of AuNPs. (b) UV-vis spectra of AuNPs, DNA and the probe. (c) Zeta potentials of AuNPs and the probe in H2O. (d) Electrophoresis image of DNA ladder (lane 1); Flare (lane 2); TS (lane 3); the duplex (lane 4) of Flare and TS; the mixture of the duplex, dNTPs and cell extraction (lane 5) or heat-treated extraction (lane 6) after incubation at 37 ºC for 1 h.

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Figure 2. Detection of telomerase activity in cell extracts. (a) Fluorescence emission spectra of the designed probe in response to telomerase from different numbers of MCF-7 cells. (b) The relationship between the fluorescence ratio of acceptor to donor (FA/FD) and the number of cells. Inset: linear relationship between the fluorescence ratio and cell numbers. (FA/FD)0: the fluorescence ratio of the probe which was not incubated with cell extracts. Error bars indicate standard deviation of triplicate tests. (λex = 485 nm, λem = 517 nm and 576 nm)

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Figure 3. DNase I effects on the fluorescence signal of the FRET probe (a) and TAMRA-labeled probe (b). (a) Plots of fluorescence ratio values of the FRET probe in PBS in the presence of 20 U/L DNase I (circle, red) and absence of DNase I (square, black) vs. incubation time (λex = 485 nm, λem = 517 nm and 576 nm). Inset: the fluorescence spectra of two groups at 120 min. (b) Plots of fluorescence emission maximum of the single-dye probe vs. incubation time (λex = 530nm, λem = 576 nm). Inset: the fluorescence spectra of the two groups at 100 min (black and red) and the single-labeled probe treated with mercaptoethanol (blue) which can release the oligonucleotides completely.

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Figure 4. Z-stack images of the merged fluorescence of FITC and TAMRA in the HeLa cell after incubated with the probe for 5 h and degraded by pancreatin. The images were taken in a series of 30-step (0.65 µm-step-sizes) measurements.

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Figure 5. Semi-quantification of telomerase activity in living cells. (a) Confocal images of HeLa, MCF-7, HepG2 and L-O2 cells after incubated with 30 µL probe for 5 h. (b) Fluorescence ratio values of different cell lines. The data were processed by Image J software. (c) Flow cytometric analysis of various cell lines after incubated with or without the probe. A-E correspond to the control groups of A549, HeLa, MCF-7, L-O2, HepG2 cells, respectively.

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Figure 6. Confocal images of HeLa cells pretreated with 0, 50, 100, 150, 200 µg/mL EGCG (a ~ e) and then incubated with 30 µL probe for 5 h.

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Reliable FÃ¶rster Resonance Energy Transfer Probe Based on

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