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FAM: 6-carboxyfluorescein; TAMRA: 6-carboxy-tetramethylrhod-
amine; HEG linker: [(-CH2-CH2-O-)6]). The oligonucleotides were syn-
thesized and HPLC purified by Shanghai Sangon, China.
control primer), and 0.4 mL of 5 UmLÀ1 Taq polymerase. The mix-
tures were incubated at 948C for 90 s, then subjected to 30 cycles
of PCR (948C for 30 s, 528C for 30 s and 728C for 45 s). The PCR
products were separated on 8.0% polyacrylamide gels, run at ap-
proximately 250 V for 100 min. The final gels were stained with
10000 gel red (Biotium, USA) for approximately 15 min, then vi-
sualized under UV illumination (Gel image system, Tanon 1600,
China).
For each measurement, a 50 mm stock of fluorescently labeled DNA
oligonucleotides in water was made, and then diluted to 0.8 mm in
a buffer of 60 mm potassium cacodylate, 10 mm Tris-HCl (pH 7.4).
The DNA was annealed by heating at 958C for 5 min, then slowly
cooled to room temperature and incubated overnight. Each
sample was measured in a total volume of 20 mL, with the final
concentration of DNA being 0.5 mm and small molecules (L or Pt2L)
being 1.0 mm. Melting curves were obtained using a Roche Light-
Cycler 2.0 real-time PCR machine. Fluorescence data were recorded
with an excitation wavelength at 470 nm, detection wavelength at
530 nm, at intervals of 18C during a temperature range of 37–
998C, with each temperature being maintained for 30 s. Data anal-
ysis was carried out with Origin 8.0 (OriginLab Corp.).
Continuous-wave EPR spectroscopy
The hTel DNA (5’-AGGGTTAGGGT TAGGGTTAGGG-3’) was obtained
commercially (Integrated DNA Technology, Coralville) and purified
by HPLC.[18c] To anneal the DNA, hTel DNA was first heated at 958C
for 5 min, then appropriate buffer was added to a final concentra-
tion of 100 mm NaCl, 10 mm Tris-HCl (pH 7.4). The mixture was
cooled to room temperature and then incubated at 48C overnight.
To analyze the binding of Pt2L to hTel, the desired amount of an-
nealed hTel was incubated with 100 mm of Pt2L in a solution of
100 mm NaCl, 10 mm Tris-HCl at pH 7.4. Incubations were carried
out for 30 min at room temperature, following which cw-EPR spec-
tra were acquired.
Telomere-repeat amplification protocol assays
Materials (dNTP mix, RNase inhibitor, and Taq polymerase) were
obtained from TaKaRa Biotechnology. DNA oligonucleotide primers
were synthesized and PAGE purified by Invitrogen Technology,
Shanghai, China. The primer sequences are: TS, 5’-AATCCGTCGA
GCAGAGTT-3’; ACX, 5’-GCGCGGCTTA CCCTTACCC TTACCCTAACC-3’;
NT, 5’-ATCGCTTCTC GGCCTTTT-3’; and TSNT, 5’-AATCCGTCGA GCA-
GAGTTAA AAGGCCGAGA AGCGAT-3’. The experiments included
three steps.
Each cw-EPR spectrum was obtained using a 5 mL sample loaded
in a round glass capillary (0.6 mm i.d.0.8 mm o.d., Vitrocom, Inc.,
Mountain Lakes) sealed at one end. Measurements were carried
out at room temperature on an X-band Bruker EMX spectrometer
using an ER4119HS resonator. The incident microwave power was
2 mW, and the field modulation was 1 G at a frequency of 100 kHz.
Each spectrum was acquired with 512 points, corresponding to
a spectral range of 100 G. All spectra were corrected for back-
ground and baseline, and if needed, normalized following reported
procedures.[22]
Step 1: primer elongation by telomerase in the presence of
Pt2L: Telomerase extract was prepared from HeLa cells using the
NP-40 lysis buffer, which contained 10 mm Tris-HCl, pH 8.0, 1.0 mm
MgCl2, 1.0 mm ethylene glycol tetraacetic acid (EGTA), 1.0% NP-40,
0.25 mm sodium deoxycholate, 10% glycerol, 150 mm NaCl,
0.1 mm phenylmethanesulfonylfluoride (PMSF) and 5.0 mm b-mer-
captoethanol (b-ME). Each reaction was carried out in a volume of
50 mL. The reaction mixture included 5.0 mL of 10TRAP buffer,
1.0 mL of 0.05 mgmLÀ1 BSA, 4.0 mL of 2.5 mm dNTP mix, 1.0 mL of
100 ngmLÀ1 TS primer, 0.5 mL of 2 UmLÀ1 RNase inhibitor, 1.0 mL tel-
omerase extract (obtained from ~500 cells), 32.5 mL diethyl pyro-
carbonate-treated (DEPC) water, and 5.0 mL of Pt2L at the desired
concentration. The reaction mixture was transferred to a thermal
cycler (Bio-Rad S1000, USA), then incubated at 308C for 30 min,
948C for 10 min, and a final maintenance temperature of 208C. A
number of negative controls were included. Negative control 1: tel-
omerase was heated inactivated by incubation at 858C for 10 min
prior to primer elongation; negative control 2: telomerase was
omitted, and the reactions were carried out using 1.0 mL lysis
buffer instead of 1.0 mL telomerase extract; negative control 3: Pt2L
was omitted, and the reactions were carried out using 5.0 mL of
DEPC-treated water instead of the Pt2L complex.
Double electron electron resonance spectroscopy
Measurements were carried out on samples of Pt2L-bound hTel. To
prepare a sample, appropriate amount of Pt2L and annealed hTel
DNA (see above) were mixed in a solution containing 100 mm NaCl
and 10 mm Tris-HCl (pH 7.4). The mixture was incubated at room
temperature for 30 min, and then glycerol solution with the proper
amount of salt was added to obtain a sample with a final concen-
tration of 100 mm NaCl, 10 mm Tris-HCl (pH 7.4) and 50% glycerol
(v/v). The sample was immediately loaded into a quartz capillary
and flash frozen by submerging in liquid nitrogen, and then used
immediately for measurement.
DEER measurements were carried out at 78 K on a Bruker ELEXSYS
E580 X-band spectrometer equipped with a MD4 resonator. Previ-
ously reported acquisition parameters and procedures[29] were
used with slight modifications (d1=200 ns instead of 128 ns). Spe-
cifically, a dead-time-free four-pulse scheme was used,[30] with the
pump pulse frequency set at the center of the nitroxide spectrum
and the observer frequency at approximately 70 MHz higher. The
observer p pulse was 32 ns, and the pump p pulse was optimized
using a nutation experiment, usually set at 16 ns. The video band-
width was fixed at 20 MHz. The shot repetition time was set at
1000 ms. Accumulation time in each measurement was approxi-
mately 24 h with 100 shots per point. Inter-spin distance distribu-
tions were computed from the resulting dipolar evolution data
using DEER analysis 2013.[31]
Step 2: removal of Pt2L: The QIA quick nucleotide purification kit
(Qiagen, 28304) was used to purify the elongated products and to
remove Pt2L according to the manufacturer’s instructions. As re-
ported, ethanol was found to inhibit the PCR amplification. To
avoid the inhibition effect of residual ethanol in the kit, the final
samples eluted in water were freeze-dried.
Step 3: PCR amplification: PCR was carried out to amplify the tel-
omerase elongation products. For each reaction the purified elon-
gation products were dissolved in 37.6 mL water, then mixed with
5.0 mL 10TRAP buffer (200 mm Tris-HCl, pH 8.3, 15 mm MgCl2,
630 mm KCl, 0.5% Tween-20 and 10 mm EGTA in DEPC-treated
water), 1.0 mL of 5.0 mgmLÀ1 BSA, 4.0 mL of 2.5 mm dNTP mix, 1.0 mL
of 100 ngmLÀ1 TS primer, 1.0 mL of primer mix (100 ngmLÀ1 ACX re-
verse primer, 100 ngmLÀ1 NT primer, and 4.010À14 m TSNT internal
Circular dichroism spectroscopy
Circular dichroism measurements were carried out using the hTel
DNA (5’-AGGGTTAGGGT TAGGGTTAGGG-3’). The DNA was synthe-
Chem. Eur. J. 2016, 22, 3405 – 3413
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