S 1
Supplementary Information
Electrical Detection of TATA Binding Protein at DNA
-Modified
Microelectrodes
Alon A. Gorodetsky, Ali Ebrahim, and Jacqueline K. Barton.
Department of Chemistry and Chemical Engineering, California Institute of Technology,
Pasaden
a, California 91125
Supporting Figure 1: Schematic illustration of the coupling of Nile Blue to the NHS
-
Carboxy
-dT modified DNA on solid support. The solvent was 9:1 N,N
-
dimethylformamide/N,N
-diisopropylethylamine. The overall yields of the rea
ction ranged
from 30% to 80%.
S 2
A.
B.
C.
0
0.3
0.6
230
480
730
Wavelength (nm)
Absorbance (a. u.)
Supporting Figure 2: Characterization and purification of the NB
-DNA conjugate. (A)
Analytical HPLC chromatogram of the pure NB
-DNA conjugate with an acetonitrile
gradient of 5% to 75% over 30 mi
nutes. (B) MALDI mass spectrometry of the pure NB
-
DNA conjugate with the MW
found
= 5498 amu and MW
expected
=5499 amu. (C)
Corresponding UV
-visible absorption spectrum showing the Nile Blue peak at 600 nm
and the DNA peak at 260 nm.
S 3
0
0.25
450
750
Wavelength (nm)
Absorbance (a. u.)
Single Stranded NB
.5 Equivalent
1 Equivalent
5 Equivalents
10 Equivalents
Supporting Figure 3: U
V-
visible absorption spectrum of the Nile Blue modified ssDNA
(sequence
5’-U
GC GTG CTT TAT ATC TC
-3’) recorded
before and after addition of
increasing equimolar amounts of the complement. Significant hypochromicity is
observed for the Nile Blue moiety, th
e location of which is indicated by the U.
S 4
A.
B.
Supporting Figure 4: Square wave voltammetry of a NB
-DNA modified Au on silicon
electrodes of 130 μ m radius (A) and 13 μ m radius (B) in p
H = 7 phosphate buffer
cont
aining 5 mM
P
i
and 50 mM NaCl. Note that the two electrodes were fabricated
together in parallel and run minutes apart yet still did not yield identical potentials.
S 5
.
B.
Supporting Figure 5: The effect of oxygen on cyclic voltammetry at (A) a m
acroelectrode
and (B) a 10 μ m diameter microelectrode at 50 mV/s. Voltammetry in oxygen saturated
buffers is shown in black with voltammetry after ten to fifteen more minutes of vigorous
argon bubbling into the solution in blue. Trace amounts of oxygen hav
e a more
significant effect on voltammetry observed at microelectrodes. The gray to orange traces
in B represent successive voltammograms taken on the same electrode open to air in an
initially degassed solution with a slow, steady increase observed over t
hirty minutes.
S 6
A.
B.
C.
Supporting Figure 6: Cyclic voltammetry of a NB
-DNA modified 10 μ m diameter
electrode at 50 mV/s before addition of TBP in blue and after addition of 300 nM TBP in
red. Initial background voltammetry of the electrode i
s shown in (A) with significant
signal loss observed after addition of TBP. The electrode is subsequently rinsed in
succession (5 minutes each) with 1 M KCl in MQ H
2
O, 1 M KCl in Tris buffer, and
TATA storage buffer. Background voltammograms are recorded f
or the same electrode
before it is used to detect TBP again (B). The electrode can then be taken through another
KCl rinse cycle before being used for detection a third time (C).
S 7
A.
B.
C.
Supporting Figure 7: Cyclic voltammetry of NB
-DNA modi
fied 25 μ m diameter
electrodes at 50 mV/s before addition of TBP in blue and after addition of TBP in red.
The signal attenuation shown is for addition of (A) 3 nM, (B) 15 nM, and (C) 30 nM.
S 8
Protein
Preparation
(1) Human
TATA binding protein was custom
ordered from ProteinOne, Inc.
in
pH = 7,
5 mM NaP
i
, 50 mM NaCl, 4 mM MgCl
2
, 4 mM spermidine, 50 μ M EDTA, 10 %
glycerol buffer. The protein concentration was confirmed using the Bradford Assay Kit
from Bio
-Rad Laboratories. Stock solutions of the protein we
re stored at
-80ºC and
aliquoted under an argon atmosphere. The TATA binding protein utilized for detection
experiments was taken through no more than two freeze/thaw cycles.
(2) Bovine serum albumin was purchased from New England Biolabs, Inc. and utiliz
ed
for experiments as received.
(3) Endonuclease III
was prepared according to established literature procedures (see
Boal, A. K.; Yavin, E.;
Lukianova, O.
A.; O’Shea, V. L.; David, S. S.;
Barton, J. K.
Biochemistry
,
2005
,
4
, 8397.). The enzyme was quanti
fied by UV
-visible spectroscopy as
described in the references therein.
(4) Bam
HI Methyltransferase was purchase
d from New England Biolabs, Inc. Prior to
experiments, the protein was dialyzed overnight in pH = 7, 5 mM NaP
i
, 50 mM NaCl, 4
mM MgCl
2
, 4 mM s
permidine, 50 μ M EDTA, 10 % glycerol buffer to remove excess
dithiothreitol.