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Facile, Fast-Responsive and Photostable Imaging of Telomerase Activity in Living Cells with a Fluorescence Turn-on Manner Yuan Zhuang, Fujian Huang, Qi Xu, Mengshi Zhang, Xiaoding Lou, and Fan Xia Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.5b04756 • Publication Date (Web): 12 Feb 2016 Downloaded from http://pubs.acs.org on February 18, 2016
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Analytical Chemistry
Facile, Fast-Responsive and Photostable Imaging of Telomerase Activity in Living Cells with a Fluorescence Turn-on Manner Yuan Zhuang, Fujian Huang, Qi Xu, Mengshi Zhang, Xiaoding Lou* and Fan Xia Hubei Key Laboratory of Bioinorganic Chemistry & Materia Medica, School of Chemistry and Chemical Engineering, Huazhong University of Science and Technology, Wuhan 430074, P. R. China. ABSTRACT: In situ detecting and monitoring intracellular telomerase activity is significant for cancer diagnosis. In this work, we report a facile and fast-responsive bioprobe for in situ detection and imaging of intracellular telomerase activity with superior photostability. After transfected into living cells, quencher group labeled TS primer (QP) can be extended in the presence of intracellular telomerase. Positive charged TPE-Py molecules (AIE dye) will bind to primer as well as extension repeat units, producing a telomerase activity-related turn-on fluorescence signal. By incorporating positive charged AIE dye and substrate oligonucleotides, in situ light-up imaging and detection of intracellular telomerase activity were achieved. This strategy exhibits good performance for sensitive in situ tracking of telomerase activity in living cells. The practicality of this facile and fast-responsive telomerase detection method was demonstrated by using it to distinguish tumor cells from normal cells and to monitor the change of telomerase activity during treatment with antitumor drugs, which shows its potential in clinical diagnostic and therapeutic monitoring.
Telomerase is upregulated in ≥85% of cancer cells, while there is no detectable telomerase activity in normal cells.1-5 Therefore, telomerase could be regarded as a valuable tumor marker, and the evaluation of telomerase activity is of significant importance to cancer diagnosis, therapy, and monitoring.6-13 Over the past thirty years (telomerase was discovered in 1985), different strategies to assay telomerase activity were developed.14-23 Among these strategies, three main categories can be divided. (1) The most frequently used method is polymerase chain reaction (PCR)-based classic telomeric repeat amplification protocols (TRAPs).24,25 (2) Enzyme assisted isothermal amplification26,27 and (3) nanomaterial-based sensing platform28-32 are also developed accordingly. The above mentioned strategies have achieved satisfactory sensitivity and specificity, however, most of them still have shortcomings, including complicated manipulation, time consuming or need elaborate instruments and expensive fluorescent labels. For example, the initial TRAP is environmentally unfriendly due to use of radioisotope 32P in the process. Other strategies, such as electrochemistry methods, usually require complicated synthesis of probe, complex pretreatment of samples, and multistep reaction. Fluorescence enjoys the advantages of high sensitivity and selectivity, non-invasion, and less interference, and is thus widely used in sensor, imaging, and screening applications.33-35 Taking advantage of aggregation-induced emission (AIE) phenomenon (a species of propeller-shaped molecules, which emit faintly in their solutions, but fluoresce intensely in the aggregated state),36-38 our previous work designed a simple (label-free), highly sensitive (down to 10 cells) and rapid (within 1 h) method to light up telomerase by AIEgens.39 High specificity detection of telomerase activity was achieved by combining AIEgens with quencher.40 However, the AIEgens used in our previous works were blue emission. For biological applications, luminogens with longer-wavelength emissions
are favored as these luminescent materials suffer little interferences from optical self-absorption and autofluorescence from the background.41-43 Furthermore, in most of the present telomerase detection method, extract from cultured cells or urine specimens were used for telomerase activity analysis, which fails to provide telomerase information in complex biological environments particularly in living cells. To overcome this limitation, in this study, we made use of the AIE property and developed a facile and fast-responsive bioprobe for in situ imaging with superior photostability and detection of intracellular telomerase activity. A yellowemissive AIE dye (TPE-Py, Scheme 1) synthesized by attaching a pyridinium unit to tetraphenylethene salt (TPE) through vinyl functionality was chosen in this strategy.44 The positive charged TPE-Py is one of a typical AIE molecular and presents its multi-functional properties including high fluorescence quantum yield and long-wavelength emissions. TPE-Py can spontaneously bind to the negatively charged quencher group labeled TS primer (QP) via electrostatic interaction.45 Telomerase can add specific sequence (extension repeat units; TTAGGG) for several times to 3’-end of the primer (AATCCG TCGAGC AGAGTT). Before telomerase extension reaction, TPE-Py aggregate emission is efficiently quenched because of the fluorescence resonance energy transfer (FRET) from TPE-Py aggregate to the quencher. The quencher group labeled TS primer can be extended in the presence of telomerase. TPE-Py molecules will bind to extension repeat units which is relatively far away from quencher, producing a telomerase-activity-related fluorescence signal. This strategies was free from complicated synthesis and sample treatment due to the electrostatic attraction between TPEPy and QP. Moreover, only one step was required to complete detection. By incorporating positive charged AIE dye and
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Analytical Chemistry
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substrate oligonucleotides, in situ light-up imaging and detection of intracellular telomerase activity were achieved. Scheme 1. Schematic illustration of the AIE-based in situ telomerase activity detection and imaging. Insets: HeLa cancer cells after transfection and dyeing for different time.
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washed three times using PBS buffer before imaging observation. Laser scanning confocal microscopy (LSCM) imaging. All cellular fluorescent images were collected with an Olympus biological confocal laser scanning microscope (model: FV1200). A 405 nm laser was used for the excitation of TPEPy, and the fluorescence emission was collected at 525-625 nm. A 488 nm laser was used for the excitation of LysoTracker and NBD, the fluorescence emission were collected at 500600 nm and 510-580 nm, respectively. The images were acquired using 60× or 100× oil immersion objective lens. The fluorescence intensity values in the LSCM imaging assay were analyzed by quantifying the average fluorescence intensity of LSCM images with the image processing software (Olympus FV10-ASW V4.0).
RESULTS AND DISCUSSION
EXPERIMENTAL SECTION Materials. Oligodeoxynucleotides were synthesized by TaKaRa Bio Inc. (Dalian, China). MiRNA (miR-21) was synthesized by Sangon Biotech. Water was purified by a Millipore filtration system. The recombinant RNase inhibitor (RRI), deoxynucleotide solution mixture (dNTPs), S1 nuclease (S1 Nase), adenosine triphosphate (ATP) and RNase-free water were purchased from TaKaRa Bio Inc. The 1× CHAPS lysis buffer was purchased from Millipore (Bedford, MA). E-J cells, T24 cells, MCF-7 cells, HepG2 cells and HeLa cells were obtained from Xiangya Central Experiment Laboratory. HLF cells were obtained from China Center for Type Culture Collection. Telomerase extension reaction buffer was consist of 1.5 mM MgCl2 and 20 mM Tris (pH = 7.76). 0.05% Trypsin/EDTA and penicillin-streptomycin (10000 IU penicillin and 10000 µg/mL streptomycin) were purchased from Multicell Technologies. Fetal calf serum (FBS) was purchased from HyClone. 3'-Azido-3'-deoxythymidine (AZT), (-)epigallocatechin gallate (EGCG) and thrombin (from human plasma) were purchased from Sigma-Aldrich (Shanghai) Trading Co., Ltd. 30% Arc-Bis (29:1) was purchased from Biosharp. Bst 2.0 WarmStart DNA polymerase (Bst DNA polymerase) was purchased from Biotium. Bull serum albumin (BSA) was purchased from Kayon. Glutathione (GSH, 98%) was purchased from Acros Organics. Lipofectamine 2000 reagent was purchased from Invitrogen. Telomerase extracted from cultured cells. Telomerase was extracted according to the previous literature.39,40 In situ imaging of telomerase activity. 0.5 mL HeLa (E-J, T24, MCF-7, HepG2, HLF) cells of 4×104 mL-1 were seeded in a 20 mm confocal dish for 24 h. 3’-Azido-3’deoxythymidine (AZT) diluted in the corresponding culture medium was then added into control group for 48 h with the concentration of 20 µM and total volume of 1 mL, meanwhile the culture medium without AZT was added into experimental group with the total volume of 1 mL. The QP (TP, NBD-primer) was transfected using 10 µL lipofectamine 2000 in 250 µL of Opti-MEM at 37 ºC for 1 h. The QP (TP, NBD-primer) was transfected with the concentration of 3.6 µM. Subsequently, the Opti-MEM transfection mixtures were removed from the cells dish. Cells were then
Working Mechanism Interpretation. To verify this method and retrieve optimal conditions for further living cell experiments, we performed a number of in vitro tests. Sequences of oligonucleotides used in this assay are shown in Table S1. As shown in Figure 1a, the fluorescence intensities of TPE-Py increase rapidly in the presence of QP and active telomerase during Thus 1 hour was chosen as optimized reaction time in subsequent cell experiments. With constant TPE-Py, dNTPs and QP quantity, various amounts of telomerase extracted from cancer cells (human bladder cancer cells E-J, cervical cancer cells HeLa, and breast cancer cells MCF-7) were coincubated, followed by measurement of fluorescence spectra. The results shown in Figure 1b (Figure S1) revealed that fluorescence intensity gradually increased with the increasing amounts of telomerase (equal to cell number from 0 to 10000). For the purpose of proving the specificity of this method that the fluorescence enhancements are only related with telomerase activity, control experiments were conducted involved in inactive telomerase, telomerase extracted from normal cells and HeLa cells which were pretreated with 100 µM 3’-azido3’-deoxythymidine (AZT, a typical telomerase inhibition) for 48 h before extraction, lysis buffer, trypsin, thrombin, BSA, and Bst DNA polymerase. The results show notable difference between active telomerase and others, certifying the specificity of our method effectively (Figure 1c). To verify the influence of biomolecules such as ATP, GSH, lipofectamine, and RNA, specificity study was carried out. Obviously, TPE-Py exhibited very weak fluorescence intensity in the presence of ATP, GSH, lipofectamine 2000, and miR-21 with high concentration (Figure 1c), indicating the negligible influence of these anionic biomolecules to TPE-Py aggregate in living cells. Afterwards, several telomerase inhibitors (AZT, EGCG, SODN, and ASODN) were used to inhibit the activity of telomerase. The fluorescence intensity decreases after adding any inhibitor, suggesting the potential application of the assay for screening telomerase inhibitors (Figure 1d and Figure S2). Moreover, S1 Nuclease (S1 Nase, a single-strand-specific endonuclease) was selected to digest the DNA strand. (Figure S3) By conducting cleaved reaction, the fluorescence intensity decreases gradually. It suggests that most of the signal strand DNA is cut into fragmentation, resulting in less efficient aggregation emission of TPE-Py. Finally, we employ nondenaturating polyacrylamide gel electrophoresis (PAGE) (Figure S4) to monitor the resultant DNA and compare with our method. In addition, a commercial human telomerase ELISA Kit was used to test the telomerase extracted from E-J and
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Analytical Chemistry
MCF-7. The concentration from ELISA Kit was in according with (I/I0) value from this method (Figure S5). These results totally proved the credibility of our method. Intracellular Feasibility Study. Having demonstrated the capability and credibility of our telomerase detection method in buffer solution, the capability for telomerase detection and imaging was further demonstrated in living cells. Prior to intracellular usage, the transfection effect of modified TS primer was tested using NBD-labeled TS primer (NBD-primer, 250 µL, 3.6 µM). As shown in Figure S6, green fluorescence signal from E-J cells was observed, showing that the NBD-primer was successfully transfected into the cells. To demonstrate cell-specific light-up telomerase imaging, we incubated the probe with different cancer and normal cell lines using E-J cells (bladder cancer cells), T24 cells (bladder cancer cells), HeLa cells (cervical cancer cells), MCF-7 cells (breast cancer cells), HepG2 cells (liver cancer cells) and HLF cells (human lung fibroblast) as the models. After transfection of QP, TPEPy was added then cleaned, and the fluorescence images of different cells were taken using confocal microscopy. Confocal microscopy images of cancer cells showed a higher fluorescence intensity than those of other cells, indicating that the telomerase activities in cancer cells were higher than those in HLF cells as well as the AZT-
Figure 1. Working mechanism interpretation of the low background fluorescence assay to detect activity of telomerase. (a) Relationship between fluorescence intensity (at 575 nm) and telomerase extension reaction time in the presence (black) and absence (red) of telomerase from 10000 E-J cells. (b) Emission spectra of TPE-Py in response to telomerase extracts from different numbers of bladder cancer cells (E-J). (c) Fluorescence responses of this sensing system to telomerase extracts from 10000 E-J (A), HeLa (B), and MCF-7 cells (C), heat-inactivated telomerase extracts from 10000 E-J (D), HeLa (E), and MCF-7 cells (F), human lung fibroblast cells (HLF) (G), HeLa cells which were pretreated with 100 µM AZT for 48 h (H), lysis buffer (I), trypsin (J), thrombin (K), BSA (L), Bst DNA polymerase (M), ATP (N), GSH (O), lipofectamine 2000 (P), and miR-21 (Q), respectively. (d) Inhibition of telomerase activity in extracts from 10000 E-J cells by 1 mM AZT. Error bars indicate standard deviation of triplicate tests.
treated cancer cells (Figure 2a, Figure S7). It is reported that telomerase mostly exists in nucleus, but a part of telomerase still present in cytoplasm near the nucleus.12,46 In this work, we detected the telomerase in cytoplasm. The fluorescence intensity in T24 exhibited 31.12 times higher than that in AZTtreated ones, and 15.52 times higher than that in HLF (Figure 2b, Table S2). Figure 2c showed telomerase activity of different cell lines followed in descending order is MCF-7, T24, EJ, HeLa, and HepG2, keeping consistent to the telomerase activity of extracts tested in solutions (MCF-7, E-J, and HeLa; Figure 1c). As a result, the fluorescence intensity could reflect the difference of telomerase activity in living cells. Thus the designed strategy could be applied for various cell types and distinguishing cancer cells from normal cells. Photobleaching Telorance. The above results clearly demonstrate the utility of TPE-Py and QP for sensing and imaging of telomerase activity in living cells. To further evaluate its value for bioapplications, photostability of this method was further tested. As shown in Figure S8, even after 10 scans with a total irradiation time of ∼2 min, the signal remaining of TPE-Py is >94%, while the fluorescence signals of commercialized LysoTracker almost disappear after only 10 scans (with
