Catalytic Hairpin Assembly-Assisted Rolling Circle Amplification for High-Sensitive Telomerase Activity Detection.

Telomerase is a promising biomarker and a potential therapeutic target of malignant tumors. Reliable, facile, and sensitive telomerase activity analysis is of vital importance for both early diagnosis and therapy of malignant tumors. Herein, we proposed a novel fluorescent assay termed catalytic hairpin assembly-assisted rolling circle amplification (CAR) for both in vitro and in situ high-sensitive telomerase activity detection. In the presence of active telomerase, the extension of a designed telomerase primer was limited to five bases (GGGTT), thus forming short telomerase products. Afterward, the obtained telomerase extension products cyclized Padlock and subsequently initiated the rolling circle amplification (RCA). In order to maintain a higher sensitivity, an ingeniously designed catalytic hairpin assembly (CHA) was attached for both signal amplification and result readout. The highlights of the CAR method were concluded as follows: (i) dual signal amplification from CHA and RCA ensures high sensitivity and (ii) the CAR method has the potential for both in vitro and intracellular imaging of telomerase activity. We believe that the CAR method would be of great potential for the diagnosis and therapy of various human diseases.

Introduction

Telomerase is a kind of specialized reverse transcriptase composed by two main components: human telomerase reverse transcriptase and human telomerase RNA. Telomerase could protect the stability of a eukaryotic chromosome from undesired degradation recombination or end-to-end fusion and maintain cell viability through adding telomeric repeats (5′-TTAGGG-3′) to the 3′ end of telomeres.[1,2] It is well-known that telomerase is highly expressed in more than 80% cancer cells, while reduced or absent in normal human cells.[3] Consequently, telomerase is considered to be a potential universal biomarker for early cancer diagnosis and prognosis and even a potential therapeutic target for cancer therapy.[4] Therefore, it is of paramount significance to develop both reliable and sensitive telomerase activity detection approaches.

Various analytical methods for telomerase activity detection have been developed.[5−7] Among them, the most classic strategy for telomerase activity detection is the telomeric repeat amplification protocol (TRAP). The TRAP exhibits excellent sensitivity for telomerase activity detection based on the polymerase chain reaction (PCR) technique.[8,9] However, it is also criticized for its tedious operation, high costs, and requirements for rapid thermal cycling.[10,11] In recent years, numerous PCR-free methods have been elegantly designed and have made progress to avoid part of the drawbacks from the TRAP method.[12] Especially, isothermal amplification has attracted abundant attention in telomerase activity detection because of its stable signal output and easy-to-operate characteristics, such as exponential isothermal amplification, DNAzyme-mediated double-cycling amplification, and telomerase-triggered exponential rolling circle amplification (RCA) methods.[13−15] However, requirements of relatively expensive instruments; time-consuming, complicated preparation; and susceptibility to reaction conditions limit their further application. Therefore, development of a facile method with comparable or even better detection sensitivity and specificity is an urgent demand. Furthermore, signal amplifications in these methods are always triggered by telomerase extension products. As we all know, the telomerase extension products are a mixture of products with variable repeated GGGTTA units, and the telomerase products with less repeated units account for a higher proportion compared with the more repeated ones.[16,17] However, few of the former developed methods focused the detection of telomerase extension products with less repeated units. Therefore, it is significant to develop a new strategy for accurate and dependable detection of telomerase activity and thus contribute to early diagnosis of malignant tumors. RCA has emerged as a highly specific isothermal gene amplification approach that could be performed at a constant temperature (at 30 °C or even room temperature)[18,19] in the presence of thermally stable DNA polymerase without sophisticated instruments and may provide a new idea for developing a facial telomerase activity detection method.

In this paper, we have proposed a catalytic hairpin assembly (CHA)-assisted RCA (CAR) method for high-sensitive telomerase activity detection and successfully applied for high-sensitive in vitro and in situ telomerase activity detection. In the proposed strategy, the primer is extended by telomerase and the obtained products, especially these products with less repeated units, assist the formation of cyclized padlock and thus subsequently trigger the RCA. In order to maintain a higher sensitivity, an ingeniously designed CHA was attached for both signal amplification and readout. We consider that the proposed CAR method would have the potential for high-sensitive in situ telomerase activity imaging in living systems and thus be an important approach for the diagnosis and therapy of various human diseases.

Results and Discussion

Principle of the Proposed Method

The high-sensitive telomerase activity detection and imaging strategy were designed by integrating the RCA triggered by short telomerase extension products and CHA (Scheme ). To realize the high-sensitive telomerase activity detection and imaging, we divided the whole biosensing process into two steps: RCA for telomerase activity sensing and CHA for attached signal amplification. In the RCA step, we first designed telomerase substrate (TS) primer and padlock probe (Padlock). In detail, the middle region (M region, red) in the Padlock is responsible for transcription of CHA initiation sequences, and the two terminals were for hybridization by telomerase extension products. When telomerase existed, the TS primer was recognized and extended through adding telomeric repeats (5′-TTAGGG-3′) to the 3′ end by telomerase. However, if only deoxyguanosine triphosphate (dGTP) and deoxythymidine triphosphate (dTTP) were added, a tail with only five bases of “GGGTT” is joined to the 3′-termini of the TS primer because of the lack of dATP. Afterward, the obtained telomerase extension products could cyclize the Padlock through simultaneously hybridizing with the two terminals. On the contrary, no cyclized Padlock would be produced without telomerase due to the lack of telomerase products. With the cooperation of both short telomerase products and cyclized Padlock, the isothermal RCA reaction could be initiated. In the next step, the obtained RCA products composed of repeated M region transcription sequences were responsible for initiating the attached CHA. In the CHA amplification step, we designed two hairpin structure probes (H1 and H2). In detail, the 5′ terminal of hairpin structure H1 probe was a hybridizing section, while the two terminals of H2 probes were labeled with a fluorescent group (Cy3) and corresponding quenching group (BHQ) to maintain a relatively low fluorescence background through the Förster resonance energy transfer (FRET).[20] When telomerase extension products initiated the RCA, the H1 probes could be recognized by RCA products in the hybridizing section and trigger the CHA reaction. Subsequently, H2 probes would open and cause significant increase in fluorescence signals. Eventually, the displaced RCA products proceeded to the next CHA amplification cycle, enriching the H1 and H2 hybridization products, and the fluorescent signal gradually increased. As a result, the CAR method was proposed.

Scheme 1

Working Principle of the Proposed Telomerase Activity-Sensing Platform

Feasibility of Our Proposed Telomerase Activity-Sensing Strategy

To test whether the RCA could be selectively triggered by the telomerase extension products, PAGE was performed. As shown in Scheme , we still observed an 80 ≈ 100 bp band in the electrophoresis for the solution containing Padlock and a synthetic telomerase extension product-like (TEPL) sequence (Table S1), suggesting that telomerase extension products could hybridize with two terminals of Padlock and cyclize it. When phi29 enzyme and deoxyribonucleoside 5′-triphosphate (dNTP) were added into the abovementioned mixture, a pronounced RCA products band appeared in the gel. This result hinted that telomerase extension products were essential for RCA initiation. Afterward, we investigated the feasibility of the proposed method for telomerase activity detection through a fluorescence assay. As shown in Scheme , a significantly enhanced fluorescence was observed through the mixture of active telomerase, which was extracted from HeLa cells, indicating the successful performance of the CAR method. Furthermore, neglectable fluorescence was observed when the Padlock was removed. The same results were obtained when T4 DNA ligase and phi29 enzymes were absent, indicating that Padlock, T4 DNA ligase, and phi29 enzymes were all essential for CAR methods. Meanwhile, fluorescence intensity was reduced by almost 54.7% when the H1 probe was absent, confirming that CHA was essential for improved sensitivity in the CAR method. Furthermore, the heat-inactivated cell extracts through heating at 95 °C for 20 min showed a slightly enhanced fluorescence intensity compared with the negative control, indicating that fluorescence response was actually caused by the active telomerase.

Scheme 2

Feasibility of the CAR Method; (a) PAGE Electrophoresis Result of TEPL Sequence-Triggered RCA; (b) Fluorescence Intensity of the CAR Method When Free from Part of Components

Data are represented as the means ± SD (n = 3).

Optimization of Experimental Conditions

The length of the telomerase extension products was first optimized. In order to avoid the “GGGTTA” repeats, which may lead to the various length of telomerase extension products, dATP was not added in the telomerase-catalyzed primer extension step. As shown in Scheme , weak fluorescence intensity increase was observed when dATP was added in the telomerase-catalyzed primer extension step. However, when dATP was absent, a rapid fluorescence increase (3 times fluorescence rise) was monitored, indicating that the fluorescence signal given by long telomerase extension products was much weaker than that given by short telomerase extension products. The same result was also reported by DeMing Kong and explained that if a telomerase extension product 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.[21]

Scheme 3

Optimization of Experimental Conditions; (a) Fluorescence Spectra of the CAR Method When dATP Utilized in the Telomerase-Catalyzed Primer Extension Step or Not; Control Refers to the Initial Fluorescence of H2 Probes; (b) Fluorescence Intensity of the Different Padlock Probe in the Presence of Active Telomerase; Three Telomerase Samples Duplicates Were Used for Three Padlock Optimizations; (c) Fluorescence Spectra of the CHA Process with RCA Products Existed or Not; Inset shows a Histogram of the Fluorescence Intensity of the Corresponding Group at Em = 560 nm

Data are represented as the means ± SD (n = 3).

Given that the CAR method is a RCA-dependent process, the effects of Padlock length were investigated. Therefore, three Padlock strands (Padlock 1–3), whose 3′-ends have 0, 2, 3 bases that are complementary with the TS primer, were proposed. In the presence of active telomerase, the fluorescence intensity of the Padlock 3-triggered CAR method was significantly higher than the other two and more reliable methods (Scheme ). Hence, Padlock 3 was chosen as the optimal one for telomerase elongation.

We then investigated the CHA process through a fluorescence assay. The result showed that the presence of RCA products could induce a significant increase in the fluorescent signal, suggesting that it can be recognized by H1 probes and triggering the attached CHA (Scheme ).

Sensitivity of Telomerase Activity Detection

As telomerase is broadly expressed in cancer cells, the CRA method was applied to detect the activity of telomerase in extracts from HeLa (human cervical cancer cells) under the optimized conditions. As shown in Scheme , a dramatic fluorescence increase was observed with the addition of cell concentration from 0 to 10,000 cells μL–1. Scheme illustrates the relationship between the fluorescence intensity at 560 nm and the concentration of HeLa cell extracts. The calibration equation is Y = 0.6390(C) + 195.3 with a correlation coefficient (R2) of 0.9704 (C refers the concentration of the HeLa cell extracts). The excellent sensitivity is mainly ascribed to the dual signal amplification of CHA-assisted RCA. Furthermore, we compared the CAR method with enzyme-linked immunosorbent assay (ELISA) on telomerase activity detection in five samples. As shown in Scheme , telomerase activity detected by the CAR method maintained a high consistency with ELISA results (R2 = 0.9767), demonstrating that this method has a high application potential in the detection of clinical specimens.

Scheme 4

Sensitivity of CAR for Telomerase Activity Detection; (a) Fluorescence Spectra of the CAR Method When Incubated with Different Concentrations of Telomerase Extracted from 0, 50, 100, 200, 400, 800, 1000, 1500, 2000, 2500, 3000, 3500 HeLa Cells μL–1; (b) Linear Relationship between the Fluorescence Intensity and Concentrations of Cell Extracts; (c) Correlation of Telomerase Activity Detection through the CAR Method and ELISA

CAR for Intracellular Telomerase Activity Imaging

The CAR method was then applied for intracellular telomerase imaging by facilitating the intracellular delivery of all components through lipofectamine-2000. Here, HeLa cell was selected as a model. EGCG, which has been reported to induce the apoptosis of cancer cell lines, could effectively reduce the telomerase activity in cancer cells. Scheme shows the fluorescence images of HeLa cells after treating with different amounts of EGCG in the culture medium for 48 h and then incubating with components of the CAR method for 2 h. With the addition of 200 μg mL–1 EGCG, the fluorescence intensity of EGCG-treated HeLa cells gradually weakened, indicating that the intracellular telomerase activity was effectively inhibited by EGCG because the fluorescence recovery of Cy3 was related to the telomerase activity.

Scheme 5

Confocal Images of Telomerase Activity in HeLa Cells When EGCG Existed or Not

Scale bar = 10 μm. From left to right, Hoechst (blue), Cy3 (green), and merged images.

Herein, we propose a new telomerase activity detection strategy through the integration of RCA and CHA. Compared to other reported telomerase activity detection methods, our telomerase sensing assay has the following distinctive advantages: (i) Only five base extension is needed at the 3′-termini of the TS primer and short TEP makes telomerase own high translocation efficiency; (ii) elaborately designed CAR method endows the sensing platform with a high signal amplification efficiency; and (iii) compared with some of the former reported RCA-based strategies, CAR exhibits greatly simplified experimental operation. Although the established CAR demonstrated a satisfactory performance in telomerase activity detection, some deficiencies potentially exist. Because of the differences in telomerase extraction efficiency, the obtained calibration equation is not perfect but acceptable.

Conclusions

In summary, by taking advantage of telomerase extension products triggered RCA and attached CHA, a novel strategy termed CAR is designed for both highly sensitive in vitro detection of telomerase and in situ imaging of intracellular telomerase activity. Compared to the existing methods for the detection or tracking of telomerase activity, CAR possesses higher sensitivity based on the dual signal amplification and could be both applied for in vitro and in situ telomerase activity detection. We believe that the CAR method would open a new perspective for the development of a highly selective, stable, and sensitive disease diagnosis and treatment system.

Experimental Section

Materials and Reagents

All oligonucleotides used in this experiment (see Table S1) were synthesized and purified from Sangon Biotech. Co. Ltd. (Shanghai, China). T4 DNA ligase and Phi29 DNA polymerase were obtained from New England Biolabs (NEB, Beijing, China). dNTPs, dGTP, dTTP, ethidium bromide, and 20 bp DNA ladder were obtained from Tiangen Biotech. Co. Ltd. (Beijing, China). The other reagents in this experiment are shown in part 1 of the Supporting Information. Diethypyrocarbonate-treated water (DNase, RNase free) obtained from Beyotime Institute of Biotechnology (Shanghai, China) was used in all experiments.

Cell Culture and Liposome-Mediated Transfection

HeLa cells (human cervical cancer cell line) were cultured in Dulbecco’s modified Eagle’s medium (Gibco) with 1% penicillin streptomycin (Gibco) and 10% fetal calf serum (Sijiqing). We took the frozen HeLa cells out of liquid nitrogen and immediately put them into a 37° C water bath and shook them slightly. After the liquid has melted (about 1–1.5 min), the HeLa cells were centrifuged at 1000 rpm for 5 min. We then discarded the supernatant and added 1 mL of the medium to resuspend the cells. Afterward, the cells were aspirated into a Petri dish containing 10 mL of the culture medium. When the cell coverage in the culture dish reached 80–90%, appropriate trypsin (only the cells can be covered) was added and digested for 1–2 min. After the cells are rounded, add an equal volume of serum-containing medium to terminate the digestion. HeLa cells were cultured in a humidified atmosphere containing 5% CO2 at 37 °C.

Nuclease-free water (400 μL) was added to the tube containing liposomes and shaken for 10 s to dissolve the lipid. The ingredients required for liposomes and CAR are then mixed in a 1:1 ratio. The mixture was left at room temperature for 10–15 min. The medium was aspirated in HeLa cell culture plates and washed once with phosphate buffered saline or serum-free medium. The mixture was added, and the cells were kept in the incubator for 1 h.

Telomerase Extraction

We selected and collected HeLa cells in the exponential phase of growth after trypsinization, and they were washed with phosphate buffered saline (137 mM NaCl, 2.7 mM KCl, 10 mM phosphate buffer, pH = 7.4). They were then precipitated at 4° C, 2000 rpm for 10 min. We then resuspended approximately 1 × 106 cells in 200 μL of ice-cold 1 × CHAPS lysis buffer (0.5% CHAPS, 10 Mm Tris-HCl, pH = 7.5, 1 mM MgCl2, 1 mM EGTA, 5 mM β-mercaptoethanol, 0.1 mM medium PMSF, 10% glycerol). The cells were then incubated on ice for 30 min and centrifuged at 4° C (12,000 rpm, 20 min). After that, the supernatant was carefully transferred to a new tube and stored at −80° C until use. For control experiments, the telomerase extract was pre-treated for 10 min by incubating 20 μL of active cell extract (equivalent to one thousand cells) at 95° C before detection.

Feasibility of the CAR Method

We first mixed 2 μL of 10 μM TS primers and 1 μL of 10 μM Padlock in 20 μL of telomerase extension reaction buffer. The mixture was then heated in a PCR apparatus at 95° C for 5 min and then slowly cooled to room temperature. Cell extracts from different cell numbers were then added to the above mixture with 0.5 μL of 100 mM dGTP, 0.5 μL of 100 mM dTTP, and 4 U RNase inhibitor. The mixture was then allowed to react at room temperature for 1 h to perform a telomerase-catalyzed extension reaction. Next, we added 2 μL of 10× Ligation Buffer (50 mM KCl, 10 mM Tris-HCl (pH 7.4), 0.1 mM EDTA, 1 mM DTT, 200 μg/mL BSA), Milli-Q water, and 0.5 μL of T4 DNA ligase and diluted into 50 μL. The obtained 50 μL of the mixture was incubated at room temperature for 2 h. The mixture was then reacted at 60° C for 10 min to inactivate the T4 ligase, and then 10 μL of Phi29 DNA polymerase buffer, 5 μL of H1 probes (10 μM), and 5 μL of H2 probes (10 μM) were added to a final concentration of 0.25 mM dNTP and 5 U Phi29 DNA polymerase. The obtained mixture was incubated at 30° C for 180 min. After the reaction was completed, the fluorescence signal was detected using a Shimadzu RF-5301PC fluorescence spectrometer.

Quantification of Telomerase Activity Using a Commercial ELISA Kit

Telomerase activity measured by ELISA was determined according to the instructions. A standard curve was drawn by measuring the optical density of standard telomerase samples at different concentrations in the range of 0–40 IU/L. Then, the telomerase activity of the cultured HeLa cells was evaluated against a standard curve.

Data Analysis

SPSS 21.0 statistical software (SPSS Inc.) was used for all statistical analyses. The qualitative data were compared using the independent sample chi-square test or Fisher exact test. The quantitative data were analyzed using independent samples t-test, and P < 0.05 was considered statistically significant.