Ultrasensitive detection of telomerase activity in a single cell using stem-loop primer-mediated exponential amplification (SPEA) with near zero nonspecific signal† †Electronic supplementary information (ESI) available. See DOI: 10.1039/c6sc00802j

A SPEA strategy is developed for the detection of telomerase activity in a single cell with a near zero nonspecific signal.

Ltd. (Shanghai, China). The SLTSR8 oligonucleotide was synthesized by Integrated DNA Technologies. The other oligonucleotides used in this work, RNase inhibitor, dNTPs, RNase-free water, 20 bp DNA ladder and 6 nucleic acid sample loading buffer were purchased from TaKaRa Biotechnology Co. Ltd. (Dalian, China). All the reagents were of analytical grade and were used as received without further purification. All the oligonucleotides were purified by PAGE. The sequences of the oligonucleotides were given in the Table S1

Preparation of telomerase extracts
The cultured Hela cells or other cells were centrifuged at 700 rpm for 5 minutes to remove the culture medium after trypsinization (0.2% trypsin, 1 mM EDTA, Invitrogen), followed by washing three times with the cold D-PBS buffer (10 mM sodium phosphate buffer, 0.1 M NaCl, pH 7.4 @25°C). 10 6 adherent Hela cells or other cells were collected respectively into a centrifuge tube and then the cells were pelleted by centrifuged at 2000 rpm for 10 min.
Each cell pellet (containing 10 6 cells) was suspended in the 200 μL cold CHAPS lysis buffer (5000 cells/μL), incubated on ice for 30 min and then centrifuged for 30 min at 12000 rpm at 4°C. The telomerase extracts in the supernatant were frozen and stored at -80°C as the stock extracts. The samples for detection of telomerase activity were prepared by appropriate dilution of the stock extracts with CHAPS buffer.

Preparation of cell lysates
The cultured Hela cells or other cells were centrifuged at 700 rpm for 5 minutes to remove the culture medium after trypsinization (0.2% trypsin, 1 mM EDTA, Invitrogen), washed three times with cold D-PBS buffer, and then resuspended in the cold D-PBS buffer at a concentration of 10 4 cells per μL. For the telomerase activity analysis of 100 cells, the cell suspension was serially diluted to 100 cells per μL in the cold D-PBS buffer. 1 μL diluted cell suspension containing 100 cells was pipetted to 1 μL cold CHAPS lysis buffer, and then incubated for 30 min on ice.
For the telomerase activity analysis of 10 cells or 1 cell, firstly, the cell suspension was serially diluted to 10 cells per μL or 1 cell per μL in the cold D-PBS buffer, 1 μL diluted cell suspension (containing 10 cells or 1 cell) was transferred to the microslide, and then 10 cells or 1 cell was counted and manipulated by using Narishige micromanipulator system equipped on an Olympus IX53 inverted microscope with a monitor. Subsequently the 10 cells or 1 cell were dropped into 1 μL cold CHAPS lysis buffer, incubated for 30 min on ice. The cell lysates were then immediately used as the samples to detect the telomerase activity.

S3
The cell lysates or 1 μL diluted extracts were added to the reaction mixture containing 0.2 μM stem-loop telomerase substrate (SLTS) primer, 0.5 mM dNTPs, 1.6 U/μL RNase inhibitor and telomerase reaction buffer (20 mM Tris-HCl, 1.5 mM MgCl 2 , 63 mM KCl, 1 mM EGTA, 0.05% Tween-20) with the final volume of 5.0 μL. Then the mixture was incubated for 30 min at 37 °C to elongate the SLTS primer, and finally heated to 95 °C for 5 min to inactivate telomerase activity. For the detection of SLTSR8 model target, SLTS was replaced by SLTSR8 of different concentrations without the cell lysates or the diluted extracts. The CHAPS buffer was used as the blank, which was detected with the same procedures in the absence of cell lysates, diluted extracts or SLTSR8.
After the telomerase reaction, the reaction products were mixed with a standard SPEA reaction solution

Optimization of the amount of Bst DNA polymerase
The SPEA reaction relied on auto-cycling strand displacement DNA synthesis that was catalyzed by Bst DNA polymerase with displacement activity. So the amount of Bst DNA polymerase was an important parameter for the SPEA-based telomerase assay. As described in the paper, a synthetic telomerase-elongated product SLTSR8, corresponding to SLTS elongated with eight telomeric repeats (TTAGGG), was employed as a model to optimize the experiment conditions. Therefore, the effect of amount of Bst DNA polymerase was investigated by detection of

Optimization of the amount of stem-loop primer (SLP)
To investigate the influence of the amount of SLP, 10 aM, 100 aM and 1 fM SLTSR8 were respectively detected with the SPEA-based assay by using SLP of different concentrations. As depicted in Fig. S2 (A-C), with increasing the SLP concentration from 50 nM to 200 nM, the SPEA reaction was gradually speeded up. However, the POI values of SLTSR8 decreased very little. The differences of the POI values between 10 aM, 100 aM, and 1 fM SLTSR8 reached their maximum when 100 nM SLP was used. Therefore, 100 nM SLP was employed for the SPEA-based assay.

Optimization of the temperature of SPEA reaction
The temperature of SPEA reaction was closely associated with the reaction speed and the sensitivity for detection of telomerase activity. The influence of temperature of SPEA reaction was investigated by simultaneously detecting the blank, 10 aM, 100 aM and 1 fM SLTSR8 with the SPEA-based assay at different reaction temperatures. As demonstrated in Fig. S3(A-B), when the reaction temperature was elevated from 60 °C to 65 °C, the POI values of SLTSR8 were shortened about 15 min and the difference of the POI values between 10 aM, 100 aM and 1 fM SLTSR8 were increased. However, when the SPEA was performed at 70 °C, as shown in Fig. S3C, no detectable fluorescence signals could be observed, indicating that no SPEA reaction took place at 70 °C. It should be reasonable that the melting temperature (Tm) of the double stranded stem in SLP and SLTSR8 is less than 70 °C. So the stemloop structure of SLP and SLTSR8 will be destroyed. Therefore, 65 °C was selected as the optimized temperature of SPEA reaction in this work.  The efficient preclusion of non-specific amplification in this work is mainly due to the ingenious probe design in the SPEA system. Firstly, in the proposed SPEA system, the double stem-loop DNAs are the essential starting materials for the subsequent loop-mediated isothermal signal amplification. As illustrated in Fig. 1, if telomerase is absent, the SLTS cannot be elongated to produce the telomere repeat, and thus the telomere repeat-templated extension of SLP cannot occur at all. Consequently, no double stem-loop DNAs can be formed to initiate the subsequent signal amplification reaction. Secondly, in the proposed SPEA system, the sequences of FIP and BIP are also rationally designed to avoid nonspecific signal amplification. FIP has the same sequence with F1c and F2 of SLTS, and BIP has the same sequence with B1c and B2 of SLP. As such, when telomerase is absent, the four coexisted nucleic acid sequences (SLTS, SLP, FIP, BIP) in the SPEA system will not hybridize with each other to produce any nonspecific amplification reaction due to their stringent sequence design.

Evaluation and explanation of the near-zero nonspecific amplification
On the other hand, for the conventional thermal cycle-based PCR amplification, the formation of primer-dimer (self-dimer or hetero-dimer) of the PCR primers during the thermal cycles is the main source of nonspecific amplification. The primer-dimers can form in the annealing step of PCR when some bases in the primers are complementary. As illustrated in Fig. S5, even the formation of very few primer-dimers may ultimately generate significant false positive results after exponential amplification with the PCR thermal cycles. In contrast, in the SPEA system, the exponential amplification is performed under isothermal conditions. As illustrated in Fig. S6, even if the primers of the SPEA could also form the self-dimer or hetero-dimer, the self-dimer or hetero-dimer of SPEA primers can only extend one time and then the primer extension will be stopped under the constant temperature, which cannot initiate the subsequent signal amplification and cannot generate obvious nonspecific signals. Therefore, from above we can concluded that the near zero nonspecific signal in this study is reasonable due to the rational probe design and the isothermal nature of the exponential amplification in SPEA.

Charaterization of SPEA products with gel electrophoresis
To further support the proposed SPEA-based telomerase assay, the amplification products of SPEA reaction were characterized by polyacrylamide gel electrophoresis (PAGE). As shown in Fig. S7, the SLTS shows a defined band corresponding to 52 bp (lane 2). After incubation of SLTS with the Hela cell extraction, as depicted in lane 3, the amplification products of SPEA reaction showed S9 multiple bands in ladder-like patterns. According to the principle of SPEA discussed in Fig. 1, the SPEA products are a large amount of the mixture of stem-loop DNAs with various stem lengths. So the ladder-like patterns of PAGE were well consistent with the prediction of SPEA products. Meanwhile, the negative control (lane 4) and the blank (lane 5), in which the Hela cell extract is heat-inactivated or absent, did not produce any observable bands except for the SLTS band. On the other hand, the synthetic SLTSR8 shows a band at the position corresponding to about 160 bp of dsDNA marker (lane 6). When the SLTSR8 was amplified by SPEA reaction, as shown in lane 7, the multiple bands with the ladder-like pattern could be observed from the amplification products. More importantly, the ladderlike pattern of the SLTSR8 amplification products was almost the same as that of Hela cell extract-incubated SLTS amplification products, indicating that the amplification products shown in lane 3 were indeed originated from the telomerase-elongated SLTS and the proposed assay was reliable for detection of telomerase activity.

HCT-116, MCF-7 and MRC-5 cell lines
To test whether the proposed assay is generally applicable to the detection of cellular heterogeneity arising from cell-to-cell variations in different cell lines, the SPEA strategy was further applied to the detection of telomerase activities in different number of cancer cells (CEM, S10 HCT-116 and MCF-7 cell lines) as well as normal cell line MRC-5. For each tested cell line, diluted extracts equivalent to 1, 10, 100 cells and cell lysates from 1, 10, 100 cells are repectively tested according to procedures described in the standard assay protocols. The CHAPS buffer was employed as the blank while the diluted extract corresponding to 100 cells or cell lysates of 100 cells was preheated at 95 °C for 5 min to inactivate telomerase activity to be used as the negative control. As depicted in Fig. S8, Error bars indicate standard deviation of ten replicative tests for 1 cell, 5 replicative tests for 10 cells, and 3 replicative tests for 100 cells, respectively. The CHAPS buffer was employed as the blank and the diluted extract corresponding to 100 cells (in image (I)) or crude lysates of 100 cells (in image (II)) is preheated at 95 °C for 5 min to inactivate telomerase activity to be used as the negative control.