S
1
SUPP
ORTING
INFORMATION
Multiplexed Electrochemistry of DNA
-
bound Metalloproteins
Catrina G. Pheeney, Anna R. Arnold, Michael A. Grodick,
and Jacqueline K. Barton*
Division of Chemistry and Chemical Engineering, California Institute of Technology,
Pasadena
, California 91125, USA
Email
jkbarton@caltech.edu
Primer Sequences
Mutation
Primer
Y205H
5'
-
TGT GAA
CAC
AAA GAG AAA GTT GAC ATC TGA GGA TCC GGC TGC
TAA C
-
3' (Forward)
5'
-
GTT AGC AGC CGG ATC CTC AGA TGT C
AA CTT TCT CTT T
GT
G
TT
CAC A
-
3' (Reverse)
E200K
5'
-
GCC CCG CTG TGG CTC TTG TAT TAT T
AA
A
GA TCT TTG TGA ATA C
-
3' (Forward)
5'
-
GTA TTC ACA AAG ATC
TTT
AAT AAT ACA AGA GCC ACA GCG GGG C
-
3' (Reverse)
K208E
5'
-
GTG AAT ACA AAG AG
G
AA
G TTG ACA TCT GAG GA
T CCG GCT GCT
AAC
-
3' (Forward)
5'
-
GTT AGC AGC CGG ATC CTC AGA TGT CAA C
TT
C
CT CTT TGT ATT
CAC
-
3' (Reverse)
S
2
Wild Type and Mutant EndoIII
Purification Protocol
A stock of BL21star
-
(DE3)pLysS containing a pET11
-
ubiquitin
-
His
6
-
nth
construct was used to
inoculate a 100 mL culture of LB containing 100
μ
g/mL of ampicillin and 35
μ
g/mL of chloramphenicol.
This culture was grown overnight at 37 °C with shaking. Then, 1 mL of the overnight culture was used to
inoculate each of four 1L cultures of LB containin
g the same amount of ampicillin and chloramphenicol
as the overnight culture. The 1L cultures were shaken at 37 °C until the OD
600
reached ~0.6
-
0.8. Enough
i
sopropyl
β
-
D
-
1
-
thiogalactopyranoside
(IPTG) was then added to bring the total concentration of IPTG to
300
μ
M. The cultures were subsequently shaken at 150 rpm for ~3.5 hours at 30° C. The cells were
collected by centrifugation at 5,500 rpm for 15 minutes
, flash
-
frozen, and stored at
-
80 ºC. All
subsequent steps were carried out at 4 °C or on ice. On the day of the purification the pellet was
resuspended in 250 mL of Buffer A (20 mM sodium phosphate, pH 7.4, 250 mM NaCl, 5 mM DTT, 5%
glycerol, DNase (Roc
he), RNase (Roche), and EDTA
-
free protease inhibitor cocktail tablets (Roch
e)).
The resuspended cells were
lysed via microfluidization. The cell lysate was clarified by centrifugation at
17,000
g
for 30 minutes. Enough NaCl was added to the resulting super
natant to bring the NaCl
concentration to 500 mM. The supernatant was then loaded onto a Histrap HP column (GE Healthcare)
that had been equilibrated with buffer B (20 mM sodium phosphate, pH 7.4, 500 mM NaCl, 1 mM DTT).
After washing with buffer B, EndoII
I was eluted using a gradient from 0
-
100% buffer C (20 mM sodium
phosphate, pH 7.4, 500 mM NaCl, 500 mM imidazole, 1 mM DTT) over 20 column volumes. A yellow
band that eluted at ~100 mM imidazole was collected. This imidazole
-
containing buffer was then
i
mmediately exchanged into buffer D (20 mM sodium phosphate, pH 7.4, 500 mM NaCl, 0.5 mM EDTA,
and
20 %
glycerol) using a HiPrep 26/10 desalting column (GE Healthcare). The protein solution, now in
buffer D, was concentrated down to ~5 mL using 10,000 MWCO
Amicon Ultra
-
15 centrifugation filter
units (Millipore) and was loaded onto a HiLoad Superdex 16/600 75 pg size exclusion column (GE
Healthcare) that had been equilibrated with the protein storage buffer (20 mM sodium phosphate, pH 7.4,
100 mM NaCl, 0.5 mM
EDTA,
20 %
glycerol). A clean peak that eluted after approximately 50
-
55 mL of
buffer had passed through the column was collected and concentrated to achieve a final concentration of
S
3
~
100
μ
M as quantified using an
ε
410
of 17,000 M
-
1
cm
-
1
(30
)
. The protein
was then aliquoted, flash
-
frozen
in liquid nitrogen and stored at
-
80 °C. The approximate yield was 6 mg/L. The purity of the protein was
determined to be >95% as analyzed by SDS
-
PAGE (data not shown).
S
4
Equation
S
1.
Circular dichroism (CD) thermal
denaturation data was fit using a n
on
-
linear
least
-
squares regression to a simple two
-
state unfolding model (32), where
Y
is the fractional
ellipticity,
T
is the temperature in Celsius,
Δ
H
m
is the enthalpy at the unfolding transition,
T
m
is
the melting temperature (°C),
R
is the ideal gas constant, while
m
n
and
y
n
describe the pre
-
transition (native protein) slope and y
-
intercept and
m
d
and
y
d
describe the
equivalent values
post
-
transition (denatured protein). Reported errors in T
m
values are derived from this fitting.
S
5
Figure
S1.
Cyclic voltammetry (scan rate = 100 mV/s) of EndoIII (30
μ
M) incubated on closely
packed well
-
matched duplex
DNA
monolaye
rs acquired
in phosphate buffer (
20 mM sodium
phosphate, 100 mM NaCl, 0.5 mM EDTA, 20
% glycerol
,
pH 7.4
) are presented
.
Three different
thicknesses for the Buna N gasket, utilized during the multiplexed chip assembly, were tested:
0.
0
64
′′
(green),
0.
0
32
′′
(red), and 0.
0
20
′′
(red).
With decreasing gasket thickness th
e
signal
generated from EndoIII is shown to increase
while the background contributions decrease.
Inset
:
Background signals acquired prior to the addition of EndoIII in spermid
ine buffer (
5 mM
phosphate, 5
0
mM NaCl, 40 mM MgCl
2
, 5 mM spermidine
,
pH 7
.0)
.
S
6
Figure
S2.
Signal accumulation of EndoIII as a function of time at various concentrations on
both closely and loosely packed dsDNA monolayers
formed
in the presence and a
bsence of 100
mM MgCl
2
during assembly
, respectively
.
The peak signal current was quantified based on the
reductive signal of EndoIII in the cyclic voltammaogram (scan rate = 100 mV/s) acquired in
phosphate buffer (
20 mM sodium phosphate, 100 mM NaCl, 0.5
mM EDTA,
20 %
glycerol
,
pH
7.4
).
S
7
Figure S3.
Enzymatic assay for EndoIII glycosylase activity. Glycosylase activity was
determined for each mutant (Y205H, K208E, and E200K) compared to wild type EndoIII based
on previously
established methods (22). For this assay, protein sample
s (1
μ
M) were incubated
for 15 min at 37 ºC with 5
′
-
32
P
-
radiolabeled 35
-
mer duplex DNA
(100 nM)
modified with
5
-
hydroxy
uracil
, a substrate for EndoIII, in 10 mM Tris HCl, 1 mM EDTA, 50 mM NaCl, pH 7.6
.
Reaction
s
were
then
quenched
by adding of 1 M NaOH
t
o a final concentration of 100 mM
NaOH
, dried, and electrophoresed through 20% denaturing PAGE at 90 W for 1.5 hours
.
Glycosylase activity of EndoIII results in the appearance of the 14
-
mer cleavage product in the
denaturing gel. The glycosylase activity
was determined as the fraction of 14
-
mer product
observed relative to the total quantity of DNA and the percent activity compared to WT (purple)
was
then calculated
for Y205H (green), K208E (blue), and E200K (red). Two control lanes
containing the 35
-
mer
duplexed DNA, yet lacking the enzymatic protein, were
either
untreated
or
treated with the 1 M NaOH.
S
8
Figure
S
4
.
Electrochemical s
tability of EndoIII mutants
. (
Left
)
Protein concentrations were
normalized for electrochemistry coupling to yield
appro
ximately
equivalent signal sizes at early
time points (~ 4 hours).
Cyclic voltammetry (scan rate =100 mV/s) acquired in phosphate buffer
(
20 mM sodium phosphate, 100 mM NaCl, 0.5 mM EDTA,
20 %
glycerol
,
pH 7.4
)
for wild type
(purple), Y205H (green), K208E
(blue), and E200K (red) EndoIII
are presented
.
(
Right
) After
extended incubation (~20
hours) on the multiplexed chip
, the electrochemical signal from the
electrostatic EndoIII mutants and
wild type
protein diminished based on their CVs
. The degree
of si
gnal loss directly correlates with the stability and DNA CT profi
ci
ency of the protein
s
, with
the
remaining
signal size
decreasing in the following order: E200K (
red
), wild type (
purple
),
K208E (
blue
), and finally Y205H (
green
)
which had
no discernible sig
nal remaining.