Technical Note pubs.acs.org/ac
PCR-Free Telomerase Assay Using Chronocoulometry Coupled with Hexaammineruthenium(III) Chloride Shinobu Sato and Shigeori Takenaka* Department of Applied Chemistry, Kyushu Institute of Technology, Sensui-cho 1-1, Tobata-ku, Kitakyushu-shi, Fukuoka 804-8550, Japan ABSTRACT: An electrochemical method based on chronocoulometry coupled with hexaammineruthenium chloride (RuHex) is proposed for simple and rapid assay of telomerase without relying on PCR and gel electrophoresis. Thus, DNA extended by telomerase in extracts of as small as 5−1 000 HeLa cells on the TS primer-immobilized electrodes was quantified successfully. This method is suitable for quick screening of drug candidates which inhibit telomerase. When 10 compounds were tested, the multiplicity of extension (x in (TTAGGG)x) varied from 11 to 0, suggesting that there is more than one mechanism of inhibition. IC50 values of telomerase inhibitors TMPyP4 and PIPER were determined as 5.5 and 15 μM, respectively, though their mechanisms of inhibition are different. This method is capable of discriminating two possible mechanisms of telomerase inhibition: direct binding of inhibitors to telomerase and indirect inhibition through their binding to the quadruplex generated by telomerase. As this method is easy and quick to run, it will be useful for high-throughput screening of drug candidates which inhibit telomerase.
T
screening drug candidates which inhibit telomerase is also assessed.
elomerase is a ribonucleoprotein which elongates telomere DNA.1 Telomerase is expected to serve as a cancer marker because of its specific expression in cancer cells.2 At the same time, specific inhibitors of telomerase will serve as an anticancer drug.3 Telomerase activity has been detected by the telomerase repeat amplification protocol (TRAP)4 and the expression analysis of the human telomerase reverse transcriptase (hTERT) gene coding for the telomerase catalytic unit.5 The former, known as the TRAP assay, is more widely adopted and runs as follows. First, a TS-primer, a telomerase substrate DNA fragment, is mixed with a sample solution. Where telomerase activity is present, telomere repeating units of TTA GGG are added at its termini repeatedly. After amplification of the product by polymerase chain reaction (PCR), ladders of six-base pairs apart are observed in gel electrophoresis. Telomerase activity is evaluated from the occurrence of the ladders and their intensity. The TRAP assay is useful also to evaluate the inhibitory ability of drug candidates which inhibit telomerase.6 However, many drugs bind to double stranded DNA strongly and some of them inhibit the polymerase reaction during PCR, obscuring the inhibition occurring at the telomerase or polymerase level.7 Herein, we attempt to quantify DNA extended by telomerase on the solid support by chronocoulometry (CC) with hexaammineruthenium(III) chloride (RuHex) without relying on PCR. RuHex binds electrostatically to the phosphate backbone of DNA immobilized on the electrode to give rise to an electrochemical signal whose intensity is proportional to the amount of DNA.8 The principle of this telomerase detection is depicted in Figure 1. The feasibility of the present assay in © 2012 American Chemical Society
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EXPERIMENTAL SECTION Materials. Wherever possible, nuclease-free buffers and reagents were used to minimize RNase contamination. Hexaammineruthenium(III) chloride (RuHex) and Tris-HCl buffer (pH 7.4 and 8.5) were obtained from Sigma-Aldrich (St. Louis, MO). RNase-free water, 2 M MgCl2, and 3 M KCl were purchased from Life Technologies (Carlsbad, CA). RNase inhibitor and 10 mM dNTP mixture were obtained from Toyobo (Shiga, Japan). Oligonucleotide TS-primer 5′-HO(CH2)6SS(CH2)6-TTT TTT TTA ATC CGT CGA GCA GAG TTA GGG-3′ was custom-synthesized by Genenet (Fukuoka, Japan). To immobilize on the gold electrode, a thiol moiety was introduced into the TS primer at its 5′terminus. Dyes TMPyP4,9 PIPER,10 3′-azido-3′-deoxythymidine triphosphate (AZT),11 5′-d(TTAGGG)-3′ (inhibitor III),3 and 2,6-bis[3-(N-piperidino)propionamido]anthracene-9,10-dione (inhibitor V)12 were purchased from Merck KGaA (Darmstadt, Germany). Ethidium bromide (EtBr) and guanidine thiocyanate13 were from Wako Pure Chemical (Osaka, Japan) and Tokyo Chemical Industry (Tokyo Japan), respectively. FND3, FND7,14 and tris naphthalene diimide (TND)15 were the same as those used previously.14,15 HeLa cells (1.0 × 106 cells) obtained from Millipore (Billeria, MA) were suspended in 200 Received: August 24, 2011 Accepted: January 6, 2012 Published: January 7, 2012 1772
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Figure 1. Electrochemical telomerase assay based on chronocoulometry with RuHex. This system enables one to estimate telomerase activity from the DNA length elongated from the TS-primer.
μL of lysis buffer (Millipore) and incubated on ice for 30 min. After spinning in a microcentrifuge at 12 000 rpm for 20 min at 4 °C, the supernatant containing telomerase was collected and its activity was estimated by the TRAP assay described below. The protein concentration of the supernatant was determined on the basis of tetrazolium WST-8 binding16 with the Proteostain Protein Quantification Kit-Wide Range (Dojindo, Kumamoto, Japan). Extracts from 100 HeLa cells were found to contain 0.2 μg of protein. TRAP Assay. This was conducted using a TRAPeze kit (Millipore). PCR was run as follows: after standing at 30 °C for 1 h, the following cycles were repeated 33 times; 95 °C for 30 s, 59 °C for 1 min, 72 °C for 30 s, and then kept at 72 °C for 20 min. Gel electrophoresis on 12.5% polyacrylamide prepared in 1.25× TBE (89 mM Tris base, 89 mM borate, and 1 mM EDTA, pH 8.0) was run at 200 V for 1 h in 1× TBE. After electrophoresis, the gel was stained with 1× GelStar Nucleic Acid Stain (Takara Bio, Shiga, Japan) in 1× TBE for 30 min and photographed. Preparation of TS Primer-Immobilized Electrodes. Disposable chips carrying a gold electrode (Hano Manufacturing, Fukuoka, Japan) were prepared as described previously.17 The holder of the chip was washed with 70% ethanol and ElectroZap water (Life Technologies) quickly to avoid RNase contamination of the electrodes and dipped in a solution containing hydrophobic polymer N102 (NOF Corp., Tokyo, Japan), washed with Milli-Q water, and dried. The chip was set in the holder. A volume of 10 μL of 5 or 25 nM TS primer in 50 mM NaCl was added on the gold electrode and kept at 37 °C for 3 h. After the addition of 300 μL of 1 mM 6mercaptohexanol, the chip was incubated at 37 °C for 1 h and washed with 300 μL of Milli-Q water twice to yield TS primerimmobilized electrodes. Electrochemical Measurements and Enzyme Reaction. The electrochemical measurement was made with a threeelectrode configuration in a portable apparatus described previously,18 where a platinum electrode acted as the counter electrode, Ag/AgCl as the reference electrode, and the TS primer-immobilized electrode as the working electrode. CC was measured in 10 mM Tris-HCl (pH 7.4) containing 50 μM RuHex, and the electrode was washed with 300 μL of Milli-Q water twice and subsequently washed with 100 μL of Milli-Q water containing 40 units/mL RNase Inhibitor. For the telomerase reaction, 20 μL of a reaction solution prepared above was placed on the electrode and incubated at 37 °C for 30 min. After washing the electrode with 300 μL of Milli-Q water twice, CC was taken again under the same conditions. In the Cottrell equation, eq 1, for chronocoulograms (plot of Q vs t1/2) in the absence and presence of RuHex, the y-intercept
at time zero represents Qdl (capacitive charge, □ in Figure 2A) or Qbefore (charge before elongation reaction, ■ in Figure 2A)
Figure 2. (A) Chronocoulometric traces for the TS-primer immobilized electrode before (squares) and after elongation reaction (circles) in the absence (open symbols) and presence of RuHex (closed symbols). (B) The correlation of the number (x) of elongation in (TTAGGG)x with the TS-primer density on the electrode.
and ΓDNA is derived from eqs 2 and 3 using the obtained Qdl and Qbefore. After telomerase reaction of this electrode, the yintercept at time zero in the chronocoulogram in the presence of RuHex gives Qafter (charge after elongation reaction, ● in Figure 2A). Accordingly, the elongation time x in (TTAGGG)x is given by eqs 4 and 5 using the obtained Qbefore and Qafter.
Q=
2nFAD01/2C0+ 1/2 + Q dl + nFA Γ0 t π1/2
(1)
Γ0 =
(Q before − Q dl) nFA
(2)
⎛z⎞ ΓDNA = Γ0⎜ ⎟NA ⎝m⎠ ΔΓ =
(3)
(Q after − Q before) nFA
(TTAGGG)x =
ΔΓ(z /6)NA ΓDNA
(4)
(5)
The parameters used are as follows: n, number of electrons per molecule for reduction (n = 3); F, Faraday constant (C/ equiv); A, electrode area (cm2); Qdl, capacitive charge (C); Γ0, 1773
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amount of redox marker, RuHex (mol/cm2); ΓDNA, probe surface density (molecules/cm2); m, the number of bases in the probe DNA (m = 30); ΔΓ, elongated products per electrode area (mol/cm2); z, charge of the redox molecule (z = 3); NA, Avogadro’s number (molecules/mol); Qbefore, charge before elongation reaction; Qafter, charge after elongation reaction, and (TTAGGG)x, average elongation time per hexanucleotide per primer molecule. The condition of CC measurements was as follows: potential step from 0.1 to −0.4 V, step = 1, pulse width = 0.25 s, sample interval = 5 ms, quiet time = 2.0 s, sensitivity = 1× 10−5 A/V. The telomerase reaction solution (20 μL) consisted of 20 mM Tris-HCl (pH 8.5), 1.5 mM MgCl2, 63 mM KCl, 0.05% Tween 20, 20 μM dNTP mixture, and 25-HeLa cell extracts. Telomerase Inhibitor Assay. Compounds used in telomerase inhibitor assay were the followings: TMPyP4, PIPER, AZT, inhibitor III, inhibitor V, EtBr, guanidine thiocyanate, FND3, FND7, and TND. A volume of 10 μL of telomerase reaction solution of 25-cell extracts containing 2.5 μM each of the compounds was incubated at 37 °C for 30 min and the extended degree evaluated electrochemically. For TMPyP4 and PIPER, the inhibitor assay was run by the TRAP with a TRAPeze kit and CC at varied dye concentrations. Thus, 20 μL of telomerase reaction solution of 100-cell extracts containing 0, 6.8, 13, 25, or 50 μM TMPyP4 or PIPER were incubated at 37 °C for 30 min and analyzed by gel electrophoresis. The electrochemical measurement was conducted under identical conditions and the extended degree evaluated electrochemically.
total of 11 ladders were observed in this gel, indicating that as long as 66-meric oligonucleotides were formed mainly under these conditions. This is in agreement with the result obtained from the electrochemical assay (Figure 3B, 100-cell extracts). In summary, electrochemical assay can detect elongated telomere DNA without PCR and gel electrophoresis. Effect of TS Primer Density on the Electrode. Electrodes carrying varied densities of TS primer were prepared by placing different concentrations of TS-primer on the electrode. They were treated with extracts of 25 HeLa cells and the length of the oligonucleotide elongated was plotted against the density of the TS primer immobilized. As shown in Figure 2B, elongation was suppressed as the surface density of the TS primer increased. Elongation was observed at and below 1.0 × 1011 molecules/cm2, where the average distance between the TS primers was about 20 nm. Human telomerase consists of 127 kDa of the catalytic subunit hTERT, 150 kDa (451 nt) of RNA units (TR), and 240 kDa of hTEP1,11 but the exact size of holo enzyme is not known yet, whereas the size of TERT (70 kDa) of Tribolium castaneum enzyme was reported to be 13 nm.19,20 Taken together, the surface density of the TS primer on the electrode required was at least 4.0 × 1010 molecules/ cm2, but electrodes of this low density were difficult to prepare reproducibly and those carrying the TS primer at 4.0 × 1010 molecules/cm2 (distance between TS primers is about 50 nm) were used in the following experiments, where a 120 mer of the elongated oligonucleotide was observed after treatment with 25-cell extracts. Dependence on the Number of HeLa Cells. The elongated oligonucleotide was estimated by CC with various amounts of HeLa cell extracts. The length of the elongated oligonucleotide increased over 5−1 000 cells as shown in Figure 3B. The elongated oligonucleotides of 40−80 mer were observed at 37 °C for 30 min in this system, indicating the elongation rate of 1.3−2.7 mer/min. The fact that the elongated oligonucleotide of 66 mer was observed in the gel electrophoresis in the TRAP assay points also to an elongation rate of 2.2 mer/min. The telomerase elongation rate of 0.2−0.9 mer/min was estimated by surface plasmon resonance.21 Given various differences in experimental conditions in terms of the type of tumor cells used and the sequence of the TS primer on the surface, the rates obtained here and in the related system may be comparable. Electrochemical Telomerase Inhibition Assay for Drugs. The telomerase inhibiting ability of 10 arbitrarily selected drug candidates was examined with 25-cell extracts by the electrochemical telomerase assay as well as by TRAP. Under conditions where the TTAGGG sequence was extended 11 times on average, AZT, FND7, and guanidine thiocyanate at 2.5 μM each hardly affected the extension (Figure 4). Addition of TMPyP4,9 inhibitor III, EtBr, TND, or FND3 suppressed the extension to 4−5.7 times, while the extension was smaller than 4 with inhibitor V and PIPER.10 Especially, no telomerase extension was observed with 2.5 μM inhibitor V whose IC50 (50% inhibitory concentration) was 4.5 μM.11 Incidentally, when analyzed by TRAP, neither the ladders nor the markers observed in their absence were visible in the presence of 6.8, 13, 25, or 50 μM TMPyP4 or PIPER, indicating PCR was inhibited indeed by either of them.7 There are two mechanisms of telomerase inhibition envisaged: Inhibitors may bind to telomerase directly to inhibit its activity. Inhibitor V and PIPER10 in which the extension was smaller than 4 times seem to fall in this class. Alternatively,
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RESULTS AND DISCUSSION Elongation Reaction on the Electrode by Telomerase. The TS primer was immobilized on the electrode through a thymine octamer as the linker. The resulting electrode was treated with 10 μL of extracts from 25 HeLa cells at 37 °C for 30 min and CC measured before and after the treatment. As shown in Figure 2A, the charge increased after the elongation reaction. When the reaction solution did not contain cell extracts, virtually no charge increase was observed (background levels of increase ratio, (Qbefore − Qdl)/(Qafter − Qdl), were lower than 2%). To prove that the increased charge derived from the DNA elongated on the electrode, the sample after the telomerase reaction (100-cell extracts) was subjected to PCR amplification and gel electrophoresis. Bands were observed only in the presence of HeLa cell extracts as shown in Figure 3A. A
Figure 3. (A) Gel electropherogram after PCR amplification of the cut disk electrode treated with telomerase in the absence (a) and presence of 100-cell extracts (b). (B) Plot of elongated product against the number of HeLa cells. Immobilization density of the TS-primer was 4.0 × 1010 molecules/cm2 in this experiment. 1774
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candidates for telomerase and for studying the mechanism of inhibition.
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AUTHOR INFORMATION
Corresponding Author
*Phone: +81-93-884 3322. Fax: +81-93-884 3322. E-mail:
[email protected].
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ACKNOWLEDGMENTS We thank Hiroki Kondo for reading the manuscript. This work was supported in part by Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) and the Japan Society for the Promotion of Science to S.S.
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Figure 4. Telomerase inhibition assay by the electrochemical method with 2.5 μM each of inhibitor III, inhibitor V, TMPyP4, PIPER, AZT, guanidine thiocyanate, EtBr, FND3, FND6, or TND in the presence of 25-cell extracts at 37 °C for 30 min. Immobilization density of the TSprimer was 4 × 1010 molecules/cm2 in this experiment.
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compounds such as TMPyP4 bind to the quadruplex DNA generated by telomerase,3,7,9,10,12 thereby blocking further DNA extension.22 Unlike other methods, our electrochemical method is capable of discriminating these two possibilities. This was further explored with TMPyP4 and PIPER by changing their concentration with extracts of 100 HeLa cells. In the electrochemical telomerase assay, the lengths of the elongated oligonucleotides decreased reciprocally in the presence of TMPyP4 or PIPER to give IC50 values of 5.5 and 15 μM, respectively (Figure 5). These values compare fairly
Figure 5. Telomerase inhibition assay by the electrochemical method with 6.8, 13, 25, and 50 μM TMPyP4 (A) or PIPER (B) in the presence of 100-cell extracts at 37 °C for 30 min. Immobilization density of the TS-primer was 4 × 1010 molecules/cm2 in this experiment.
well with 8−10 μM6 or 8.9 μM for TMPyP4.7 Likewise, the IC50 of PIPER was reported as >20 μM.7 It is noted that these literature values were obtained with 2 μg and 0.5 μg of protein prepared from HEK293T and A2780 cells, respectively, whereas 0.2 μg of protein from 100 HeLa cells were used in this experiment. In other words, our electrochemical method is comparable or even higher in analytical sensitivity than the existing methods. It is also noted that all of these assays were completed in an hour, as long as TS primer-immobilized electrodes and samples to be analyzed are ready. Likewise, each of the dose−response curves illustrated in Figure 5 was obtained within an hour. Hence, this electrochemical method will be useful for high-throughput screening of inhibitor 1775
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