S1
Supporting Information
Redmond Red as a Redox Probe for the DNA-mediated Detection of Abasic Site
s
Marisa C. Buzzeo and Jacqueline K. Barton
Division of Chemistry and Chemical Engineering, Cal
ifornia Institute of Technology
Pasadena, California 91125
1. Synthesis of modified DNA
2. Electrochemical experimental conditions
3. Characterization data
S1. HPLC chromotagrams of Redmond Red modified DNA
S2. HPLC chromotagrams of DNA containing abasic site
S3. MALDI-TOF mass spectra
S4. UV-vis spectra of Redmond Red modified DNA
S5. Melting temperature analyses
1. Synthesis of modified DNA
All syntheses were performed using standard phosphoramidite chemis
try. Thiol-modified DNA
strands were synthesized as previously described using a C
6
-disulfide phosphoramidite, available
from Glen Research. Treatment with excess dithiothreitol in 10 mM
Tris buffer (pH 8) for 30
min at ambient temperature resulted in cleavage of the disulfide bond
and concomitant removal
of the dimethoxytrityl (DMT) protecting group. Nap-5 Sephadex columns (
GE Healthcare) were
used to isolate the thiolated strands, and reversed-phase HPLC was
used to purify the DNA
before and after disulfide cleavage (see Figure S1). Redmond Red wa
s incorporated into the
DNA sequence at the desired position as a phosphoramidite (also ava
ilable from Glen Research).
Given the sensitivity of Redmond Red to typical cleavage and deprotect
ion conditions (NH
4
OH
at 60 ºC), less harsh conditions were employed (K
2
CO
3
/MeOH at ambient temperature for 12 –
17 h) for strands containing this modification. Accordingly, phosphoramidite
s containing more
labile protecting groups than standard reagents were required for sy
nthesis (Glen Research). All
synthesized DNA strands were purified by reversed-phase HPLC
before and after removal of
DMT protecting groups using a Varian DYNAMAX C18 semi-preparative
column. Isolated
products were characterized by MALDI-TOF mass spectrometry,
UV-visible spectroscopy, and
melting temperature analysis of hybridized duplexes. Representati
ve purification and
characterization data are shown below
2. Electrochemical experimental conditions
Prior to self-assembly, gold macroelectrodes (1.6 mm nominal diamet
er, BAS) were polished on
a clean, damp microcloth (Buehler) with alumina slurries of decrea
sing particle size (1.0
μ
m,
0.05
μ
m, Buehler). Electrodes were then etched in H
2
SO
4
to expose a fresh, clean surface as well
as determine the working area, and rinsed copiously with deionized H
2
O. Immediately following,
a 20
μ
l droplet of 50
μ
M duplex DNA was immobilized on the gold surface and monolayers
were allowed to self-assemble overnight at 5 ºC. Duplexes were
incubated in the presence of 100
mM MgCl
2
in order to encourage the formation of densely packed films. Ele
ctrodes were rinsed
thoroughly in phosphate buffer (5 mM sodium phosphate, 50 mM NaCl, pH 7) prior to
electrochemical measurement.
S2
0
500
1000
1500
2000
2500
0
10
20
30
40
50
time (min)
absorbance (mAU) .
0
400
800
1200
1600
2000
0
10
20
30
40
50
60
time (min)
absorbance (mAU) .
0
40
80
15
25
35
time (min)
absorbance (mAU) .
3. Characterization data
Figure S1a.
HPLC chromatogram for Redmond Red-modified DNA (SH- 5’ AGT
ACT GCA GTA
RR
GCG 3’), prior to disulfide cleavage and DMT deprotection (black
: 260nm, blue: 295 nm, red: 580 nm).
Failed oligonucleotide syntheses appear around 10 min, while the product elutes around 28 min. Gr
adient:
100% 50mM NH
4
OAc / 0% MeCN to 50% 50 mM NH
4
OAc / 50% MeCN in 50 min (1% / min), at 3.5
ml / min.
Figure S1b.
HPLC chromatogram for Redmond Red-modified DNA, following disulf
ide cleavage
achieved by treatment with DTT (black: 260nm, blue: 295 nm, red: 580 nm
). The first main peak,
centered around 24 min, corresponds to the product. Mass spectrometry conf
irmed that the side peaks that
follow are not the desired product. Gradient: 100% 50mM NH
4
OAc / 0% MeCN to 40% 50 mM NH
4
OAc
/ 60% MeCN in 80 min (0.5% / min), at 3.5 ml / min. Inset: magnified
view of the spectrum recorded at
580 nm.
S3
0
400
800
1200
1600
2000
0
5
10
15
20
25
time (min)
absorbance (mAU) .
0
100
200
300
400
500
600
0
4
8
12
16
time (min)
absorbance (mAU) .
Figure S2a.
HPLC chromatogram for DNA strand containing an abasic site (
5’ CGC __ TAC TGC AGT
ACT 3’ where “__” indicates the abasic site), prior to remov
al of DMT protecting group (black: 260nm,
red: 294 nm). Product peak appears around 14 min. Gradient: 95% 50mM NH
4
OAc / 5% MeCN to 35%
50 mM NH
4
OAc / 65% MeCN in 40 min (2% / min), at 3.5 ml / min.
Figure S2b.
HPLC chromatogram for DNA strand containing an abasic site, (
5’ CGC __ TAC TGC AGT
ACT 3’) following deprotection (black: 260nm, red: 294 nm). Deprotection i
s achieved by incubating
DNA in 80% CH
3
HOOH for 30 min and then precipitating with cold EtOH. Gradient: 95
% 50mM
NH
4
OAc / 5% MeCN to 75% 50 mM NH
4
OAc / 25% MeCN in 40 min (0.5% / min), at 3.5 ml / min.
Product confirmed by mass spectrometry.