Ultrasensitive Detection of Telomerase Activity at the Single-Cell Level

Nov 10, 2013 - Kim , N. W.; Piatyszek , M. A.; Prowse , K. R.; Harley , C. B.; West , M. D.; Ho , P. L.; Coviello , G. M.; Wright , W. E.; Weinrich , ...
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Ultrasensitive Detection of Telomerase Activity at the Single-Cell Level Li-juan Wang,† Yan Zhang,† and Chun-yang Zhang* Single-Molecule Detection and Imaging Laboratory, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, Guangdong 518055, China

ABSTRACT: Human telomerase is a ribonucleoprotein complex which adds the repeats of TTAGGG on the telomeric ends of chromosomal DNA. Even though the vast majority of human cancers express telomerase activity, most human somatic cells lack the telomerase activity; consequently, telomerase has been regarded as both a biomarker for early cancer diagnosis and a therapeutic target. Here we develop a simple, rapid and highly sensitive method for the detection of telomerase activity at the single-cell level using telomeres-induced two-stage isothermal amplification-mediated chemiluminescence assay. In the presence of telomerase, the telomere repeats of (TTAGGG)n are added to the 3′ end of telomerase substrate primer, which can be converted to the template of strand displacement amplification (SDA) for the generation of short oligonucleotides, catalytic DNAzymes, and the telomere repeats of (TTAGGG)n. The short oligonucleotide might function as a new trigger to bind the free telomerase substrate primer and consequently initiate an isothermally exponential amplification reaction (EXPAR), generating a large number of catalytic DNAzymes. Both the DNAzymes and the G-rich telomeric repeat units can interact with hemin to form the catalytic hemin-G-quadruplex nanostructures, which can catalyze the generation of amplified chemiluminescence signals in the presence of luminol and H2O2. While in the absence of telomerase, the two-stage isothermal amplification cannot be initiated, and no chemiluminescence signal is observed. Owing to the high amplification efficiency of two-stage isothermal amplification as well as the high sensitivity and wide dynamic range of the chemiluminescence assay, the proposed method can sensitively measure the synthetic telomerase product TPC8 with a detection limit of as low as 0.1 aM and a large dynamic range of 10 orders of magnitude from 0.1 aM to 1 nM and can even detect the telomerase activity from a single HeLa cancer cell without the need for any labeled DNA probes. The proposed method can be further used to screen the anticancer drugs and might provide a promising approach for the discovery of new anticancer drugs.

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regarded as both a cancer marker for early cancer diagnosis and a therapeutic target.1,7,9,10 Over the past decades, a variety of methods have been developed to detect telomerase activity.11 Polymerase chain reaction (PCR)-based telomere repeat amplification protocol (TRAP) is one of the most general approaches for the detection of telomerase activity.6−8,12 Even though it exhibits excellent sensitivity and high throughputs, TRAP is limited because it is time-consuming and requires precise control of temperature cycling. Alternatively, several PCR-free methods have been developed for the detection of telomerase activity,

uman telomerase is a ribonucleoprotein complex which adds the repeats of (TTAGGG)n on the telomeric ends of chromosomal DNA by using the telomerase RNA component (hTR) of the complex as a template.1,2 Telomeres are tandem noncoding repeats of sequence (TTAGGG)n, which protect the chromosome ends from undesired degradation, recombination, and end-to-end fusion.3 In proliferating cells with the lack of telomerase activity, telomeres progressively shorten with every cell division, resulting in a critical telomere length and ultimately the triggering of cell cycle arrest or cell death.4,5 Even though telomerase activity is highly depressed in most human somatic cells, over 85% of human cancers express either up-regulation or reactivation of telomerase activity.6−8 Due to its strong association with cell immortalization and tumorigenesis, telomerase has been © 2013 American Chemical Society

Received: August 29, 2013 Accepted: November 10, 2013 Published: November 10, 2013 11509

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Table 1. Sequences of the Oligonucleotidesα note

sequence (5′−3′)

reverse primer telomerase substrate primer synthetic telomerase product (TPC8)

CCC TTA CCC TTA CCC TTA CCC TAA AAA CCT ACA ATC CGT GGA AGA GCC GAG CAG AGT TGC TCT TCC GGG TAG GGC GGG TTG GGG CTC TTC CAA ACC TAC AAT CCG TCG AGC AGA GTT AAA CCT ACA ATC CGT GGA AGA GCC GAG CAG AGT TGC TCT TCCGGG TAG GGC GGG TTG GGG CTC TTC C AA ACC TAC AAT CCG TCG AGC AGA GTT AGG GTT AGG GTT AGG GTT AGG GTT AGG GTT AGG GTT AGG GTT AGG G

α

The underlined letters symbolize the recognition sequences of nicking endonuclease Nt. BspQI. The italic bold letters in the TPC8 indicate the telomeric repeats.

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including optical fiber sensors,13 surface Plasmon resonance,14 fluorescence,15 electrochemistry,16,17 electrochemiluminescence,18 magnetomechanical,19 and nanotechnology-based methods.20 Although they have made some improvements to some extent, these methods suffer from poor sensitivity,17,18 complicated manipulation,20 time-consuming protocol,15 and the requirement for the immobilization of the telomerase substrate primer on solid supports.13,16−18,20 The G-quadruplex DNAzyme is a type of functional nucleic acid, which can fold into a G-quadruplex structure with the cofactor hemin and exhibits catalysis similar to horseradish peroxidase (HRP).21 DNAzyme as a catalytic label for biosensing is attractive due to its advantages of high sensitivity, simple protocols, the elimination of nonspecific adsorption caused by protein labels, and the stability and reversibility during thermal denaturation.21 Recently, the hemin/Gquadruplex horseradish peroxidase (HRP)-mimicking DNAzyme has been used as an amplifying label for the detection of telomerase activity,22,23 cations,24−26 proteins,27,28 nucleotides,29−32 and cancer cells33 through either catalyzing the H2O2mediated oxidation of ABTS2− to the colored product ABTS−22 or catalyzing the oxidation of luminol by H2O2 to yield chemiluminescence.33 Notably, the G-rich telomeric repeat units can fold up into four-stranded G-quadruplex structures34 and incorporate hemin in catalytically active structures, functioning as biocatalytic amplifying labels for the detection of telomerase activity.3 However, so far, a chemiluminescence assay with the capability of ultrasensitively detecting telomerase activity has not been reported, to our knowledge, due to the lack of an efficient amplification method for telomere repeats. Here we develop a simple, rapid, and highly sensitive method for the detection of telomerase activity at the single-cell level using telomeres-induced two-stage isothermal amplificationmediated chemiluminescence assay. Isothermal amplification techniques, such as strand displacement amplification (SDA) 3 5 , 3 6 and exponential amplification reaction (EXPAR),37−39 are frequently used for DNA and RNA detection. The isothermal amplification reaction proceeds at a constant temperature and has distinct advantages of high amplification efficiency and rapid amplification kinetics with 106∼109-fold amplification of short oligonucleotides in minutes.38 Taking advantage of the high amplification efficiency of the two-stage isothermal amplification reaction (SDA and EXPAR) and the intrinsically high sensitivity of DNAzymedriven chemiluminescence, the proposed method exhibits a detection limit of as low as 0.1 aM for the detection of the synthesized telomerase product TPC8 and can even detect the telomerase activity at the single-cell level without the need for any thermal cycling, multiple separation, and washing steps. Moreover, the proposed method can be used to screen the anticancer drugs.

EXPERIMENTAL SECTION Materials. All oligonucleotides (Table 1) were synthesized by TaKaRa Bio. Inc. (Dalian, China). GRN163 (a N3′→P5′ thio-phosphoramidate olignucleotide) was obtained from Integrated PNA Technologies. The Bst 2.0 WarmStart DNA polymerase (8000 units mL−1), nicking endonuclease Nt. BspQI (10000 units mL−1), and deoxynucleotide triphosphates (dNTPs, 200 μM) were purchased from New England Biolabs (Beverly, MA). SYBR Green I (10000×) was obtained from Xiamen Bio-Vision Biotechnology (Xiamen, China). Ribonuclease, transfection reagent FuGENE6, magnesium chloride (MgCl2), TWEEN 20, potassium chloride (KCl), ethylene glycol tetraacetic acid (EGTA), ethylenediaminetetraacetic acid (EDTA), trizma hydrochloride (pH 8.3, pH 7.9, and pH 9.0, respectively), hemin, luminol, peroxidase (H2O2), 4-(2hydroxyethyl) piperazine-1-ethanesulfonic acid sodium salt (HEPES), and sodium chloride (NaCl) were obtained from Sigma-Aldrich Company (St. Louis, MO). TRAPeze 1× CHAPS lysis buffer was purchased from Millipore (Bedford, MA). The luminol solution and hemin solution were prepared by using 0.1 mM NaOH and dimethysulfoxide (DMSO) as the solvents, respectively, and stored at −20 °C in the dark. Other analytical grade chemicals were used without further purification. Ultrapure water obtained from a Millipore filtration system was used in all experiments. Preparation of Telomerase Extracts. HeLa cells and MDA-MB-231 cells were cultured with 10% fetal bovine serum (FBS) in Dulbecco’s modified Eagle’s medium (DMEM). MRC-5 cells was cultured with 15% fetal bovine serum and 1% −1 L-glutamine in DMEM supplemented with 50 units mL penicillin and 50 mg mL−1 streptomycin in a humidified chamber, containing 5% CO2 at 37 °C. Cells were collected with trypsinization in the exponential phase of growth, washed twice with ice-cold PBS (pH 7.4), and pelleted at 2000 rpm at 4 °C for 10 min. About 1 × 106 million cells were resuspended in 200 μL of ice-cold 1× CHAPS lysis buffer, incubated on ice for 30 min, and then centrifuged at 12000g at 4 °C for 20 min. After centrifugation, the supernatant was carefully transferred into a fresh tube and stored at −80 °C. For the control experiments, the telomerase extracts were pretreated by incubating 20 μL of active cell extracts (one thousand cells equivalence) at 85 °C for 10 min prior to the detection. Telomeres-Induced Two-Stage Isothermal Amplification. The two-stage reaction system (20 μL) contains 1 μL of cell extracts/2 μL of TPC8, 100 nM telomerase substrate primer, 50 nM reverse primer, 30 mM Tris-HCl (pH 8.3), 3 mM MgCl2, 70 mM KCl, 1 mM EGTA, 0.05% (v/v) Tween 20, dNTPs (200 mM each), 1 unit Bst 2.0 WarmStart DNA polymerase, and 5 units Nt.BspQI NEase. After incubation at 37 °C for 10 min for the telomerase extension, the telomeresinduced two-stage isothermal amplification reaction was performed at 55 °C for 20 min. 11510

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Scheme 1. Schematic Illustration for the Detection of Telomerase Activity Using Telomeres-Induced Two-Stage Isothermal Amplification-Mediated Chemiluminescence Assay

Real-Time Fluorescence Detection and Chemiluminescence Detection. The real-time fluorescence measurements of telomeres-induced two-stage isothermal amplification reaction were performed in a Roche Light Cycler Nano (Switzerland) with 1× SYBR Green I as the fluorescent indicator, and the fluorescence intensity was monitored at a time interval of 30 s. The luminol solution (0.25 mM) and hemin solution (75 nM) were prepared by adding luminol and hemin to the mixture of isothermal amplification reaction products (15 μL), ddH2O (50 μL), and 30 μL of reaction buffer (40 mM HEPES, pH 8.0, 300 mM NaCl, 20 mM KCl). Then the mixture was incubated at room temperature for 30 min to make the nucleotides fold into active quadruplex structures of DNAzymes. With the addition of 25 μL of H2O2 (10 mM) to the mixture, the chemiluminescence signals were generated and recorded on a GloMax 96 Microplate Luminometer (Promega, Madison, WI) with a time interval of 1.5 s. Gel Electrophoresis. A 12% nondenaturating polyacrylamide gel electrophoresis (PAGE) was used to analyze the products of two-stage isothermal amplification reaction in 1× TBE buffer (9 mM Tris-HCl, pH 7.9, 9 mM boric acid, 0.2 mM EDTA) at a 110 V constant voltage for 50 min at room temperature, and SYBR Green I was used as the fluorescent indicator. The stained gel was scanned by a Kodak Image Station 4000 MM (Rochester, NY). Inhibition Assay. The cells were transfected by GRN163 using the lipid carrier of FuGENE6. After a 24 h transfection, the cells were collected and the telomerase activity of 100 cell equivalents was measured. The chemiluminescence signals in response to different-concentration GRN163 were obtained by

a GloMax 96 Microplate Luminometer (Promega, Madison, WI), and the relative activity (RA) of telomerase was quantitatively calculated according to the following equation:40 RA =

Ii − Io × 100% It − Io

Where Io, It, and Ii are the chemiluminescence intensity in the absence of telomerase, in the presence of telomerase, and in the presence of both telomerase and GRN163, respectively. The IC50 value of GRN163 in the presence of the lipid carrier (FuGENE6) was obtained from the curve-fitting equation.

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RESULTS AND DISCUSSION

Principle of Telomeres-Induced Two-Stage Isothermal Amplification-Mediated Chemiluminescence Assay. The principle of telomeres-induced two-stage isothermal amplification-mediated chemiluminescence assay is illustrated in Scheme 1. The designed telomerase substrate primer consists of seven regions (AXBX′CX′D). The sequence of A plus B is similar to the sequence of D, which telomerase can recognize as a substrate and catalyze the addition of telomeric repeats (TTAGGG)n onto the 3′-end of sequence D to generate the telomerase product. Region X is complementary to Region X′, both of which are the “heart” of the template, wherein recognition sites for Nt.BspQI are generated upon the formation of a double-stranded DNA (dsDNA). Region C is the DNAzyme sequence, which catalyzes the generation of an amplified chemiluminescence signal for the detection of telomerase activity. The telomeres-induced two-stage isother11511

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Figure 1. (A) Variance of the chemiluminescence (CL) intensity with the amount of Bst 2.0 WarmStart DNA polymerase at a fixed amount of Nt.BspQI nickase (5 units) in the presence of 1 × 10−11 M TPC8 (gray column) and the telomerase extracts equivalent to 100 HeLa cells (black column). (B) Variance of the chemiluminescence intensity with the amount of Nt.BspQI nickase at a fixed amount of Bst 2.0 WarmStart DNA polymerase (1 unit) in the presence of 1 × 10−11 M TPC8 (gray column) and telomerase extracts equivalent to 100 HeLa cells (black column). (C) Variance of the chemiluminescence intensity with the concentration of the reverse primer in the presence of 1 × 10−8 M TPC8. (D) Variance of the chemiluminescence intensity with the incubation time in the presence of telomerase extracts equivalent to 100 HeLa cells (black) and the negative control (red). (E) Variance of the chemiluminescence value ratio of I/I0 with the concentration of hemin. I and I0 are the chemiluminescence signals with and without 1 × 10−11 M TPC8, respectively. Error bars show the standard deviations of three experiments.

mal amplification-mediated chemiluminescence assay involves three steps: (1) telomerization reaction, (2) telomeres-induced two-stage isothermal amplification reaction, and (3) chemiluminescence assay. In the first step, the repeat units of (TTAGGG)n are incessantly added to the 3′ end of the telomerase substrate primer in the presence of telomerase and dNTPs to generate the telomerase product, whose amount is proportional to the telomerase activity. In the second step, the extended telomerase substrate primer functions as the template of isothermal amplification, which hybridizes with the reverse primer and initiates the polymerization in the presence of Bst 2.0 WarmStart DNA polymerase and dNTPs, generating a stable double-stranded DNA duplex with three recognition sites for the nicking endonuclease Nt.BspQI. The cleavage of recognition site of X can produce a short oligonucleotide of A′, which is complementary to A, through a polymerization and displacement reaction cycle. The cleavage of recognition site of X′ results in the initiation of a secondary polymerization cycle and the release of DNAzyme (C) and G-rich telomeric repeat units (R). Each strand displacement amplification (SDA) reaction cycle produces a DNAzyme (C), G-rich telomeric repeat units (R), and a short oligonucleotide (A′). The linear SDA reaction can be turned into an exponential amplification with the short oligonucleotide (A′) as the trigger of EXPAR. The new DNA trigger (A′) can bind to the free telomerase substrate primer and initiate the second switch in the

amplification circuit, generating a large number of DNAzymes (C) and new DNA triggers (A′) through polymerization, nicking, and displacement. As a result, the new DNA trigger (A′) can bind to another template and initiate a new cycle of polymerization, nicking, and displacement, eventually leading to an exponential amplification and the generation of a large number of DNAzymes (C) and DNA triggers (A′). In the third step, the amplified reporter oligonucleotides of DNAzymes and G-rich telomeric repeat units bind with hemin to form the hemin-G-quadruplex nanostructures, generating an amplified chemiluminescence signal in the presence of luminol/H2O2.23 The two-stage isothermal amplification has significant advantages of isothermal condition, label-free, low-cost, rapid, and simple, without multiple separation and washing steps. In addition, the chemiluminescence assay has the advantages of high sensitivity, simple protocol, and the elimination of nonspecific adsorption. Taking advantage of the high amplification efficiency of two-stage isothermal amplification reaction and the intrinsically high sensitivity of DNAzymedriven chemiluminescence, the proposed method can be used to rapidly, simply, and sensitively monitor the telomerase activity. Optimization of Experimental Condition. The detection sensitivity of the proposed method relies on the amount of the hemin molecule bound to the products of two-stage isothermal amplification reaction, which is influenced by the amount of 11512

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Figure 2. Two-stage isothermal amplification reaction monitored through real-time fluorescence. (A) Amplification curves in response to different concentrations of TPC8:1 × 10−9 M (curve 1), 1 × 10−11 M (curve 2), 1 × 10−13 M (curve 3), 1 × 10−15 M (curve 4), 1 × 10−17 M (curve 5), 1 × 10−18 M (curve 6), and in the absence of TPC8 (curve 7). (B) Linear relationship between the POI values and the logarithm of TPC8 concentration. (C) Amplification curves obtained from 1 × 10−11 M TPC8 (curve 1), various telomerase extracts equivalent to 1000 cells (curve 2), 200 cells (curve 3), 100 cells (curve 4), 10 cells (curve 5), and 1 cell (curve 6), heat-inactivated telomerase extracts equivalent to 1000 cells (curve 7), and the control without cell extracts (curve 8). (D) The relationship between the POI values and the logarithm of cell number. Error bars show the standard deviations of three experiments.

basis of the above results, the optimal concentration is 1 unit for Bst 2.0 WarmStart DNA polymerase and 5 units for Nt.BspQI nickase, respectively. The concentration of reverse primer has a crucial effect on both the amplification efficiency and chemiluminescence signal. On one hand, low concentration of the reverse primer might adversely affect the amplification efficiency because the amplification efficiency of strand displacement amplification (SDA) is highly dependent on the ability of the reverse primer to hybridize with the extended telomerase substrate primer for driving the construction of a newly formed exponential amplification template. On the other hand, high concentration of the reverse primer might adversely influence the chemiluminescence signal because the excess reverse primers can hybridize with the amplification reaction products of G-rich telomeric repeat units, preventing the formation of hemin-Gquadruplex nanostructures which are prerequisites for the chemiluminescence signal. Therefore, the concentration of the reverse primer should be optimized carefully. As shown in Figure 1C, the chemiluminescence intensity increases with the increase of reverse primer concentration from 0 to 50 nM, followed by the decrease beyond the reverse primer concentration of 50 nM. Thus, the 50 nM reverse primer is used in the subsequent research. To investigate the influence of incubation time for telomerase extension upon the chemiluminescence signal, a

polymerase, nicking endonuclease, the concentrations of reverse primer and hemin, and the telomerase incubation time for telomerase extension. To optimize the experimental condition of two-stage isothermal amplification reactionmediated chemiluminescence assay, synthetic telomerase product TPC8, which corresponds to the telomerase substrate primer extended with eight telomeric repeats of (TTAGGG)n, was used as a positive control to estimate the telomerase activity from given cell extracts. The cooperation of two enzymes, Bst 2.0 WarmStart DNA polymerase and Nt.BspQI nickase, is crucial to the amplification efficiency.41,42 In the absence of either templates or priming DNA strands, high background amplification by thermophilic polymerases can be accelerated in the presence of nicking endonucleases.41 The precise mechanism for enzymatic interaction, cooperation, and competition with one another remains unclear, while the reaction might be simply modulated by changing the ratio of polymerase and nicking endonuclease.42 Figure 1A indicates that no significant change in the chemiluminescence signal is observed with the variance of the concentration of Bst 2.0 WarmStart DNA polymerase. Thus, we investigated the influence of Nt.BspQI nickase on the chemiluminescence signal at a fixed amount of Bst 2.0 WarmStart DNA polymerase (1 unit). As shown in Figure 1B, the chemiluminescence signal increases with the increasing amount of Nt.BspQI nickase from 2 to 5 units and reaches the highest value at 5 units. On the 11513

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different incubation time was evaluated. As shown in Figure 1D, the chemiluminescence signal increases rapidly with the incubation time from 0 to 10 min followed by the decrease beyond the incubation time of 10 min. Thus, 10 min incubation time is used for telomerase extension in the subsequent research. The influence of hemin concentration on the chemiluminescence signal was further investigated. The chemiluminescence signal increases with the increasing hemin concentration, no matter if it is in the presence (I) or in the absence (I0) of the synthetic telomerase product TPC8, due to the dependence of catalytic kinetics of luminol−H2O2 reaction upon hemin concentration.43 To obtain the optimal concentration of hemin, the variance of the chemiluminescence ratio value of I/I0 in response to the hemin concentration was evaluated (Figure 1E). The maximum value of I/I0 is obtained at the hemin concentration of 75 nM. Thus, 75 nM hemin is used in the subsequent research. Real-Time Fluorescence Monitoring of TelomeresInduced Two-Stage Isothermal Amplification. To evaluate the two-stage isothermal amplification reaction, the fluorescence in response to different concentrations of synthetic telomerase product TPC8 and telomerase extracts from a different number of HeLa cells was monitored in real time by using SYBR Green I as the fluorescent indicator. As shown in Figure 2 (panels A and B), TPC8 is detected quantitatively in the range from 10−9 M to 10−18 M by the real-time fluorescence measurement of the amplification products. The real-time fluorescence intensity increases in a sigmoidal fashion as TPC8 is converted from a single-stranded to a partially doublestranded DNA duplex (Figure 2A). The point of inflection (POI), which is defined as the time corresponding to the maximum slope in the sigmoidal curve,37 is used for the quantitative detection of the telomerase product. As shown in Figure 2B, a linear correlation is obtained between the POI values and the logarithm (log) of TPC8 concentration in the range from 10−9 to 10−18 M. The correlation equation is POI = −4.66 − 0.90 log10 C with a correlation coefficient of 0.9930, where C is the concentration of TPC8. The two-stage isothermal amplification reaction is further tested on a series of diluted HeLa cell extracts obtained from telomerase-positive human cervical carcinoma (Figure 2C). As shown in Figure 2D, the POI value decreases monotonically with the increase of the cell number, and a linear correlation is obtained between the POI values and the logarithm (log) of the cell number. The correlation equation is POI = 21.58 − 1.52 log10 N with a correlation coefficient of 0.9968, where N is the cell number. These results clearly indicated the process of two-stage amplification reaction. The amplification specificity of the proposed method was further verified by nondenaturating PAGE (Figure 3). The characteristic bands of DNAzyme (25 nt) and G-rich telomeric repeat units (32∼68 nt with an increment of 6 nt) are observed in the presence of telomerase extracts equivalent to 100 cells (Figure 3, lane 6). In contrast, neither the characteristic band of DNAzyme nor those of G-rich telomeric repeat units are observed in the negative control groups with either the presence of heat-inactivated telomerase extracts equivalent to 1000 cells (Figure 3, lane 4) or the absence of cell extracts (Figure 3, lane 1). These results demonstrated the high amplification specificity of the proposed method. Sensitivity of the Proposed Method. To demonstrate the improved sensitivity of the proposed method, we measured the chemiluminescence signals in response to either the

Figure 3. Nondenaturating PAGE analysis of telomeres-induced twostage isothermal amplification in the presence of a negative control with the lysis buffer (lane 1), DNA marker (lane 2), only reverse primer (lane 3), negative control with the presence of heat-inactivated telomerase extracts equivalent to 1000 cells (lane 4), positive control with only DNAzyme (25 nt) (lane 5), and the presence of telomerase extracts equivalent to 100 cells (lane 6), respectively. Note: the observed band in lanes 1 and 4 is attributed to the presence of the telomerase substrate primer.

synthetic telomerase product TPC8 or a series of diluted HeLa cells from telomerase-positive human cervical carcinoma. As shown in Figure 4A, the chemiluminescence intensity improves with the increase of TPC8 concentration due to the generation of a large number of DNAzymes and G-rich telomeric repeat units induced by TPC8. Notably, the chemiluminescence intensity exhibits a linear correlation with the logarithm of the TPC8 concentration over a large dynamic range of 10 orders of magnitude from 0.1 aM to 1 nM (Figure 4A). The correlation equation is Y = 9.59 E6 + 476478.71 log10 C with a correlation coefficient of 0.9909, where Y is the chemiluminescence intensity and C is TPC8 concentration. Notably, the dynamic range of the current assay is about 1 order of magnitude larger than the doubly labeled molecular beacon-based real-time EXPIATR assay.44 Figure 4B shows that the chemiluminescence intensity increases monotonically with the increasing number of HeLa cells, indicating that the amount of DNAzymes and G-rich telomeric repeat units are proportional to the number of cancer cells due to a high level of telomerase activity in high-density cells and, consequently, a fast extension speed at the same given time.45 Interestingly, the chemiluminescence intensity has a linear relationship with the logarithm of HeLa cell number in the range from 1 to 1000 cells. The correlation equation is Y = 1.74 E6 + 966534.79 log10 N with a correlation coefficient of 0.9676, where Y is the chemiluminescence intensity and N is the number of cells. Notably, the telomerase activity from even a single HeLa cell can be sensitively detected. The sensitivity of the proposed method is comparable with the EXPIATR assay44 and is superior to most of the previously reported methods, including the amplification-based telomere repeat amplification protocol (TRAP) (10 cells)12 and the PCR-free methods such as the gold nanoparticle-based surface plasmon resonance assay (18 cells),46 the ion-sensitive field effect-based transistor device (65 11514

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Figure 5. Chemiluminescence signal in response to telomerase extracts equivalent to (A) 100 HeLa, (B) MDA-MB-231, and (C) MRC-5 cells and (D) the control group with only the lysis buffer. Error bars show the standard deviations of three experiments.

Telomerase has been regarded as an important target for the development of new anticancer drugs on the basis of reversing the tumor growth by telomerase inhibition. A large number of telomerase inhibitors targeting different sites of the telomerase complex and telomeres themselves have been discovered.48 Among them, GRN163 is a N3′ → P5′ thio-phosphoramidate olignucleotide and has been proved to be effective in telomerase inhibition, telomere shortening, and inhibition of cancer cell growth.49 The inhibition effect of GRN163 upon both HeLa cells and MDA-MB-231 cells was investigated in the presence of the transfection reagent of FuGENE6.49 As shown in Figure 6, the inhibition of telomerase activity by GRN163 is Figure 4. Chemiluminescence detection of (A) synthetic telomerase product TPC8 and (B) telomerase activity of human HeLa cells. (A) Variance of chemiluminescence signal with TPC8 concentration. The TPC8 concentration is 1 × 10−9, 1 × 10−11, 1 × 10−13, 1 × 10−15, 1 × 10−17, and 1 × 10−19 M, respectively. (B) Variance of chemiluminescence signal with the cell number. The cell number is 1000, 100, 10, 1, and 0 cells, respectively. Error bars show the standard deviations of three experiments.

cells),46 and the quantum dot-based optical method (270 cells).46 Such significant improvement in the sensitivity might be attributed to the following three factors: (1) the two-stage isothermal amplification reaction leads to a high amplification efficiency (106∼109-fold amplification),38 (2) both DNAzymes and G-rich telomeric repeat units produced from two-stage isothermal amplification reaction can bind with hemin to form hemin-G-quadruplex nanostructures, generating an amplified chemiluminescence signal,23,24 and (3) the high sensitivity and wide dynamic range of the chemiluminescence method.47 To evaluate the potential of the proposed method for clinical diagnosis, we further measured the telomerase activity in another cancer cell line MDA-MB-231 and a normal cell line MRC-5. As shown in Figure 5, the MRC-5 cells cannot generate any chemiluminescence signal, similar to the control group with only the lysis buffer, due to the lack of telomerase activity in normal cells.4,5 In contrast, a distinct chemiluminescence signal is observed in both HeLa cells and MDA-MB231 cancer cells, consistent with the overexpressed telomerase in human tumors.6−8 These results demonstrate the feasibility of the proposed method for the discrimination of cancer cells from normal cells in clinical diagnosis. Telomerase Inhibition Assay. Telomerase is overexpressed in over 85% of all known human tumors, while telomerase activity is absent in human somatic cells.6−8

Figure 6. Variance of the relative activity of telomerase with GRN163 concentration in HeLa cells (black) and MDA-MB-231 cells (red). Error bars show the standard deviation of three experiments.

dose-dependent in both HeLa cells and MDA-MB-231 cells. The relative activity of telomerase decreases with the increase of GRN163 concentration. The IC50 value is the inhibitor concentration required to reduce the enzyme activity by 50%. On the basis of the plot of relative activity of telomerase versus GRN163 concentration, the IC50 of GRN163 is calculated to be 0.44 ± 0.05 μM for HeLa cells and 0.34 ± 0.05 μM for MDAMB-231 cells, respectively. Therefore, the proposed method might provide a new approach for the screening of telomerase inhibitors.

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CONCLUSION In summary, we have developed a simple, rapid, and ultrasensitive method for the detection of telomerase activity using the telomeres-induced two-stage isothermal amplifica11515

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tion-mediated chemiluminescence assay. In comparison with the telomere repeat amplification protocol (TRAP),12 the proposed method is more simple, highly sensitive, and rapid with an isothermal condition but without the need for any thermal cycling, washing, and separation steps. In comparison with the EXPIATR assay,44 the proposed method has a significant advantage of low cost without the requirement for expensive fluorescent-labeled nucleotides. Owing to the high amplification efficiency of two-stage isothermal amplification as well as high sensitivity and wide dynamic range of chemiluminescence assay, the proposed method can sensitively measure the synthetic telomerase product TPC8 with a detection limit as low as 0.1 aM and a large dynamic range of 10 orders of magnitude from 0.1 aM to 1 nM and can even detect the telomerase activity from a single HeLa cancer cell. More importantly, the proposed method can be used for the screening of telomerase inhibitors and might provide a promising approach for the discovery of new anticancer drugs.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel: +86 755 86392211. Fax: +86 755 86392299. Author Contributions †

L.-j.W. and Y.Z. contributed equally.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the National Basic Research Program 973 (Grants 2011CB933600 and 2010CB732600), the National Natural Science Foundation of China (Grants 21325523 and 21075129), the Award for the Hundred Talent Program of the Chinese Academy of Science, the Guangdong Innovation Research Team Fund for Low-cost Healthcare Technologies, the Natural Science Foundation of Shenzhen City (Grant JCYJ20130401170306879), the Funds for both Guangdong Key Laboratory of Nanomedicine and Shenzhen Engineering Laboratory of Single-molecule Detection and Instrument Development [Grant (2012) 433].

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