Intercalation of DNA by Ruthenium(II) Pteridinyl Complexes
Shannon R. Dalton, Samantha Glazier, Alanna Albano, Courtney Megatulski & Sharon J. Nieter Burgmayer*
Department of Chemistry, Bryn Mawr College, Bryn Mawr, PA 19010
Introduction
Ruthenium(II) Pteridinyl Complexes
Hydrogen bonding Capabilities
Molecules can bind to DNA through several
types of interactions, resulting in various
binding strengths and specificities.
Electrostatic interaction of molecules with
the negatively charged DNA surface and
within major and minor grooves is weaker
than intercalation interactions where planar
portions of a molecule slip between the
planes of GC and AT base pairs. Intercalators
typically have an aromatic, heterocyclic ring
structure which can insert between the base
pairs of a DNA helix. Intercalative binding
of a molecule forces the base pairs apart and
can distort the helical shape of DNA.
Resultant distortions of the DNA helix
include bending, lengthening, and stiffening.
These changes can prevent replication
enzymes such as polymerase, helicase, and
topoisomerase from recognizing their
specific binding sites, thus inhibiting
replication. For this reason, intercalators
have substantial pharmaceutical applications
and warrant extensive study. Ruthenium
tris-chelate complexes, such as
[Ru(II)(bpy)2DPPZ]2+, are known
intercalators which have been extensively
studied. Their inherent photochemical
stability, as well as redox and photophysical
properties, make it possible to utilize
multiple techniques to study DNA
intercalation.
Reported here are the results of fluorescence titrations, viscometry, circular
dichroism, plasmid unwinding and thermal denaturation studies on DNA in the
presence of four new Ru(II)(bpy)2 complexes chelated by a phenanthroline
ligand extended with dimethylalloxazine (phen-DMA), alloxazine (phenalloxazine), diaminopteridine (phen-diaminopteridine) and pterin (phen-pterin).
The resulting complexes are shown below. These experiments differentiate
between electrostatic and intercalative binding mechanisms. While all four of the
new complexes shown DNA interactions by fluorescence and absorbance
titrations, viscometry, CD spectroscopy, DNA melting and plasmid unwinding
experiments give evidence that only three complexes, [Ru(bpy)2(phendimethylalloxazine)]2+, [Ru Ru(bpy)2(phen-alloxazine)] 2+, and [Ru(bpy)2(phendiaminopteridine)]2+ intercalate to calf thymus (CT) DNA. Future experiments
include photocleavage induced by the ligands, and polyacrylamide gel
electrophoresis to show sequence specific intercalation due to the unique
hydrogen bonding properties of the pterin derivative phenanthroline ligands.
The structural relationship of pteridines to the nucleic acids of DNA has been
exploited previously in other labs where pteridine binding at abasic sites along
duplex DNA is made possible by matched H-bonding profiles of the pteridine and
the nucleic acid. We similarly hypothesized that pteridine extensions onto the
robust phenanthroline ligand might introduce new aspects into Ru complex binding
studies. The pteridine match to the purines and pyrimidines of DNA (shown at
right) might be envisioned to disrupt base paring or to form triplex-like
interactions, as well as allow for intercalation.
Base interactions in Triple helical DNA
C H3
H
R
N
H
N
H
N
N
O
N
N
N
N
N
NH
N
N
N
N
N
N
N
N H2
N
N
N
N
R
H
N
N
N
[Ru(bpy)2(phen-diaminopteridine)]2+
N
O
N
N
N
NH
N
N
H
H
N
N
R
N
O
N
H
N
Compare
T-A-T triad
vs.
[Ru(bpy)2(phen-alloxazine)]-A-T triad
R
N
N
N
N
H
O
[Ru(bpy)2(phen-alloxazine)]2+
O
H
N
N
N
Ru
N
H
N
N
N
H
N
N
C H3
N
N
H
O
N
O
N
O
H
N
H
O
H
R
H
H
N
O
N
N
R
N
N
R
N
O
N
N
N
N
N
CH3
N
H
N
O
N
Ru
N
N
N
N
N
Ru
H
H
N
N
O
N
N
N
N
H
N
Ru
N
N
H
NH2
N
N
O
N
N
N
[Ru(bpy)2(phen-pterin)]2+
O
Compare
C-G-C triad
vs.
[Ru(bpy)2(phen-pterin)]-G-C triad
N
Ru
Ru
N
N
N
O
NH2
N
R
O
H
O
R
2+
H
N
N
O
H
N
H
N
R
CH3
[Ru(bpy)2(phen-dimethylalloxazine)]2+
Results and Discussion
Fluorescence Titration of 20 M
Viscosity
Fluorescence Titrations
Saturation Curve
5
3x10
5
2x10
5
1x10
50
100
150
[CT DNA]:[Dye]
2.0x10
5
0.0
550
600
650
700
750
Wavelength (nm)
EtBr
1.15
Allox
Bpy3
1.1
Diamino
1.05
DMA
Pterin
1
0.95
0.00
0.05
0.10
0.15
1.72
CT DNA
1.65
1.62
1.55
1.52
Poly
dAT*dAT
(bpy)3
1.35
Pterin
1.3
1.25
1.2
h /ho
1/3
Diaminopteridine
Alloxazine
Dimethylalloxazine
DPPZ
1.15
1.1
1.05
1
0.95
25 30 35 40 45 50 55 60 65 70 75 80 85 90 95
Temperature (o C)
Poly
dGC*dGC
1.25
CT DNA
64.5 deg C
[Ru(bpy)3]2+
64.5 deg C
Ru(bpy)2pterin]2+
64.5 deg C
[Ru(bpy)2(phendiaminopteridine)]2+
67.5 deg C
[Ru(bpy)2(phenalloxazine)]2+
75 deg C
[Ru(bpy)2(phendimethylalloxazine)]2+
78 deg C
As DNA is heated, the two strands of
the helix pull apart, and denatured
single-stranded DNA is the result.
When an intercalator is bound to
the helix, it stabilizes the DNA, and
Increases the melting temperature (Tm)
This can be measured by recording the
absorbance at 260 nm, since this is the
wavelength that DNA bases absorb.
With the exception of [Ru(bpy)3]2+ and
[Ru(bpy)2pterin]2+, the Tm of intercalated
DNA was increased compared to a shift of
64.5 to 80 oC for intercalator [Ru(bpy)2DPPZ]2+.
0.95
-0.02
Molar Ellipticity (deg cm 2 dmol-1)
+Ru(bpy)3
2.0E+05
+RuDPPZ
1.0E+05
250
270
290
310
-2.0E+05
-3.0E+05
Wavelength (nm )
10 CT DNA:Ru(bpy)2 - pterin, DMA, DAP, ALX
Since surface and groove binding have little or no
effect on DNA structure, intercalation can be
distinguished by changes in CD signal. The bands
due to base stacking at 275 nm and helicity at 245
nm are easily altered by an intercalating molecule,
making circular dichroism (CD) a useful technique
for determining binding mode.
The top plot illustrates the changes made by a known
intercalator, [Ru(bpy)2DPPZ]2+, where the positive
signal centered at 275 nm in CT-DNA shifts to 270
nm and a negative band at 291 nm appears. This
change is diagnostic of a B to Z transition. In
contrast, the CD of non-intercalator [Ru(bpy)3]2+
added to DNA is identical to CT-DNA alone.
CT DNA
2.00E+05
+Rupterin
1.50E+05
+RuDMA
+RuDAP
1.00E+05
+RuALX
5.00E+04
0.00E+00
-5.00E+04230
250
270
290
-1.00E+05
-1.50E+05
-2.00E+05
Wavelength (nm)
Poly
dGC*dGC
1.12
0.92
-0.02
0.68
[Ru-DMA]: [base pairs DNA]
[RuDiamino]: [base pairs DNA]
Viscosity Titration: 0.724 mM Ru-Alloxazine
in Acetonitrile & 0.14 mM DNA in buffer
in
Acetonitrile and DNA in buffer
CT DNA
(0.18 mM)
1.45
CT DNA
1.72
1.62
1.52
Poly
dG*dC
(0.15 mM)
1.35
1.25
CT DNA
(0.09 mM)
1.15
Poly
dAT*dAT
1.42
1.32
1.22
Poly
dGC*dGC
1.12
1.02
0.92
1.05
0.95
-0.02
0.03
0.08
0.13
0.18
0.23
0.28
0.33
0.38
-0.02
Poly
dA*dT
(0.09 mM)
0.43
0.68
[RuAlloxazine]: [base pairs DNA]
Preliminary experiments to test
for DNA sequence specificity
by the intercalating
[Ru(bpy)2L]2+complexes where
L= phen-alloxazine, phendimethylalloxazine and phendiaminopteridine gave the
results at left. There are distinct
differences among the three
intercalating molecules. The
phen-diamino and phendimethylalloxazine complexes
bind more strongly to poly
dGC-dGC than to poly dATdAT. In contrast the phenalloxazine complex binds
equally well to poly dGC-dGC
and poly dAT-dAT.
[Ru-Alloxazine]: [base pairs DNA]
Plasmid Unwinding
[Ru(bpy)2(phendimethylalloxazine)]2+
2
3
4
5
6
7
8
1
2
3
4
5
6
[Ru(bpy)2(phen-alloxazine)]2+
7
8
1
2
3
4
5
6
7
8
[Ru(bpy)2(phendiaminopteridine)]2+
1
2
3
4
5
6
7
8
Circular Dichroism Results
CT DNA
0.0E+00
230
-1.0E+05
1.22
0.68
Viscosity Titrations: Ru-Alloxazine
1
10 CT DNA:Ru(bpy)3 & Ru(bpy)2DPPZ
3.0E+05
Poly
dAT*dAT
1.32
1.02
[Ru(bpy)2(DPPZ)]2+
Circular Dichroism
CT DNA
1.42
1.05
h /h o 1/3
1.4
Molar Ellipticity (deg cm 2 dmol-1)
Relative Abs @ 260 nm
CT DNA
1.35
1.15
Tm Values
DNA:Ru 15
Viscosity Titration: 0.702 mM Ru-DMA in
Acetonitrile & ~0.14 mM DNA in buffer
Viscosity Titration: 0.745 mM Ru-Diamino in
Acetonitrile & 0.14 mM DNA in buffer
1.45
Thermal Denaturation Results
0.20
Drug:Calf Thymus DNA
1.75
Thermal Denaturation
h= viscosity
t = flow time (seconds)
t0 = flow time of buffer alone (seconds)
h 0 = viscosity of DNA alone
Dppz
1/3
0
When DNA is intercalated by a molecule, the helical
structure is altered. Due to the shape distortion and
lengthening of the helix, the viscosity of the DNA
solution is increased. This is easily monitored by flow
time of the solution. All compounds tested (see legend in
plot at left), except for the known non-intercalator
[Ru(bpy)3]2+ and new complex [Ru(bpy)2pterin]2+,
showed significantly increased viscosity.
h=(t-t0)/t0
1.2
h /ho
Emission Intensity
5
1/3
Emission Intensity (cps)
4.0x10
M
Viscometry Results
Viscosity Titration: ~2.5 mM Drug in Acetonitrile &
0.90 mM CT DNA in buffer
Initial fluorescence titrations with all four
Ru(bpy)2phen-pteridine complexes showed
increased emission intensity suggestive of
DNA interactions. However, only three
complexes produce saturation curves
typical of intercalation or significant DNA
interaction. These three complexes are:
[Ru(bpy)2phen-alloxazine]2+,
[Ru(bpy)2phen-dimethylalloxazine]2+,
[Ru(bpy)2phen-diaminopteridine] 2+
A representative saturation curve is shown
at left.
The complex with the pterin component,
[Ru(bpy)2phen-pterin] 2+, shows little
increase in emission and a linear saturation
plot indicating this complex does not
intercalate or interact strongly with DNA.
1/3
[Dye] = 20
4x10
h /ho
[Ct DNA] is Increasing
5
h /ho
Ru(bpy)2phen-Alloxazine & Calf Thymus DNA
310
The CD spectra of solutions of CT-DNA with each of
the four [Ru(bpy)2phen-pteridine]2+complexes
indicates qualitatively the extent of intercalation. The
three complexes with phen-dimethylalloxazine ,
phen-alloxazine, and phen-diaminopteridine show
CD spectral changes indicative of intercalation while
[Ru(bpy)2phen-pterin]2+shows no spectral changes
from disruption of DNA structure and hence, no
intercalation.
Lanes 1-5: Supercoiled plasmid + increasing [Ru compound] + Topoisomerase
Lane 6: Supercoiled + highest [Ru compound]
Plasmid Unwinding Results
Plasmid DNA is negatively supercoiled (-Lk). Upon
intercalation, DNA is unwound, and Lk becomes less
negative. At high concentrations of intercalator, positive
supercoils (+Lk) are induced.
Lane 7: Relaxed plasmid
Lane 8: Supercoiled plasmid
Lk: Linking number,
a: Topoisomerase I
relaxation,
b: Ligand removal
[Ru(bpy)2(phen-diaminopteridine)]2+, [Ru(bpy)2(phenalloxazine)]2+ and [Ru(bpy)2(phendimethylalloxazine)]2+ all showed plasmid unwinding
behavior similar to DPPZ.
Conclusions
All five methods point to the same interpretation: three complexes— [Ru(bpy)2phen-alloxazine]2+,
[Ru(bpy)2phen-dimethylalloxazine] 2+, and [Ru(bpy)2phen-diaminopteridine] 2+— can intercalate
DNA nearly as well as ethidium bromide or [Ru(bpy)2DPPZ]2+, while [Ru(bpy)2phen-pterin]2+ shows
little ability to disrupt the DNA structure and hence, no intercalation.