ABSTRACT
Intercalating molecules can distort the helical shape of DNA, thereby inhibiting replication
enzymes and in turn have pharmaceutical applications. Due to their inherent photochemical
stability, as well as redox and photophysical properties, transition metal complexes make it
possible to utilize multiple techniques to study intercalation.
One such complex,
[Ru(phen)2DPPZ]2+ has been shown in previous work to intercalate due to its large heterocyclic
ring system. Previous fluorescence and absorbance titrations give evidence that four similar
ruthenium
compounds,
[Ru(II)(bpy)2(phen-pterin)]2+,
[Ru(II)(bpy)2(phen-alloxazine)]2+,
[Ru(II)(bpy)2(phen-dimethylalloxazine)]2+, and [Ru(II)(bpy)2(phen-diaminopteridine)]2+ bind to
calf thymus DNA. These studies cannot specify the mode of binding, so additional experiments
are required. Reported are the results of viscometry, plasmid unwinding, thermal denaturation
and circular dichroism, studies which can differentiate between binding mechanisms.
combination
of
experimental
data
from
each
method
show
that
The
[Ru(bpy)2(phen-
dimethylalloxazine)]2+ , [Ru(bpy)2(phen-alloxazine)]2+ and [Ru(bpy)2(phen-diaminopteridine)]2+
do bind to DNA via an intercalative mode. Additional study of these compounds leading to
further characterization of binding could result in the development of stable complexes for use as
DNA probes and the discovery of life saving pharmaceuticals.
1
TABLE OF CONTENTS
Abstract
1
Acknowledgements
3
Introduction
Modes of Binding
4
Metallointercalators
7
Oxidative Reactions
11
Ruthenium Intercalators
13
New Ruthenium Complexes
27
Our Contribution to DNA Binding Characterization
of Ruthenium(II) Pteridinyl Compounds
32
Results
34
Discussion
45
Materials
56
Methods
57
Works Cited
59
Abbreviations
61
2
ACKNOLEDGEMENTS
I would like to thank everyone I have met in the Bryn Mawr community for helping me
through some difficult times. There are some specific people I would like to thank. Dr. Sharon
Burgmayer, is without a doubt, the “coolest” adviser out there! Dr. Samantha Glazier was great
to work with, and willing to listen to all of my research ideas. All of the students in the
Burgmayer Lab have been amazing, especially those I spent a lot of time with: Ying Hou, Mica
Grantham and Alanna Albano. Dr. Xenia Morin and Dr. Tamara Davis have taken time to help
me with my experiments and offer their advice, and Jim Schweppe and Cheryl Selah showed me
the “ropes” of biochem techniques.
I am, as always, thankful for my family. I received so much support and love from my
mother, and I would not have had the confidence to come to Bryn Mawr without her. It is great
to have a sister who you can talk chemistry with, not to mention anything and everything else. I
have to give a lot of credit to Jason Drury, who came to lab with me at midnight to start
experiments, stayed up late with me grading lab reports, and made sure all the figures on my
posters lined up.
He was and is there for me every time I need him, academically and
emotionally. Last but not least, I should mention my two best friends, who double as cats, Boots
and Missy. For the last twelve years, they have always known how to cheer me up when I am
upset, and remind me what life is about when I am frustrated and overwhelmed.
3
INTRODUCTION
Modes of Binding
Life depends on the ability of molecules to bind to DNA. Enzymes binding to nucleic
acids are necessary for essential processes such as DNA replication. Each molecule that binds
does so with a specific mode, specificity and strength. The three common modes are surface
binding, groove binding, and intercalation. The colored molecules in Figure 1 represent the
different binding modes; surface binding represented by green, groove binding by red, and
intercalation by yellow.
Figure 1: Colored molecules represent three modes of binding; green is surface
binding, yellow is intercalating, and red is groove binding1
Surface binding relies on electrostatic and van der Waals forces, and as the name
suggests, molecules using this binding mode are simply attracted to the surface of a DNA helix.
Groove binding occurs in the major or minor groove of a helix. While groove binding can be
fairly stable and is closely associated with the helix, a molecule utilizing this mode does not
perturb the shape of the helix. For example, the structure of well studied groove binder Hoechst
4
33258 is shown in Figure 2a. Hoechst 33258 is known to groove bind to DNA as depicted in
Figure 2b.
(a)
(b)
Figure 2: (a) Structure of Hoechst 33258, a standard groove binder and
(b) Representation of Hoechst 33258 binding to a DNA helix2
Intercalation occurs when a planar heterocyclic portion of a molecule inserts itself
between base pairs of DNA. This is a mode that brings the binding molecule in close proximity
to the helix, but also results in physical changes of the DNA, including lengthening, stiffening,
and unwinding.3 The structure of a standard intercalator, ethidium bromide, is shown in Figure
3a, and Figure 3b illustrates a molecule of ethidium bromide intercalating between DNA base
pairs.
5
(a)
(b)
Figure 3: (a) Structure of ethidium bromide, a standard intercalator and
(b) a representation of two molecules of ethidium bromide intercalating
between two DNA base pairs.
The change in DNA structure induced by intercalation is what makes these molecules
popular targets of research. Since the shape of DNA is a common criterion for the successful
binding of many enzymes, intercalators can be engineered to inhibit DNA replication when
desirable. Such is the case for many drugs on the market today, including daunomycin and
adriamycin.4 These drugs cause DNA to “buckle” and prevent the binding of helicase and
topoisomerase, preventing protein synthesis, and finally cell division. By this prevention of
replication, the growth of cancerous cells is inhibited. Figure 4 shows the structures of these
closely related anti-cancer drugs, and depicts the buckle formed along the DNA background as a
result of daunomycin intercalating. Although the two drugs only differ by one hydroxyl group,
they effectively treat different types of cancer. For example, daunomycin is used for medicating
patients with leukemia, and adriamycin is most effective for treating solid tumors.5
6
(a)
(b)
Figure 4: (a) Structures of cancer treating intercalating drugs, daunomycin and adriamycin and
(b) a representation of daunomycin inducing buckle in DNA helix6
Metallointercalators
History
Transition metal complexes have been useful as probes to study intercalation. These
compounds are easy to work with because they are inert, stable and fairly soluble.
The
spectroscopic properties of transition metals provide various means for monitoring the effect of
intercalation through absorption and fluorescence studies. Figure 5 shows the structures of
cobalt and ruthenium tris(phenanthroline) ([M(phen)3]2+). These compounds were among the
first octahedral transition metal compounds used to study intercalation.
7
Figure 5: Structures of the first transition metal compounds used to study intercalation. The
top two complexes are left () and right () handed enantiomers of [Ru(phen)3]2+.
The lower two complexes show bulkier derivatives of the phenanthroline ligands.
These compounds, while not demonstrating the strong binding affinities of known
intercalators such as daunomycin and ethidium bromide, provoked interest in probing the
interaction of redox and photo active compounds with DNA. One discovery based on early
experiments that continues to be prevalent in current studies is enantiomeric specificity. Studies
showed that -isomers were more likely to intercalate into right-handed DNA. Despite the
enatiospecificity, researchers concluded that compounds with increased intercalating properties
were necessary since slight changes in salt concentration, DNA sequence and temperature
resulted in a variation of binding affinities.7
8
Intercalators were found to have in common a large aromatic planar moiety. Therefore,
increasing the surface area of this portion should increase the affinity for that ligand to
intercalate. Figure 6 shows the structures of bidentate, bis-nitrogen donor intercalating ligands
such as 9,10-phenanthrenequinone diimine (phi) and dipyrido[3,2-a:2’,3’-c]phenazine (dppz)
complexed (not shown as metal complexes) with transition metals and ancillary ligands such as
bipyridyl (bpy) and phenanthroline (phen).
Phi and dppz were shown to have a greater
intercalating affinity and photophysical changes upon binding to DNA compared to phen and
bpy. The enantioselectivity of these chiral compounds binding to DNA is a significant focus of
study.7
Figure 6: Structures of ancillary ligands phen and bpy, and those showing intercalative properties, dppz and phi
Transcription Inhibition
As mentioned earlier, intercalators have the capability of halting replication of DNA.
Transcription is the process of synthesizing RNA using DNA as a template. RNA is responsible,
in part, for the production of essential proteins and enzymes.
In order for this to occur,
transcription factors must bind specifically to sites on the DNA so that RNA polymerase can
bind. An example of an interfering intercalator would be -1-[Rh(MGP)2phi]5+ (MGP = 4(guanidinylmethyl)-1,10 phenanthroline) as shown in Figure 7a. This complex has been shown
9
using NMR to be site-specific to an engineered domain containing the binding site preferred by a
transcription factor. This ability is due to the hydrogen bonding capabilities of the asymmetrical
MGP ligand.7
(a)
(b)
Figure 7: (a) shows the structure of -1-[Rh(MGP)2phi]5+ and (b) shows the
structure of -3-[Rh(MGP)2phi]5+, a regio-isomer
Transcription factor yAP-1 (yeast protein activator protein 1) was incapable of binding to
its domain when -1-[Rh(MGP)2phi]2+ was present, occupying the same binding site. Inhibition
by the complex required a concentration of at least 120 nM. This inhibition is sensitive to
structure. When the guanidinium groups face away from the phi ligand, as in the isomer -3[Rh(MGP)2phi]5+ shown in Figure 7b, there is no competitive binding.
These significant results suggest that metallointercalators can be designed to be sitespecific and be competitive with naturally occurring proteins. Future applications include gene
regulation,7 a process that can result in the lack of expression of a certain protein or enzyme. For
example, by controlling the effect of a given transcription factor, synthesis of a disease-causing
protein can be prevented.
10
Oxidative Reactions
Not only does the ability to design preferred enantiomers allow the use of
metallointercalators as biological probes, but researchers have learned to exploit the inherent
redox chemistry of these compounds. Several significant reactions, most of which are based on
oxidation by metallointercalators, have been reported.
Due to easy oxidation of guanine
compared to the other bases, reactions of redox active metallointercalators and DNA are usually
selective to guanine.
However, several mechanisms exist for oxidative damage to DNA.
Oxidation of bases has been seen to increase in reactivity when intercalating ligand dppz is
coordinated with a Ru(II) complex such as [Ru(bpy)2dppz]2+. The ruthenium metal center can be
activated electrochemically to initiate direct oxidative damage to guanine.7
Singlet Oxygen
Many metal complexes with affinity for DNA have the ability to sensitize oxygen. The
sensitization process occurs when singlet oxygen is formed by way of a triplet energy transfer
from the metal excited state. Non-intercalative [Ru(bpy)3]2+ has demonstrated oxidative damage
to DNA because the singlet O2 produced by sensitization can diffuse along the helix and
preferentially reacts with guanine. This reaction cannot be used to locate binding sites on DNA
because the complex is not intercalated or bound to a specific site. However, a similar reaction
does occur with intercalated complexes containing the dppz ligand. This reaction can therefore
be used to determine the binding site of the metallointercalator by monitoring the location of the
reaction using gel electrophoresis.7
11
Long-Range Electron Transfer
In addition to metallointercalators causing oxidative damage in close proximity to their
binding site, they can be induced by flash-quench technique to cause reactions from a distance.
As mentioned above, when Ru(II) is sensitized to form singlet oxygen, it then reacts with
guanines in close proximity to the binding site. However, electron transfer to a surface bound
quencher can result in the oxidized intercalator having sufficient oxidative potential to oxidize
guanine. Therefore, electron transfer allows for reaction at a guanine up to 200 angstroms away.
Figure 8 shows the two possibilities for oxidation by the bound intercalator, through singlet
oxygen-dependent oxidation of an adjacent guanine (left), and quencher dependent, oxidation of
a remote guanine (right).7
Figure 8: Oxidation of either adjacent (left) or remote (right) guanines by the tethered complex.
The position of reaction depends on the pathway, singlet-oxygen (left) or quencher (right).
Oxidative Repair
Not only can oxidative abilities of photoexcited complexes cause damage to DNA, but
photoexcitation can also repair damaged DNA.
Cyclobutane thymine dimers are the most
common form of UV induced damage to DNA.7 While some organisms such as fish, birds and
12
marsupials have an enzyme, photolyase, to repair these lesions, humans do not, and therefore
extensive research is taking place on methods of repair.
Barton, et al. has demonstrated the ability of a metallointercalator, [Rh(phi)2bpy’]3+, to
oxidatively repair photolesions. Figure 9 illustrates a strand of DNA containing a single UV
induced thymine dimer annealed to a strand to which the rhodium intercalator is covalently
bound. It was arranged so that the intercalation site was 19-26 angstroms away from the lesion.
Irradiation at 400 nm induced oxidation and cleavage of the thymine dimer as monitored by
HPLC. The quality of base-stacking appeared to have an effect on this process. Different
lengths of DNA used yielded different repair efficiencies, as the farther the intercalation site was
from the lesion, the greater the repair ability, indicating dependence on the more stabilized
stacking of longer DNA. Also, the repair only functioned at approximately 50 % when unpaired
bases were used to create kinks in the DNA, indicating the importance of electron transfer
through the helix.7
Figure 9: Representation of DNA strand with cyclobutane thymine dimer and tethered rhodium intercalator
Ruthenium Intercalators
Tris(phenanthroline)Ruthenium(II) ([Ru(phen)3] 2+)
Since the early 1980s, there has been an ongoing controversy between researchers
regarding the type of interaction [Ru(phen)3]2+ has with DNA. The methods that earlier studies
13
used to characterize binding mode were later disputed by results from new experimental
techniques. The literature is also unclear on what results constitute intercalative binding versus
other modes. The following section outlines results from different research groups according to
technique after a brief introduction of each group.
Early Studies: A Controversy
In 1984, Barton, et al. chose to use ruthenium(II) complexes for intercalative studies for
several reasons: a) the d6 low-spin configuration, b) they are kinetically inert, c) they have a large
metal to ligand charge-transfer band (MLCT), and d) the existence of previously established
spectroscopic properties of poly(pyridine) complexes. For example, in addition to exhibiting
efficient luminescence, [Ru(phen)3]2+ exhibits strong absorption in the visible region but not
overlapping with the DNA absorption band.4
Figure 10 shows the optical isomers of [Ru(phen)3]2+. Due to the inert nature of the
compound, the isomers can be completely purified, another characteristic that sets the Ru(II)
complexes apart from other transition metals. This proves to be a very valuable asset, since
research concludes that helical DNA has preferences for certain isomers.4
Figure 10: Left () and right () enantiomers of [Ru(phen)3]2+
14
Barton’s group used spectroscopic methods, helical unwinding, and equilibrium dialysis
to study the interactions of each enantiomer with DNA. In 1986, Barton, et al. continued her
studies of tris(phenanthroline)ruthenium(II) and its interaction with DNA. This study focused on
examining enantiomeric selectivity, sequence specificity and differentiating between binding
modes.8
Hiort, et al. studied the binding geometries of tris(phenanthroline)ruthenium(II) with
DNA using linear and circular dichroism (LD and CD) in 1990. The researchers intended to
explore the different interactions with DNA for  and  enantiomers, and the possibility that this
compound could discriminate between B and Z form DNA.9
In 1985, Kelly, et al. investigated tris(phenanthroline)ruthenium(II) as well as new
derivatives of phenanthroline shown in Figure 12b to examine the effect of ligand size and shape
on the binding affinity.
As mentioned in the section on oxidative reactions, ruthenium
complexes are good sensitizers of oxygen and have strong redox characteristics. It was for these
reasons that Kelly, et al. chose to work with them, including the assumption that the cationic
nature of the compounds would facilitate reaction with DNA. In addition to absorbance and
fluorescence data, their study looked at the ability of these compounds to unwind plasmid DNA,
increase melting temperature by stabilizing the DNA helix and be photo-activated to cleave
DNA.10
15
Figure 12: A Shows the structure of [Ru(phen)3]2+ and
B depicts the various polypyridyl ligands; a: 2,2’-bipyridyl (bpy);
b: 1,10-phenanthroline (phen); c: 2,2’,2”-terpyridyl (terpy)
Chaires, et al. cites the work of the Barton group and Hiort, et al. in a 1992 publication
where
these
authors
disagree
with
Barton
tris(phenanthroline)ruthenium(II) bind by intercalation.
that
either
enantiomer
of
They believe that the small
spectroscopic changes observed by the Hiort group are not results from an intercalative
interaction. Instead, Chaires, et al. suggests that the compound may, in addition to groove
binding, partially intercalate DNA.11
Fluorescence Studies
In a 1986 publication by the Barton group, it is stated that intercalation of DNA results in
large changes of the photophysical properties of the metal compound, which can be monitored
by emission lifetimes, steady-state polarization, and emission polarization. Also, the chirality of
the complex, matching the shape of the complex to the topography of the helix, and the groove
size of the DNA play a significant role in binding by intercalation. On the other hand, surface
binding is a result of electrostatic and hydrophobic interactions.
16
While topography and
stereochemistry might still have an effect on this mode of binding, the compound would be free
to diffuse along the helix, rather than a stationary intercalative compound.8
The study claims that emission polarization can distinguish between surface binding and
intercalation, since an intercalative molecule will be anchored within the helix and not free to
move about. This characteristic would result in long emission lifetimes and finite polarization,
unlike a surface bound molecule, which would not affect the polarization. Experimental results
showed an increase in polarization when the DNA concentration was increased, as well as more
polarization for the -isomer, indicating it is preferred. By selecting specific quenchers, it was
possible to selectively reduce emission from either the bound or the free ruthenium complex.
When the free compound was quenched, the polarization of bound compound increased, and
quenching of the bound compound resulted in a decrease of polarization. These results indicate
the presence of both free and bound forms of compound.8
The same techniques as described above were used to test the affinity of the compounds
for varying sequences of DNA. Steady-state polarization results showed an increase in binding
of -[Ru(phen)3]2+ with increasing GC content. The researchers speculate that this may result
since sequences with more GC pairs may stack more closely, providing a tighter groove for
secure intercalation.8
Barton, et al. found a 17% decrease in the MLCT intensity was observed upon binding
and an increase in luminescence due to changes in stacking, and lessened mobility of the
complex when bound to the helix respectively. The magnitude of hypochromic shifts observed
were similar to those due to  to * transitions of organic intercalators, and a larger change in
luminescence for the -isomer was interpreted as an indication that more -[Ru(phen)3]2+ binds
than its counterpart. Experimental lifetimes of each enantiomer with and without DNA present
17
were the same as the lifetimes recorded for the racemic mixture. This shows that the -isomer
has a greater affinity for DNA, not that the two isomers bind by different modes.2
Kelly, et al. also found that the fluorescence of [Ru(phen)3]2+ at 595 and 610 nm was
enhanced upon addition of CT DNA, most likely due to protection of the compound by the helix
from solvent quenching.
Further experimentation revealed that the emission observed is
sensitive to ionic strength, and even more sensitive to the concentration of Mg2+ over other
cations. Emission intensity was also found to increase when poly[d(A-T)] DNA was used,
leading to the assumption that Ru(phen)32+ prefers A-T or T-A binding sites to G-C or C-G.10
While increased fluorescence seemed to be consistent with intercalation, luminescence
studies yielded further results. Luminescence quenching experiments by the Barton group using
ferrocyanide indicated the presence of more than one binding mode. Linear behavior would be
expected from a Stern-Volmer plot indicating a single binding mode. The curvature instead
shows a downward trend, suggesting the existence of multiple accessibilities of the ruthenium to
the quencher. Since ferrocyanide quenches unbound [Ru(phen)3]2+ at a much higher rate than
any of those calculated for these experiments, it can be determined that the curvature observed is
resulting from more than one binding mode, rather than of one binding mode and free compound
in solution. Data also showed an increased affinity for the -isomer.8
Plasmid Unwinding and Photocleavage
Researchers believed that unwinding of supercoiled plasmid DNA was an indication of
intercalative binding. Upon intercalation, lengthening of the helix would ease the tension of the
plasmid, allowing for the removal of supercoils. These experiments were conducted using the
enzyme topoisomerase, which cleaves the backbone of DNA and removes supercoils.
18
Photocleavage is a secondary experiment done by both the Barton and Kelly groups. While the
ability of a metallointercalator to cleave the DNA backbone upon irradiation does not identify
intercalation from other binding modes, it is hypothesized to require an intimate association of
the molecule with the phosphate backbone, which is more likely from an intercalated position.
Topoisomerisation experiments by Kelly, et al. in 1985 were used to estimate the amount
of unwinding induced into supercoiled pBR322 DNA by the ruthenium compounds. DNA is
incubated with compound, and then with topoisomerase to relax the plasmid and remove all
remaining supercoils. However, upon electrophoresis, the intercalators are removed from the
plasmid, reintroducing the previously unwound coils unaffected by the topoisomerase.
Therefore, the more the plasmid is unwound, the greater the mobility when electrophoresed.
This shift of topoisomer bands characterizes intercalative unwinding.
Only the ruthenium
complexed with phenanthroline ligands was shown to unwind plasmid DNA; [Ru(bpy)3]2+ and
[Ru(terpy)3]2+ shown in Figure 12 did not show unwinding ability. By measuring the distance
between mean topoisomer bands and using the ratio of the concentration of compound added to
DNA, the unwinding induced per bound molecule could be calculated. Using varying amounts
of Mg2+, believed to alter binding of some intercalators, the estimate of unwinding ranged from
0.5° to 1.4°. However the most unwinding at 22o was achieved without the addition of Mg2+,
and is close to the value of 26o for standard intercalator ethidium bromide. All compounds tested
negative for inhibition of topoisomerase.10
Irradiating solutions of supercoiled pBR322 DNA with [Ru(phen)3]2+ and [Ru(bpy)3]2+ at
430 nm resulted in cleavage of the plasmid. Longer irradiation periods generated more cleavage,
and eventual fragmentation. More cleavage was also observed for short irradiation times and
increased compound concentrations.10
19
Additionally, electrophoresis results from the Barton group in 1984 indicated that both
enantiomers of [Ru(phen)3]2+ are able to unwind plasmid DNA. Since a lower concentration of
the -isomer compared to the -isomer removed all supercoils of the plasmid, the -isomer
shows a greater affinity for the DNA. In contrast to the photocleavage results by Kelly, et al.,
irradiation of the mixture with ultraviolet light showed that the DNA was not cleaved by the
ruthenium compound.2
Circular and Linear Dichroism
Equilibrium dialysis experiments were carried out with calf thymus DNA with a racemic
mixture of [Ru(phen)3]2+ by Barton, et al. in 1984. Following dialysis, a diluted solution of the
dialysate was characterized by circular dichroism. The results shown in Figure 11 indicate a
larger concentration of the -isomer in solution, and the conclusion again is an increased affinity
for -[Ru(phen)3]2+. The researchers then used circular dichroism to examine the spectra of lefthanded DNA combined with the ruthenium compound. The results showed that the addition of
[Ru(phen)3]2+ did not transform the DNA from a Z to B form, so no binding preference could be
determined.2
20
Figure 11: (□) represents the CD spectra of -[Ru(phen)3]2+. After dialysis: (+)
is the compound against B DNA and (■) show the spectra against Z DNA. Dialysis
against the B DNA leads to an increase of the -isomer in solution.
Using CT DNA, the LD spectra published by Hiort, et al. showed two distinct bands of
opposite signs at 470 and 380 nm for the -isomer. Binding of the -isomer yielded similar
spectra, although a smaller negative value at 480 nm, indicating that it may cause less
perturbation of the helix than the other. Since both enantiomers had very similar LD spectra for
both AT and GC rich DNA, it was determined that they bind by the same geometry. CD spectra
of DNA with a racemic mixture, however, did show a change in amplitude of the peaks with
different types of DNA,9 similar to the results found in the Barton group.
For the LD used in this study, hydrodynamic flow is used to align the axis of the DNA
such that the incident beams are parallel and then perpendicular to the axis. When they are
aligned perfectly, the orientation factor is said to equal one, while random orientation would
equal zero. A decrease in the signal in the region of base absorbance indicates that the -isomer
lowers the DNA orientation value, while its counterpart only affects it slightly.9
21
Equilibrium Dialysis
Equilibrium dialysis of [Ru(phen)3]2+ against DNA was used to determine dependence of
binding on ionic strength. Experiments showed that as ionic strength increased, binding affinity
decreased. On a plot of log of binding constant versus log of the concentration of positive ion
concentration (a measure of ionic strength), a slope value of 2.2 from the electrostatic component
suggests that 2.2 monovalent counterions from polymer DNA are released upon binding of the
ruthenium dication. This is the same slope value reported by Howe-Grand and Lippard for
binding of the divalent metallointercalator (phen)Pt(en)2+8.
Thermal Denaturation
Double stranded DNA is denatured into single stranded DNA upon the addition of heat.
When intercalators are bound, the helix is stabilized and a greater temperature is needed to
denature the DNA. Denaturation is monitored by taking the absorbance of the solution at 260
nm. With temperature plotted against absorbance, the melting temperature (Tm) is found by the
mean absorbance value.
Poly[d(A-T)] and calf thymus (CT) DNA were used to determine the effects of helix
stabilization by the polypyridyl ruthenium compounds studied by Kelly, et al. Similar results
were obtained with both types of DNA. At DNA/compound ratios of 50, 25, 12 and 6 of
Ru(phen)32+, the melting temperatures were increased by 4°, 10°, 18° and 20°C respectively.10
Chronological Conclusions of the Binding of Tris(phenanthroline)Ruthenium(II) to DNA
Barton, 1984. Based on a schematic view of each enantiomer bound to B form DNA,
there is steric hindrance between the ligands and the phosphate backbone in the case of the -
22
isomer, whereas its counterpart fits easily. The luminescence and helical unwinding assays
result in the suggestion that -[Ru(phen)3]2+ binds between 30 and 50% stronger than the other
isomer, while the arguably more sensitive circular dichroism studies suggest a 10 to 30%
increase in affinity. This relatively early study demonstrates the importance of researching the
interaction between different enantiomers of intercalating compounds in relation to the
handedness of DNA.4
Kelly, 1985. The study concludes that [Ru(phen)3]2+ may intercalate DNA, although the
process is difficult due to steric interference produced by the other two phenanthroline ligands.
Kelly, et al. also believed that in order for intercalation to occur, contribution from electrostatic
binding is also necessary. At the time of this study, the effect of Mg2+ concentration on
intercalation had not previously been reported, and the observation that sufficient concentrations
of this cation could prevent efficient plasmid unwinding was a novel idea. Due to enhancement
of emission intensity upon the addition of AT rich DNA versus GC rich leads to a comparison
between the observed preference of [Ru(phen)3]2+ for AT rich sequences, and the similar
preference for the intercalating drug daunomycin. Lastly, photolysis experiments show that
intercalation is not necessary for the cleavage of DNA, and that it is a result of singlet oxygen
production and redox reaction. The experiment shows future applications for determining site
specificity, based on the specific photolysis at guanine by methylene blue observed by Friedman
and Brown.10
Hiort, 1990. The authors conclude that the binding geometries for [Ru(phen)3]2+ with
different sequences of DNA are the same, but different for each enantiomer. The authors make a
direct comparison of their results to the claim of Barton, et al. that one enantiomer intercalates
while the other surface binds. The LD results do not exclude the possibility of two binding
23
modes, but only if the ratio of bound forms stay the same as binding increases. Since it had been
established that increasing ionic strength alters the binding ratio, increasing salt concentration,
would require the same free energy changes to take place for both binding modes. This does not
seem likely, since it could be expected that between intercalation and surface binding
electrostatic contributions would differ. While there seems to be a preference of the -isomer
for AT-rich DNA and the -isomer for GC-rich DNA, binding of the former is weakened upon
binding of the latter. It is also hypothesized that both enantiomers may bind in the major groove,
albeit with different binding geometries.9
Viscometry Data Induces More Controversy
Not all researchers agree that the studies previously used such as fluorescence and
plasmid unwinding are able to determine intercalation. Equilibrium dialysis and fluorescence
experiments done by Chaires, et al. in 1992 were in agreement.
Calculations of binding
constants from the experiments yielded that the -isomer binds 1.8 times more tightly than its
racemate, which was not enough to be considered selective by the authors.11
The binding dependence on salt concentration was also investigated and compared to that
of known intercalators ethidium bromide and daunomycin. The results were analyzed by the
polyelectrolyte theory of Record, et al. The slope of the lines in Figure 13 generated an estimate
of Zwhere  is the fraction of counterions associated with each phosphate of DNA and Z is
the ligand charge. The values calculated for ethidium bromide and daunomycin were 0.75 and
0.84. Theoretically calculated values for the ruthenium compound should then be at least 1.76,
however data showed much lower values, 1.38 and 1.24 for the  and -isomers.
The
contribution of nonelectrostatic forces to the binding constant can be found from further
24
calculation. The data showed that the binding of ethidium bromide and daunomycin is largely
due to non-electrostatic forces. The calculated values for [Ru(phen)3]2+ were 1-2 orders of
magnitude lower, indicating less favorable binding free energy. This finding suggests that the
binding of the ruthenium compound is predominately electrostatic.11
Figure 13: Salt dependence of binding constants, the following compounds are represented by
A: Daunomycin, B: Ethidium bromide, C: -[Ru(phen)3]2+, and D: -[Ru(phen)3]2+
In 1961 Lerman proposed that the lengthening of the DNA helix is an unambiguous result
of intercalation. One way to monitor this change is by measuring the viscosity of the solution
containing the intercalated DNA by observing the hydrodynamic flow. Figure 14 shows the
results of a comparative viscosity study of both [Ru(phen)3]2+ enantiomers using ethidium
bromide as the positive control, and groove binder Hoechst 33258 for the negative. As expected,
ethidium bromide increases the viscosity of the solution, resulting in a positive slope. Hoechst
33258 has essentially no slope, characteristic for a groove binding molecule which would not
alter the shape or length of a DNA helix. The absence of positive slope for the -isomer is
consistent with a nonintercalative binding mode. Interestingly, a negative slope is observed with
binding of the -isomer. This corresponds to results seen by Kapicak and Gabbay in 1975 who
25
speculated the decrease in viscosity was due to partial intercalation. This form of binding could
induce a bend or kink in the DNA which would in turn reduce the length of the helix.11
Figure 14: Viscosity of calf thymus DNA with increasing amounts of compound. The following compounds are
represented by A: Ethidium bromide, B: -[Ru(phen)3]2+, C: Hoechst 33258 and E: -[Ru(phen)3]2+
Chaires, et al. concludes the study by again citing previous publications. The authors
disagree that photophysical techniques can differentiate between binding modes. They instead
reiterate the importance of the viscosity experiments which clearly show that neither isomer
binds to DNA by full intercalation. They interpret their results to mean that the non-intercalated
phenanthroline ligands prevent complete intercalation by the other, resulting in a kink or bend in
the helix. The change in the shape of the helix is what the authors believe is responsible for the
decrease in orientation observed by Hiort, et al.
The conclusion also discredits helical
unwinding results by Barton, et al. in 1984 as a criterion for intercalation. It is stated that the
compound Irehidiamine A has been shown to unwind plasmid DNA but not increase the length,
as observed by Waring in 1970. Also, Wakelin, et al. in 1981 showed that crystal violet unwinds
26
DNA, but decreases viscosity. The authors speculate that plasmid unwinding may be a result of
intercalation, but cannot alone be considered proof of this binding mode.11
New Ruthenium Complexes
Bis(phenanthroline)dipyridophenazineRuthenium(II) ([Ru(phen)2dppz] 2+)
A 1993 publication by Hiort, et al. proposed to study the mode of interaction between
DNA and the enantiomers of [Ru(phen)2dppz]2+ shown in Figure 15 using linear dichroism,
steady-state and time-resolved luminescence spectroscopy.
Figure 15: Left () and right () enantiomers of [Ru(phen)2dppz]2+
Luminescence titrations yielded perfectly linear curves, suggesting that all compound
added over a range of 0.08 to 0.20 binding ratios was bound to DNA. Calculations of quantum
yield show a value 5.8 times greater for the -isomer than for its racemate. While a near perfect
fit of the non-cooperative binding equation by McGhee and von Hippel was obtained for the isomer, the titration curve for the -isomer does fall within a 5% margin of the mean of the
intensities calculated. So, although there is a lower quantum yield for the latter, it may still
compete for binding sites with the former. This observation also suggests that the binding
constants for both isomers are similar, with no enantioselectivity.12
Single-exponential decays of fluorescence were observed for pure enantiomers in
acetonitrile, however upon addition of DNA, two exponential decays were observed. Steady-
27
state and time-resolved data for conditions where all compound should be bound are in
agreement. At low binding ratios quantum yields decrease, but the ratio between enantiomers
remains essentially unchanged. Data show, however, that the -isomer has a quantum yield ten
times larger than the -isomer, which differs from the initial value of 5.8 from the luminescence
titration. The fact that the -isomer has a larger quantum yield seems to correlate with a much
longer excited state lifetime for the -isomer at the same compound/DNA ratio than for the isomer. Interestingly, when binding ratios are increased from 0.02 to 0.25, the longest lifetime is
not altered for the -isomer, but increases dramatically for the -isomer. Also for the latter, at
ratios above 0.25, the lifetime and population of long life component increase, suggesting the
presence of another binding mode.12
The shapes of the LD spectra obtained are similar, suggesting similar binding geometries
for the enantiomers. It was observed that altered orientation was responsible for varied LD
amplitude in the UV region upon binding of the compound. The orientation was increased by
both enantiomers, more so by the D-isomer. However, when the signal from the DNA was
removed, the LD spectra showed similar contribution from the enantiomers, again suggesting the
two bind with similar geometries. The authors state that because the shape of the LD spectra is
essentially the same for each enantiomer, it is reasonable to believe that the compounds bind
with the same geometry. If there is more than one binding mode, it would mean that they must
occur at the same ratios during the titration, which is not likely.12
They speculate that the reason behind the luminescence of the compound is due to a
delocalized charge transfer excitation which is restricted to the dppz ligand itself. This results in
the formation of an anion radical, which can be protonated in solution by water and thereby
quenched. The emission enhancement and larger lifetimes suggest that the radical is being
28
protected upon binding. The only reasonable explanation the authors give is that the ligand must
be completely inserted into the helix as in an intercalative mode.12
Barton,
et
al.
deemed
it
necessary
to
study
the
interaction
of
bis(phenanthroline)dipyridophenazineRuthenium(II) by modes other than fluorescence in order
to better understand the structural aspect of its binding. The researchers used 1H and 31P NMR
and thermal denaturation studies to support the model given by fluorescence studies. They
believed in the existence of two binding geometries, based on earlier quenching experimentation
by the group which yielded a bi-exponential decay in emission for both enantiomers. The group
proposed that there are two models for intercalation of the dppz ligand into DNA. The first
being a head-on mode in which the long axis of the ligand is parallel to the axis of the base pairs,
providing full protection of both nitrogens on the ligand. The second mode is proposed to be
more slanted, where the axis of the ligand forms an acute angle with the base pair axis, resulting
in one side of the ligand being protected more. Barton, et al. states that the previous observation
of differences in excited state lifetimes and quantum yields for the enantiomers suggests the
presence of two intercalative modes.13
1D NMR spectra obtained for the two isomers when bound to DNA were drastically
different, suggesting enantioselective binding. Also, perturbations of the phenanthroline ligand
protons were much smaller than for those of the dppz ligand, indicating that the dppz is
selectively involved in binding to the DNA for each enatiomer. However broader lines in the
spectra for the -isomer could indicate lower specific binding.13
The remainder of NMR experiments in the study focuses on the measurement of
intermolecular nuclear Overhauser effects (NOEs). NOEs are measured on the basis of a two
spin system. For instance, resonance of spin I is being monitored, while resonance of spin S is
29
saturated. These spins are close enough to experience a through-space interaction. The NOE is a
measure of the change in intensity of spin I on the saturation of spin S.14
The NOEs observed for protons on the sugar of the DNA and protons near the metal
center of the -isomer dppz complex indicate a very intimate binding, consistent with
intercalation. Also, because two NOEs representative of two protons on the dppz ligand are
present simultaneously, it is possible that there are two intercalative orientations. It is also
notable that there were no NOEs present for protons of the phenanthroline ligands. While the
cross-peak pattern is similar for the-isomer, no NOEs could be assigned.13
Melting temperature experiments also indicate differences in the binding affinities
between the two enantiomers. While the -isomer increased the melting temperature of the
DNA five degrees Celsius, its counterpart caused a shift of sixteen degrees. However, both
results indicate intercalative binding.13
Bis(phenanthroline)dipyridoquinoxalineRuthenium(II) ([Ru(phen)2dpq] 2+)
In 1997, Greguric, et al. studied the binding of a dppz analog, dipyridoquinoxaline (dpq)
shown in Figure 16, to DNA using NMR. While most metallointercalators have been proposed
to bind through the major groove, the authors believe that this compound intercalates through the
minor groove.15
30
Figure 16: Structure of [Ru(phen)2dpq]2+
Using 1D NMR, the resonances for the bound dpq ligand protons 11, 12 and 13 show
large upfield shifts, while those of the phenanthroline are very small, suggesting that the dppz
analog is selectively involved in binding to the DNA. Also, the shifts for the minor groove
protons were larger than those of the major groove.15
NOEs between the DNA and metal complex were observed for protons in the minor
groove associated with protons in positions 4, 10 and 13. The results suggest that this compound
is one of the few metallointercalators to bind through the minor groove.15
Intercalation by the ligand dpq is further supported in 1999 by Collins, et al. using 1 and
2D NMR, melting, and viscosity experiments. The compound was very similar to that studied by
Greguric, et al., with the addition of two methyl groups on the phenanthroline ligand
(dimethylphenanthroline).16
The NOE data showed that both enantiomers of the complex intercalated through the
minor groove.
The melting experiments showed that the -isomer shifted the melting
temperature eight degrees Celsius, while the -isomer showed a thirteen degree change.
Viscosity data showed that the latter increased the viscosity of solution, indicating intercalation,
while the former decreased the viscosity, leading to the conclusion that it only partially
intercalates. This result is similar to the observations by Chaires, et al. for the -isomer of
31
[Ru(phen)3]2+. Both studies performed on the dipyridoquinoxaline ligand indicate that the isomer intercalates completely, and through the minor groove.16
Our Contribution to DNA Binding Characterization of Ruthenium(II) Pteridinyl
Compounds
DNA Binding Studies Reported
Presented in this thesis are the results of fluorescence, viscometry, plasmid unwinding,
thermal denaturation and circular dichroism experiments performed on the compounds depicted
in Figure 17. These techniques were selected to focus on the determination of the intercalative
binding mode versus groove and surface binding modes.
It was hypothesized that the
compounds may bind to DNA via an intercalative mode given the similarity in structure of the
pteridinyl ligands to that of known intercalators such as dppz.
By positioning varying
constituent groups on the pterin ligand that match hydrogen bonding sites on Watson-Crick base
pairs increases the potential for hydrogen bonding to the nitrogenous bases.
The role of
hydrogen bonding in the mode of binding could be investigated. Results indicate that each
compound intercalates DNA with the exception of Ru(bpy)2(phen-pterin)]2+.
32
Figure 17: Structures of ruthenium pteridinyl complexes used for study
33
RESULTS
Reported in the following section are the results of fluorescence, viscometry, plasmid
unwinding, thermal denaturation and circular dichroism experiments. Studies show enhanced
emission, increased viscosity, plasmid unwinding ability, helix stabilization and changes in the
base stacking and helicity of DNA upon binding of[Ru(bpy)2(phen-dimethylalloxazine)]2+,
[Ru(bpy)2(phen-alloxazine)]2+ and [Ru(bpy)2(phen-diaminopteridine)]2+.
Fluorescence
Fluorescence experiments for each compound indicated an increase in emission intensity
with increasing concentration of calf thymus (CT) DNA, for example, Figure 18 with
[Ru(bpy)2(phen-alloxazine)]2+.
[Ru(bpy)2(dppz)]2+.
The data is very similar to titrations done with intercalator
This is a positive result of significant interaction with DNA, since
protection from solvent quenching is provided, meaning that the compounds have close
interaction with the helix. The data does not give information on mode of binding; however the
positive result gives reason for further study.
34
Figure 18: Fluorescence titration of [Ru(bpy)2(phen-alloxazine)]2+ at 20 M and increasing [calf thymus DNA]
Viscometry
Viscosity measurements were used to probe the mode of binding between the ruthenium
complexes and CT DNA. Others have shown that DNA is lengthened when an intercalator is
added which has the effect of increasing the viscosity.3,
17, 18
As seen in Figure 19, a slope
significantly greater than 0 was observed for all dye complexes except [Ru(bpy)3]2+ and
Ru(bpy)2(phen-pterin)]2+.
Two
other
complexes,
[Ru(bpy)2(phen-alloxazine)]2+
and
[Ru(bpy)2(phen-dimethylalloxazine)]2+, exhibited behavior identical to ethidium bromide and
[Ru(bpy)2(dppz)]2+ which are known to intercalate into CT DNA.3,
13, 19
One compound,
[Ru(bpy)2(phen-diaminopteridine)]2+, exhibited an intermediate slope suggesting that no single
binding mode predominates. Through many trials, it was determined that viscometry studies are
35
very sensitive to temperature changes, and this variable must be kept constant throughout the
experiment to provide reliable data.
Viscosity Titration: ~2.5 mM Drug in Acetonitrile &
0.900.90
mMmM
Calf
bufferwith
DNA in DNA
CTThymus
increasing concentration of complex
1.2
EtBr
h /ho
1/3
1.15
Allox
Bpy3
1.1
Diamino
1.05
DMA
Pterin
1
0.95
0.00
Dppz
0.05
0.15
0.10
0.20
DNA
Drug:Calf
Complex:Thymus
CT DNA
Figure19: Viscosity plot of [Ru(bpy)2(phen-alloxazine)]2+ (Allox), [Ru(bpy)2(phen-pterin)]2+ (Pterin),
[Ru(bpy)2(phen-dimethylalloxazine)]2+ (DMA) and [Ru(bpy)2(phen-diaminopteridine)]2+ (Diamino), including
negative control [Ru(bpy)3]2+ (Bpy3), and positive controls [Ru(bpy)2(dppz)]2+ (Dppz) and ethidium bromide (EtBr)
Plasmid Unwinding
Determination of Topoisomerase activity.
Varying experiments with topoisomerase were performed to determine conditions
necessary to acquire complete relaxation of the plasmid. Bacterial topoisomerase is reported to
function most efficiently at a pH of 7.5. Since Tris-HCl is known to lower pH as it is heated, it
was thought that the buffer capacity might be exhausted upon incubation. However, upon
36
varying the starting pH from 7.8 to 7.5 and incubation times of 30 to 90 minutes for different
batches of enzyme, no difference in activity was seen. Sigma reports that the addition of Mg2+ to
the bacterial topoisomerase can enhance enzyme activity. Adding up to 2.5 mM Mg2+ to the
enzyme buffer at 1 to 3 times the calculated concentration of enzyme necessary shows a slight
increase in relaxation, however not complete function. Complete relaxation of the supercoiled
plasmid, as seen in lanes 3 and 6 in Figure 20, was only achieved at 10 times the calculated
concentration with 2.5 mM Mg2+ present for an incubation time of 90 minutes.
1
2
3
4
5
6
Figure 20: Lanes 1-3: 1, 5 and 10x concentrations of topoisomerase
Lanes 4-6: 1, 5 and 10 x concentrations of topoisomerase
spiked half way through incubation
Test for Enzyme Inhibition: Relative Unwinding.
Since an intercalator can have the ability to inhibit topoisomerase, false negative results
can be obtained.20 All ruthenium compounds and intercalator controls, ethidium bromide and
[Ru(bpy)2(dppz)]2+, were each added at 1.5 M to supercoiled plasmid in an unwinding
experiment. The results showed that all compounds were able to resupercoil to some degree,
with the exception of [Ru(bpy)2(phen-pterin)]2+, which did not show any unwinding activity.
The data indicate that none of the compounds inhibit relaxation by topoisomerase, since each
resupercoiled to a lesser degree than the untreated plasmid.
37
The assay also gives information about the relative abilities of the compounds to unwind
the supercoiled plasmid as shown in Figure 21. For comparison, controls were used of relaxed
plasmid in lane 1, supercoiled plasmid in lane 2 and positive intercalator controls ethidium
bromide (EtBr) in lane 3 and [Ru(bpy)2(dppz)]2+ in lane 4.
Relative unwinding could be
determined because all the ruthenium compounds were at the same concentration. Based on the
mobility of the bands, it can be interpreted that [Ru(bpy)2(dppz)]2+ and [Ru(bpy)2(phendimethylalloxazine)]2+ interact with DNA similarly and to the same degree, while
[Ru(bpy)2(phen-alloxazine)]2+ and [Ru(bpy)2(phen-diaminopteridine)]2+ have significantly less
resupercoiling. [Ru(bpy)2(phen-pterin)]2+ does not show any ability to unwind DNA, and
therefore no further unwinding experiments using this compound will be presented.
1
2
3
4
5
6
7
8
Relaxed
Topoisomer
Supercoiled
Figure 21: Lane 1: Relaxed; Lane 2: Supercoiled; Lanes 3-8: EtBr, [Ru(bpy)2(dppz)2+, [Ru(bpy)2(phen-L)]2+
where L is dimethylalloxazine, alloxazine, diaminopteridine and pterin) at 1.5 M
Unwinding Results.
While the ability to unwind supercoiled plasmid DNA is not a definitive method for
determining intercalation,11 it is a commonly observed trait.4, 10 Results are shown in Figures 22
though 25. The mobilities shown in these figures are slightly different than those shown in
Figure 21 due to differences in sample purity. After sample preparation, the samples were
electrophoresed on a 1% agarose gel containing 0.1% SDS to resolve the bands. The following
38
concentrations of metal compound were present in samples run in lanes 1-5: [Ru(bpy)2(dppz)]2+,
0.44, 0.66, 0.88, 1.1 and 1.3 M; [Ru(bpy)2(phen-alloxazine)]2+, 0.25, 0.5, 1.0, 1.3, 1.6 M;
[Ru(bpy)2(phen-dimethylalloxazine)]2+, 0.33, 0.66, 0.88, 1.1, 1.5 M; [Ru(bpy)2(phendiaminopteridine)]2+, 0.2, 0.25, 0.3, 0.45, 0.6 M along with the appropriate concentration of
topoisomerase necessary to achieve complete relaxation. Lane 6 contained supercoiled plasmid
with the same concentration of metal complex in lane 5. Lane 7 in Figures 23 and 25 contains
completely relaxed plasmid, and lane 8 contains supercoiled plasmid alone. As compared to
[Ru(bpy)2(dppz)]2+, all complexes except for [Ru(bpy)2(phen-pterin)]2+ (not shown) display the
same unwinding capabilities.
With increasing concentrations of complex, the plasmid
resupercoils to an increasing degree, with an increased mobility through the gel. This result
indicates
that
[Ru(bpy)2(phen-alloxazine)]2+,
[Ru(bpy)2(phen-dimethylalloxazine)]2+
and
[Ru(bpy)2(phen-diaminopteridine)]2+ all have the ability to unwind supercoiled plasmid DNA,
one of the effects of intercalation as shown by [Ru(bpy)2(dppz)]2+.
No
Topo blank
[Ru]
1
2
3
4
5
6
7
SC
alone
8
Figure 22: Positive control: Lanes 1-5: supercoiled + Topo and increasing concentrations of
[Ru(bpy)2(dppz]2+. Lane 8: Supercoiled alone
39
No
Topo
[Ru]
1
2
3
4
5
Rel
6
SC
alone
7
8
Figure 23: Lanes 1-5, SC + Topo and increasing concentrations of [Ru(bpy)2(phen-alloxazine)]2+
Lane 6: SC+ Ru, Lane 7: Relaxed only, Lane 8: Supercoiled only
No
SC
Topo blank alone
[Ru]
1
2
3
4
5
6
7
8
Figure 24: Lanes 1-5, SC + Topo and increasing concentrations of [Ru(bpy)2(phen-dimethylalloxazine)]2+
Lane 6: SC+ Ru, Lane 8: Supercoiled only
40
No
Topo
[Ru]
1
2
3
4
5
6
Rel
SC
only
7
8
Figure 25: Lanes 1-5, SC + Topo and increasing concentrations of [Ru(bpy)2(phen-diaminopteridine)]2+
Lane 6: SC+ Ru, Lane 7: Relaxed only, Lane 8: Supercoiled only
Thermal Denaturation
Intercalating molecules have many effects on the DNA helix, one being to stiffen the
helix and increase its rigidity. Therefore an increase in the melting temperature (Tm) of the
DNA with increasing concentrations of a binding molecule can signify intercalation.16 For
example, intercalating ligand Dppz has been published to cause a shift in Tm of more than 8
°C.17
The melting curves at a constant ratio of 15 for [DNA]:[Complex] in 2 mM phosphate
buffer and 3 mM NaCl are shown in Figure 26. The melting points calculated from these curves
using the mean absorbance of the DNA are listed in Table 1. Each compound except for
[Ru(bpy)2(phen-pterin)]2+ shifted the melting temperature of the DNA. Increasing shifts were
observed for increasing concentrations of complex.
The data suggest that the other three
complexes intercalate DNA, stiffening the helix, and require a larger melting temperature. It is
also possible to estimate the relative strengths of the compounds to intercalate. Based on Table
1, the trend in strength of binding appears to be [Ru(bpy)2(dppz)]2+ (dppz) > [Ru(bpy)2(phendimethylalloxazine)]2+ (dimethyl alloxazine) > [Ru(bpy)2(phen-alloxazine)]2+ (alloxazine) >
[Ru(bpy)2(phen-diaminopteridine)]2+ (diamino).
41
DNA:Ru 15
[DNA]:[Ru Complex] = 15
Compounds
Melting
temperature (°C)
Relative Abs @ 260 nm
1.4
1.35

CT DNA
64.5
1.3

(bpy)3
64.5

pterin
64.5
1.15

diamino
67.5
1.1

alloxazine
75.0

dimethyl
78.0

dppz
80.0
1.25
1.2
1.05
1
0.95
50
55
60
65
70
75
80
85
90
95
o
Temperature ( C)
Table 1
Figure 26: Plot of temperature vs. absorbance at 260 nm for CT DNA alone and with the addition of each
Table
compound at [DNA]:[Complex] of 15
1: Melting temperatures obtained for each compound as compared to DNA alone, a negative control
[Ru(bpy)3]2+, and a positive control [Ru(bpy)2(dppz)]2+
Circular Dichroism
The CD signals are sensitive to base stacking at 275 nm and helicity at 245 nm are easily
altered by an intercalating molecule; making circular dichroism a useful technique for
determining binding mode. Since surface and groove binding have little or no effect on DNA
structure, intercalation can be distinguished by changes in CD signal.21 As seen in Figures 27
and 28, upon addition of each metal complex at [DNA]:[Complex] of 10 with the exceptions of
[Ru(bpy)3]2+ and [Ru(bpy)2pterin]2+, the positive signal at 275 nm disappears and a negative band
at 291 nm appears. This change is indicative of a B to Z transition, as seen for metal complexes
containing the known intercalating ligand, Dppz.17 Variation in amplitude of the signals can be
attributed to the amount of DNA transformed. Therefore, a trend in the ability of the compounds
42
to efficiently change B form DNA to Z form can be established as follows: [Ru(bpy)2(dppz)]2+
(RuDPPZ)
>
[Ru(bpy)2(phen-dimethylalloxazine)]2+
(RuDMA),
[Ru(bpy)2(phen-
diaminopteridine)]2+ (RuDAP) > [Ru(bpy)2(phen-alloxazine)]2+ (RuALX).
Molar Ellipticity (deg cm 2 dmol-1)
10 CT DNA:Ru(bpy)3 & Ru(bpy)2DPPZ
CT DNA
3.0E+05
+Ru(bpy)3
2.0E+05
+RuDPPZ
1.0E+05
0.0E+00
230
-1.0E+05
250
270
290
310
-2.0E+05
-3.0E+05
Wavelength (nm )
Figure 27: Controls: Circular dichoic spectra of CT DNA alone and with addition of a positive control
[Ru(bpy)2(dppz)]2+ (RuDPPZ) and a negative control [Ru(bpy) 3]2+ (Ru(bpy)3) at [DNA]:[Complex] of 10
43
10 CT DNA:Ru(bpy)2 - pterin, DMA, DAP, ALX
Molar Ellipticity (deg cm 2 dmol-1)
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
310
-1.00E+05
-1.50E+05
-2.00E+05
Wavelength (nm)
Figure 28: Circular dichoic spectra of CT DNA alone and with addition of [Ru(bpy)2(phen-pterin)]2+ (Rupterin),
[Ru(bpy)2(phen-dimethylalloxazine)]2+ (RuDMA),[Ru(bpy)2(phen-diaminopteridine)]2+ (RuDAP), and
[Ru(bpy)2(phen-alloxazine)]2+ (RuALX) at [DNA]:[Complex] of 10
44
DISCUSSION
Preliminary fluorescence data indicates that each of the compounds synthesized by the
Burgmayer lab interact with DNA in some fashion. Since the majority of researchers in this field
place doubt on the ability of fluorescence to distinguish binding mode, additional
experimentation was required.
The results of viscometry, plasmid unwinding, thermal
denaturation and circular dichroism are in agreement that each of the three compounds
[Ru(bpy)2(phen-dimethylalloxazine]2+,
[Ru(bpy)2(phen-alloxazine]2+
and
[Ru(bpy)2(phen-
diaminopteridine]2+ bind to DNA by an intercalative mode. The data from each experiment also
show, surprisingly, that [Ru(bpy)2(phen-pterin]2+ does not exhibit intercalation.
Fluorescence Results Provoke Additional Experimentation
Fluorescence and absorbance studies cannot distinguish between binding mechanisms.17,
22
They do, however, give information about the proximity of a fluorescent compound to DNA,
and an indication of affinity.
Exploratory fluorescence experiments in the Burgmayer lab
indicated positive results for DNA interactions, as in Figure 18 there is enhanced emission
intensity upon increasing concentrations of CT DNA.
Each ruthenium compound has an
inherent fluorescence, which is diminished by quenching through hydrogen bonding from the
solvent. When the complex is held in close proximity to the DNA, the helix provides protection
from solvent quenching, thus enhancing the inherent fluorescence of the compound to be
monitored. Since each compound displays this enhancement, early conclusions assumed that the
compounds are interacting with the DNA in some fashion.
Reports question the validity of fluorescence experiments to determine what binding
mode compounds utilize to complex to the DNA helix. It is expected that a simple electrostatic
interaction would be present, given the 2+ charge on the ruthenium compounds and the high
45
negative charge provided from the phosphates of the DNA back bone. Further experimentation
was therefore necessary to determine whether the compounds were surface binding, groove
binding or intercalating.
Due to the similarity of the pteridine ligands planar aromatic structure to that of known
intercalating ligand dipyridophenazine (dppz), it was hypothesized that the initial electrostatic
attraction may bring the compounds in close proximity to the DNA, enabling them to intercalate
between the base pairs.
It was for this reason experimental methods used to monitor the
perturbations of the DNA helix induced by intercalating molecules were chosen.
Increased Viscosity as a Positive Marker for Intercalation
Others have published that an increase in viscosity is an unambiguous result of
intercalative molecules have on DNA in solution.
3, 17 , 18
There are several methods that are
sensitive to hydrodynamic changes of solution in addition to viscometry such as rotational
diffusion and sedimentation.
Viscometry, however, is a relatively simple, inexpensive and
reportedly reliable method to monitor these changes. A marked increase in viscosity is seen due
to the overall lengthening of the DNA helix brought about by base pairs being pushed apart as a
molecule intercalates between them.17 This effect should not be seen for groove or surface
binding molecules as they do not disturb the shape of the helix as seen in Figure 14 for the
standard groove binder Hoechst 33258.11
Results shown in Figure 19 clearly show a substantial increase in slope with increasing
concentration of the three novel ruthenium compounds, [Ru(bpy)2(phen-dimethylalloxazine]2+,
[Ru(bpy)2(phen-alloxazine]2+ and [Ru(bpy)2(phen-diaminopteridine]2+
similar to those of
positive control compounds ethidium bromide and [Ru(bpy)2(dppz)]2+. This result indicates that
46
these compounds increase the length of the helix, which can only be accomplished via
intercalative binding.
Viscometry experiments with [Ru(bpy)2(phen-diaminopteridine]2+
produced a slightly smaller slope than the other compounds, which could indicate weaker
intercalative binding or a second binding mode. [Ru(bpy)3]2+ and [Ru(bpy)2(phen-pterin)]2+ do
not show an increase in slope upon binding. [Ru(bpy)3]2+ has previously shown to be incapable
of intercalative behavior due to the lack of the large planar moiety which stacks between the base
pairs. Therefore, it is not surprising that it has a nearly zero slope. The most surprising result
was that in this, as in the other techniques explained below, [Ru(bpy)2(phen-pterin)]2+ did not
display intercalative characteristics. It is apparent, based on results from this reportedly reliable
method, this compound does not bind to DNA via an intercalative mode. However, fluorescence
results indicate that the compound does interact with DNA in some fashion. In order to further
characterize the binding properties of these novel compounds to DNA, additional
experimentation was done.
Plasmid Unwinding: A Characteristic of Intercalative Binding
While some authors claim an ability of intercalating compounds to remove supercoils
from plasmid DNA,4, 10 others dispute it as a criterion for intercalation.11 It is a consensual view
that plasmid unwinding is a common observation for intercalative compound, however not a
definitive method. The theory behind the experiment is depicted in Figure 29.23
47
Figure 29: Helical unwinding by increasing concentrations of intercalator (B-D) Lk is the linking number, or the
number of times the strand crosses itself. a is the addition of topoisomerase, and b is the removal of the intercalator
by electrophoresis23
In order to understand the concept of the experiment, it is necessary to describe the
supercoiling of a plasmid by the following equation: Lk = Tw + Wr, where Lk is the linking
number, Tw is the twist, and Wr is the writhe. The linking number is a fixed integer that
describes the number of times a strand is linked, or crosses over itself. It is independent of the
other variables, and can only be altered by breaking and annealing the strands. Twist is the
arrangement of the strands twisting around each other in space. Writhe is a way to describe the
change of the helical axis in space. Twist and writhe are variable parameters.
The experiment relies on the ability of the enzyme, topoisomerase I, to relax and remove
supercoils from supercoiled plasmid DNA. Topoisomerase I actually cleaves one DNA strand,
and allows it to unwind around the other to relieve tension. The strands are then ligated,
resulting in removal of supercoils. Therefore, the addition of topoisomerase I is the only way to
reduce the linking number of a plasmid. To input the effect of topoisomerase into the above
equation, as the plasmid is relaxed, the Wr approaches zero, and therefore Lk = Tw.
Intercalation results in the reduction of Tw. Topoisomerase is activated by the change in Tw, and
48
adjusts the Lk to account for it. Now, Lk is a measure of the Tw of the plasmid induced by the
intercalated compound.24
In Figure 29, as the intercalator (I) is added, the Lk becomes less negative (Lk is assigned
a negative value to reflect the direction of the supercoils: by convention, right handed DNA has
negative supercoiling), a result of the lowering of the Tw parameter. Upon the addition of
topoisomerase (a), the remaining supercoils are removed, and the Lk is equal to zero. A high
enough concentration of intercalator can actually induce supercoiling in the opposite, or positive
direction, as in situation D. When the DNA is electrophoresed on an agarose gel, the intercalator
is removed (b). The result is that the plasmid is now allowed to resupercoil, increasing the Tw
that was removed upon intercalation. Thus, a positive marker for helical unwinding would be an
increase in supercoiled plasmid with increasing concentrations of intercalator. This can be
visualized in the gel by an increased mobility of supercoiled plasmid, versus the lower mobility
of more relaxed plasmid, as seen for intercalator [Ru(bpy)2(dppz)]2+ in Figure 22.
In Figures 23 through 25, it can be seen that there is an increase of supercoiled plasmid
with increasing concentrations of the ruthenium compounds, shown by the futher migrating
bands. While the concentrations of each compound necessary to show a gradual shift varied
slightly, they were all on the same order. Therefore, it can be determined that [Ru(bpy)2(phendiaminopteridine)]2+, [Ru(bpy)2(phen-alloxazine)]2+ and [Ru(bpy)2(phen-dimethylalloxazine)]2+
all unwind plasmid DNA with similar strength as [Ru(bpy)2(dppz)]2+. In contrast, lane 8 in
Figure 21 shows that [Ru(bpy)2(phen-pterin)]2+ did not show plasmid unwinding ability. This
observation is in agreement with viscometry data.
49
Thermal Denaturation: Stabilization of the Helix
Researchers have attributed an increase in melting point of DNA to stabilization of base
stacking by intercalated molecules.21 Since buffer and salt conditions can also stabilize DNA,
this is an ambiguous indicator for intercalation; however, proven intercalators such as the dppz
ligand have been reported to increase the melting temperature of CT DNA by upwards of 8
degrees, Celcius17 and derivatives of the ligand have shown a shift of up to 13 degrees.21 The
melting point of DNA is found by monitoring the absorbance at 260 nm over a range of
temperatures. The temperature is plotted against the absorbance, in which the mean absorbance
corresponds to the melting temperature (Tm) of the DNA.
The Tm values calculated based on the graph in Figure 26 are shown in Table 1. The CT
DNA at a concentration of 100 M alone was shown to have a Tm of 64.5°C in 2 mM phosphate
buffer and 3 mM NaCl. The same Tm value of 64.5 was obtained with the addition of nonintercalators [Ru(bpy)3]2+ and [Ru(bpy)2(phen-pterin)]2+at 6.7 M.
The other compounds
increased the Tm by a range of 3 to 12 degrees. [Ru(bpy)2(dppz)]2+ increased the Tm of the CT
DNA by 15 degrees under these conditions. While [Ru(bpy)2(phen-diaminopteridine]2+ showed
the smallest shift in Tm, [Ru(bpy)2(phen-alloxazine]2+ and [Ru(bpy)2(phen-dimethylalloxazine]2+
shifted the Tm by 10 and 12 degrees respectively. This data is in agreement with the viscometry
and plasmid unwinding data. It can be interpreted from these results that [Ru(bpy)2(phendiaminopteridine]2+
has
less intercalative binding than the other compounds, and
[Ru(bpy)2(phen-pterin]2+ does not intercalate DNA.
50
Circular Dichroism Sensitive to Base Stacking and Helicity Changes
The use of circular dichroism (CD) to monitor changes in helical structure has been used
by several researchers to monitor intercalation.17 This method is sensitive to binding mode
because surface and groove binding molecules which do not alter the structure of the helix, also
do not alter the CD signal. Intercalating molecules have been shown to change DNA from a B
form to a Z form.17 These two forms of DNA shown in Figure 30 are drastically different. Most
DNA is naturally occurs in the B form. This form of DNA is characterized by a right handed
helix which makes a turn every 3.4 nm and encompasses 10 base pairs per turn. Z form DNA is
a left handed helix which turns every 4.6 nm and includes 12 base pairs for each turn.25
Figure 30: B and Z form DNA which differ in handedness, length and base pairs per turn25
The resulting CD spectra of these two forms of DNA are also very different. For B form
DNA, the main points of interest are a positive band around 273 nm distinctive of base stacking
and a negative band around 242 nm representing helicity.
A change to Z form DNA is
characterized by the disappearance of the positive band, and the development of both a negative
band around 285 nm and a positive band around 260 nm. Figure 31 shows the CD spectrum for
51
B form CT DNA changing to Z form upon the addition of intercalating [Cr(dppz)2Cl2]+ at 52
M.17
Figure 31: B form CT DNA (____) and Z form DNA (-----) upon the addition of [Cr(dppz)2Cl2]+ 17
This spectrum bares a strikingly similar shape to those shown in Figures 27 and 28. The
data collected for the pteridinyl ruthenium compounds all show a change from B to Z form DNA
with the exceptions of [Ru(bpy)3]2+ and [Ru(bpy)2(phen-pterin)]2+, which have no effect on the
DNA, and remains in a B form. The relative amplitude of the peaks corresponds to the amount
of sample changed from a B form to a Z form. The data obtained from the CD spectra agree
with that of the viscometry, plasmid unwinding and thermal denaturation data. While all the
novel ruthenium compounds induce enough change in the shape of the DNA helix to transform
CD signal, [Ru(bpy)2(phen-pterin]2+ does not.
52
Evaluation of DNA Binding Properties of Pteridinyl-Phenanthroline Complexes of Ru(II)
Based on prior research, it is apparent that one method alone cannot provide a good case
for intercalative binding. It is for this reason that the data from several different methods should
be taken into account and compared. Viscometry, plasmid unwinding, thermal denaturation, and
circular dichroism experiments performed on the four ruthenium pteridinyl complexes are all in
agreement. Each compound intercalates DNA with the exception of [Ru(bpy)2(phen-pterin]2+.
Each method reproducibly suggests the same rough estimate of the relative binding strengths for
the
compounds
as
follows:
[Ru(bpy)2(phen-dimethylalloxazine)]2+
>[Ru(bpy)2(phen-
alloxazine)]2+ >[Ru(bpy)2(phen-diaminopteridine)]2+. This order agrees with all the data except
the CD spectra, in which the [Ru(bpy)2(phen-diaminopteridine)]2+ appears to change the helical
shape the most.
It is clear that the binding of the other three complexes is very different from that of
[Ru(bpy)2(phen-pterin]2+. Fluorescence data indicates that the compound does interact with
DNA, but the experimentation shows that the binding mode is not intercalative. To find a reason
for this difference, it is essential to study the structural differences of the ligands in Figure 17.
The characteristics of these compounds allow for many different possibilities for association.
Each compound has an overall positive charge, which would be expected to be attracted to the
negative charge on the phosphates comprising the DNA backbone. The compounds each have
planar heterocyclic structures that would fit between base pairs of DNA without steric hindrance.
Lastly, each ligand has its own combination of functional groups.
While the four compounds are all based on the same basic bicyclic pteridine structure, the
constituent groups are different. The compounds have different placement of oxygen, amine and
methyl groups. The main differences between these groups are the polarity and hydrogen
53
bonding capabilities they possess. The difference in binding mode between [Ru(bpy)2(phenpterin]2+ and the three intercalative compounds therefore must be based on this characteristic.
Figure 32 depicts the similarities between the nitrogenous base pairs and intercalative alloxazine
and the non-intercalative pterin. The placement of the hydrogen bond donors and acceptors for
Alloxazine
Pterin
Figure 32: Comparison between the hydrogen bond donors and acceptors of pteridine ligands and
nitrogenous bases26
the alloxazine ligand are in the same placement as those of thymine, which is capable of making
two hydrogen bonds to adenine. The placement of the hydrogen bond donors and acceptors of
the pterin ligand are the same as those of cytosine, which makes three hydrogen bonds to
guanine. It could be possible that it is more favorable for the pterin ligand to make three
hydrogen bonds somewhere along the helix to unpaired bases or portions of single stranded DNA
rather than intercalate, whereas only making two hydrogen bonds for the other ligands is less
54
favorable than intercalation. This could be a contributing factor as to why the [Ru(bpy)2(phenpterin]2+ complex shows such different binding characteristics to the other three compounds,
especially [Ru(bpy)2(phen-alloxazine]2+.
Conclusions
The combination of viscometry, plasmid unwinding, thermal denaturation and circular
dichroism data show that [Ru(bpy)2(phen-dimethylalloxazine)]2+ , [Ru(bpy)2(phen-alloxazine)]2+
and [Ru(bpy)2(phen-diaminopteridine)]2+ do bind to DNA via an intercalative mode. These
experiments with the combination of fluorescence data indicate that the parent compound,
[Ru(bpy)2(phen-pterin]2+ does not bind to DNA by intercalation, but by some other association.
It remains to be seen whether the three intercalative compounds have sequence specificity, what
role the hydrogen bonding capabilities play in their binding to DNA, or if the interaction is
enantiospecific. Additional study of these compounds leading to further characterization of
binding could result in the development of stable complexes for use as DNA probes and the
discovery of life saving pharmaceuticals.
55
MATERIALS
[Ru(bpy)2(phen-pterin)](PF6)2,
[Ru(bpy)2(phen-alloxazine)](PF6)2,
[Ru(bpy)2(phen-
dimethylalloxazine)](PF6)2, and [Ru(bpy)2(phen-diaminopteridine)](PF6)2 were synthesized in
the Burgmayer lab. [Ru(bpy)2(dppz)](PF6)2 was a gift from Prof. Stefan Bernhard, Princeton
University.
Calf thymus (CT) DNA was purchased from Sigma-Aldich Co. The solid DNA was
added to a 10 mM phosphate buffer with 50 mM NaCl and sonicated for ~1.5 hours. The
solution was vortexed, centrifuged for 12 minutes, and then vortexed again. Solutions of CT
DNA used for fluorescence, viscometry and circular dichroism were made with 10 mM
phosphate buffer and 50 mM NaCl, and 2 mM phosphate buffer and 3 mM NaCl for thermal
denaturation experiments.
pGEM-4Z DNA (2.7kb) was purchased from Promega Corporation at a concentration of
1 mg/mL. Electrophoresis 5X sample loading buffer, EZ Load 500 base-pair Molecular Ruler
and electrophoresis 10X TBE buffer were purchased from Bio Rad, and bovine serum albumin
(BSA) from New England Biolabs. Topoisomerase I (Topo) from Vaccinia virus was purchased
from Sigma-Aldrich Co. The enzyme had an activity such that one unit converts 1 g of
supercoiled plasmid to relaxed form in one hour at pH 7.5 at 30 oC. All other chemicals,
including Proteinase K at a concentration of 22 mg/mL, were purchased from Sigma-Aldrich Co.
Reactions for plasmid unwinding experiments were done in relaxation buffer as prepared
by Webb and Ebeler.20 The buffer contained 50 mM Tris-HCl, 20 mM KCl, 1 mM EDTA, 1
mM dithiothreitol, and 0.3 mg/mL BSA. The pH of the solution was adjusted to 7.5. Proteinase
K was diluted to the appropriate concentration with a solution of 50 mM Tris-HCl and 1 mM
CaCl2.
56
METHODS
Fluorescence. Fluorescence titrations were performed using a SPEX Fluoromax-3.
Ratios of
[CT DNA]:[Complex] between 1 and 75 were achieved using CT DNA as the titrant with
concentrations of complex between 5 and 20 M.
Viscometry. An Ostwald viscometer in a non-circulating water bath at 23oC was used to measure
the relative viscosity of ethidium bromide and all ruthenium complexes. A 2.5 mM stock
solution of each ruthenium complex, prepared in acetonitrile due to limited solubility, was
titrated with 0.90 mM CT DNA. The solution was bubbled in the viscometer with N2 for 1
minute to mix prior to measurements. The theory of Cohen and Eisenberg27 was used to plot the
data as (h/h)1/3 versus time. The concentrations of the Ru complex and CT DNA were chosen
to minimize the volume of Ru complex added to a solution of DNA concentrated enough to
make changes in the slope maximally distinguishable.
Plasmid Unwinding. Supercoiled pGEM plasmid was diluted with relaxation buffer to 4.4 ng/L
and 2.5 mM MgCl2. For complete relaxation, 10 units of Topo were added to an aliquot of 115
L of supercoiled plasmid and incubated at 30oC for 90 minutes.
In sterilized 0.5-mL
microcentrifuge tubes, 2 L of metal complex was added to 17 L of relaxed or supercoiled
DNA.
Ru compounds were diluted using relaxation buffer to appropriate concentrations
depending on compound to show optimum distribution of topoisomers. The samples were
incubated in the dark at room temperature (~25°C) for 20 minutes. One unit of Topo was then
added to each 19 L sample, and incubated at 30°C in the dark for 90 minutes.
The
topoisomerization was stopped by immediately adding 2.5 L of 10% sodium dodecyl sulfate
57
(SDS). The addition of 2.5 L of 1 mg/mL Proteinase K to each sample digested the protein
when incubated in the dark at 45°C for 60 minutes.
Samples were refrigerated prior to
electrophoresis for no more than one day.
Electrophoresis was carried out using a horizontal Hoefer HE33 submarine agarose gel
unit. The samples were electrophoresed at 2.2 V/cm for 10 hours on gels containing 1% agarose
and 0.1% SDS. The gels were stained for 40 minutes in 1 g/mL ethidium bromide (EtBr), and
destained in water for 1 hour. A Bio Rad Geldoc imager was used to visualize the gels by UV
illumination.
Thermal Denaturation. Continuously monitoring the absorbance at 260 nm from a sample, the
temperature was increased from 25° to 92°C and recorded by a PerkinElmer Lambda Bio
UV/Visible spectrophotometer. The temperature of the samples was controlled by an Endocal
RTE-110 water bath and monitored by an Omega DP701 thermometer. The heating rate was
approximately 1°C/minute. The samples had a ratio of 15 [CT DNA]:[Complex]. CT DNA was
used at a concentration of 100 M and complexes at 6.7 M.
Circular Dichroism. An AVIV 202-01 spectropolarimeter at Haverford College operating at
25°C was used to collect spectra between 200 and 350 nm. Samples contained a ratio of 10 [CT
DNA]:[Complex] using a 1cm pathlength cell. Molar ellipticity values [] were calculated using
the formula: [] = ____ x 100 where  is the observed ellipticity in degrees, c is the
cl
concentration in molarity, and l is the pathlength in decimeters.
Fluorescence experiments were conducted predominately by Samantha Glazier and
Alanna Albano, and recent viscometry studies were completed by Alanna Albano.
58
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15. Greguric, I., Aldrich-Wright, J, Collins, J, A 1H NMR study of the binding of delta[Ru(phen)2DPQ]2+ to the hexanucleotide d(GTCGAC)2. Evidence for intercalation from the minor
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60
ABBREVIATIONS
ALX, Allox : Alloxazine
Bpy : Bipyridyl
BSA : Bovine serum albumin
CD : Circular dichroism
CT DNA : Calf thymus DNA
DAP, Diamino : Diaminopteridine
DMA, Dimethyl : Dimethylalloxazine
Dppz : Dipyridophenazine
Dpq : Dipyridoquinoxaline
EtBr : Ethidium bromide
LD : Linear dichroism
Lk : Linking number
MGP : 4-(guanidinylmethyl)-1,10 phenanthroline
MLCT : Metal to ligand charge transfer
Phen : Phenanthroline
Phi : phenanthrenequinone diimine
Rel : Relaxed
SC : Supercoiled
Tm: Melting temperature
Topo : Topoisomerase I
Tw : Twist
Wr : Writhe
yAP-1 : yeast protein activator protein 1
61