S1
SUPPORTING INFORMATION
[Ru(Me
4
phen)
2
dppz]
2+
, a Light Switch for DNA Mismatches
Adam N. Boynton, Lionel Marcélis, and Jacqueline K.
Barton
Division of Chemistry and Chemical Engineering, Cal
ifornia Institute of Technology, Pasadena,
California 91125, United States
Email: jkbarton@caltech.edu
S2
Materials
All chemicals and starting materials were purchase
d from commercial vendors and used
as received. Dipyrido[3,24
a
:2’,3’4
c
]phenazine (dppz) was prepared according to the lit
erature.
1
UV4Visible spectra were recorded on a Beckman DU 74
00 UV4Visible spectrophotometer
(Beckman Coulter). Oligonucleotides were synthesize
d using standard phosphoramidite
chemistry at Integrated DNA Technologies (Coralvill
e, IA) and purified by HPLC using a C
18
reverse4phase column (Varian, Inc.) on a Hewlett4Pa
ckard 1100 HPLC. The copper complex
Cu(phen)
2
2+
was generated
in situ
by combining CuCl
2
with phen ligand in a 1:3 ratio.
Synthesis
Ru(Me
4
phen)
2
Cl
2
:
Following a modified literature report,
2
RuCl
3
•
n
H
2
O (0.217 g, 0.830
mmol), 3,4,7,84Tetramethyl41,104phenanthroline (0.4
94 g, 2.09 mmol), and LiCl (0.298 g, 7.03
mmol) were combined in a Schlenk flask under argon.
The contents were dissolved in anhydrous
DMF (5 mL), and the solution was heated to 140°C an
d stirred for 4 h while being protected
from light. The contents were cooled to room temper
ature, diluted with acetone (20 mL), and
stored in the freezer overnight. The black precipit
ate was collected by vacuum filtration, washed
three times with 5 mL portions of H
2
O and three times with 5 mL portions of diethyl eth
er, and
dried. The product was used subsequently without fu
rther purification (0.495 g, 73%).
Ru(Me
2
bpy)
2
Cl
2
:
RuCl
3
•
n
H
2
O (0.281 g, 1.07 mmol) was reacted with 5,5’4Dimeth
yl4
2,2’4dipyridine (0.500 g, 2.71 mmol) and LiCl (0.38
5 g, 9.08 mmol) in DMF (15 mL) under the
conditions described for the synthesis of Ru(Me
4
phen)
2
Cl
2
. The product was isolated and used
subsequently without further purification (0.304 g,
63%).
S3
[Ru(Me
4
phen)
2
dppz]X
2
(X = PF
6
or Cl): Dppz ligand (0.025 g, 0.089 mmol) was
combined with Ru(Me
4
phen)
2
Cl
2
(0.057 g, 0.089 mmol) in ethylene glycol (8 mL) an
d heated to
130°C and stirred for 5 h. The reaction was cooled
to room temperature and diluted with H
2
O (8
mL). Excess NH
4
PF
6
was added to precipitate the product, which was co
llected by filtration,
washed copiously with H
2
O and diethyl ether, and dried. (0.084 g, 82%). ESI
(+)MS (
m/z
):
[M/2]
+
found 428.2. The complex was converted to its wate
r4soluble Cl salt by anion exchange
chromatography (Sephadex QAE) and further purified
by preparative HPLC using an isocratic
method of 65% MeOH and 35% H
2
O (containing 0.1% TFA) over 60 min.
1
H NMR (500 MHz,
DMSO4d
6
) δ 9.58 (dd,
J
= 8.2, 1.3 Hz,
2H), 8.54 (dd,
J
= 6.3, 3.4 Hz, 2H), 8.52 (d,
J
= 0.9 Hz,
4H), 8.22 (dd,
J
= 6.6, 3.4 Hz, 2H), 8.13 (dd,
J
= 5.4, 1.3 Hz, 2H), 7.90 (m, 4H), 7.76 (s, 2H),
2.82 (d,
J
= 1.3 Hz, 12H), 2.27 (d,
J
= 3.7 Hz, 12H). The complex was again converted to
its Cl
salt by anion exchange chromatography to remove TFA
anions present from the HPLC
purification.
[Ru(Me
2
bpy)
2
dppz]X
2
(X = PF
6
or Cl): Dppz ligand (0.240 g, 0.851 mmol) was
combined with Ru(Me
2
bpy)
2
Cl
2
(0.304 g, 0.563 mmol) in ethylene glycol and react
ed as
described for the Me
4
phen complex, and the product was collected as its
PF
6
salt (0.521 g,
88.8%). ESI(+)MS (
m/z
): [M/2]
+
found 376.2. The complex was converted to its wate
r4soluble
Cl salt by anion exchange chromatography (Sephadex
QAE) and further purified by preparative
HPLC using a gradient of H
2
O (with 0.1% TFA) to CH
3
CN over 60 min.
1
H NMR (500 MHz,
DMSO4d
6
) δ 9.62 (dd,
J
=
8.2, 1.3 Hz, 2H), 8.71 (d,
J
= 8.4 Hz, 2H), 8.67 (d,
J
= 8.4 Hz, 2H),
8.52 (m, 2H), 8.21 (m, 4H), 8.04 (m, 4H), 7.94 (dd,
J
= 8.4, 1.9 Hz, 2H), 7.54 (dt,
J
= 1.8, 0.8
Hz, 2H), 7.48 (dt,
J
= 1.4, 0.7 Hz, 2H), 2.26 (d,
J
= 0.7 Hz, 6H), 2.08 (d,
J
= 0.7 Hz, 6H). The
Figure S1
: Steady4state luminescence titrations of [Ru(Me
4
phen)
2
dppz]
2+
with well4matched (blue)
and mismatched (red) DNA.
Left plot
: samples prepared in 5 mM tris, 50 mM NaCl, pH 7.5.
Right
plot
: samples prepared in 5 mM tris, 200 mM NaCl, pH 7.5. [Ru] = 2
M,
λ
ex
= 440 nm. Emission
spectra were integrated from 5644820 nm.
S4
complex was again converted to its Cl salt by anion
exchange chromatography to remove TFA
anions present from the HPLC purification.
Steady-State Luminescence Measurements
Luminescence spectra were recorded on an ISS4K2 spe
ctrofluorometer at 25°C. The Ru
complex was excited at 440 nm, and emission spectra
were integrated from 5644820 nm. The Cl
salt of the complex was used for all experiments. I
n all cases, [DNA] is defined as the
concentration of full sequence.
Titrations of [Ru(Me
4
phen)
2
dppz]
2+
with the well4matched and mismatched duplexes
(Figure S1) were used to determine the binding affi
nity of the complex for well4matched and
mismatched sites. For the titration with well4match
ed DNA, the binding affinity is evaluated
using the McGhee4Von Hippel method;
3
a value of 6.75 10
4
M
41
per base pair is obtained with an
occupational factor, n, of 2.3.
In order to evaluate the binding affinity of the co
mplex for the mismatched site, we first
must consider two competing equilibria, expressed b
elow.
+ ⇌ [_]
=
[_]
[
]
[
]
S5
+ ⇌ [_]
=
[_]
[
]
[
]
K
ass
describes the binding equilibrium between the comp
lex,
C
, and the well4matched base pair
sites,
BP
, in the DNA.
K
MM
describes the binding equilibrium between the compl
ex and the
mismatched site,
MM
.
Next, we will express the total concentration of co
mplex as
C
c
; this is kept constant
throughout the titration. We can then define the va
rious molar fractions for the complex as
follows:
=
[]
, the molar fraction of free complex.
=
[_]
, the molar fraction of complex bound to WM base pa
irs.
=
[_]
, the molar fraction of complex bound to MM sites.
Additionally, we express the total concentration of
duplex as
C
ODN
; this value increased
throughout the titration. The variable
R
is introduced as being equal to the ratio
C
ODN
/C
C
, and in
our titration the luminescence of the complex is me
asured as a function of this ratio
R
. The
luminescence intensity,
I
, can be expressed as a function of
R
as follows:
= +
where
and
are equal to the relative emissivity of complex as
sociated with
BP
and
MM
,
respectively.
We must define two final parameters:
x
, the the ratio of well4matched sites to mismatched
sites in the duplex, and
p
, the occupational factor which takes into account
the possible inhibition
of binding by two complexes in close vicinity. We a
re now ready to express the equilibrium
S6
concentrations of free BP and MM sites as follows:
[
]
=
1 −
!"
− #
[
]
=
1 −
!"
− #
[
]
=
!"
−
[
_
]
=
!"
−
Thus,
[]
=
1 −
$ − #
and
[]
= $ −
The binding equilibrium equations are thus rewritte
n as:
=
%
&
'
()*
+), %
and
=
-
&
' * +)-
The expression of
b
and
m
as functions of
f
can thus be obtained:
1 −
$ − #
− = 0
=
1 −
$
1 +
#
$ −
− = 0
=
$
1 +
With
1 = + +
0 = − 1 +
1 −
$
1 +
#
+
$
1 +
S7
0 =
− 1
1 +
#
1 +
+
1 −
$ 1 +
+
$
1 +
#
The expression of the intensity of luminescence,
, can be written as follows:
=
1 −
$
1 +
#
+
$
1 +
The fitting process is realized by an iterative sol
ving to the expression of
f
using the previous
equation.
A global fitting on the data obtained from the well
4matched and mismatched titrations is
performed (occupational factor set to 2) and yield
the values of K
ass
= 6.8 10
4
M
41
per base pair
and K
MM
=
1.8 10
6
M
41
per mismatched site for the 200 mM NaCl condition
and K
ass
= 1.1 10
5
M
4
1
per base pair and K
MM
= 9.7 10
6
M
41
per mismatched site for the 50 mM NaCl condition.
The
errors are evaluated to be equal to 10 %.
When comparing titrations in 50mM and 200mM NaCl, w
e observe two changes. First
for both duplexes, the emission intensities at satu
rating values for 200mM NaCl are
approximately half those obtained at 50 mM NaCl, co
nsistent with the increase in ionic strength
leading to a decrease in emission intensities obser
ved previously. Second we find that the ionic
strength affects the shape of the binding curve. At
50 mM NaCl we see a maximum emission
intensity upon titration followed by a decrease in
emission as DNA/Ru increases; at 200 mM
NaCl this effect is less dramatic. We attribute thi
s difference to less non4specific DNA
association at higher ionic strength.
S8
Time-Resolved Luminescence Measurements
Time4resolved spectroscopic measurements were carri
ed out at the Beckman Institute
Laser Resource Center, and were conducted using ins
trumentation that has been described.
4
Briefly, a 460 nm light produced by OPO pumped with
a 10 Hz, Qswitched Nd:YAG laser
(Spectra4Physics Quanta4Ray PRO4Series) was used as
an excitation source (pump pulse
duration ≈8 ns). The emitted light was detected at
660 nm with a photomultiplier tube
(Hamamatsu R928) following wavelength selection by
a double monochromator (Instruments
SA DH410). Scattered laser light was removed from t
he detectors using suitable filters. The
samples were held in 1 cm path length quartz cuvett
es (Starna) equipped with stir bars and
irradiated at 460 nm with 500−1000 laser pulses at
3 mJ/pulse. Kinetic traces were fit to
exponential equations of the form
I
(
t
) =
a
0
+ Σ
a
n
exp(−
t
/
τ
n
), where
I
(
t
) is the signal intensity as a
function of time,
a
0
is the intensity at long time,
a
n
is a pre4exponential factor that represents the
relative contribution from the
n
th component to the trace, and
τ
n
is the lifetime of the
n
th
component, convoluted with a Gaussian function to t
ake into account the Instrument Response
Function (fwmh = 8ns). The errors are evaluated to
be equal to 5%, but the incertitude on the
short component (associated with complexes bond to
well4matched DNA,
i.e.
33435 ns) being
close to the IRF time characteristic is subject to
a greater error (+/4 8 ns).
Models of [Ru(Me
4
phen)
2
dppz]
2+
Binding to Well-matched and Mismatched DNA
The binding constants obtained for the complex with
the mismatched and well4matched
sites indicate that the complex preferentially bind
s to mismatched DNA. The relative emissivity
determined during the fitting process, i.e. 40 for
complexes associated with well4matched sites
and 380 for complexes associated with mismatched si
tes, also correlates with the differential
S9
luminescence lifetimes. As such, ruthenium bound to
the mismatched site should be more
protected from quenching than complex bound
via
intercalation at well4matched sites. To gain
more insight regarding the local environment around
the DNA4bound complex, we used
published crystal structures
5,6
of DNA to model the binding of [Ru(Me
4
phen)
2
dppz]
2+
. In one
model, we inserted
4[Ru(Me
4
phen)
2
dppz]
2+
into an AC mismatch
via
the minor groove (Figure
4 of main text and Figure S2), and oriented the com
plex in such a way as to maximize the
protective environment around the dppz ligand while
avoiding steric clashes with the DNA. As
seen in Figures 4 and S2, the dppz ligand is capabl
e of being deeply inserted into the mismatch
site, allowing greater protection from quenching. I
n the other model, we intercalated the complex
at a well4matched site from the major groove, again
minimizing steric clashes between the
Me
4
phen ligands and the DNA. In this model, we oriente
d the complex in both a head4on fashion
(Figure S2) and a side4on orientation (Figure 4). I
n the head4on orientation, both phenazine
nitrogen atoms are relatively well4surrounded by th
e duplex, but in the side4on approach, we see
that dppz is more exposed to quenching.
S10
Figure S2:
Top
: Axial views, down the helical axis, of
4[Ru(Me
4
phen)
2
dppz]
2+
modeled into
the crystal structures of DNA duplexes.
Top left:
Metalloinsertion at the mismatch site from the
minor groove; the extruded mismatch bases are shown
in orange.
Top right:
Head4on
intercalation at a well4matched site from the major
groove.
Bottom:
Side4views of (
left
)
metalloinsertion at the mismatch from the minor gro
ove and (
right
) head4on intercalation at a
well4matched site from the major groove. The optimi
zation and visualization were carried out
using UCSF Chimera
program.
S11
MTT Cytotoxicity Assay
This assay was performed as described previously.
7
HCT116N and HCT116O cells were
plated in 964well plates (50,000 cells/well), treat
ed with the Ru concentrations indicated in
Figure 3 of the main text, and incubated for 72 hou
rs (37
°
C, 5% CO
2
, humidified atmosphere).
After this incubation period, MTT was added to the
cells (Roche Cell Proliferation Kit I) and the
cells were incubated for an additional 4 hours. Ins
oluble formazan crystals were dissolved in
solubilizing reagent (Roche) over 24 hours (37
°
C, 5% CO
2
, humidified atmosphere). The
solubilized formazan was quantified at 570 nm with
690 nm as the reference wavelength. Percent
cell viability was calculated as a function of form
azan formed in the Ru4treated cells relative to
untreated cells.
Figure S3:
Steady4state NaI quenching of [Ru(Me
4
phen)
2
dppz]
2+
(2
M) bound to well4matched
(left, blue) and mismatched (right, red) DNA (2
M). Solid lines indicate no NaI present, and
dotted lines represent increasing NaI concentration
s of 25, 50, and 75 mM, respectively.
λ
ex
=
440 nm. Samples prepared in 5 mM tris, 200 mM NaCl,
pH 7.5. The DNA sequences are as in
Figure 1 of the main text.
S12
Figure S4
: (Left) Schematic of [Ru(Me
2
bpy)
2
dppz]
2+
. (Right) Steady4state luminescence
titrations of [Ru(Me
2
bpy)
2
dppz]
2+
with well4matched (blue) and mismatched (red) DNA.
Samples prepared in 5 mM tris, 200 mM NaCl, pH 7.5.
[Ru] = 2
M,
λ
ex
= 440 nm. Emission
spectra were integrated from 5504850 nm.
Figure S5
: Differential cytotoxicity of [Ru(Me
4
phen)
2
dppz]
2+
towards HCT116N and HCT116O
cell lines. Cells were plated in a 964well format (
5 x 10
4
cells/well) and treated with the indicated
Ru concentrations for 72 h. Following this incubati
on period, the cells were labeled with MTT
for 4 h. Metabolically active cells reduce the MTT
to produce its insoluble formazan, which is
then solubilized and quantified by its absorbance a
t 570 nm. The percent viability is the ratio of
formazan absorbance in cells treated with ruthenium
to the absorbance in untreated cells.
S13
Supporting References
1.
Dickerson, J.E.; Summers, L.A.
Aust. J. Chem.
1970
,
23
, 102341027.
2.
Nakabayashi, Y.; Watanabe, Y.; Nakao, T.; Yamauchi,
O.
Inorg. Chim. Acta
2004
,
357
,
255342560.
3.
McGhee, J.D.; von Hippel, P.H.
J. Mol. Biol.
1974
,
86
, 469 4 489.
4.
Dempsey, J. L.; Winkler, J. R.; Gray, H. B.
J. Am. Chem. Soc.
2010
,
132
, 1060−1065.
5.
Pierre, V.C.; Kaiser, J.T.; Barton, J.K.
Proc. Natl. Acad. Sci.
2007
,
104
, 4294434.
6.
Kielkopf, C.L.; Erkkila, K.E.; Hudson, B.A.; Barton
, J.K.; Rees, D.C.
Nat. Stuct. Biol.
2000
,
7
, 1174121.
7.
Mosmann, T.J.
J. Immunol. Methods
1983
,
65
, 55463.