Label-Free Telomerase Detection in Single Cell using a Five-Base

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Label-Free Telomerase Detection in Single Cell using a Five-Base Telomerase Product-Triggered Exponential Rolling Circle Amplification Strategy Xiao-Yu Li, Yunxi Cui, Yi-Chen Du, An-Na Tang, and De-Ming Kong ACS Sens., Just Accepted Manuscript • DOI: 10.1021/acssensors.9b00334 • Publication Date (Web): 04 Apr 2019 Downloaded from http://pubs.acs.org on April 5, 2019

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ACS Sensors

Label-Free Telomerase Detection in Single Cell using a Five-Base Telomerase Product-Triggered Exponential Rolling Circle Amplification Strategy XiaoYu Li†, YunXi Cui†, YiChen Du†, AnNa Tang†, DeMing Kong†,‡* State Key Laboratory of Medicinal Chemical Biology, Tianjin Key Laboratory of Biosensing and Molecular Recognition, Research Center for Analytical Sciences, College of Chemistry, Nankai University, Tianjin, 300071, P. R. China Fax: (+)8622-2350245, E-mail: [email protected] ‡Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), Tianjin, 300071, P. R. China. †

ABSTRACT: Telomerase is a universal biomarker of malignant tumors. Sensitive and reliable analysis for telomerase activity is of vital importance for both early diagnosis and therapy of malignant tumors. Herein, a novel fluorescent strategy was proposed for sensitive and label-free detection of telomerase activity. One highlight of this strategy is that an exponential signal amplification can be triggered by a very short telomerase extension product (TEP). Without adding dATP, the designed telomerase primer can be easily controlled to extend five bases (GGGTT) to give short TEP with definite length. The resulted short TEP can then be constructed as a circular rolling circle amplification (RCA) template and thus initiate a nicking enzyme-mediated exponential RCA, producing G-rich amplification products that can be sensitively probed via specific binding between the fluorescent dye Thioflavin T (ThT) and the nucleic acid G-quadruplexes. Elevated telomerase translocation efficiency, combining with exponential signal amplification and specific probing of RCA products by ThT, endow the sensing platform with extraordinary high detection sensitivity. The requirement for short TEP increases the possibility to analyze telomerase with low activity. The proposed sensing platform can achieve sensitive telomerase activity detection in individual cell, even with the interference of accumulated normal cells. It was also demonstrated to show excellent capability in screening for the inhibitors of telomerase. Therefore, the proposed sensing platform has great potential for not only clinical diagnosis but also anticancer drug development. KEYWORDS: Telomerase; Rolling circle amplification; Label-free; Telomerase inhibitor; G-quadruplex; Single-cell

Human telomerase is a ribonucleoprotein in the essence, it contains two major components, one is human telomerase reverse transcriptase (hTERT) and the other part is human telomerase RNA (hTR).1-3 Telomerase binds to the telomere region at the end of each chromosome through the hTR template and then catalyzes the extension of telomere with a repeat sequence (GGGTTA)n from the 3′-end of telomere, preventing chromosomes from senescence and apoptosis and thus leading to cell immortalization.4,5 It is well known that telomerase activity is up-regulated in more than 85-90% of primary tumors,6,7 but reduced in most healthy somatic cells.810 This character makes telomerase a universal diagnostic biomarker for early diagnosis, prognosis evaluation and a promising therapeutic target of cancer therapy. Therefore, sensitive and reliable quantitation of telomerase activity is of critical importance to bioanalytical research, clinical diagnosis and therapeutics. One of the most classical methods for telomerase activity detection is telomeric repeat amplification protocol (TRAP), which is based on polymerase chain reaction (PCR) technique.11 This assay is very sensitive, but complicated, time-consuming and subjects to PCR-derived issues (e.g. risk of false positive results,12 requirements for rapid thermal cycling and thermostable DNA polymerases). In recent, a variety of new strategies for telomerase activity detection with

good sensitivity have been developed based on various signaloutput mechanisms, such as fluorescence,13-19 colorimetry,20-23 electrochemistry,24,25 chemiluminescence,26 27,28 electrochemiluminescence and Raman scattering spectroscopy.29 These free-PCR methods are designed elegantly and have made some progress to some extent. In particular, fluorescent assay has been attracted more attention due to its stable signal output and easy-to-operate characteristic. For examples, Jiang et al. proposed a method for telomerase activity detection using designed smart DNA tweezers.17 Zhang et al. introduced a strategy based on tripleamplification process for sensitive telomerase activity analysis.15 The above methods were developed on the basis of costly and carefully designed dual-labelled nucleic acid probes, which are easily affected by reaction conditions (e.g. pH), thus inevitably increasing the experimental complexity, cost and risk of false-positive results. Therefore, development of a label-free method with comparable or even better detection sensitivity and specificity is in urgent demand. In addition, regular telomerase detection methods were mostly constructed on the basis of telomerase extension products (TEPs)-triggered signal amplifications. Nevertheless, it is well known that the TEPs are the mixture of extension products with varying lengths of repeated GGGTTA units, and TEPs with less repeated units usually show higher frequency than

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those with more repeated ones.17,30-32 However, in most of the reported telomerase-sensing methods, extremely very short TEPs cannot participate in signal amplification or only give very weak signal outputs, thus resulting that the telomerase with low activity cannot be well characterized. This is a major challenge of most amplification-based methods, which greatly hampers the improvement of sensitivity and thus limits their applications. Therefore, developing a new strategy to detect short TEPs is significant for accurate and dependable detection of low telomerase activity and thus for early diagnosis of malignant tumors. In this paper, a novel fluorescent sensing platform in label-free mode, named as five-base telomerase product-triggered exponential rolling circle amplification (RCA), has been introduced in ultrasensitive detection of telomerase activity. In the proposed strategy, only five bases of “GGGTT” are needed to be extended by telomerase. The resulted short TEP can then trigger a nicking enzyme-mediated exponential RCA to produce thousands of nucleic acid G-quadruplexes, which are specifically probed by a commercial fluorescent dye in a labelfree mode. The proposed method showed an ultrahigh sensitivity, thus the detection of telomerase activity at single cell level can be achieved. EXPERIMENTAL SECTION Materials and Reagents. All oligonucleotides used in this experiment (see Table S1) were purchased from Sangon Biotech. Co. Ltd. (Shanghai, China). Thioflavin T (3,6dimethyl-2-(4-dimethylaminophenyl)benzo-thiazolium cation, ThT) were obtained from Sigma-Aldrich (Shanghai, China). Nicking endonuclease Nb.BbvCI, T4 DNA ligase and Phi29 DNA polymerase were obtained from New England Biolabs (NEB, Beijing, China). Deoxyribonucleoside 5'-triphosphate mixture (dNTPs), deoxyguanosine triphosphate (dGTP), deoxythymidine triphosphate (dTTP), ethidium bromide (EB) and 20bp DNA ladder were obtained from Tiangen Biotech. Co. Ltd. (Beijing, China). The other reagents in this experiment are shown in part 1 of supporting information. Diethypyrocarbonate (DEPC)-treated water (DNase, RNase free) obtained from Beyotime Institute of Biotechnology (Shanghai, China) was used in all experiments. Cell Culture. HepG2 cells (human liver cancer cell line), HeLa cells (human cervical cancer cell line), 293T cells (embryonic kidney cell line) and MCF-7 cells (human lung cancer cell line) were all cultured in Dulbecco’s modified Eagle’s medium (DMEM, Gibco) with 1% penicillinstreptomycin (Gibco) and 10% fetal calf serum (Sijiqing). CCRF-CEM cells (human acute lymphoblastic leukemia cell line) and HL-7702 cells (human normal hepatic cell line) were cultured in RPMI-1640 (Gibco) with 1% penicillinstreptomycin and 10% fetal calf serum. All kinds of cells were cultured in a humidified atmosphere containing 5% CO2 at 37 °C. Preparation of telomerase extracts. Telomerase extracts were prepared according to previous protocol.33,34 Briefly, cells were collected with trypsinization in the exponential phase of growth and 1  106 cells were dispensed in a 1.5 mL RNase-free EP tube, washed twice with cooled PBS solution (pH 7.4), and pelleted at 800 rpm at 4 °C for 3 min. The cells were resuspended in 200 μL of ice-cold 1  CHAPS lysis buffer and mixed gently with a pipette. After being kept on ice for 30 min, the mixture was centrifuged at 14000 rpm at 4 °C

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for 20 min. Without disturbing the pellet, the supernatant was carefully transferred to a new RNase-free EP tube, immediately used for detection of telomerase or frozen at 80 °C. Heat-inactivated control was prepared by heating the cell extracts at 95 °C for 20 min prior to the detection. Detection of telomerase activity. 2 μL of 10 μM TS primer and 1 μL of 10 μM Padlock were mixed in 20 μL telomerase extension reaction buffer. The above mixture was heated at 95 oC for 5 min and 37 oC for 30 min. Then serially diluted cell extracts with respective numbers of cells were added into above mixture together with 0.5 μL of 100 mM dGTP, 0.5 μL of 100 mM dTTP and 4 U of RNase inhibitor. The above mixture was kept at 37 oC for 40 min for telomerase-catalyzed extension reaction. Second, 2 μL of 10  T4 DNA ligase buffer, 20 U of T4 DNA ligase were added to the above solution. The obtained mixture with 40 μL was incubated at 16 °C for 180 min. Third, 6 μL of 10  Phi29 DNA polymerase buffer, final concentration of 0.25 mM of dNTPs, 5 μM of ThT, 5 U of Nb.BbvCI and 5 U of Phi29 DNA polymerase were added in to a total volume of 100 μL. The obtained mixture was incubated at 30 °C for 180 min. After reaction was completed, fluorescence signal was detected using a Shimadzu RF-5301PC fluorescence spectrometer. Excited at 425 nm wavelength, the emission spectra were collected from 450 nm to 620 nm, and the fluorescence intensity at 485 nm was employed for quantitative analysis of telomerase activity. In real-time detection mode, 30 μL of mixture was prepared and cultured at 30 °C for 3 h. The reaction was held using a commercial StepOnePlus™ RealTime PCR instrument. Polyacrylamide gel electrophoresis (PAGE) analysis. 2 μL of loading buffer was firstly mixed with 10 μL of RCA reaction solution and then the solution was loaded onto a 10% polyacrylamide gel. Then PAGE analysis was carried out in 1  TBE buffer, under stable voltage of 120 V for 1 h. After that, the gel was stained by EB solution for 20 min, then it was imagined using a gel documentation system (Huifuxingye, Beijing, China). RESULTS AND DISCUSSION Principle of telomerase activity detection. The working mechanism of the proposed telomerase activity-sensing strategy is depicted in Scheme 1. In this strategy, only two oligonucleotides (telomerase substrate (TS) primer and padlock probe (Padlock), Table S1) were used. The TS primer can be recognized and extended by telomerase after adding of dNTPs. However, if only dGTP and dTTP are added, a tail with only five bases of “GGGTT” are jointed to the 3′-termini of TS primer because of the lacking of dATP. The obtained short TEP can then simultaneously bind to regions I and IV of Padlock, making the 3′- and 5′-ends of Padlock adjacent and producing cyclized Padlock catalyzed by T4 DNA ligase. Using short TEP and cyclized Padlock as primer and template, respectively, isothermal RCA reaction can be initiated. If a Crich sequence (region III) is introduced in Padlock, RCA products, which included thousands of G-rich fragments, are produced. However, such a linear amplification method still maintains relatively limited efficiency. To increase the amplification efficiency, two nicking enzyme recognition sites (region II) are introduced into the Padlock template. During the process of the RCA, the generated DNA can be recognized and cleaved into short fragments via nicking enzyme


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ACS Sensors Nb.BbvCI. A part of fragments can, in turn, be used to trigger a new round of cyclization reaction of linear Padlocks and subsequent RCA amplification, thus initiating the cyclizationpolymerization-nicking cycle. So the RCA reaction could be enlarged continuously and exponential amplification mode with significantly enhanced efficiency can be achieved.35 The amplified product fragments could fold into G-quadruplex, a special DNA secondary structure that can be specifically recognised by commercial fluorescent dye thioflavin T (ThT),36-38 achieving the highly sensitive, label-free detection of RCA amplification products. Different from other DNAprobing fluorescence dyes, like SYBR Green I, SYBR Gold or SYBR Green II, which response to both single- and doublestranded DNAs, ThT specifically recognizes G-quadruplexes but is insensitive to single- and double-stranded DNAs,36 thus can endow the sensing system with greatly minimized background and improved signal-to-noise ratio. Without the target telomerase, however, TS primer would not be extended and cyclized Padlock will not be formed. Certainly, RCA reaction will not be initiated so that there is no fluorescence enhancement can be observed. In this telomerase-sensing strategy, a very short TEP with only five-base extension can trigger highly efficient signal amplification reaction, achieving highly sensitive telomerase activity detection in a label-free mode. As a result, we named this method as five-base telomerase product-triggered exponential RCA strategy. Since only five-base extension is needed, this method is very suitable for the conditions in which telomerase activity is low. In addition, telomerase-catalyzed extension reaction is terminated after five bases. Then, telomerase has to dissociate from the TEP and search for other TS primers. That is, the requirement of short TEP might be helpful to increase the translocation efficiency of telomerase and thus benefit for the improvement of detection sensitivity.

telomerase (Figure 1a, Lines 2 and 3), which was extracted from HeLa cells, indicating the successful initiation of RCA reaction by telomerase. The sensing system with Nb.BbvCI (Line 2) gave about 2.2 times fluorescence signal than that without Nb.BbvCI mediation (Line 3), which is consistent with the principle which nicking enzyme-mediated exponential RCA has higher amplification efficiency than traditional one. In contrast, none of the negative controls lacking Padlock, Phi29 DNA polymerase, T4 DNA ligase or ThT exhibited observable fluorescence signals (Lines 4-7). Meanwhile, the heat-inactivated cell extracts through heating at 95 oC for 20 min could only give a weak fluorescence signal (Line 8) that was only a little higher than that given by blank lysis buffer (Line 1), thus verifying the positive fluorescence response was actually caused by the active of the telomerase. PAGE analysis was applied for further validation of the proposed mechanism. As shown in Figure 1b, when telomerase extract was absent, no RCA products could be observed in the PAGE gel (Lane 1). However, very long RCA products, whose band was stuck at the sample-loading site (Lane 2), were observed in the presence of active telomerase, revealing RCA amplification reaction had been successfully triggered by telomerase. Further addition of Nb.BbvCI resulted in a great decrease in band intensity (Lane 3), which was consistent with the expectation that products of the RCA has been cleaved into short oligos with different lengths. More important, when the 3′-end of TS primer was tailed with GGGTT sequence to mimic TEP, identical results were given no matter Nb.BbvCI was present or not (Lanes 4 and 5), thus giving a direct evidence for our proposed five-base telomerase product-triggered exponential RCA strategy.

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Figure 1. (a) Fluorescence emission spectra of different sensing systems. Lines 2, 3 and 8 contain cell extracts from 5000 HeLa cells, but in Line 8, the telomerase is heatinactivated. The insert shows the photographs of reaction systems containing heat-inactivated or active telomerase under the irradiation of 425 nm light. (b) Non-denaturing PAGE analysis of telomerase-triggered RCA reactions.

Scheme 1. Working mechanism of the proposed telomerase activity-sensing platform. To confirm the feasibility of our proposed telomerase activity-sensing strategy, fluorescence assay was first performed. As shown in Figure 1a, a significantly high enhanced fluorescence responses were observed in the presence of active

Optimization of the experimental condition. Above experiments have implied that our proposed strategy is ability to detect extracted telomerase from cancer cells. To achieve the best assay performance, we investigated several experimental parameters. First, the length of TEP was optimized. To make sure that only five bases of “GGGTT” could be extended from the 3′-end of TS primer, dATP wasn't added in the telomerase-catalyzed primer extension step. If dATP was also added, longer TEPs with different numbers of “GGGTTA” repeats might be obtained. Experimental results showed that the fluorescence signal given by long TEPs was much weaker than that given


ACS Sensors was observed in the range of 1 to 100 HeLa cells. Notably, obviously different fluorescence signals were given by one HeLa cell and heat-inactivated control, indicating that the proposed method performs well in telomerase activity detection at single-cell level. Such a high sensitivity was comparable with previously reported methods (Table S2),1315,20,24,28,44,45 and was the best in the reported label-free ones. Correspondingly, traditional RCA reaction without Nb.BbvCI addition could only detect telomerase activity in more than 100 HeLa cells (Figure S8), thus supporting the greatly increased signal amplification capacity of nicking enzymemediated exponential RCA. Compared to the slopes of the calibration curves obtained from sensing systems with or without Nb.BbvCI addition, it could be calculated that the mediation of Nb.BbvCI could improve the detection sensitivity about 19 times.

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by short TEP (Figure S1). The reasons are as follows: (1) If a TEP contains two or more than two GGGTTA repeats, the 3′end of Padlock has several binding sites on the TEP, and only one of them can be recognized by T4 DNA ligase to give cyclized Padlock (Scheme S1). That is, the production efficiency of circular RCA template will be greatly reduced. (2) Even if cyclized Padlock was produced with the help of long TEP, the TEP cannot be directly used as primer for the initiating of the RCA reaction since its 3′-end is noncomplementary with the circular template. Although we and other groups have demonstrated that the tails protruding from the 3′-end of RCA primers can be digested by the 3′→5′ exonuclease activity of Phi29 DNA polymerase.39-43 The presence of this digestion step will inevitably affect the RCA reaction efficiency. Therefore, only dGTP and dTTP were selected to be added in telomerase-catalyzed TS primer extension step. To simplify the telomerase-sensing procedure, telomerase was not inactivated after telomerase-catalyzed extension reaction step. Thus, the leftover telomerase activity might also work on unreacted TS primers during subsequent RCA reaction to give long TEPs. By comparing the sensing systems with or without telomerase inactivation step, one could find that comparable fluorescence signals were given by them (Figure S2), suggesting that the elimination of this step had no obvious effects on telomerase activity detection, and the sensing system could still mainly follow the working principle in Scheme 1. Next, the sequence of Padlock was optimized. A desired Padlock should meet such a requirement: it cannot be cyclized by unextended TS primer, but can be by TEP. Therefore, three Padlock strands (Padlock 1-3) were designed. 3′-ends of them have 0, 2, 3 bases that are complementary with TS primer, respectively. When five bases of “GGGTT” were added to TS primer, the number of complementary bases would increase to 5, 7 and 8, respectively (Figure S3). By comparing the fluorescence signals given by these three Padlock strands in the presence of active telomerase or not, we found that Padlock2 showed the best signal-to-noise ratio. Although Padlock3 had the highest signal output in the presence of active telomerase, high background was also given when telomerase was heat-inactivated, indicating a part of Padlock3 could also be ligated to circular template in the presence of unextended TS primer. Therefore, Padlock2 was selected as the optimal one. In addition, other conditions, including telomerase-catalyzed extension reaction time, amounts of Padlock2, TS primer, and nicking endonuclease Nb.BbvCI (Figure S4 ~ Figure S7), were optimized. Correspondingly, 40 min of extension time, 100 nM of Padlock2, 200 nM of TS primer and 5 U of Nb.BbvCI, were chosen in the followed experiments. Sensitivity of telomerase activity. Under the optimized conditions, the sensitivity of the telomerase-sensing platform was evaluated (Figure 2). Telomerase was extracted from HeLa cells. As expected, more and more TS primer and Padlock strands participated in subsequent RCA with the increase of HeLa cell number, producing long DNA with repeated G-rich parts, which were reflected through the gradual increase in the fluorescence intensity (Figure 2a). Similarly, the fluorescence intensity at 485 nm progressively increased with the telomerase activity equivalent to 1 ~ 5000 HeLa cells in Figure 2b. A good linear correlation between the fluorescence intensity value and cell number (R2 = 0.9950)

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Figure 2. Telomerase activity detection in (a,b) end-point mode or (c,d) real-time mode. (a) Fluorescence emission spectra in response to telomerase extracted from increasing HeLa cell number. (b) HeLa cell number-dependent change of fluorescence intensity at 485 nm. Inset: the linear correlation between the fluorescence intensity and HeLa cell number ranging from 1 to 100 HeLa cells. (c) Fluorescence ~ time and (d) log(F) ~ time plots obtained from the sensing systems in response to telomerase extracted from increasing HeLa cell number. Inset: the linear correlation between RTt value and the logarithm of HeLa cell number ranging from 1 to 100 HeLa cells. Heat-inactivated control was prepared by heating the extracts of 5000 HeLa cells at 95 oC for 20 min. Besides above the fluorescence signal was measured after RCA reaction, the telomerase activity detection might also be performed in a high throughput and convenient real-time detection mode. Our previous studies have demonstrated that fluorescent dye ThT could give a rapid and specific fluorescent response to G-quadruplexes,46 thus real-time monitoring of the amplification via ThT fluorescence change possible. As shown in Figure 2c, time-dependent signal increase could be observed, and the rate of the fluorescence increasing was highly dependent on the HeLa cells number. To quantitatively evaluate the telomerase activity, the logarithm of the fluorescence (log(F)) ~ time plots (Figure 2d) were easily calculated from the F ~ time ones (Figure 2c). With certain RTt values, which is the reaction time at set


threshold of log(F), a linear correlation (R2 = 0.9899) was obtained between RTt value and telomerase activity rangeing from 1 to 100 HeLa cells. By using this detection mode, single HeLa cell could be more easily distinguished from the heatinactivated control, which might be benefit for the dynamic monitoring of RCA reaction. This real-time detection mode, compared with the end-point one, needs a more simplified operation procedure due to the combination of signal amplification and detection steps. In addition, such a combined procedure guarantees that the materials could be discarded directly within original container, thus reducing the risk of pollution of amplification products, which is a special challenge always suffered in exponential nucleic acid amplification reactions.

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Figure 3. Telomerase activity analysis of different cell lines. (a) Fluorescence emission spectra and (b) fluorescence intensities at 485 nm in response to the blank control group and cell extracts (equivalent to 1000 cells) of different cancer cell lines. Telomerase activity detection in different cell lines. Although telomerase is up-regulated in more than 85 ~ 90% of malignant tumors, telomerase expression levels in diverse cancer cells are greatly different. To further demonstrate the potential of our proposed assay to apply in telomerase activity analysis, the telomerase activities in varies of cell lines, including HeLa, 293T, HepG2, CCRF-CEMMCF-7 and HL7702, were tested and compared.

fluorescence signal enhancement were given by MCF-7, HepG2, CEM, 293T and HeLa cells, confirming that the activity of telomerase among most of malignant tumor cells was up-regulated.6,7 The tested telomerase activity levels in different cancer cells followed the order of 293T > HeLa > CEM > HepG2 > MCF-7, which was consistent with other studies.13,24 These results provided the proof for the feasibility of our proposed assay to discriminate different levels of telomerase activity among various cell lines. Cancer cell detection in cell mixture. In tumor microenvironment, cancer cells always coexist with adjacent normal cells. In clinical samples for cancer diagnosis, cancer cells are also mixed with accumulated normal cells. To evaluate the clinical diagnosis applicability of our sensing strategy, its ability to recognize cancer cells in cell mixture was investigated. To achieve this, cancer cells (HeLa) were premixed with normal cells (HL-7702) according to the ratios of 1:999, 1:99, 1:9, 1:4 and 1:1 by fixing the total cell number at 1000, and the cell extracts of these mixtures were used for telomerase activity analysis. The fluorescence intensity at 485 nm raised correspondingly with the increase of HeLa cell percentage (Figure 4). The fluorescence intensity given by the 1:999 mixture, which was consist of 1 HeLa cell and 999 HL7702 cells, was obviously distinguishable from that given by the group containing 100% of HL-7702 cells as the blank control. These results suggested the proposed method could detect very few cancer cells from high concentrated normal cells. This is of critical importance for early diagnosis of malignant diseases. 100

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ACS Sensors

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Figure 4. Measurement of telomerase activity in the mixtures of HL-7702 and HeLa cells. The ratios of HeLa/HL-7702 are 1:999, 1:99, 1:9, 1:4 and 1:1, respectively. The total cell number is 1000. As shown in Figure 3, almost no signal enhancement was observed for HL-7702 cells compared to the heat-inactivated control, revealing the negative expression of telomerase activity in normal cells.8-10 By contrast, different levels of

Inhibition evaluation of telomerase activity. Telomerase activity is highly expressed for most of malignant tumor cells, and the inhibition of its activity would repress the unlimited proliferation of malignant tumor cells.47 Thus, telomerase can be considered to be both an important biomarker and a therapeutic target of cancer treatment, and telomerase inhibitors might be developed as telomerase-targeted anticancer drugs. The screening of telomerase inhibitors is significant for developing new anti-cancer drugs.48 Our proposed strategy could also be applied for evaluating inhibitory activity and thus for screening telomerase inhibitors. To verify this, BIBR1532 was chose as an example of the inhibitors. As shown in Figure 5, the fluorescence signal decreased according with BIBR1532 concentration. Notably, if the inhibitor was added after telomerase-catalyzed primer extension reaction, the fluorescence signal obtained was


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similar as the one without adding inhibitor (Figure S9), thus demonstrating that the inhibitor nearly had no effects on other materials maintained in the system. Therefore, the fluorescence decrease observed in Figure 5 was actually caused by the repression of telomerase activity by the inhibitor. Based on the calibration curve (Figure 5), the IC50 value (defined as the concentration of the inhibitor when it can suppress about 50% telomerase activity) was calculated to be 0.40 μM, which is approximate to those reported previously.49,50 This result demonstrated that the proposed sensing strategy is performing well in the application of telomerase inhibitors screening and their inhibitory capabilities evaluating, thus showing a great potential in anticancer drug discovery.

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AUTHOR INFORMATION Corresponding Author

*[email protected]; Fax: +86-22-23502458 Notes The authors declare no competing ﬁnancial interest.

ACKNOWLEDGMENT This work was supported by the National Natural Science Foundation of China (Nos. 21874075, 21728801), the National Natural Science Foundation of Tianjin (No. 16JCYBJC19900) and the Fundamental Research Funds for Central University (China). This work is dedicated to 100th anniversary of Nankai University.

CONCLUSION In summary, we have proposed a new strategy, named as fivebase telomerase product-triggered exponential RCA, for the ultrasensitive detection of telomerase activity. Compared to other reported telomerase detection methods, our telomerasesensing assay has the following distinctive advantages: (1) Only five bases extension is needed at the 3′-termini of TS primer. Short TEP makes telomerase own high translocation efficiency. Definite TEP length can simplify the design of Padlock strand and increase the production efficiency of circular template. All of these are helpful for the improvement of detection sensitivity. In addition, the requirement for short TEP endows the proposed method with the ability to detect telomerase with very low activity, which is of critical importance for early diagnosis of malignant diseases. (2) Elaborately designed exponential RCA strategy endows the sensing platform with extraordinarily high signal amplification efficiency. (3) Via the fluorescence response of Gquadruplexes to ThT, telomerase activity detection can be performed in a cost-efficient “label-free” mode. Highly recognised specificity of ThT towards G-quadruplexes can also benefit for the reduction of background, thus eliminating the generation of false-positive signals. (4) Comparing with some reported RCA-based assays,14,15 our method has greatly simplified experimental operation due to the elimination of other signal amplification steps and the requirement of circle template preparation in advance via complex gel separation steps. The proposed sensing platform could achieve highly sensitive telomerase activity detection at single-cell level, even with the interference of accumulated normal cells. It was also confirmed that our proposed method was suitable for screening telomerase inhibitor and evaluating inhibitory activity, thus showing great potential for not only clinical diagnosis but also anticancer drug development.

ASSOCIATED CONTENT Supporting Information

Supporting Information Available: The following files are available free of charge. The oligonucleotides used in the experiment; Comparison of different lengths of TEPs; Comparison of the sensing systems with or without telomerase inactivation step; Optimization of experimental conditions; Comparison of our proposed strategy with other reported ones; Telomerase activity detection using traditional RCA; Effects of BIBR1532 on the activities of nicking endonuclease, ligase and polymerase.

REFERENCES (1) Autexier, C.; Lue, N. F. The structure and function of telomerase reverse transcriptase. Annu. Rev. Biochem. 2006, 75, 493-517. (2) Blackburn, E. H. Structure and function of telomeres. Nature 1991, 350, 569-573. (3) Schmidt, J. C.; Cech, T. R. Human telomerase: biogenesis, trafficking, recruitment, and activation. Genes Dev. 2015, 29, 1095-1105. (4) Walsh, K. M.; Codd, V.; Smirnov, I. V.; Rice, T.; Decker, P. A.; Hansen, H. M.; Kollmeyer, T.; Kosel, M. L.; Molinaro, A. M.; McCoy, L. S. Variants near TERT and TERC influencing telomere length are associated with high-grade glioma risk. Nat. Genet. 2014, 46, 731-735. (5) Xu, Y. Chemistry in human telomere biology: structure, function and targeting of telomere DNA/RNA. Chem. Soc. Rev. 2011, 40, 2719-2740. (6) Kimura, M.; Koida, T.; Yanagita, Y. A study on telomerase activity and prognosis in breast cancer. Med. Oncol. 2003, 20, 117-126. (7) Wang, X. L.; Sun, L.; Wang, X. J.; Kang, H. F.; Ma, X. B.; Wang, M.; Lin, S.; Liu, M.; Dai, C.; Dai, Z. J. Acidified bile acids enhance tumor progression and telomerase activity of gastric cancer in mice dependent on c-Myc expression. Cancer Med. 2017, 6, 788-797. (8) Masutomi, K.; Yu, E. Y.; Khurts, S.; Ben-Porath, I.; Currier, J. L.; Metz, G. B.; Brooks, M. W.; Kaneko, S.; Murakami, S.; De Caprio, J. A.; Weinberg, R. A.; Stewart, S. A.; Hahn, W. C. Telomerase maintains telomere structure in normal human cells. Cell 2003, 114, 241-253. (9) Broccoli, D.; Young, J. W.; de Lange, T. Telomerase activity in normal and malignant hematopoietic cells. Proc. Natl. Acad. Sci. U S A 1995, 92, 9082-9086. (10) Blasco, M. A. Telomere length, stem cells and aging. Nat. Chem. Biol. 2007, 3, 640-649. (11) Kim, N. W.; Piatyszek, M. A.; Prowse, K. R.; Harley, C. B.; West, M. D.; Ho, P. L. C.; Coviello, G. M.; Wright, W. E.; Weinrich, S. L.; Shay, J. W. Specific association of human telomerase activity with immortal cells and cancer. Science 1994, 266, 2011-2015. (12) Krupp, G.; Kuhne, K.; Tamm, S.; Klapper, W.; Heidorn, K.; Rott, A.; Parwaresch, R. Molecular basis of artifacts in the detection of telomerase activity and a modified primer for a more robust ‘TRAP’ assay. Nucleic Acids Res. 1997, 25, 919921. (13) He, C. T.; Liu, Z. J.; Wu, Q. L.; Zhao, J.; Liu, R. Y.; Liu, B. H.; Zhao, T. T. Ratiometric fluorescent biosensor for visual discrimination of cancer cells with different telomerase expression levels. ACS Sens. 2018, 3, 757-762. (14) Ma, F.; Wei, S.-H.; Leng, J. H.; Tang, B.; Zhang, C.-Y. A simple “mix-and-detection” method for the sensitive detection of telomerase from cancer cells under absolutely isothermal conditions.Chem. Commun. 2018, 54, 2483-2486.


Page 7 of 9 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Sensors (15) Li, C.-C.; Zhang, Y.; Liu,W.-J.; Zhang, C.-Y. A tripleamplification strategy for sensitive detection of telomerase at the single-cell level. Chem. Commun. 2018, 54, 9317-9320. (16) Zhuang, Y.; Shang, C. L.; Lou, X. D.; Xia, F. Construction of AIEgens-based bioprobe with two fluorescent signals for enhanced monitor of extracellular and intracellular telomerase activity. Anal. Chem. 2017, 89, 2073-2079. (17) Xu, X. W.; Wang, L.; Li, K.; Huang, Q. H.; Jiang, W. A smart DNA tweezer for detection of human telomerase activity. Anal. Chem. 2018, 90, 3521-3530. (18) Tian, L. L.; Weizmann, Y. Real-time detection of telomerase activity using the exponential isothermal amplification of telomere repeat assay. J. Am. Chem. Soc. 2013, 135, 1661-1664. (19) Wang, H. H.; Wang, H.; Liu, C. H.; Duan, X. R.; Li, Z. P. Ultrasensitive detection of telomerase activity in a single cell using stem-loop primer-mediated exponential amplification (SPEA) with near zero nonspecific signal. Chem. Sci. 2016, 7, 4945-4950. (20) Wang, J. S.; Wu, L.; Ren, J. S.; Qu, X. G. Visualizing human telomerase activity with primer-modified Au nanoparticles. Small 2012, 8, 259-264. (21) Xu, X. L.; Wei, M.; Liu,Y. J.; Liu, X.; Wei, W.; Zhang, Y. J.; Liu, S. Q. A simple, fast, label-free colorimetric method for detection of telomerase activity in urine by using hemingraphene conjugates. Biosens. Bioelectron. 2017, 87, 600-606. (22) Li, H., Fu, H.-W.; Zhao, T.; Kong, D.-M. Simple, PCR-free telomerase activity detection using G-quadruplex-hemin DNAzyme. RSC Adv. 2015, 5, 6475-6480. (23) Wang,Y. C.; Yang,L. Z.; Wang,Y. J.;Liu, W.; Li,B. X.;Jin, Y.An ultrasensitive colorimetric assay for reliable visual detection of telomerase activity. Analyst 2017, 142, 3235-3240. (24) Liu, X.; Wei, M.; Xu, E. S.; Yang, H. T.; Wei, W.; Zhang, Y. J.; Liu, S. Q. A sensitive, label-free electrochemical detection of telomerase activity without modification or immobilization. Biosens. Bioelectron. 2017, 91, 347-353. (25) Liu, X. J.; Li, W.; Hou, T.; Dong, S. S.; Yu, G. H.; Li, F. Homogeneous electrochemical strategy for human telomerase activity assay at single-cell level based on T7 exonuclease-aided target recycling amplification. Anal. Chem. 2015, 87, 4030-4036. (26) Wu, L.; Wang, J. S.; Feng, L. Y.; Ren, J. S.; Wei, W. L.; Qu, X. G. Label-free ultrasensitive detection of human telomerase activity using porphyrin-functionalized graphene and electrochemiluminescence technique. Adv. Mater. 2012, 24, 2447-2452. (27) Zhang, H.-R.; Wang, Y.-Z.; Wu, M.-S.; Feng, Q.-M.; Shi, H.-W.; Chen, H.-Y.; Xu, J.-J. Visual electrochemiluminescence detection of telomerase activity based on multifunctional Au nanoparticles modified with G-quadruplex deoxyribozyme and luminol. Chem. Commun. 2014, 50, 12575-12577. (28) Wang, L.-J.; Zhang, Y.; Zhang, C.-Y. Ultrasensitive detection of telomerase activity at the single-cell level. Anal. Chem. 2013, 85, 11509-11517. (29) Ma, W.; Fu, P.; Sun, M. Z.; Xu, L. G.; Kuang, H.; Xu, C. L. Dual quantification of MicroRNAs and telomerase in living cells. J. Am. Chem. Soc. 2017, 139, 11752-11759. (30) Huard, S.; Moriarty, T. J.; Autexier, C. The C terminus of the human telomerase reverse transcriptase is a determinant of enzyme processivity. Nucleic Acids Res. 2003, 31, 4059-4070. (31) Ren, X.; Li, H.; Clarke, R. W.; Alves, D. A.; Ying, L.; Klenerman, D.; Balasubramanian, S. Analysis of human telomerase activity and function by two color single molecule coincidence fluorescence spectroscopy. J. Am. Chem. Soc. 2006, 128, 4992-5000. (32) Su, X.; Li, Z. H.; Yan, X. Z.; Wang, L.; Zhou, X.; Wei, L.; Xiao, L. H.; Yu, C. Y. Telomerase activity detection with amplification-free single molecule stochastic binding assay. Anal. Chem. 2017, 89, 3576-3582. (33) Kim, N. W.; Piatyszek, M. A.; Prowse, K. R.; Harley, C. B.; West, M. D.; Ho, P. L. C.; Coviello, G. M.; Wright, W. E.; Weinrich, S. L.; Shay, J. W. Specific association of human

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(36)

(37)

(38)

(39) (40)

(41)

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(48) (49)

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telomerase activity with immortal cells and cancer. Science 1994, 266, 2011-2015. Kong, D.-M.; Jin, Y.-W.; Yin, Y.-J.; Mi, H.-F.; Shen, H.-X. Real-time PCR detection of telomerase activity using specific molecular beacon probes. Anal. Bioanal. Chem. 2007, 388, 699709. Liu, H. Y.; Li, L.; Duan, L. L.; Wang, X.; Xie, Y. X.; Tong, L. L.; Wang, Q.; Tang, B. High specific and ultrasensitive isothermal detection of microRNA by padlock probe-based exponential rolling circle amplification. Anal. Chem. 2013, 85, 7941-7947. Mohanty, J.; Barooah, N.; Dhamodharan, V.; Harikrishna, S.; Pradeepkumar, P. I.; Bhasikuttan, A. C. Thioflavin T as an efficient inducer and selective fluorescent sensor for the human telomeric G-Quadruplex DNA. J. Am. Chem. Soc. 2013, 135, 367-376. Wang,H. X.;Li,Y.;Zhao,K. L.; Chen, S. Y.;Wang,Q.;Lin, B.; Nie, Z.;Yao,S. Z. G-quadruplex-based fluorometric biosensor for label-free and homogenous detection of protein acetylationrelated enzymes activities. Biosens. Bioelectron. 2017, 91, 400407. Du, Y.-C.; Zhu, L.-N.; Kong, D.-M. Label-free thioflavin T/Gquadruplex-based real-time strand displacement amplification for biosensing applications. Biosens. Bioelectron. 2016, 86, 811817. Khare, V.; Eckert, K. A. The proofreading 3′ → 5′exonuclease activity of DNA polymerases: a kinetic barrier to translesion DNA synthesis. Mutat. Res. 2002, 510, 45-54. Larsson, C.; Koch, J.; Nygren, A.; Janssen, G.; Raap, A. K.; Landegren, U.; Nilsson, M. In situ genotyping individual DNA molecules by target-primed rolling-circle amplification of padlock probes. Nat. Methods 2004, 1, 227-232. Liu, M.; Zhang, W. Q.; Zhang, Q.; Brennan, J. D.; Li, Y. F. Biosensing by tandem reactions of structure switching, nucleolytic digestion, and DNA amplification of a DNA assembly. Angew. Chem. Int. Ed. 2015, 54, 9637-9641. Du, Y.-C.; Zhu, Y.-J.; Li, X.-Y.; Kong, D.-M. Amplified detection of genome-containing biological targets using terminal deoxynucleotidyl transferase-assisted rolling circle amplification. Chem. Commun. 2018, 54, 682-685. Li, X.-Y.; Du, Y.-C.; Zhang, Y.-P.; Kong, D.-M. Dual functional Phi29 DNA polymerase-triggered exponential rolling circle amplification for sequence-specific detection of target DNA embedded in long stranded genomic DNA. Sci. Rep. 2017, 7, 6263-6272. Ding, C. F.; Li, X. Q.; Wang, W.; Chen, Y. Y. Fluorescence detection of telomerase activity in cancer cell extracts based on autonomous exonuclease III-assisted isothermal cycling signal amplification. Biosens. Bioelectron. 2016, 83, 102-105. Li, X. Q.; Wang, W.; Chen, Y. Y.; Ding, C. F. Fluorescence detection of telomerase activity in high concentration of cell lysates based on strand displacement mediated recycling. Analyst 2016, 141, 2388-2391. Jiang, H.-X.; Liang, Z.-Z.; Ma, Y.-H.; Kong, D.-M.; Hong, Z.Y.G-quadruplex fluorescent probe-mediated real-time rolling circle amplification strategy for highly sensitive microRNA detection. Anal. Chim. Acta 2016, 943, 114-122. Hahn, W. C.; Stewart, S. A.; Brooks, M. W.; York, S. G.; Eaton, E.; Kurachi, A.; Beijersbergen, R. L.; Knoll, J. H. M.; Meyerson, M.; Weinberg, R. A.Inhibition of telomerase limits the growth of human cancer cells. Nat. Med. 1999, 5, 1164-1170. Cohn, E. P. M. T.; Wu, K.-L.; Pettus, T. R. R.; Reich, N. O.A new strategy for detection and development of tractable telomerase inhibitors. J. Med. Chem. 2012, 55, 3678-3686. Wardi, L.; Alaaeddine, N.; Raad, I.; Sarkis, R.; Serhal, R.; Khalil, C.; Hilal, G. Glucose restriction decreases telomerase activity and enhances its inhibitor response on breast cancer cells: possible extra-telomerase role of BIBR 1532. Cancer Cell Int. 2014, 14, 60-73. Shi, Y.; Sun, L.; Chen, G.; Zheng, D. Y.; Li, L.; Wei, W. G. A combination of the telomerase inhibitor, BIBR1532, and


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paclitaxel synergistically inhibit cell proliferation in breast cancer cell lines. Targ. Oncol. 2015, 10, 565-573.

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Ultrasensitive detection of telomerase activity using a five-base telomerase product-triggered exponential rolling circle amplification strategy 64x60mm (300 x 300 DPI)


Label-Free Telomerase Detection in Single Cell using a Five-Base

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