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Sensitive detection of telomerase activity in cells using a DNA-based fluorescence resonance energy transfer nanoprobe
Journal Pre-proof Sensitive detection of telomerase activity in cells using a DNA-based fluorescence resonance energy transfer nanoprobe Guohai Yang, Qian Zhang, Lin Ma, Youwei Zheng, Fei Tian, Haitao Li, Peng Zhang, Lu-Lu Qu PII:
S0003-2670(19)31379-0
DOI:
https://doi.org/10.1016/j.aca.2019.11.035
Reference:
ACA 237245
To appear in:
Analytica Chimica Acta
Received Date: 2 September 2019 Revised Date:
27 October 2019
Accepted Date: 12 November 2019
Please cite this article as: G. Yang, Q. Zhang, L. Ma, Y. Zheng, F. Tian, H. Li, P. Zhang, L.-L. Qu, Sensitive detection of telomerase activity in cells using a DNA-based fluorescence resonance energy transfer nanoprobe, Analytica Chimica Acta, https://doi.org/10.1016/j.aca.2019.11.035. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. 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. © 2019 Elsevier B.V. All rights reserved.
Sensitive detection of telomerase activity in cells using a DNA-based fluorescence resonance energy transfer nanoprobe Guohai Yanga‡, Qian Zhanga‡, Lin Mab, Youwei Zhenga, Fei Tiana, Haitao Lia, Peng Zhang*, b, Lu-Lu Qu*, a [a]
School of Chemistry and Materials Science, Jiangsu Normal University, Xuzhou, Jiangsu,
221116, China [b]
School of Life Science, Jiangsu Normal University, Xuzhou, Jiangsu, 221116, China
[*]
Corresponding authors, E-mail address: [email protected]; [email protected]
[‡]
These authors contributed equally to this work.
ABSTRACT Telomerase activity is inhibited in normal somatic cells but abnormally high in the majority of cancer cells. Maintenance of active telomerase in cancer cells promotes proliferation and immortalization. With the difference in telomerase activity between cancer and normal cells in mind, we designed a nanoprobe based on quantum dot (QD) and fluorescence resonance energy transfer (FRET). The nanoprobe consisted of a specific sequence of DNA with the two ends labeled with QD as a fluorescent donor and Alexa488 as a fluorescent acceptor, respectively. FRET signal tracking was performed by adjusting the distance between donors and acceptors, and changes in the FRET signal shown to be related to telomerase activity. Incubation of cells with the nanoprobe facilitated sensing of intracellular telomerase activity, and consequent discrimination between normal and cancer cells. Our novel DNA nanoprobe based on QD-FRET achieved sensitive detection of telomerase in cells up to a detection limit of 1
one cell, and quantitative detection of telomerase activity in different numbers of cells. The nanoprobe generated in this study is expected to allow dynamically monitoring of the changes in telomerase activity in cells under treatment with drugs, providing a potential basis for early diagnosis and management of cancer. ARTICLEINFO Keywords: Cells; Fluorescence resonance energy transfer (FRET); DNA; Nanoprobe; Telomerase activity 1. Introduction The biology of telomeres and telomerase has attracted significant interest and gradually become a research hotspot since the discovery of telomerase in 1985 [1-5]. A portion of the repeat nucleotide sequence (TTAGGG) present at the end of the chromosome is referred to as "telomere" [6]. Telomerase, an enzyme responsible for telomere elongation in cells, is a basic nuclear protein reverse transcriptase [7]. Abnormal activation of telomerase leads to cell immortality and cancer [8], while inactivation may accelerate cell senescence [9]. In addition, recent studies have shown positive telomerase expression in cancer stem cells and some stem cell-like cells [10-12]. The mechanism underlying maintenance of active telomerase in cancer cells facilitates proliferation and immortalization [13]. Therefore, research on telomeres and telomerase is of significant interest. At present, telomerase detection methods are mainly divided into two types, detection of telomerase activity in vitro and telomerase imaging in vivo. In vitro 2
telomerase activity can be detected using various methods, such as fluorescence [14,15], electrochemistry [16], colorimetry [17], electrochemiluminescence [18], and surface-enhanced Raman scattering (SERS) [19]. For example, a DNA sensor with a functional hairpin structure containing a “coged” inactive G-quadruplex sequence and zinc(II)-protoporphyrin IX (ZnPPIX) fluorophore has been designed to detect telomerase activity [20]. A multi-nanometer scaled plasma assembly (Py-SERS) probe was employed in an earlier study to detect intracellular telomerase activity in situ by enhancing the SERS signal [21]. Moreover, a number of efficient imaging methods have been explored for in vivo detection of telomerase activity. Zhuang and co-workers used a positively charged TPE-Py molecule to study imaging of telomerase in living cells [22]. Qian et al. [23] performed in situ monitoring of intracellular telomerase activity using a telomerase-responsive probe. In vivo telomerase imaging can be effectively utilized to track the dynamic processes of telomerase in living cells in real time, which is beneficial to investigate its physical role in disease development and drug reaction [24,25]. Although these experiments have achieved the detection of intracellular telomerase, several problems persist, such as low sensitivity and complex operations, highlighting an urgent need to develop biological nanoprobes with high sensitivity and specificity to achieve real-time monitoring of intracellular telomerase activity. Fluorescence resonance energy transfer (FRET) is a phenomenon of non-radiative energy transfer between two closely spaced fluorescent dye molecules. The dynamics of a single molecule can be observed by tracking the FRET signal [26-28]. FRET 3
technology is a ratiometric method that is used for telomerase detection due to its low interference and high accuracy [29]. Semiconductor quantum dots (QDs) have unique optical properties, including high quantum yield [30], good photobleaching resistance [31]and narrow luminescent bands [32], and are widely used in optical sensing and bioanalysis [33-35]. Here, we generated a nanoprobe combining the properties of QD with the advantages of FRET for detection of intracellular telomerase activity. The telomerase sensing principle of our DNA nanoprobe based on QD-FRET is shown in Scheme 1A. The nanoprobe contains DNA with specific sequences and complementary base sequences at both ends. The 5' and 3' ends are labeled with QD as a fluorescent donor and Alexa 488 dye molecule as a fluorescent acceptor, respectively. After DNA hybridization, a hairpin structure is formed, resulting in shorter distance between the donor and acceptor, and a FRET signal generated. In the presence of telomerase and dNTP, the 3' end of the telomerase primer (TSP) is extended to produce a telomere repeat [36]. As the extended portion is exactly complementary to the hairpin structure formed, the ring structure opens and the FRET signal disappears. The fluorescence intensity is related to telomerase activity, and therefore allows sensing of telomerase activity in cells incubated with nanoprobes (Scheme 1B). Owing to the significant differences in telomerase activity between normal cells and cancer cells, nanoprobe incubation facilitates telomerase sensing to distinguish malignant cells and provides a potential tool for cancer identification and early diagnosis.
2. Experimental 2.1. Modification of Quantum Dots 4
QD (10 µL; 0.1 mg·mL-1) solution was added to 10 µL of DNA1 solution, and shaken in an air bath at 37 °C for 3 h to ensure that the quantum dots were sufficiently modified to DNA1. The solution obtained was designated "MB".
2.2. Preparation of Cell Extracts The A549 cell line was cultured in a cell culture flask containing DMEM supplemented with 10% fetal bovine serum, penicillin (100 µg·mL-1) and streptomycin (100 µg·mL-1) in a humidified atmosphere containing 5% CO2 at 37 °C. To obtain the telomerase-containing cell extracts, well-grown A549 cells were initially collected. In total, 3 × 105 A549 cells were dispersed in a centrifuge tube, washed twice with iced PBS (pH 7.4), and incubated on ice in lysis buffer (10 mM Tric-HCl, 1 mM EGTA, 1mM MgCl2, 0.1 mM PMSF, 0.5% CHAPS, 10% glycerol). The mixture was allowed to stand in an ice water bath for 20 min and subsequently centrifuged at 14,000 rpm for 30 min. The supernatant comprising cell lysates containing telomerase was collected and diluted for in vitro detection of telomerase or stored at -80 °C until use. 2.3. Telomerase-Responsive Fluorescence Mechanism TSP (10 µL), dNTPs (10 µL) and 10 µL telomerase standard solution (32 IU·L-1) were added to 10 µL MB solution and the fluorescence intensity of MB observed at 37 °C in the mixed solution. Similarly, 10 µL TSP and 10 µL dNTPs were added to 10 µL MB solution in the absence of telomerase standard solution as a control, and fluorescence intensity measured after mixing. 2.4. Gel Electrophoresis 5
TSP solution (10 µL) and dNTP solution (10 µL) were mixed with 10 µL cell extract or 10 µL telomerase standard solution, and reacted at 37 °C for 1 h, designated Samples 1 and 2, respectively. Samples 3 and 4 were obtained by reacting 10 µL DNA1 solution, 10 µL TSP solution, and 10 µL dNTPs with 10 µL cell extract or 10 µL telomerase standard solution at 37 °C for 1 h. For gel electrophoresis, we mixed TSP solution, Sample 1, sample 2, DNA1 solution, Sample 3, Sample 4 and ladder DNA solution used as an indicator with the DNA loading solution, respectively, and injected these mixtures into the agarose gel in Tris-EDTA (TE) buffer. Electrophoresis was conducted for 40 min at 120 V in TE buffer and images of the gel obtained under UV irradiation. 2.5. Detection of Telomerase Activity in Cell Extracts An aliquot (10 µL) of TSP, 10 µL dNTPs and 10 µL cell extract were added to 10 µL MB solution, and fluorescence intensity changes of the mixture measured at 37 °C. The fluorescence response mechanism under the action of telomerase was examined by observing the change in the fluorescence intensity of the mixed solution added to the cell extract within 1 h. 2.6. Confocal Imaging of Cells under the Action of Probes Activity A549 cells (0.5 mL, 1 × 106·mL-1) were incubated in a 20-mm confocal culture dish for 24 h and 10 µL of probe solution added. Cells containing the probe were incubated at 37 °C for different time-periods and detected via fluorescence confocal imaging. 2.7. MTT assay First, cells (100 µL, 1.0×104·mL-1) were placed in a 96-well plate, and after incubation for 24 h, the culture solution was discarded. The culture solution containing the probe was incubated with A549 cells for different time-periods. Simultaneously, 6
cells incubated in culture medium in the absence of the probe were used as the control group. MTT reagent (10 µL, 5 mg·mL-1) was added separately to each well. After incubation at 37 °C for 4 h, the solution in the well was discarded, and 100 µL DMSO was added to each well. The 96-well plates were shaken at room temperature for 15 min to dissolve the crystals formed by living cells. Finally, plates were placed in a Hitachi/Roche System Cobas 6000 (Tokyo, Japan) microplate reader, and the absorbance value of each well at 490 nm was read. Relative cell activity (%) was calculated using the formula: (Atest/Acontrol) × 100%.
3. Results and discussion 3.1. Characterization of QD and MB
TEM observation of QD shows an average particle size of ~10 nm and good dispersibility (Fig. 1A). When the QD was excited at 326 nm, the QD showed a maximum emission peak at 480 nm. Fluorescence intensity of QD at 480 nm was about 13,000 and that of DNA1 at 535 nm was about 2100 (Fig. 1B, C). Under similar measurement conditions, when DNA1 was modified with QD, the fluorescence intensity of QD decreased to about 1700 and that of Alexa488 increased to about 5700 owing to FRET from the donor, QD, to the acceptor, Alexa 488 (Fig. 1D). Our FRET results indicated that QD was successfully modified onto DNA1. 3.2. Telomerase-Responsive Fluorescence Mechanism To validate the effectiveness of our probes in detection of cellular telomerase activity, we initially examined the telomerase response mechanism of the probe using standard solution. To verify the fluorescence recovery mechanism under telomerase 7
response, TSP, dNTP and telomerase standard solutions were added to MB and changes in fluorescence intensity observed at 37°C. As shown in Fig. 2A, the fluorescence intensity of QD at 480 nm gradually recovered and that of Alexa488 at 535 nm was decreased significantly in the first 10 min, confirming rapid telomerase reaction. At ~40 min, the fluorescence intensity of the probe showed almost no change, which was attributed to saturation of the binding site occupancy of QD37,38. TSP is reported to be prolonged under the action of telomerase and the prolonged region is complementary to the formed hairpin structure. This binding step leads to opening of the hairpin structure, thereby increasing the distance between the receptors and inducing FRET signal disappearance. In addition, the concentration of telomerase had an effect on the fluorescence response. As shown in Fig. 2B, fluorescence recovery or decreasing trend of the probe was more obvious with increasing concentrations of telomerase standard solution after 60 min reaction. To determine the effect of telomerase on the fluorescence response mechanism, we used the mixed solution in the absence of telomerase standard solution as a control. The fluorescence intensity of the probe was not significantly altered in absence of telomerase, validating the fluorescence conversion mechanism of the telomerase response (Fig. 2C).
TSP elongation and reaction of its extended product with MB was confirmed via gel electrophoresis. The TSP solution was mixed with dNTP solution, reacted with cell extract or telomerase standard solution at 37°C for 1 h and subjected to gel electrophoresis. As observed on the electropherogram, the extended product of TSP after the reaction showed a broad band ~20 bases in length (Fig. 2D; lanes d and e), which was extended by approximately 2–3 telomere repeat sequences, compared to the 8
original TSP (Fig. 2D, lane f). Our findings demonstrated recognition and catalytic elongation of TSP by telomerase. The DNA1 solution was mixed with TSP and dNTPs, reacted with cell extract or telomerase standard solution at 37°C for 1 h, and subjected to gel electrophoresis. On the electropherogram, the number of bases of the product after the reaction was about 70 (Fig. 2D; lanes a, b) and increased by about 20, compared with DNA1 (Fig. 2D, lane c), confirming hybridization of MB with TSP elongation products. 3.3. Detection of Telomerase Activity in Cell Extracts To establish the mechanism of action of the probe, cell extracts were mixed with MB solution, TSP and dNTPs to test for fluorescence intensity. The results disclosed the existence of a fluorescence response mechanism under the action of cell extracts and similar fluorescence response effects under the action of telomerase standard solution. As shown in Fig. 3A, the fluorescence intensity of QD at 480 nm gradually recovered while that of DNA1 at 535 nm decreased with increasing time after addition of cell extracts. Similarly, the fluorescence intensity of Alexa488 at 535 nm was stable at around 40 min, with a similar fluorescence response to that with the telomerase standard. In proportion to increasing incubation times, the fluorescence intensity ratio (I480/I535) gradually increased until it was stable at about 40 min (Fig. 3B). Fluorescence spectra of the probe solution after incubation with extracts of different numbers of cells for 60 min are presented in Fig. 3C. Notably, the fluorescence intensity ratio (I480/I535) gradually increased with cell number, with a linear range from 1–100 and 1,000–5,000, respectively (Fig. 3D). The limit of detection was one cell. Compared with previously work, our nanoprobe has the advantages of high efficiency and high sensitivity with a low detection limit of telomerase activity with 9
the proposed FRET-based ratio assay (Table S1), demonstrating its utility in detection of actual telomerase activity in cells with high sensitivity. 3.4. Stability Assay of the Nanoprobe We further investigated the stability of the nanoprobe with the aid of several controlled experiments. First, the probe solution containing TSP, dNTPs, and telomerase standard solution was mixed with different media and the changes in fluorescence intensity of the mixed solution measured. Our results showed that fluorescence intensity changes at 480 nm and 535 nm as a function of time were similar in PBS, DMEM, RPMI1640 and water (Fig. S1). To further confirm that the production of the intracellular fluorescent signal is indeed caused by the catalytic action of telomerase and no other substances in cells, we used the telomerase inhibitor, 3'-azido-3'-deoxythymidine (AZT), as a control and examined fluorescence of the probe under different concentrations of AZT. AZT is a reverse transcriptase inhibitor that promotes the termination of DNA synthesis during the replication process, thereby inhibiting elongation of telomerase primers. The probe solution to which the cell extract was added was pretreated with AZT for 1 h and subjected to a fluorescence test. As shown in Fig. 4A, fluorescence recovery intensity at 480 nm was gradually weakened. The fluorescence intensity ratio (I480/I535) gradually decreased with increasing AZT concentrations, clearly indicating more pronounced inhibition of telomerase activity with higher concentrations of AZT (Fig. 4B). Accordingly, we propose that production of intracellular fluorescent signals is attributable to the action of telomerase and our probes have important application potential in the screening of telomerase inhibitors. In addition, no telomerase response mechanism was observed in heat-inactivated cell extracts, as determined from comparison of the fluorescence changes of heat-inactivated cell extracts at 95°C and normal cell extracts (Fig. S2). 10
This finding can be explained by the fact that telomerase in cell extracts lost enzymatic activity at high temperatures. Our results suggested that the nanoprobe generated in this study had high stability and reliability in the detection of cellular telomerase activity. 3.5. Selectivity Assay of Nanoprobes To determine the selectivity of the probe solution for telomerase, we examined the effects of adding a range of biologically relevant samples to the probe, including bovine serum albumin (BSA), immunoglobulin G (IgG), lysozyme and thrombin, using fluorescence detection. Fluorescence of the probe solution did not change significantly at 480 nm and 535 nm after the addition of BSA, IgG, lysozyme or thrombin, as shown in Fig. 5A. However, fluorescence intensity recovery at 480 nm and decrease in fluorescence at 535 nm were observed in mixed solution containing telomerase, indicating disappearance of the FRET signal. The fluorescence intensity ratios (I480/I535) of the mixed solution after adding different sample solutions are presented in Fig. 5B. This value was significantly higher in the presence of telomerase relative to the other agents examined, further demonstrating the high selectivity of our nanoprobe for telomerase. 3.6. Confocal Imaging of Intracellular Telomerase with Nanoprobes To confirm that the nanoprobes can be used for sensing telomerase in living cells, we incubated them into cells and investigated its cytotoxicity using the MTT assay. After incubating the probes with cells for different time periods, relative cell viability was calculated by measuring absorbance at each time-point. Our results showed that after incubation with nanoprobes for 12 h, A549 and HBE cells had high relative survival rates (A549: 91.2%, HBE: 94.5%), clearly indicating that the probe was low 11
in cytotoxicity and suitable for detection of telomerase activity in cells (Fig. S3). In situ Fluorescence imaging experiments were further performed on intracellular telomerase activity using the prepared probes. Fluorescence signals of cells were observed under a confocal microscope. A549 cells without the probe solution were used as the control group, as observed from Fig. 6. After incubation with the probe, the blue fluorescence showed obvious enhancement with increasing time, while the green fluorescence decreased, which is due to the opening of the hairpin structure of the MB under the action of telomerase, consistent with data obtained from previous fluorescence spectroscopy experiments. The result clearly demonstrated the presence of telomerase predominantly in the cytoplasm. In summary, our novel probe could be efficiently utilized to detect intracellular telomerase activity through monitoring changes in fluorescence signal intensity.
4. Conclusions Here, we designed a nanoprobe based on QD-FRET for detecting intracellular telomerase activity. The designed nanoprobe contained a specific DNA sequence, which could form a hairpin structure after hybridization and thus decrease the distance between QD as a fluorescence donor and Alexa488 as a fluorescence acceptor to generate FRET. Telomere primers were prolonged under the action of telomerase. The extended region was complementary to the formed hairpin structure, leading to opening of the structure and disappearance of the FRET signal. Telomerase activity was determined by detecting the changes in fluorescence intensity and observing fluorescence signals of cells incubated with probes. The nanoprobe was further used for quantitative analysis of telomerase activity in different numbers of cells. Sensitive detection of telomerase activity in cells was achieved. The nanoprobe generated in our 12
study has the advantage of high efficiency and sensitivity with a low detection limit of telomerase activity (one cell) and can be effectively applied to dynamically monitor changes in telomerase activity in cells under the action of telomerase drugs, thus providing
a
useful
tool
for
the
discovery
and
screening
of
potential
telomerase-targeted anticancer drugs.
Acknowledgements This research was supported by the National Natural Science Foundations of China (21505057, 21605062, and 51504242), the Priority Academic Program Development of Jiangsu Higher Education Institutions, the Brand Major of Universities in Jiangsu Province, and the Top-notch Academic Programs Project of Jiangsu Higher Education Institution (TAPP).
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Figures and captions
Scheme 1. (A) Schematic illustration of the DNA nanoprobe based on QD-FRET for telomerase sensing. (B) Detection of telomerase activity in cells under the action of probes.
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Fig. 1. (A) TEM image and (B) fluorescence spectrum of the QD. (C) Fluorescence spectrum of the DNA1. (D) Fluorescence spectrum of the QD-DNA1.
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Fig. 2. (A) Fluorescence spectra of the probe solution containing TSP, dNTPs, and telomerase standard solution in response to reaction time. (B) Fluorescence spectra of the probe solution after incubation with different concentrations of telomerase standard solution for 60 min. (C) Fluorescence spectra of the probe solution containing TSP, dNTPs in absence of telomerase standard solution in response to reaction time. (D) Electrophoresis image of (a) mixture of DNA1, TSP, dNTPs and telomerase standard solution after incubation for 1 h, (b) mixture of DNA1, TSP, dNTPs and cell extract after incubation for 1 h, (c) DNA1, (d) mixture of TSP, dNTPs and telomerase standard solution after incubation for 1 h, (e) mixture of TSP, dNTPs and cell extract after incubation for 1 h, (f) TSP, and (g) Ladder DNA.
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Fig. 3. (A) Fluorescence spectra of the probe solution containing TSP, dNTPs, and cell extract in response to reaction time. (B) Plot of the fluorescence intensity ratio (I480/I535) vs incubation time. (C) Fluorescence spectra of the probe solution containing TSP, dNTPs after incubation with extract of different numbers of cells for 60 min. (D) The linear relationship between the fluorescence intensity ratio (I480/I535) and number of the cell.
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Fig. 4. (A) Fluorescence spectra of the probe solution containing TSP, dNTPs, and cell extract after incubation with different concentrations of AZT for 60 min. (B) Plot of the fluorescence intensity (I480/I535) vs different concentrations of AZT.
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Fig. 5. (A) Fluorescence spectra of probe solutions containing TSP and dNTPs after 60 min reaction with BSA, IgG, lysozyme, thrombin and telomerase. (B) Fluorescence recovery intensity at 480 nm of probe solution containing TSP and dNTPs after addition of (a) BSA, (b) IgG, (c) lysozyme, (d) thrombin, (e) telomerase standard solution for 60 min.
25
Fig. 6. Fluorescence images of telomerase in living A549 cells.
26
Highlights A DNA-based fluorescence resonance energy transfer nanoprobe was designed. Incubation of cells with the nanoprobe facilitated sensing of intracellular telomerase activity. The nanoprobe achieved sensitive detection of telomerase in cells up to a detection limit of one cell.
Declaration of interests The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. ☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:
S0003-2670(19)31379-0
DOI:
https://doi.org/10.1016/j.aca.2019.11.035
Reference:
ACA 237245
To appear in:
Analytica Chimica Acta
Received Date: 2 September 2019 Revised Date:
27 October 2019
Accepted Date: 12 November 2019
Please cite this article as: G. Yang, Q. Zhang, L. Ma, Y. Zheng, F. Tian, H. Li, P. Zhang, L.-L. Qu, Sensitive detection of telomerase activity in cells using a DNA-based fluorescence resonance energy transfer nanoprobe, Analytica Chimica Acta, https://doi.org/10.1016/j.aca.2019.11.035. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. 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. © 2019 Elsevier B.V. All rights reserved.
Sensitive detection of telomerase activity in cells using a DNA-based fluorescence resonance energy transfer nanoprobe Guohai Yanga‡, Qian Zhanga‡, Lin Mab, Youwei Zhenga, Fei Tiana, Haitao Lia, Peng Zhang*, b, Lu-Lu Qu*, a [a]
School of Chemistry and Materials Science, Jiangsu Normal University, Xuzhou, Jiangsu,
221116, China [b]
School of Life Science, Jiangsu Normal University, Xuzhou, Jiangsu, 221116, China
[*]
Corresponding authors, E-mail address: [email protected]; [email protected]
[‡]
These authors contributed equally to this work.
ABSTRACT Telomerase activity is inhibited in normal somatic cells but abnormally high in the majority of cancer cells. Maintenance of active telomerase in cancer cells promotes proliferation and immortalization. With the difference in telomerase activity between cancer and normal cells in mind, we designed a nanoprobe based on quantum dot (QD) and fluorescence resonance energy transfer (FRET). The nanoprobe consisted of a specific sequence of DNA with the two ends labeled with QD as a fluorescent donor and Alexa488 as a fluorescent acceptor, respectively. FRET signal tracking was performed by adjusting the distance between donors and acceptors, and changes in the FRET signal shown to be related to telomerase activity. Incubation of cells with the nanoprobe facilitated sensing of intracellular telomerase activity, and consequent discrimination between normal and cancer cells. Our novel DNA nanoprobe based on QD-FRET achieved sensitive detection of telomerase in cells up to a detection limit of 1
one cell, and quantitative detection of telomerase activity in different numbers of cells. The nanoprobe generated in this study is expected to allow dynamically monitoring of the changes in telomerase activity in cells under treatment with drugs, providing a potential basis for early diagnosis and management of cancer. ARTICLEINFO Keywords: Cells; Fluorescence resonance energy transfer (FRET); DNA; Nanoprobe; Telomerase activity 1. Introduction The biology of telomeres and telomerase has attracted significant interest and gradually become a research hotspot since the discovery of telomerase in 1985 [1-5]. A portion of the repeat nucleotide sequence (TTAGGG) present at the end of the chromosome is referred to as "telomere" [6]. Telomerase, an enzyme responsible for telomere elongation in cells, is a basic nuclear protein reverse transcriptase [7]. Abnormal activation of telomerase leads to cell immortality and cancer [8], while inactivation may accelerate cell senescence [9]. In addition, recent studies have shown positive telomerase expression in cancer stem cells and some stem cell-like cells [10-12]. The mechanism underlying maintenance of active telomerase in cancer cells facilitates proliferation and immortalization [13]. Therefore, research on telomeres and telomerase is of significant interest. At present, telomerase detection methods are mainly divided into two types, detection of telomerase activity in vitro and telomerase imaging in vivo. In vitro 2
telomerase activity can be detected using various methods, such as fluorescence [14,15], electrochemistry [16], colorimetry [17], electrochemiluminescence [18], and surface-enhanced Raman scattering (SERS) [19]. For example, a DNA sensor with a functional hairpin structure containing a “coged” inactive G-quadruplex sequence and zinc(II)-protoporphyrin IX (ZnPPIX) fluorophore has been designed to detect telomerase activity [20]. A multi-nanometer scaled plasma assembly (Py-SERS) probe was employed in an earlier study to detect intracellular telomerase activity in situ by enhancing the SERS signal [21]. Moreover, a number of efficient imaging methods have been explored for in vivo detection of telomerase activity. Zhuang and co-workers used a positively charged TPE-Py molecule to study imaging of telomerase in living cells [22]. Qian et al. [23] performed in situ monitoring of intracellular telomerase activity using a telomerase-responsive probe. In vivo telomerase imaging can be effectively utilized to track the dynamic processes of telomerase in living cells in real time, which is beneficial to investigate its physical role in disease development and drug reaction [24,25]. Although these experiments have achieved the detection of intracellular telomerase, several problems persist, such as low sensitivity and complex operations, highlighting an urgent need to develop biological nanoprobes with high sensitivity and specificity to achieve real-time monitoring of intracellular telomerase activity. Fluorescence resonance energy transfer (FRET) is a phenomenon of non-radiative energy transfer between two closely spaced fluorescent dye molecules. The dynamics of a single molecule can be observed by tracking the FRET signal [26-28]. FRET 3
technology is a ratiometric method that is used for telomerase detection due to its low interference and high accuracy [29]. Semiconductor quantum dots (QDs) have unique optical properties, including high quantum yield [30], good photobleaching resistance [31]and narrow luminescent bands [32], and are widely used in optical sensing and bioanalysis [33-35]. Here, we generated a nanoprobe combining the properties of QD with the advantages of FRET for detection of intracellular telomerase activity. The telomerase sensing principle of our DNA nanoprobe based on QD-FRET is shown in Scheme 1A. The nanoprobe contains DNA with specific sequences and complementary base sequences at both ends. The 5' and 3' ends are labeled with QD as a fluorescent donor and Alexa 488 dye molecule as a fluorescent acceptor, respectively. After DNA hybridization, a hairpin structure is formed, resulting in shorter distance between the donor and acceptor, and a FRET signal generated. In the presence of telomerase and dNTP, the 3' end of the telomerase primer (TSP) is extended to produce a telomere repeat [36]. As the extended portion is exactly complementary to the hairpin structure formed, the ring structure opens and the FRET signal disappears. The fluorescence intensity is related to telomerase activity, and therefore allows sensing of telomerase activity in cells incubated with nanoprobes (Scheme 1B). Owing to the significant differences in telomerase activity between normal cells and cancer cells, nanoprobe incubation facilitates telomerase sensing to distinguish malignant cells and provides a potential tool for cancer identification and early diagnosis.
2. Experimental 2.1. Modification of Quantum Dots 4
QD (10 µL; 0.1 mg·mL-1) solution was added to 10 µL of DNA1 solution, and shaken in an air bath at 37 °C for 3 h to ensure that the quantum dots were sufficiently modified to DNA1. The solution obtained was designated "MB".
2.2. Preparation of Cell Extracts The A549 cell line was cultured in a cell culture flask containing DMEM supplemented with 10% fetal bovine serum, penicillin (100 µg·mL-1) and streptomycin (100 µg·mL-1) in a humidified atmosphere containing 5% CO2 at 37 °C. To obtain the telomerase-containing cell extracts, well-grown A549 cells were initially collected. In total, 3 × 105 A549 cells were dispersed in a centrifuge tube, washed twice with iced PBS (pH 7.4), and incubated on ice in lysis buffer (10 mM Tric-HCl, 1 mM EGTA, 1mM MgCl2, 0.1 mM PMSF, 0.5% CHAPS, 10% glycerol). The mixture was allowed to stand in an ice water bath for 20 min and subsequently centrifuged at 14,000 rpm for 30 min. The supernatant comprising cell lysates containing telomerase was collected and diluted for in vitro detection of telomerase or stored at -80 °C until use. 2.3. Telomerase-Responsive Fluorescence Mechanism TSP (10 µL), dNTPs (10 µL) and 10 µL telomerase standard solution (32 IU·L-1) were added to 10 µL MB solution and the fluorescence intensity of MB observed at 37 °C in the mixed solution. Similarly, 10 µL TSP and 10 µL dNTPs were added to 10 µL MB solution in the absence of telomerase standard solution as a control, and fluorescence intensity measured after mixing. 2.4. Gel Electrophoresis 5
TSP solution (10 µL) and dNTP solution (10 µL) were mixed with 10 µL cell extract or 10 µL telomerase standard solution, and reacted at 37 °C for 1 h, designated Samples 1 and 2, respectively. Samples 3 and 4 were obtained by reacting 10 µL DNA1 solution, 10 µL TSP solution, and 10 µL dNTPs with 10 µL cell extract or 10 µL telomerase standard solution at 37 °C for 1 h. For gel electrophoresis, we mixed TSP solution, Sample 1, sample 2, DNA1 solution, Sample 3, Sample 4 and ladder DNA solution used as an indicator with the DNA loading solution, respectively, and injected these mixtures into the agarose gel in Tris-EDTA (TE) buffer. Electrophoresis was conducted for 40 min at 120 V in TE buffer and images of the gel obtained under UV irradiation. 2.5. Detection of Telomerase Activity in Cell Extracts An aliquot (10 µL) of TSP, 10 µL dNTPs and 10 µL cell extract were added to 10 µL MB solution, and fluorescence intensity changes of the mixture measured at 37 °C. The fluorescence response mechanism under the action of telomerase was examined by observing the change in the fluorescence intensity of the mixed solution added to the cell extract within 1 h. 2.6. Confocal Imaging of Cells under the Action of Probes Activity A549 cells (0.5 mL, 1 × 106·mL-1) were incubated in a 20-mm confocal culture dish for 24 h and 10 µL of probe solution added. Cells containing the probe were incubated at 37 °C for different time-periods and detected via fluorescence confocal imaging. 2.7. MTT assay First, cells (100 µL, 1.0×104·mL-1) were placed in a 96-well plate, and after incubation for 24 h, the culture solution was discarded. The culture solution containing the probe was incubated with A549 cells for different time-periods. Simultaneously, 6
cells incubated in culture medium in the absence of the probe were used as the control group. MTT reagent (10 µL, 5 mg·mL-1) was added separately to each well. After incubation at 37 °C for 4 h, the solution in the well was discarded, and 100 µL DMSO was added to each well. The 96-well plates were shaken at room temperature for 15 min to dissolve the crystals formed by living cells. Finally, plates were placed in a Hitachi/Roche System Cobas 6000 (Tokyo, Japan) microplate reader, and the absorbance value of each well at 490 nm was read. Relative cell activity (%) was calculated using the formula: (Atest/Acontrol) × 100%.
3. Results and discussion 3.1. Characterization of QD and MB
TEM observation of QD shows an average particle size of ~10 nm and good dispersibility (Fig. 1A). When the QD was excited at 326 nm, the QD showed a maximum emission peak at 480 nm. Fluorescence intensity of QD at 480 nm was about 13,000 and that of DNA1 at 535 nm was about 2100 (Fig. 1B, C). Under similar measurement conditions, when DNA1 was modified with QD, the fluorescence intensity of QD decreased to about 1700 and that of Alexa488 increased to about 5700 owing to FRET from the donor, QD, to the acceptor, Alexa 488 (Fig. 1D). Our FRET results indicated that QD was successfully modified onto DNA1. 3.2. Telomerase-Responsive Fluorescence Mechanism To validate the effectiveness of our probes in detection of cellular telomerase activity, we initially examined the telomerase response mechanism of the probe using standard solution. To verify the fluorescence recovery mechanism under telomerase 7
response, TSP, dNTP and telomerase standard solutions were added to MB and changes in fluorescence intensity observed at 37°C. As shown in Fig. 2A, the fluorescence intensity of QD at 480 nm gradually recovered and that of Alexa488 at 535 nm was decreased significantly in the first 10 min, confirming rapid telomerase reaction. At ~40 min, the fluorescence intensity of the probe showed almost no change, which was attributed to saturation of the binding site occupancy of QD37,38. TSP is reported to be prolonged under the action of telomerase and the prolonged region is complementary to the formed hairpin structure. This binding step leads to opening of the hairpin structure, thereby increasing the distance between the receptors and inducing FRET signal disappearance. In addition, the concentration of telomerase had an effect on the fluorescence response. As shown in Fig. 2B, fluorescence recovery or decreasing trend of the probe was more obvious with increasing concentrations of telomerase standard solution after 60 min reaction. To determine the effect of telomerase on the fluorescence response mechanism, we used the mixed solution in the absence of telomerase standard solution as a control. The fluorescence intensity of the probe was not significantly altered in absence of telomerase, validating the fluorescence conversion mechanism of the telomerase response (Fig. 2C).
TSP elongation and reaction of its extended product with MB was confirmed via gel electrophoresis. The TSP solution was mixed with dNTP solution, reacted with cell extract or telomerase standard solution at 37°C for 1 h and subjected to gel electrophoresis. As observed on the electropherogram, the extended product of TSP after the reaction showed a broad band ~20 bases in length (Fig. 2D; lanes d and e), which was extended by approximately 2–3 telomere repeat sequences, compared to the 8
original TSP (Fig. 2D, lane f). Our findings demonstrated recognition and catalytic elongation of TSP by telomerase. The DNA1 solution was mixed with TSP and dNTPs, reacted with cell extract or telomerase standard solution at 37°C for 1 h, and subjected to gel electrophoresis. On the electropherogram, the number of bases of the product after the reaction was about 70 (Fig. 2D; lanes a, b) and increased by about 20, compared with DNA1 (Fig. 2D, lane c), confirming hybridization of MB with TSP elongation products. 3.3. Detection of Telomerase Activity in Cell Extracts To establish the mechanism of action of the probe, cell extracts were mixed with MB solution, TSP and dNTPs to test for fluorescence intensity. The results disclosed the existence of a fluorescence response mechanism under the action of cell extracts and similar fluorescence response effects under the action of telomerase standard solution. As shown in Fig. 3A, the fluorescence intensity of QD at 480 nm gradually recovered while that of DNA1 at 535 nm decreased with increasing time after addition of cell extracts. Similarly, the fluorescence intensity of Alexa488 at 535 nm was stable at around 40 min, with a similar fluorescence response to that with the telomerase standard. In proportion to increasing incubation times, the fluorescence intensity ratio (I480/I535) gradually increased until it was stable at about 40 min (Fig. 3B). Fluorescence spectra of the probe solution after incubation with extracts of different numbers of cells for 60 min are presented in Fig. 3C. Notably, the fluorescence intensity ratio (I480/I535) gradually increased with cell number, with a linear range from 1–100 and 1,000–5,000, respectively (Fig. 3D). The limit of detection was one cell. Compared with previously work, our nanoprobe has the advantages of high efficiency and high sensitivity with a low detection limit of telomerase activity with 9
the proposed FRET-based ratio assay (Table S1), demonstrating its utility in detection of actual telomerase activity in cells with high sensitivity. 3.4. Stability Assay of the Nanoprobe We further investigated the stability of the nanoprobe with the aid of several controlled experiments. First, the probe solution containing TSP, dNTPs, and telomerase standard solution was mixed with different media and the changes in fluorescence intensity of the mixed solution measured. Our results showed that fluorescence intensity changes at 480 nm and 535 nm as a function of time were similar in PBS, DMEM, RPMI1640 and water (Fig. S1). To further confirm that the production of the intracellular fluorescent signal is indeed caused by the catalytic action of telomerase and no other substances in cells, we used the telomerase inhibitor, 3'-azido-3'-deoxythymidine (AZT), as a control and examined fluorescence of the probe under different concentrations of AZT. AZT is a reverse transcriptase inhibitor that promotes the termination of DNA synthesis during the replication process, thereby inhibiting elongation of telomerase primers. The probe solution to which the cell extract was added was pretreated with AZT for 1 h and subjected to a fluorescence test. As shown in Fig. 4A, fluorescence recovery intensity at 480 nm was gradually weakened. The fluorescence intensity ratio (I480/I535) gradually decreased with increasing AZT concentrations, clearly indicating more pronounced inhibition of telomerase activity with higher concentrations of AZT (Fig. 4B). Accordingly, we propose that production of intracellular fluorescent signals is attributable to the action of telomerase and our probes have important application potential in the screening of telomerase inhibitors. In addition, no telomerase response mechanism was observed in heat-inactivated cell extracts, as determined from comparison of the fluorescence changes of heat-inactivated cell extracts at 95°C and normal cell extracts (Fig. S2). 10
This finding can be explained by the fact that telomerase in cell extracts lost enzymatic activity at high temperatures. Our results suggested that the nanoprobe generated in this study had high stability and reliability in the detection of cellular telomerase activity. 3.5. Selectivity Assay of Nanoprobes To determine the selectivity of the probe solution for telomerase, we examined the effects of adding a range of biologically relevant samples to the probe, including bovine serum albumin (BSA), immunoglobulin G (IgG), lysozyme and thrombin, using fluorescence detection. Fluorescence of the probe solution did not change significantly at 480 nm and 535 nm after the addition of BSA, IgG, lysozyme or thrombin, as shown in Fig. 5A. However, fluorescence intensity recovery at 480 nm and decrease in fluorescence at 535 nm were observed in mixed solution containing telomerase, indicating disappearance of the FRET signal. The fluorescence intensity ratios (I480/I535) of the mixed solution after adding different sample solutions are presented in Fig. 5B. This value was significantly higher in the presence of telomerase relative to the other agents examined, further demonstrating the high selectivity of our nanoprobe for telomerase. 3.6. Confocal Imaging of Intracellular Telomerase with Nanoprobes To confirm that the nanoprobes can be used for sensing telomerase in living cells, we incubated them into cells and investigated its cytotoxicity using the MTT assay. After incubating the probes with cells for different time periods, relative cell viability was calculated by measuring absorbance at each time-point. Our results showed that after incubation with nanoprobes for 12 h, A549 and HBE cells had high relative survival rates (A549: 91.2%, HBE: 94.5%), clearly indicating that the probe was low 11
in cytotoxicity and suitable for detection of telomerase activity in cells (Fig. S3). In situ Fluorescence imaging experiments were further performed on intracellular telomerase activity using the prepared probes. Fluorescence signals of cells were observed under a confocal microscope. A549 cells without the probe solution were used as the control group, as observed from Fig. 6. After incubation with the probe, the blue fluorescence showed obvious enhancement with increasing time, while the green fluorescence decreased, which is due to the opening of the hairpin structure of the MB under the action of telomerase, consistent with data obtained from previous fluorescence spectroscopy experiments. The result clearly demonstrated the presence of telomerase predominantly in the cytoplasm. In summary, our novel probe could be efficiently utilized to detect intracellular telomerase activity through monitoring changes in fluorescence signal intensity.
4. Conclusions Here, we designed a nanoprobe based on QD-FRET for detecting intracellular telomerase activity. The designed nanoprobe contained a specific DNA sequence, which could form a hairpin structure after hybridization and thus decrease the distance between QD as a fluorescence donor and Alexa488 as a fluorescence acceptor to generate FRET. Telomere primers were prolonged under the action of telomerase. The extended region was complementary to the formed hairpin structure, leading to opening of the structure and disappearance of the FRET signal. Telomerase activity was determined by detecting the changes in fluorescence intensity and observing fluorescence signals of cells incubated with probes. The nanoprobe was further used for quantitative analysis of telomerase activity in different numbers of cells. Sensitive detection of telomerase activity in cells was achieved. The nanoprobe generated in our 12
study has the advantage of high efficiency and sensitivity with a low detection limit of telomerase activity (one cell) and can be effectively applied to dynamically monitor changes in telomerase activity in cells under the action of telomerase drugs, thus providing
a
useful
tool
for
the
discovery
and
screening
of
potential
telomerase-targeted anticancer drugs.
Acknowledgements This research was supported by the National Natural Science Foundations of China (21505057, 21605062, and 51504242), the Priority Academic Program Development of Jiangsu Higher Education Institutions, the Brand Major of Universities in Jiangsu Province, and the Top-notch Academic Programs Project of Jiangsu Higher Education Institution (TAPP).
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Figures and captions
Scheme 1. (A) Schematic illustration of the DNA nanoprobe based on QD-FRET for telomerase sensing. (B) Detection of telomerase activity in cells under the action of probes.
20
Fig. 1. (A) TEM image and (B) fluorescence spectrum of the QD. (C) Fluorescence spectrum of the DNA1. (D) Fluorescence spectrum of the QD-DNA1.
21
Fig. 2. (A) Fluorescence spectra of the probe solution containing TSP, dNTPs, and telomerase standard solution in response to reaction time. (B) Fluorescence spectra of the probe solution after incubation with different concentrations of telomerase standard solution for 60 min. (C) Fluorescence spectra of the probe solution containing TSP, dNTPs in absence of telomerase standard solution in response to reaction time. (D) Electrophoresis image of (a) mixture of DNA1, TSP, dNTPs and telomerase standard solution after incubation for 1 h, (b) mixture of DNA1, TSP, dNTPs and cell extract after incubation for 1 h, (c) DNA1, (d) mixture of TSP, dNTPs and telomerase standard solution after incubation for 1 h, (e) mixture of TSP, dNTPs and cell extract after incubation for 1 h, (f) TSP, and (g) Ladder DNA.
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Fig. 3. (A) Fluorescence spectra of the probe solution containing TSP, dNTPs, and cell extract in response to reaction time. (B) Plot of the fluorescence intensity ratio (I480/I535) vs incubation time. (C) Fluorescence spectra of the probe solution containing TSP, dNTPs after incubation with extract of different numbers of cells for 60 min. (D) The linear relationship between the fluorescence intensity ratio (I480/I535) and number of the cell.
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Fig. 4. (A) Fluorescence spectra of the probe solution containing TSP, dNTPs, and cell extract after incubation with different concentrations of AZT for 60 min. (B) Plot of the fluorescence intensity (I480/I535) vs different concentrations of AZT.
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Fig. 5. (A) Fluorescence spectra of probe solutions containing TSP and dNTPs after 60 min reaction with BSA, IgG, lysozyme, thrombin and telomerase. (B) Fluorescence recovery intensity at 480 nm of probe solution containing TSP and dNTPs after addition of (a) BSA, (b) IgG, (c) lysozyme, (d) thrombin, (e) telomerase standard solution for 60 min.
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Fig. 6. Fluorescence images of telomerase in living A549 cells.
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Highlights A DNA-based fluorescence resonance energy transfer nanoprobe was designed. Incubation of cells with the nanoprobe facilitated sensing of intracellular telomerase activity. The nanoprobe achieved sensitive detection of telomerase in cells up to a detection limit of one cell.
Declaration of interests The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. ☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: