of 5
Chemistry & Biology, Volume
22
Supplemental Information
DNA Electrochemistry Shows
DNMT1 Methyltransferase Hyperactivity
in Colorectal Tumors
Ariel L. Furst, and Jacqueline K. Barton
Supplemental Data
Figure S1
.
DNMT1 activity measured electrochemically without normalization to
the unmethylated substrate
, as shown in Figure 4
.
The fold excess activity measured
electrochemically shows hyperactivity in all but two of the tissue samples. Those that do
not show hyperactivity have equivalent DNMT1 activity between tumor and normal
tissue. The data for activity on the hemimethylate
d substrate for the tumor tissue are
normalized to that of the normal adjacent tissue.
C
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Figure S2
.
DNMT1 activity measured with radioactivity
without normalization to
the unmethylated substrate
, as shown in Figure 4
.
The fold excess activity mea
sured
with radioactive labeling does not show similar hyperactivity in the tumor sample as
when measured electrochemically. The data for activity on the hemimethylated substrate
for the tumor tissue are normalized to that of the normal adjacent tissue.
C
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l
s
A
B
C
D
E
F
G
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2
3
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)
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Figure S3
.
Sample
Western blots used for quantification
in Figure 5
.
Shown are the
bands used for quantification of differential DNMT1 protein content in the tumor as
compared to normal tissue. The top bars show the DNMT1 in each tissue set as
well as
cell lysate. The bottom bars show the loading control, Lamin A, to which each DNMT1
concentration is normalized. 60 μg of protein per lane were loaded onto the gels. 1
o
antibody for DNMT1 (R & D) and Lamin A (Santa Cruz) is incubated overnight.
Goat
anti
-
rabbit 2
o
antibody (Abcam) or Donkey Anti Sheep for DNMT1 (Santa Cruz) is
subsequently incubated, followed by imaging. Quantification is performed with Li
-
COR
Odyssey Image Studio software.
Supplemental Experimental Procedures
Methods
and Materials
DNA Synthesis and Purification
Hexynyl
-
modified oligonucleotides were synthesized on an Applied Biosystems
3400 DNA synthesizer, and unmodified complementary DNA was purchased from IDT.
The terminal hexynyl moiety that was incorporated into
the 5
ʹ
end of one of the strands
was purchased from Glen Research. DNA was deprotected and removed from solid
support with ammonium hydroxide (60 °C for 16 h). Following a preliminary round of
HPLC, oligonucleotides were treated with 80% acetic acid in w
ater for 20 minutes. Each
oligonucleotide was purified by high
-
performance liquid chromatography (HPLC) on a
PLRP
-
S column (Agilent) with a gradient of acetonitrile and 50 mM ammonium acetate.
Oligonucleotides were desalted by ethanol precipitation and qu
antified by ultraviolet
-
visible spectrophotometry based on their extinction coefficients at 260 nm (IDT Oligo
Analyzer). Masses were verified by matrix
-
assisted laser desorption (MALDI) mass
spectrometry. Based on the UV
-
Vis quantification of the DNA, DN
A duplexes were
formed by thermally annealing equimolar amounts of single
-
stranded oligonucleotides in
deoxygenated phosphate buffer (5 mM phosphate, 50 mM NaCl, pH 7.0) at 90 °C for 5
minutes followed by slowly cooling to 25 °C.
The following sequences we
re prepared:
Alkyne: 5
ʹ
-
C
2
-
(CH
2
)
6
-
GA CTG AGT ACT
GCG CGC
ACT GAT AGC
-
3
ʹ
Unmodified Complement: 5
ʹ
-
GCT ATC AGT
GCG CGC
AGT ACT CAG TC
-
3
ʹ
Methylated Complement: 5
ʹ
-
GCT ATC AGT
GCG C
m
GC
AGT ACT CAG TC
-
3
ʹ
The
BssHII
restriction site is shown in red.