Quantitative Determination of Telomerase Activity by Combining

Dec 6, 2017 - Telomerase is a key enzyme for maintaining the telomere length and is regarded as a versatile cancer biomarker and a potential drug targ...
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Article Cite This: Anal. Chem. 2018, 90, 1006−1013

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Quantitative Determination of Telomerase Activity by Combining Fluorescence Correlation Spectroscopy with Telomerase Repeat Amplification Protocol Di Su, Xiangyi Huang, Chaoqing Dong,* and Jicun Ren* School of Chemistry & Chemical Engineering, State Key Laboratory of Metal Matrix Composites, Shanghai Jiao Tong University, 800 Dongchuan Road, Shanghai, 200240, P. R. China S Supporting Information *

ABSTRACT: Telomerase is a key enzyme for maintaining the telomere length and is regarded as a versatile cancer biomarker and a potential drug target due to its important role in cancer and aging. It is necessary to develop a sensitive and reliable method for detection of telomerase activity due to its very low level in cells. In this Article, we propose an ultrasensitive and robust method for quantitative determination of telomerase activity by combining single molecule fluorescence correlation spectroscopy (FCS) with telomerase repeat amplification protocol (TRAP). The principle of this new method (FCSTRAP) is based on measurement of the change in characteristic diffusion time and molecule number of TRAP products by FCS. The characteristic diffusion time is related to the length of TRAP products, and the molecule number represents the concentration of TRAP products. We optimized the conditions of TRAP procedure and FCS measurements. We observed that the telomerase activities are positively correlated to characteristic diffusion time and molecule number of TRAP products at optimal conditions. This method was successfully used for determination of telomerase activity of different cells, and detection of a single cell was realized. Meanwhile, this method was used to evaluate the inhibition efficiency of inhibitors, and the IC50 values obtained were in good agreement with the references. Compared to current TRAP methods, this method shows reliable quantification, ultrahigh sensitivity, and short detection time and is without separation. We believe that the FCS-TRAP method has a potential application in clinical diagnosis and screening of telomerase inhibitors.

T

protocol (TRAP). This method possesses ultrahigh sensitivity due to the use of PCR amplification, and so far, it has become the classical and most-widely used method for determination of telomerase activity.9 In order to avoid radiation pollution, some modified methods were developed such as TRAP-polyacrylamide gel electrophoresis (PAGE) with ethidium bromide staining10 or with the silver staining assay (SS).11 PAGE can give the visualization of the products of telomerase extension, but it is only suitable for qualitative detection and is not a quantitative assay. In order to quantitatively determine the products of telomerase extension, some strategies were proposed, like TRAP-enzyme-linked immunosorbent assay,12 real-time-TRAP,13 asymmetric PCR on magnetic beads, and cycling probe technology.14 Although these methods are used to quantify TRAP products according to the fluorescent intensity of the telomerase extension products, they cannot provide the information on the length of the telomerase

elomerase is a ribonucleoprotein reverse transcriptase complex and is composed of the telomerase reverse transcriptase protein, a telomerase RNA template, and several associated proteins.1,2 Telomerase can maintain the length of telomeres and the indefinite proliferation of cells by the addition of hexamer repeats (TTAGGG)n to the end of chromosomes.3 Telomerase overexpression has been observed in over 85% of all known cancer cells;4 therefore, telomerase is regarded as a tumor marker for medical diagnosis as well as a potential therapeutic target.5,6 In 2009, the Nobel Prize was awarded to Greider, Blackburn, and Szostak due to their discovery of telomeres and telomerase.7 The determination of telomerase activity plays a crucial role in early cancer diagnosis, understanding cancer mechanisms, screening inhibitors, and therapeutic research. Up to date, a variety of strategies have been developed for determination of telomerase activity. Telomerase extension assay is an initial method for determination of telomerase activity, and this method required a high level of telomerase in tissue samples, long assay time, and radioactive precursors due to its poor sensitivity.8 Kim et al. developed the polymerase chain reaction (PCR)-based telomeric repeat amplification © 2017 American Chemical Society

Received: October 16, 2017 Accepted: December 6, 2017 Published: December 6, 2017 1006

DOI: 10.1021/acs.analchem.7b04256 Anal. Chem. 2018, 90, 1006−1013

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Analytical Chemistry

molecule number of TRAP products by FCS. In principle, the characteristic diffusion time is related to the length of TRAP products, and the molecule number represents the concentration of TRAP products. Compared to the current TRAP method, this new method can simultaneously provide information about the length and concentration of TRAP products. In this study, we systematically optimized the conditions of the conventional TRAP assay and FCS measurements. We observed that the telomerase activities are positively correlated to the characteristic diffusion time and the molecule number of TRAP products at optimal conditions. This method was successfully used for determination of telomerase activity of different cells. Furthermore, this method was used to evaluate the inhibition efficiency of inhibitors, and the IC50 values obtained were in good agreement with the references.

extension products, and it is difficult to guarantee the reliability of assay results. Recently, some PCR-free approaches were developed.15 Tian et al. proposed the exponential isothermal amplification of telomere repeat assay by the combination of warm start DNA polymerase and the nicking endonuclease.16 Liu et al. developed a method for determination of telomerase using T7 exonuclease-aided target recycling amplification with a 5′methylene blue (MB)-labeled hairpin (HP) probe. In this method, HP probes first hybridized the telomerase extension products, and then, these hybridized products were digested by T7 exonuclease, which resulted in the MB-labeled mononucleotides amplified electrochemical signal.17 Some other signal amplification assays were also proposed. Alizadeh-Ghodsi et al. measured the telomerase activity based on the liposomal signal amplification platform.18 Liu et al. developed label-free detection of telomerase activity using telomeric hemin/Gquadruplex triggered polyaniline deposition.19,20 Wang et al. studied telomerase activity using nucleic acids modified gold nanoparticles triggered mimic hybridization chain reaction dual signal amplification.21 Wang et al. detected telomerase activity via oxygen gas pressure readout based on platinum nanoparticles (PtNPs) catalyzing the decomposition of H2O2.22 Fluorescent assay in combination with nanotechnology is widely used to determine telomerase activity. Gao et al. developed a fluorescence polarization method for determination of the telomerase activity using GNPs with thiolated telomerase substrate (TS) primer as substrate and short carboxyfluorescein-modified complementary DNA as hybridization probes.23 Some imaging methods such as fluorescence imaging and transmission electron microscopy imaging were used for visualization of telomerase activity in vitro24 or in vivo.25−27 Su et al. proposed a single molecule imaging method on the basis of nucleic acid stochastic binding strategy.28 Although these methods above were designed skillfully to avoid a PCR procedure, they have not widely been used in clinical diagnosis due to having complicated steps, being timeconsuming, and not having high sensitivity. Up to date, TARP is still considered to be a “gold standard” method for determination of telomerase activity due to its ultrahigh sensitivity. However, current TRAP methods also have some drawbacks such as long assay time and complex steps, and more importantly, they cannot simultaneously provide information about the length and concentration of TRAP products. In the PCR reaction, different lengths of PCR products were amplified due to use of different lengths of TS extension products as templates, which is different from commonly used PCR (only a kind of PCR products). Herein, we want to develop a sensitive and reliable method for quantitative determination of telomerase activity by combining fluorescence correlation spectroscopy (FCS) with TRAP (FCS-TRAP) to solve the above drawbacks of current TRAP methods. FCS is a sensitive single molecule method, and its principle is based on measuring the fluorescence fluctuations in a small detection volume due to Brownian motion of single molecule.29 FCS is widely used to determine molecular concentration and study molecular diffusion, chemical kinetics, and molecular interactions like DNA−DNA,30 mRNA− protein,31 protein−protein,32 enzyme activity,33 and drug− protein interaction in vitro or in vivo.34−36 In this new method, FCS is used to quantitatively analyze DNA fragments in the TRAP reaction. The principle of FCS-TRAP is based on measurement of the changes in characteristic diffusion time and

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EXPERIMENTAL SECTION Chemicals and Materials. TaKaRa Biotechnology Co., Ltd. (Dalian, China) provides the following DNA fragments: telomerase substrate (TS) primer, 5′-AATCCGTCGAGCAGAGTT-3′; anchor reverse primer (ACX), 5′-GCGCGGCTTACCCTTACCCTTACCCTAACC-3′; synthetic TS extension product, 5′-AATCCGTCGAGCAGAGTTAGGGTTAGGGTTAGGG-3′ (control experiments). The deoxynucleotide mix (dNTPs, 10 mM), 3-[(3-cholamidopropyl)-dimethyl-ammonio]-1-propanesulfonate (CHAPS), β-mercaptoethanol, phenylmethylsulfonyl fluoride (PMSF), tris(hydroxymethyl) aminomethane (Tris), bovine serum albumin (BSA), fetal bovine serum (FBS), and trypsin were products of Sigma-Aldrich (USA). KOD-Plus-Neo DNA polymerase was from Toyobo (Japan). N,N,N′,N′-Tetraacetic acid (EGTA) and other common reagents were from Sinopharm chemical reagent Co., Ltd. (Shanghai, China). Diethyl pyrocarbonate-treated water (DEPC-treated water) and 3-(3-cholamidopropyl) acrylamide (30%) were provided by Sangon Biological Engineering Technology & Co., Ltd. (Shanghai, China). The 50 and 100 bp DNA fragments were from Dongsheng Biotech (Guangzhou, China), and 200 and 300 bp DNA fragments were purchased from Comwin Biotech Co., Ltd. (Beijing, China). Eva Green (EG) was from Biotium (USA). SYBR Green I (SG I), YOYO-1, and GeneRuler ultra low range DNA ladder were from Thermo Fisher Scientific (USA). Dulbecco’s modified Eagle’s medium (DMEM), RPMI-1640, and phosphate buffer saline (PBS, pH 7.4) were all purchased from HyClone Thermofisher (USA). Aloe-emodin (AE), 3′azido-3′-deoxythymidine (AZT), and 5,10,15,20-tetra (Nmethyl-4-pyridyl) porphine (Tmpyp4) were purchased from TCI Development Co., Ltd. (Shanghai, China). Cell lysis buffer consisted of CHAPS lysis buffer (10 mM Tris-HCl, pH 7.5, 1 mM MgCl2, 1 mM EGTA, 0.5% CHAPS, 0.1 mM PMSF, 5 mM β-mercaptoethanol, and 10% glycerol). TRAP buffer in telomerase extension reaction consisted of 20 mM Tris-HCl (pH 8.3, 1.5 mM MgCl2, 63 mM KCl, 1 mM EGTA, and 0.1 mg·mL−1 BSA). The 1× PCR reaction solution contained 200 μM dNTPs, 1.5 mM MgCl2, 0.005 U/μL of KOD-Plus-Neo polymerase, 400 nM TS primer, and 400 nM ACX primers. Gel electrophoresis buffer 1× TBE contained 100 mM Tris-HCl, 83 mM boric acid, and 1 mM EDTA (pH 8.0). All the solutions were prepared with DEPC-treated water. All chemicals were of analytical grade and used without further purification. 1007

DOI: 10.1021/acs.analchem.7b04256 Anal. Chem. 2018, 90, 1006−1013

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Analytical Chemistry Scheme 1. Principle for Determination of Telomerase Activity and Its Inhibitiona

a

The telomerase substrate was TS (5′-AATCCGTCGAGCAGAGTT-3′). The PCR reverse primer was ACX (5′-GCGCGGCTTACCCTTACCCTTACCCTAACC-3′). The TS was specifically extended by the telomerase, but the activation of telomerase was inhibited in the presence of inhibitors. FCS is used to selectively detect the different lengths and concentrations [C] of TRAP products by the measurements of diffusion time and molecule number at given experimental conditions.

FCS Setup. FCS measurements were performed with a home-built FCS system shown in Figure S1. Briefly, FCS setup was equipped with an inverted microscope (IX71, Olympus, Japan). The laser with 488 nm wavelength (Sapphire LP USB CDRH, Coherent.com, USA) (excitation power of about 100 μW) is expanded and reflected by a dichroic mirror (505DRLP, Omega optical, USA) and then focused by a water immersion objective (UplanApo, 60× NA 1.20, Olympus, Japan) into a sample solution. The excited fluorescence is collected by the same objective and passed through the same dichroic mirror and then filtered by a band-pass filter (530DF30, Omega Optical, USA). Finally, the fluorescence was focused into a pinhole (50 μm) in front of the single-photon counting module (SPCM-AQR16, PerkinElmer EG&G, Canada). The fluorescence fluctuation signals were autocorrelated with a real time digital correlator (Flex02-12D/C, Correlator.com, USA) with a period of 60 s. Rhodamine Green (RG) with the concentration of 5 nM (its diffusion coefficient: 2.8 × 10−6 cm2/s in water37) is used as a standard substance to calibrate the detection volume of the FCS system before measurements every day. On the basis of the Levenberg−Marquardt algorithm, FCS data are analyzed with the one-component model (eq 1) nonlinearly fitted with the Origin 8.0 software package. Cell Culture, Telomerase Extract Preparation, and Telomerase Extension Reaction. The cell culture and extraction of telomerase were carried out according to the modified method.9 These details were described in the Supporting Information. The procedure of the telomerase extension reaction was also described in the Supporting Information. Optimization of FCS Measurement Conditions and DNA Assay. Using 300 bp dsDNA marker with a concentration of 200 ng/mL, the optimization of the FCS

measurement conditions was carried out with different intercalating dyes (Eva Green, SYBR Green I, and YOYO-1) and measurement times. To verify the feasibility of the FCS method for detection of telomerase activity, the different concentrations of 300 bp dsDNA markers (10, 50, 250, 500, 750, 1000, and 1500 ng/mL) and lengths of dsDNA markers (50, 100, 200, and 300 bp) were used in this study. All the solutions of dsDNA markers and YOYO-1 were diluted with TE buffer (10 mM Tris-HCl, pH 8.0, 1 mM EDTA) in order to promote the dissolution of DNA and to maintain its stability. The molecule numbers (N) and characteristic diffusion times (τD) were then measured by FCS. Optimization of TRAP Procedure and Assay of PCR Products. 2.5 μL of telomerase extension reaction products was added into 22.5 μL of 1× PCR reaction solution. PCR was performed using a thermal cycler (Bio-Rad T100, USA) with the following four-step program: 94 °C for 5 min (to activate the hot start polymerase in the master mix); amplified for 30 cycles of 94 °C for 30 s, 60 °C for 30 s, and 72 °C for 30 s; 72 °C for 5 min; finally, an infinite hold at 4 °C. The optimization of the TRAP procedure was carried out with the annealing temperature (at 50, 55, 60, 65, and 70 °C), the concentration of KOD-Plus-Neo (at 0.01, 0.005, and 0.0025 U/μL), and the concentration of Mg2+ (1, 1.5, and 2 mM). After being diluted 200 times with TE buffer, the PCR products were analyzed using gel electrophoresis and FCS, respectively. Gel Electrophoresis Procedure. The gel electrophoresis with a silver staining method was described in the Supporting Information.

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RESULTS AND DISCUSSION Principle for Determination of Telomerase Activity and Its Inhibition. In this study, the conventional TRAP was 1008

DOI: 10.1021/acs.analchem.7b04256 Anal. Chem. 2018, 90, 1006−1013

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Analytical Chemistry used to amplify the substrates of telomerase, and FCS was used to detect the products of TRAP amplifications. Scheme 1 schematically shows the principle for determination of telomerase activity and inhibition. The TS is used as the substrate of telomerase. As shown in Scheme 1, this procedure includes extension reaction, PCR reaction, FCS detection, and data analysis. In the extension reaction as shown in Figure S2, the active telomerase extracted from cells containing RNA template (5′-CUAACCCUAAC-3′) first binds to TS, and then, TTAGGG repeat units are added to the 3′-end of the TS primer by complementary sequence in the presence of the dNTPs mixture. Then, telomerase translocation starts, and it continuously adds TTAGGG repeat units to the 3′-end of the TS. Finally, different lengths of single stranded DNA fragments are formed in the extension reaction. In the PCR reaction shown in Figure S3, the reverse primer ACX is first hybridized to the extension product, and the KOD-plus-Neo polymerase synthesizes DNA starting from the extension product or the ACX primer. The TRAP products undergo N times repeated PCR cycles (containing denature, anneal, and polymerize to amplify) by polymerase. After amplification, 2N double-stranded DNA TRAP products are formed in principle. As shown in Scheme 1, in the enzyme reaction without inhibitor, the double-stranded DNA TRAP products will be obtained in the presence of telomerase. In the enzyme reaction with inhibitor, the activity of telomerase is significantly inhibited by the inhibitor and the TRAP products will be dramatically decreased. In the presence of the high affinity intercalating dye YOYO-1, the formation of dsDNA/YOYO-1 complexes will dramatically enhance fluorescence signals of the solution. Due to their significant differences in the concentration and the length of dsDNA of TRAP products, the corresponding particle numbers (N) and the characteristic diffusion time (τD) can be measured by FCS. In principle, the activity of telomerase should be positively correlated with N or τD. FCS is an ultrasensitive and noninvasive single molecule method, and its principle is based on measuring the fluctuations of fluorescence signal in a highly focused detection volume (less than 1 fL) due to Brownian motion of a single fluorescent molecule. FCS can provide some important information, such as the average number of fluorescent particles and its characteristic diffusion time in the volume. The theory and applications of FCS were described elsewhere.38−43 Assuming the focal volume of FCS as a three-dimensional Gaussian profile, the one-component autocorrelation function can be expressed by the following form ⎡ −τ T exp τ 1⎢ T G (τ ) = ⎢1 + 1−T N⎢ ⎣

( ) ⎤⎥⎥

1

(

⎥ 1+ ⎦

The detection volume can be expressed below: V0 = π 3/2ω0 2z 0

According to eq 1, when τ = 0, G(0) is obtained by a nonlinear fitting autocorrelation curve of fluorescent TRAP products: G(0) =

)

1+

relative activity =

ω0 2 τ z 0 2 τD

where N is the total number of fluorescent molecules in the small observation volume, T is the fraction of fluorescent molecules in the triplet state with a relaxation time τT, and ω0 and z0 are the lateral and axial radii of the Gaussian emission light focused detection volume. The τD is the characteristic diffusion time of fluorescent molecules obtained with eq 1, and it is related to the diffusion coefficient D:

ω0 2 4D

(4)

N N0

(5)

The N and N0 express the particle numbers of TRAP products with and without inhibitors, respectively. Optimization of FCS Measurement Conditions and DNA Assay. In FCS analysis of DNA fragments, BPP of the DNA/intercalating dye complexes is related to the reproducibility of the results, and the characteristic diffusion time of the dsDNA fragments in the TRAP reaction is also used to distinguish the length of the telomerase extension products. In this case, we studied the effects of intercalating dyes on BPP. As shown in Figure S4A, BPP of the dsDNA/YOYO-1 complexes is about five times stronger than dsDNA/EG complexes or dsDNA/SG I complexes. In this study, YOYO-1 is used as DNA intercalating dye. Figure S4B displays the characteristic diffusion times of 300 bp DNA marker dramatically increased with an increase of the dilution ratio of YOYO-1. When the dilution ratio of YOYO-1 was 2×, the binding of intercalation dye to double-stranded DNA reached saturation. Figure S4C shows the effect of the FCS measurement times on the standard deviation of characteristic diffusion times. When this measurement time is over 60 s, the characteristic diffusion time nearly does not change. Therefore, we chose the 2× dilution ratio of YOYO-1 and the measurement time of 60 s in subsequent experiments for good repeatability and a rapid assay. To verify the feasibility of FCS detection of telomerase activity, the different lengths of dsDNA markers (50, 100, 200, and 300 bp) and different concentrations of 300 bp dsDNA marker (10, 50, 250, 500, 750, 1000, and 1500 ng/mL) were investigated, respectively. All the solutions of dsDNA markers and YOYO-1 were diluted with TE buffer (10 mM Tris-HCl, pH 8.0, 1 mM EDTA). N and τD were then measured by FCS. Figure 1A shows normalized autocorrelation curves and their fitting residuals under different lengths (50, 100, 200, and 300 bp) of DNA markers. These autocorrelation curves are well fitted with the mode of autocorrelation function as described in eq 1; the correlation coefficients (R2) are 0.997−0.999. The fitting residuals are less than 0.12, and their τD values are 0.949 ± 0.021, 1.37 ± 0.02, 1.83 ± 0.05, and 2.40 ± 0.04 ms, respectively. We observed that correlation curves shifted to the right with the increase of the length of DNA markers, indicating the increase of the DNA molecular weight and size. The inset shows that τD linearly increases with an increase of the length of DNA markers. The linear regression equation is expressed as τD = 0.0057X + 0.72 (X: length of DNA (bp)); the linear range is from 50 to 300 bp, and the correlation coefficient (R2) is 0.965.

(1)

τD =

1 N

The particle numbers (N) can be obtained with eq 4. Furthermore, the brightness per particle (BPP) is calculated on the basis of the acquired particle numbers (N) and average count rate. In inhibition of telomerase activity measured by FCS, the relative activity of telomerase is calculated.

1 τ τD

(3)

(2) 1009

DOI: 10.1021/acs.analchem.7b04256 Anal. Chem. 2018, 90, 1006−1013

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Analytical Chemistry

dimerization. The optimization of thermal cycling conditions was conducted, which includes different annealing temperatures and concentrations of polymerase and Mg2+ in order to increase the amplification of telomerase extension products and eliminate primer−dimer formation. As indicated in Figure S5A, when the annealing temperature increased to 60 °C, the formation of primer−dimers occurred only sporadically and the amount of long strands of telomerase products significantly increased. When the annealing temperature was over 60 °C, the telomerase extension products were significantly decreased. Figure S5B shows that the abundance of telomerase extension products and the primer−dimers increase with an increase in concentration of polymerase. We also investigated effects of the concentration of Mg2+ on the activity of polymerase. As shown in Figure S5C, the amount of telomerase products and the primer−dimer were also increasing with the concentration of Mg2+. Hence, to obtain more telomerase extension products and fewer primer−dimers, we chose the annealing temperature of 60 °C, the polymerase concentration of 0.005 U/μL, and the Mg2+ concentration of 1.5 mM in subsequent experiments. Furthermore, we optimized the telomerase extension time in the TRAP procedure. As illustrated in Figure 2A, the

Figure 1. Feasibility verification for FCS assay of dsDNA. (A) Normalized autocorrelation curves and their fitting residuals of different lengths of DNA marker (50, 100, 200, and 300 bp). The inset shows the linear relationship between characteristic diffusion time (τD) and the length of DNA marker. (B) Typical autocorrelation curves and their fitting residuals of different concentrations of 300 bp DNA marker (10, 50, 250, 500, 750, 1000, and 1500 ng/mL). The inset shows the linear relationship between the particle numbers (N) and concentrations of 300 bp DNA marker. The measurement time was 60 s. The error bars represent the standard deviation of 3 measurements.

Figure 2. Effects of telomerase extension time. (A) Gel electrophoresis assay for telomerase PCR product from different extension reaction times. (B) The effects of extension reaction time on the particle numbers (N). In this study, about 1000 Hela cells were used. The FCS measurement time was 60 s. The error bars represent the standard deviation of 3 measurements.

Figure 1B shows the typical autocorrelation curves and their fitting residuals under different concentrations (10, 50, 250, 500, 750, 1000, and 1500 ng/mL) of 300 bp dsDNA marker. These autocorrelation curves are well fitted with the theoretical mode of autocorrelation function with correlation coefficients (R2) of 0.998−0.999; the fitting residuals are 0.003−0.14, and their N values are 84 ± 0.085, 1.59 ± 0.06, 5.36 ± 0.09, 7.58 ± 0.06, 10.4 ± 0.2, 15.2 ± 0.5, and 22.2 ± 0.5, respectively. We observed that the amplitudes of correlation curves decrease with the increase of DNA concentration, indicating the increase of the numbers of DNA fragments. The inset shows that N linearly increases with an increase of DNA concentration (C). The linear regression equation is expressed as N = 0.013C + 0.98; the linear range is from 10 to 1500 ng/mL, and the correlation coefficient (R2) is 0.981. These results above indicate that FCS will be a highly sensitive and feasible method for characterization of the concentration and the length of PCR products after telomerase extension. Optimization of PCR Conditions in TRAP. The PCR method used in this study is based on the TRAP assay introduced by Kim et al.9 Heat-activated polymerase and hot start conditions were used in order to avoid primer

abundance of telomerase extension products increased with the increase of the telomerase extension time. Figure 2B clearly shows that N rapidly increases before about 30 min and then reaches a constant after 60 min. Thus, the extension time was selected as 60 min. FCS Assay of Telomerase Activity. We studied the telomerase activity of different cell numbers and cell lines by FCS, respectively. The gel electrophoresis was used as a contrast experiment. The changes of τD values and N values of telomerase PCR products were used to estimate telomerase activity of HeLa cells. As seen in Figure 3A, as the number of Hela cells increased, the length and the abundance of telomerase extension products increased in the gel electrophoresis assay, which meant an increase in the activity of telomerase. In order to confirm the specific amplification in the PCR reaction, we designed a control experiment using a synthetic TS extension product as a template (25 pM). As shown in Figure 3A, PCR products were about 50 to 75 bp in this case, which was consistent with the predicted values. The inactivation control and the CHAPS blank control were nearly 1010

DOI: 10.1021/acs.analchem.7b04256 Anal. Chem. 2018, 90, 1006−1013

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Analytical Chemistry

Figure 3. Investigation of telomerase activity of different cell numbers and control experiments. (A) Gel electrophoresis assay for telomerase PCR product from different cell numbers and control experiments. (B) A comparison of characteristic diffusion time (τD) under different conditions. The concentration of the synthesized TS extension product primer (5′-AATCCGTCGAGCAGAGTTAGGGTTAGGGTTAGGG-3′) was 25 pM. The inactivation was heated from 1000 Hela cells. (C) Normalized autocorrelation curves, linear fitting curves, and their fitting residuals of different numbers of Hela cells (10, 50, 100, 200, and 300). The inset shows the linear relationship between characteristic diffusion time (τD) and the number of Hela cells. (D) Typical autocorrelation curves, linear fitting curves, and their fitting residuals of different Hela cells (10, 200, 500, 750, 1000, and 1500). The inset shows the linear relationship between the particle numbers (N) and the number of Hela cells. The measurement time was 60 s. The error bars represent the standard deviation of 3 measurements.

Due to formation of only ACX dimers, the τD value of TRAP products is 0.197 ± 0.011 ms in this case. This result proved that the TS primer was the specific substrate of telomerase to achieve a series of extension products. Figure 3C shows normalized autocorrelation curves and their fitting residuals under different numbers of Hela cells (10, 50, 100, 200, and 300 cells). These autocorrelation curves are well fitted with the theoretical mode of autocorrelation function with correlation coefficients (R2) of 0.991−0.995 and the fitting residuals of less than 0.20, and their τD values are 0.867 ± 0.024, 0.987 ± 0.007, 1.06 ± 0.04, 1.24 ± 0.04, and 1.38 ± 0.05 ms, respectively. We observed that correlation curves shifted to the right with the increase of the length of PCR products, indicating the increase in the long length of TS extension products with the increase of cell numbers. The inset shows that the τD values linearly increase with cell numbers. The linear regression equation is expressed as τD = 0.001X + 0.895 (X: cell number); the linear range is from 10 to 300 cells, and the correlation coefficient (R2) is 0.956. Figure 3D displays typical autocorrelation curves and their fitting residuals from different cell numbers (10, 200, 500, 750, 1000, and 1500). These autocorrelation curves are well fitted with the theoretical mode of autocorrelation function as described in eq 1, and the correlation coefficients (R2) are 0.991−0.996; the fitting residuals are 0.011−0.032, and their N values are 7.78 ± 0.27, 8.99 ± 0.31, 11.20 ± 0.26, 12.42 ± 0.64, 13.52 ± 0.18, and 5.31 ± 0.36, respectively. We observed that the amplitude of correlation curves shifted downward with the increase of the numbers of cells, indicating the increase of the

the same with weak products. Only the ACX dimer band was observed in the control experiment without TS. These results illustrated that the PCR reaction was successful and the specific PCR products were obtained. Figure 3B clearly depicts the obvious change in τD values of TRAP products for 1 cell, 300 cells, and different control conditions. This figure provided some information as follows. First, we obtained the τD values of 1 cell and 300 cells (0.776 ± 0.048 and 1.38 ± 0.05 μs), respectively, according to the autocorrelation function mode (eq 1). Due to the low telomerase concentration of 1 cell, the average length of the extension product obtained was shorter. The average length of the extension product obtained was longer under a high telomerase concentration of 300 cells. Second, the τD value of PCR products was 1.09 ± 0.02 ms using synthetic TS extension product as a template. There are only three repeating sequences added in the synthesis of the TS extension product, and their τD is between 1 and 300 cells. Third, in the conditions of the inactivation and CHAPS, the τD values of TRAP products are 0.412 ± 0.050 and 0.295 ± 0.018 ms and are significantly shorter than the TRAP products in the condition of activated telomerase. The heated inactivation demonstrated that the heat-inactivated telomerase could not elongate the TS primer, and the CHAPS lysis buffer blank control demonstrated that the reaction system itself had little disturbance on the reaction and FCS assay. Finally, to further exclude the interference of cell extracts, we also executed a control experiment with the telomerase from 1000 Hela cells without TS primer. 1011

DOI: 10.1021/acs.analchem.7b04256 Anal. Chem. 2018, 90, 1006−1013

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Analytical Chemistry particle numbers. The inset shows that N values linearly increase with an increase of the numbers of cells. The linear regression equation is expressed as N = 0.005X + 8.099 (X: cell numbers); the linear range is from 10 to 1500 cells, and the correlation coefficient (R2) is 0.974. The result illustrated that the increase of τD values and N values depended on the activity of telomerase. FCS can be used for determining telomerase activity of 1 cell. A comparison of Figure 3C,D illustrated that the N value is more sensitive than τD. Meanwhile, the FCS results are well in line with the gel electrophoresis assay. These results confirm that FCS is an ultrahigh sensitive and reliable method for characterization of the concentration and the average length of PCR products after telomerase extension. Furthermore, telomerase activity of different cell lines was measured in order to evaluate the universality of the FCS method for the telomerase activity assay. As revealed in Figure 4A, the gel electrophoresis assay indicates that all four kinds of

Figure 5. Inhibition curve of telomerase activity by AE inhibitor. Inset shows the chemical structure of AE. The error bars represent the standard deviation of 3 measurements.

inhibitor concentration (AE, AZT, and Tmpyp4). The relative activity is determined by eq 5, where N is the particle number in the presence of inhibitors and N0 is the particle number in the 1000 Hela cells experiment without the inhibitor treatment. Table 1 summarized the IC50 values of three inhibitors ranging from 5.2 μM to 1.8 mM. These results are basically Table 1. Comparison of IC50 Values from Our Method and the References

Figure 4. Study of telomerase activity of different cell lines. (A) Gel electrophoresis assay for telomerase PCR product from different cell lines. (B) A comparison of characteristic diffusion time (τD) of different cell lines. Lines left to right show Hela, K562, MCF-7, and Hek293, respectively. About 1000 different cells were used, and the measurement time was 60 s. The error bars represent the standard deviation of 3 measurements.

inhibitor

experiment IC50

references IC50

AE AZT Tmpyp4

5.20 μM 1.88 mM 7.95 μM

5.89 μM23 1.85 mM44 6.50 μM45

consistent with IC50 values obtained by other methods,23,44,45 which reflect that FCS is applicable for evaluating potential inhibitors of telomerase.

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cell lines show an obvious telomerase PCR products band. It is evident that cancer cell lines such as human cervical carcinoma (Hela) and breast (MCF-7), leukemia cells (K562), and the embryonic kidney cells (Hek293) have high telomerase activity. FCS results as shown in Figure 4B also demonstrate that four cell lines have diacritical telomerase activity, and their N values are 13.16 ± 0.17, 14.29 ± 0.20, 9.74 ± 0.19, and 15.20 ± 0.55, respectively. Hence, combined with TRAP, FCS can discriminate the levels of telomerase activity among various cell lines. Assay for Inhibition of Telomerase Activity. Telomerase inhibitors are potentially anticancer drugs because the telomerase activity can be detected in most cancer cells as a universal cancer biomarker. We further investigated the feasibility of FCS for the evaluation of telomerase inhibitors. In this study, AE, AZT, and Tmpyp4 were used as models. AZT is a thymidine analogue, and it competes with mononucleotide and preferentially integrates into the telomeric region of CHO DNA to inhibit telomerase. Telomerase executes the extension reaction after its RNA template binds onto the unfolded stranded single-stranded telomeric DNA. However, when Gquadruplex-interacting agents (like AE or Tmpyp4) stabilize the G-quadruplex structure and block the binding of telomerase, the activity of telomerase will be inhibited. As illustrated in Figures 5, S6, and S7, the relative activity of telomerase gradually decreased with the increase of the

CONCLUSION

In summary, we propose a new method for determination of telomerase activity by combining FCS with TRAP (FCSTRAP). This method takes the advantages of PCR amplification of telomerase extension products and direct measurements of the length and concentration of TRAP products by FCS without separation. The established FCSTRAP method was successfully used to determine telomerase activity from different numbers of Hela cells and different types of cell lines. Furthermore, this method was applied to study the inhibition of telomerase activity and evaluate IC50 values of inhibitors. Compared to the gel electrophoresis method, this method provides reliable quantification and shows short analysis time and simple operation without separation. Compared to the real-time PCR method, this method can provide information about the length of TRAP products, which will guarantee the reliability of measurement results. However, our method needs to be further improved by the automation of sample measurement. When FCS is coupled with an automatic sampling system, we believe that this method will become a promising approach in telomerase-related studies and clinical diagnosis in the future. 1012

DOI: 10.1021/acs.analchem.7b04256 Anal. Chem. 2018, 90, 1006−1013

Article

Analytical Chemistry

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.analchem.7b04256. Experimental details and the supplemental figures (Figures S1−S7): cell culture and telomerase extract preparation; telomerase extension reaction; gel electrophoresis operation; the home-built FCS system; principle of human telomerase extension reaction; principle of TRAP method for PCR amplification of telomerase extension product; optimization of FCS measurement; optimization of conventional TRAP assay; inhibition curves (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*Phone: +86-21-54746001. Fax: +86-21-54741297. E-mail: [email protected]. *E-mail: [email protected]. ORCID

Chaoqing Dong: 0000-0002-6457-1975 Jicun Ren: 0000-0002-8157-5548 Author Contributions

All authors have given approval to the final version of the manuscript. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was financially supported by the NSFC (Grant Nos. 21327004, 21675111, and 21475087). We thank Prof. Yan Jin (Shaanxi Normal University, Xi’an, China) for help with the application of the conventional TRAP procedure.

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DOI: 10.1021/acs.analchem.7b04256 Anal. Chem. 2018, 90, 1006−1013