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Basic Canada Inc. and used without further purification. They were
dissolved in 10 mm Tris-HCl (pH 7.4) buffer in the presence of
to 30 mm followed by the addition of 10% volume of 2-propanol. A
linear ion trap-orbitrap mass spectrometer (Orbitrap Elite, Thermo
Fisher Scientific, Bremen, Germany) equipped with a home-built
nanoflow source was used to provide the mass spectra of alkyla-
tion adducts. Infusion with the injected gradient of 80% acetoni-
trile and 20% 150 mm ammonium acetate (pH 7.0) was loaded
into quartz emitters pulled in house, which were held at an ioniza-
tion voltage of ca. 1.5 kV and a capillary temperature of 1508C.
1
50 mm NaCl or KCl. All DNAs were annealed by heating at 958C
for 5 min, gradually cooled to RT, and then incubated at 48C over-
night before use. Double-stranded DNAs (dsDNA) were annealed
by using the prepared DNA solution with an equal amount of the
complementary oligonucleotide by the same heating-cooling tem-
perature program used for the preparation of DNA mentioned
above.
Nuclease digestion and LC-MS monitoring of alkylated ad-
ducts
CD and melting point measurements
CD spectra of the oligonucleotides were collected with a Jasco J-
À1
The reacted mixtures (50 mL, 20 ngmL ) were digested for 12 h ac-
7
15 spectropolarimeter (Jasco, Japan). A quartz cuvette with 1 cm
[36]
cording to a reported protocol. The enzymes were removed by
optical path length was used to obtain spectra, which were record-
ed from 210 to 350 nm at 2 nm bandwidth and an instrument
chloroform extraction at the end of enzymatic digestion. The re-
sulting aqueous layers were dried, reconstituted in doubly distilled
water, and analyzed by LC-MS. A linear ion trap-orbitrap mass
spectrometer (Orbitrap Elite, Thermo Fisher Scientific, Bremen, Ger-
many) coupled online with a UHPLC system (ACQUITY UPLC,
Waters, Millford, MA) was used. The hydrolyzed residuals were sep-
arated by using a BEH-C18 column (Waters). The column tempera-
ture was maintained at 458C. Eluting buffers were Buffer A (2%
À1
scanning speed of 50 nmmin with 8 s response time. The meas-
urements were the averages of two repetitions recorded at RT.
Spectra were baseline-corrected and the signal contributions of
the buffer were subtracted. Melting points (T ) of H26 and c-MYC
m
were measured by monitoring the CD signal at 265 nm, which is
a characteristic peak of the quadruplex, in the absence and pres-
ence of compounds at the indicated ratios. The temperature was
acetonitrile and 0.1% formic acid in ddH O) and Buffer B (0.1%
formic acid in acetonitrile) using gradients of 0 min (0.5% B), 1 min
À1
2
elevated gradually from 10 to 958C at a rate of 1.08Cmin .
(
1% B), 4 min (4% B), 10 min (95% B). The mass spectrometer was
DNA alkylation, denaturing polyacrylamide gel electropho-
resis, and gel visualization
operated in the positive ion mode and set to one full FT-MS scan
(m/z 200–1200) with 60,000 resolution.
DNA alkylation experiments were generally conducted at 378C for
the given time, and were terminated by the addition of 2-mercap-
toethanol (10 mL), and left for an additional 3 min. To the reaction
mixture was then added Herring sperm DNA (1 mL) (Invitrogen),
and precipitation was induced by the addition of cold ethanol. Pre-
cipitated DNA was then dissolved in gel loading buffer II (Ambion),
denatured at 908C for 5 min, and immediately resolved in 20%
polyacrylamide 7m urea sequencing gel run at 1500 V constant
Voltage for 3 h. The resulting gels were excited at 488 nm and the
fluorescence of FAM was detected with a Typhoon 9400 (GE
Healthcare, America) imager. The fluorescence intensity signals
were further processed by using ImageQuant 5.2 software. The al-
kylation yield was calculated from the integral area of the alkylated
bands divided by the total integral area of the alkylated bands and
the nonalkylated band. The reported values, which are given as
percentages, are the mean of at least three runs.
Determination of the alkylation sites by treatment with pi-
peridine
BMVC-C3M (0.5, 1, and 2 mm) was incubated with 5’-FAM labeled
+
+
DNA (1 mm) in K - or Na -containing buffers for 20 h at 378C, re-
spectively. Alkylation was terminated by the addition of 2-mercap-
toethanol (10 mL) and the mixture was left for an additional 3 min.
Maxam-Gilbert dG and dG+dA sequencing conditions were ap-
plied to provide the reference for the fragment migration. Dimeth-
yl sulfate (DMS) (dG lane reference): the nonalkylated DNA was dis-
solved in 4 mL of DMS for 10 s at 378C, and terminated by the ad-
dition of 2-mercaptoethanol (10 mL). Formic acid (dG+dA lane ref-
erence): the nonalkylated DNA was dissolved in 50 mL formic acid
for 3 min at 378C. Herring sperm DNA (1 mL) (Invitrogen) was then
added. After ethanol precipitation, the resulting residues were di-
rectly incubated with 50 mL of 0.7m piperidine at 908C for 25 min,
which induced cleavage at the depurinated sites. After evaporation
of piperidine, the gel running conditions and visualization de-
scribed above in the general method were used to obtain the gel
images.
Competition experiments using different molar ratios of
cold competitors
5
’-FAM labeled H26 (1 mm) and BMVC-C3M (2 mm) were mixed with
various concentrations of respective cold DNA competitors includ-
ing H26, scrambled ssR26, and dsH26 to give the molar ratios of
competitor/labeled H26 as 0.5, 1, 2, 5, and 10. After incubation at
Determination of the alkylation sites by 3’-exonuclease di-
gestion
3
78C for 20 h, standard workup, gel running conditions and visuali-
zation described above in the general method were used to obtain
the gel images. The relative yield assuming the adduct formation
in the absence of competitor as 100% in each experiment, is re-
ported. The reported values, which are given as percentages, are
the mean of at least three runs.
Nonalkylated H26 and alkylated adducts were first resolved by
20% polyacrylamide 7m urea gel, then the band of interest was vi-
sualized by UV shadowing, excised, and extracted by using the
crush-and-soak method. The eluted solutions were then concen-
trated by using an Amicon Ultra 0.5 mL 3 kDa centrifugal filter. The
resulting alkylated H26 were incubated in 10 mm Tris-HCl buffer
À1
(
pH 8.0) containing 2 mm MgCl and 0.5 mgmL transfer RNA with
2
Mass spectroscopic analysis of BMVC-C3M/DNA adduct
the 3’-exonuclease phosphodiesterase I from Crotalus adamanteus
venom (sigma) at 0.08 UmL for 30 min at 378C. The partial diges-
tion of nonalkylated H26 was incubated with 0.001 UmL . The di-
gested fragments were subjected to the gel running conditions
À1
Either 1 mm H26, H24, or c-MYC was reacted with 1 mm BMVC-C3M
at 378C for 20 h. The respective solution was exchanged by ultrafil-
tration to 150 mm ammonium acetate (pH 7.0) and concentrated
À1
Chem. Eur. J. 2015, 21, 17379 – 17390
17389
ꢀ 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim