2026–2033
Nucleic Acids Research, 2001, Vol. 29, No. 10
© 2001 Oxford University Press
Charge transport through DNA four-way junctions
Duncan T. Odom, Erik A. Dill and Jacqueline K. Barton*
Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, CA 91125, USA
Received February 13, 2001; Revised and Accepted March 21, 2001
ABSTRACT
Long range oxidative damage as a result of charge
transport is shown to occur through single cross-
over junctions assembled from four semi-comple-
mentary strands of DNA. When a rhodium complex is
tethered to one of the arms of the four-way junction
assembly, thereby restricting its intercalation into
the
π
-stack, photo-induced oxidative damage occurs
to varying degrees at all guanine doublets in the
assembly, though direct strand scission only occurs
at the predicted site of intercalation. In studies where
the Mg
2+
concentration was varied, so as to perturb
base stacking at the junction, charge transport was
found to be enhanced but not to be strongly localized
tothearmsthatpreferentiallystackoneachother.
These data suggest that the conformations of four-way
junctions can be relatively mobile. Certainly, in four-
way junctions charge transport is less discriminate
than in the more rigidly stacked DNA double cross-
over assemblies.
INTRODUCTION
DNA can both mediate and participate in long range electron
transport. As a result, oxidative damage can occur in DNA at a
distance from the site of radical introduction (1,2). Because
oxidative damage to DNA appears to play significant roles in
cancer and aging, understanding the molecular basis of genetic
degradation is an important research goal (3–5). In addition,
recent nanotechnological research has focused on the possible
application of DNA duplexes as one-dimensional quantum
wires (6–8) and as templates for directed material deposition
(9). In the same arena, larger nucleotide assemblies, like cross-
over junctions, have been used as building blocks for nanoscale
structures and signaling devices (10–14). Understanding the
molecular mechanisms of charge transport for these nanoscale
building blocks is of fundamental importance to designing
DNA microarray electronics. Finally, because DNA charge
transport is sensitive to base pair stacking, measurements of
charge transport also provide a means to explore the local varia-
tions in nucleic acid structure that can occur both statically and
dynamically.
To investigate long range charge transport in DNA, we
prepared assemblies containing the covalently tethered intercalator
Rh(phi)
2
bpy
′
3+
, a potent photooxidant (phi, phenanthrenequinone
diimine; bpy
′
, 4-butyric acid, 4
′
-methylbipyridine). Photolysis
at 365 nm of the tethered photooxidant leads to injection of an
electron hole directly into the DNA base stack (15). Subsequent
hole migration through the base stack results in localization and
irreversible damage to the 5
′
-G of a 5
′
-GG-3
′
doublet, the most
oxidatively sensitive site in DNA (16,17). This lesion can be
revealed by treatment with hot piperidine or base excision
repair proteins (18). In contrast, rhodium complexes of phi
promote direct strand scission at sites of intercalation without
chemical treatment when irradiated at 313 nm (19). Thus,
covalently tethering Rh(phi)
2
bpy
′
3+
to the end of a base stack
should also restrict the damage that occurs by photo-irradiation
at 313 nm to the end of the duplex to which the metal complex
is tethered (15–20).
Using this strategy, long range charge transport has been
shown to occur with a variety of photooxidants (15,21–25) and
at distances up to 200 Å from a site of hole injection in duplex
DNA (26–27). Similar charge transport reactions have been
observed in RNA–DNA hybrid duplexes (D.T.Odom, and
J.K.Barton, unpublished results; 28) and DNA triple helices
(29,30). In addition, the ability of charge transport to migrate
through regions of unusual structure, such as A tracts, has also
been demonstrated (31). Interruptions in the
π
-stack, such as
base bulges or insertion of aliphatic protein side chains in place
of nucleobases, attenuates radical migration (32,33).
Four-way DNA junctions have long been known in biology
as flexible intermediates during homologous recombination of
sister chromatids. Originally proposed by Holliday in 1964
(and thus often called Holliday junctions) (34), they are
composed of four strands of DNA that are partially comple-
mentary to form parallel stacks of bases that can interchange
between different stacking isomers. In nature these junctions
are unstable to migration and, when formed during recombina-
tion events, can rapidly migrate via a zippering mechanism.
Joining two four-way junctions together into a double cross-
over (DX) assembly greatly rigidifies the base stacks of DNA
(35). When in isolation, however, single four-way junctions
have dramatically different stacking and stability than do DX
assemblies (36–38). In the absence of magnesium the four-way
junctions adopt an extended conformation with an open core to
minimize negative charge localization from the backbone
strands. In the presence of >100
μ
M magnesium or other
highly charged cations the four helix arms fold into two coaxial
π
-stacks in an anti-parallel fashion.
In order to study the structural architecture of these mole-
cules, synthetic four-way junctions can be made immobile to
base pairing migration by constructing each arm from non-
complementary sequences (36–39). The location of the cross-
over junction is dictated by the complementarity of the arms.
These immobilized four-way junctions, however, retain high
conformer exchange rates. Solution studies have shown that
*To whom correspondence should be addressed. Tel: +1 626 395 6075; Fax: +1 626 577 4976; Email: jkbarton@caltech.edu
Nucleic Acids Research, 2001, Vol. 29, No. 10
2027
the rate of interconversion between the two junction isomers
and their relative abundance are critically dependent on the
sequence of the 3 bp region in each arm that flanks the cross-
over junction itself (40–43). By judicious choice of core, the
four-way junction can be made to partition evenly between
crossover isomers (43) or strongly prefer one or the other of
these isomers (40).
At present, two crystal structures have been reported in the
literature for all-DNA four-way junctions (44,45). Importantly,
the base pairs are well-stacked through the junction. Though
the sugar–phosphate backbone is severely distorted at the
crossover, the bases across the junction are parallel and a base
pair rise of 3.4 Å per step is evident. Furthermore, in both
crystal structures the alignment of the two base stacks is anti-
parallel and the angle between the two stack axes is
∼
40
°
.
These angle measurements are slightly smaller than those
predicted in previous studies. Atomic force microscopy of
arrays of non-covalently associated four-way junctions (46)
and other biophysical studies (47–50) had suggested angles
between the helical axes of
∼
60
°
. However, studies using fluo-
rescence resonance energy transport (FRET) (30) and restric-
tion nuclease analysis (38) have shown that in solution these
four-way junctions are fluxional and are stacked in both
possible anti-parallel conformations, though in differing
proportions of each.
Interest in using various DNA constructs to assemble archi-
tectural features in nanoconstruction has prompted us to investi-
gate the electronic structure of crossover junctions. Charge
transport has been seen in DNA DX assemblies and, remarkably,
hole migration in DX assemblies only occurs through the base
stack into which an electron hole is injected (51). Charge
migration does not occur across the junction between the
stacks. In addition, introduction of a double mismatch into an
otherwise properly paired DX assembly does not disrupt
radical migration; a similar disruption in the proper base
pairing of identically sequenced duplex DNA causes complete
disruption of charge migration. The different reactivity of the
two DNAs is likely due to the stabilization of base dynamics
that occurs in DX assemblies from the packing of the sugar–
phosphate backbones into the major groove of the adjacent
base stack.
Here we explore charge migration in single four-way junc-
tions. We have designed four-way junctions using core residues
that were shown previously to preferentially maintain one
isomeric stacking form (40). Charge transport experiments
employing this four-way junction were conducted with both
non-covalently bound metallointercalator and tethered metal
complex. Studies here contrast with previous results found for
much more rigid DX assemblies and highlight the critical
requirements for stable, well-defined architecture to control
pathways of long range charge transport.
MATERIALS AND METHODS
Oligodeoxyribonucleotide preparation
Oligodeoxyribonucleotides were prepared in an ABI392 DNA
synthesizer using standard phosphoramidite chemistry. DNA
was synthesized with a 5
′
-dimethoxytrityl (DMT) protective
group and purified on a Dynamax 300 Å reverse phase C4
column (10 mm i.d.
×
25 cm length) from Rainin. The DMT
group was removed using an aqueous 80% acetic acid solution
to suspend the dried, protected DNA for 20 min. After evapo-
ration
in vacuo
of the acetic acid the DNA was repurifed using
the same Rainin C4 column. The purified DNA was dissolved
into 10 mM Tris, pH 7.4, with 1 mM EDTA (TE) to a standard-
ized concentration of 100
μ
M using extinction coefficients of:
(
ε
260
,M
–1
cm
–1
) adenine (A) = 15 000; guanine (G) = 12 300;
cytosine (C) = 7400; thymine (T) = 6700. Synthesis and tethering
of Rh(phi)
2
bpy
′
3+
to DNA has been described elsewhere (52).
DNA techniques and photocleavage
In separate experiments, each strand except the one tethered to
the rhodium complex was 5
′
-labeled with [
γ
-
32
P]ATP using
polynucleotide kinase. BioRad P6 Microspin columns were
used to remove excess, unreacted [
γ
-
32
P]ATP. This desalted
solution was treated with 10% piperidine at 90
°
C for 30 min to
cleave damaged oligonucleotides. After drying
in vacuo
each
full-length oligonucleotide was purified on a 20% denaturing
polyacrylamide gel and the parent band extracted from the gel
by elution at 37
°
C into TE. All rinses were pooled, dried and
resuspended in 50
μ
l of TE and then passed through a Bio-Rad
P6 column to remove urea and salts. Four-way junctions and
duplexes were assembled by annealing equimolar amounts of
each strand using a linear gradient of 90–4
°
Cover90minona
Perkin-Elmer Cetus Gene Amplifier. These four-way junctions
were characterized by their mobilities on 10% non-denaturing
gels in 10 mM Mg(OAc)
2
and 45 mM Tris–acetate, 1 mM
EDTA, pH 7.6, (1
×
TAEMg) buffer. In general it was found
that the four-way junction comprised >90% of the species
visualized by their mobilities, regardless of the labeled strand;
the remaining species were of smaller size. Non-covalent
metallointercalators were added after annealing.
Irradiations were performed at 15
°
Con10–30
μ
l samples in
1.7 ml presilanized Eppendorf tubes using a 1000 W Hg/Xe arc
lamp equiped with a monochromator. Unless otherwise noted
all reactions were performed in 1
×
TAE. When magnesium
was present it was 10 mM as Mg(OAc)
2
. After irradiation
samples were dried
in vacuo
and samples to be treated with
piperidine (i.e. all 365 nm irradiations and dark controls) were
resuspended in 100
μ
l of 10% piperidine and heated to 90
°
C
for 30 min. These samples were then analyzed on a 20% dena-
turing gel followed by phosphorimagery. For quantitation,
lanes were first normalized for loading, and cleavage in the
dark controls was subtracted. Data from at least three trials
were used for all analyses. Sequencing reactions followed
standard protocols (53).
RESULTS
Design of four-way junctions to investigate long range
charge transport
We constructed a four-way junction that would preferentially
assume one isomeric conformation of an anti-parallel stacked
bundle of helices (40). Figure 1 shows the sequences
employed. The backbone strands are solvent exposed at the
crossover point when the junction is properly folded in the
presence of Mg
2+
. Rhodium complexes are covalently attached
to the 5
′
-end of a backbone strand, thus allowing spatial control
of radical injection into the base stack. The crossover strands,
which are tightly constrained and thus largely solvent excluded
2028
Nucleic Acids Research, 2001, Vol. 29, No. 10
inthepresenceofMg
2+
(44,45,48), were designed to incorporate
oxidatively sensitive guanine doublets at well-spaced locations
along the helix. Because these strands extend across the junc-
tion and into both stacks, oxidative damage on different arms
can be easily visualized in a single experiment.
Photo-induced cleavage of DNA four-way junctions by
non-covalently bound Rh(phi)
2
DMB
3+
Many organic intercalators, like MPE·Fe(II), Cu(I)-(
o
-phenan-
throline)
2
and Stains-All, preferentially bind to the core of
four-way junctions (54–57). To distinguish preferred binding
sites for non-covalently intercalated Rh(phi)
2
DMB
3+
,where
DMB is 4,4
′
-dimethylbipyridine, this metallointercalator (58–59)
was incubated with an equimolar amount of the four-way junc-
tion shown in Figure 1A. By sequentially 5
′
-
32
P-labeling each
strand and irradiating at 313 nm in the presence and absence of
Mg
2+
the binding location(s) of the metal complex within the
DNA assembly could be easily visualized. It was found that in
all cases, with and without magnesium ion, the metal complex
bound preferentially to the core of the crossover junctions.
The pattern and amount of reactivity depends on the concen-
tration of Mg
2+
and, thus, the preferred stacking geometry. In
the absence of magnesium ion the metal complex tightly binds
to the core of the junction and all of the strands show equal
damage at the crossover location (Fig. 2, –Mg
2+
313 nm lanes).
Upon introduction of Mg
2+
, however, the direct strand scission
seen in crossover strands b and c when irradiated at 313 nm in
thepresenceofRh(phi)
2
DMB
3+
dramatically decreased,
whereas the damage in backbone strand d barely decreased.
This variation in reactivity among the strands is consistent
with crossover strands b and c being less solvent accessible in
the presence of Mg
2+
and, hence, less accessible to
Rh(phi)
2
DMB
3+
, while backbone strand d, which remains on
the outside face of the crossover junction, is accessible to
photocleavage. Tight packing of the core can occur when Mg
2+
is present to neutralize the core junction phosphate charges.
This allows the arms that are extended and separate in the
absence of magnesium ion to condense into two coaxial, anti-
parallel base stacks (38).
The oxidative damage seen when these assemblies are irradi-
ated at 365 nm, with the notable exception of crossover strand
b in Figure 2A, generally remains localized to the 5
′
-G of
5
′
-GG-3
′
doublets. Furthermore, this oxidative damage is
largely independent of Mg
2+
concentration.
Long range charge transport through duplex DNA with
covalently bound Rh(phi)
2
bpy
′
3+
We then constructed a duplex assembly (Fig. 1C) as a positive
control for photo-induced long range oxidative damage in the
sequence used to assemble four-way junctions. This duplex
was designed to be identical in sequence to the arm I/arm II
base stack in the four-way junction shown in Figure 1. As
expected, the damage found with photolysis at 313 nm is local-
ized to the end of the helix that bears covalently tethered
Rh(phi)
2
bpy
′
3+
. Guanine oxidation is apparent at all of the 5
′
-G
of 5
′
-GG-3
′
doublets in the labeled strand complementary to
the rhodium-bearing strand (Fig. 3) upon irradiation at 365 nm
and piperidine treatment, consistent with long range charge
transport through the duplex.
Long range charge transport through the crossover
junction with covalently bound Rh(phi)
2
bpy
′
3+
Having verified that duplex DNA of the same sequence can
mediate long range charge transport, we assembled the four-way
junction shown in Figure 1B and performed photo-irradiation on
this assembly. By covalently tethering the metallointercalator to
Figure 1.
Schematic illustrations of four-way junctions. (
A
) A folded four-way
j
unction in the presence of magnesium ion, adapted from the crystal structure
(45). (
B
) The non-covalent four-way junction assembly with its strands color
coded and labeled with lower case letters. The stacked arms are labeled in
Roman numerals. (
C
) The four-way junction with covalently attached metal
complex. The structure of the appended metallointercalator, which is shown
schematically in this rendering, is given at the bottom of this figure. (
D
)
Duplex of the same base-stacked sequence as the combined arm I/II from (C)
with covalently attached metallointercalator. In all sequences of DNA assem-
blies used, guanine doublets to be oxidized are shown in red.
Nucleic Acids Research, 2001, Vol. 29, No. 10
2029
the 5
′
-end of backbone strand a, the location of radical injec-
tion into the assembly was restricted to within 3 bp of the
terminus of arm I.
This restricted binding of Rh(phi)
2
bpy
′
3+
was evident in that
the only site of photocleavage in these covalent assemblies,
with or without Mg
2+
, is the end of arm I (Fig. 4). Importantly,
the level of photocleavage damage observed was independent
of the amount of magnesium ion in solution. This Mg
2+
inde-
pendence demonstrates that the photocleavage decrease in the
core seen previously with non-covalent Rh(phi)
2
DMB
3+
was a
function of the architectural changes upon Mg
2+
addition that
sterically preclude metal complex access to the crossover and
not the result of non-specific magnesium ion binding to the
phosphate backbone.
In contrast, irradiating the covalent assemblies at 365 nm
causes oxidative damage to all the guanines on crossover
strands b and c. In the absence of magnesium ion, oxidative
damage occurs at all three 5
′
-GG-3
′
sites in crossover strand b.
When Mg
2+
was present, oxidative damage increases at all of
the guanines and guanine doublets. Strand c shows even more
interesting behavior (Fig. 4). In the absence of magnesium ion,
the oxidative damage is rather weak and evenly distributed
between the two guanine doublets located on this strand. With
Mg
2+
there is an increase in the level of damage at the guanine
doublet on the 3
′
-side of this strand, located in arm IV. Most
dramatically, however, the oxidative damage at the guanine
doublet on the 5
′
-end of strand c in arm II is greatly increased.
This increase may reflect the stacking of this arm onto the
rhodium-bearing arm I, allowing electronic coupling to occur
between the site of hole injection on arm I and the affected
guanine on arm II.
We sought to verify whether guanine oxidation occurred as a
result of intermolecular interactions. A radioactively labeled
four-way junction with no tethered rhodium complex and an
unlabeled four-way junction bearing a covalently bound metallo-
intercalator were annealed separately, mixed in equimolar
amounts and then photo-irradiated. No direct strand scission or
guanine oxidation was observed in the labeled assembly by
Rh(phi)
2
bpy
′
3+
tethered to a separately annealed four-way
junction. Thus, long range charge transport proceeds only
within an individual crossover assembly.
Figure 3.
Oxidation of a metallointercalator-bearing duplex representing
stacked arms I and II from the four-way junction shown in Figure 1. Lanes are
as described in Figure 2.
Figure 2.
Direct photocleavage and piperidine-induced cleavage of four-way junction DNA with non-covalently bound Rh(phi)
2
bpy
′
3+
as a function of magnesium
concentration. (
A
) Phosphorimagery after electrophoresis in denaturing 20% polyacrylamide gel with 5
′
-
32
P-end-labeling of strand b of the four-way junction in
Figure 1 after photo-irradiation. Lane 1, Maxam–Gilbert A+G; lane 2, C>T sequencing reactions (42); lane 3, photolysis at 313 nm for 10 min; lane 4 phot
olysis
at 365 nm for 2 h followed by piperidine treatment; lane 5, the same assembly without photolysis (dark control, DC) followed by piperidine treatment. O
xidatively
sensitive sites are bracketed to the left of the sequencing lanes and the site of the crossover junction is shown by a hollow triangle. The presence or ab
sence of 5 mM
Mg(OAc)
2
is indicated above the respective lanes and the arm locations are indicated to the left of each gel. (
B
) Phosphorimagery of strand c. Lanes as in (
A
). (
C
)
Phosphorimagery of strand d. Lanes as in (A).
2030
Nucleic Acids Research, 2001, Vol. 29, No. 10
DISCUSSION
Intercalator exclusion from the crossover core in the
presence of Mg
2+
Direct photocleavage of the four-way junction by non-covalently
bound Rh(phi)
2
DMB
3+
shows that under all circumstances and
salt concentrations the preferred binding site is the core of the
four-way junction. However, the affinity of the metal complex
for the crossover assembly depends on the concentration of
magnesium ion. In the absence of Mg
2+
the metal complex
cleaves at the crossover junction on all of the inspected strands.
By way of comparison, the addition of Mg
2+
, which induces
crossover strands to assume distorted conformations to bridge
between the two separate base stacks, also leads to greatly
diminished photocleavage of these strands. Notably, however,
backbone strand d shows photocleavage that appears to be
independent of the magnesium ion concentration.
The differing susceptibility of each of the strands to metal
complex binding in the presence of magnesium ion is
consistent with the previously established stacking preferences
of this junction. FRET experiments have demonstrated that the
sequences used here favor the conformation shown in Figure 1,
where arm I and arm II are stacked and arm III and arm IV are
stacked, by 20:1 over the alternative anti-parallel conformation
where arm I is stacked over arm III and arm II over arm IV
(40). Clearly, this preferred stacking pattern is reflected in the
photocleavage experiments described above; when Mg
2+
is
present and the two extended stacks are folded across one
another, strands b and c are inaccessible to the metal complex
but strand d remains solvent exposed.
In contrast to Rh(phi)
2
DMB
3+
, organic intercalators bind to
the core of the crossover junction in the presence of magnesium
ion (43–46). The metal complex tris(4,7-diphenylphenanthro-
line)rhodium(III) [Rh(DIP)
3
3+
] was also shown to target cruci-
forms (60). When bound to a plasmid in low salt conditions
and irradiated at 315 nm, this bulky, sterically demanding
metal complex induced single- and double-strand breaks at a
cruciform extrusion. This reactivity was found to be entirely
independent of the sodium chloride concentration. In a result
that is remarkably similar to what was found in this study,
cleavage of the cruciform site occured at Mg
2+
concentrations
of 1 mM but cleavage completely disappeared when magnesium
ion was present in 10 mM concentration.
Long range charge transport in single crossover DNA
assemblies
Next, we tethered the metallointercalator onto a duplex of the
same sequence as the Rh(phi)
2
bpy
3+
-bearing stack of the
preferred conformer of the four-way junction. In contrast to
four-way junction DNA, this duplex has a continous sugar–
phosphate backbone on the strand complementary to the metal
complex (Fig. 1C). As expected, direct photocleavage mapped
the rhodium-binding site to the end of the helix. When photo-
irradiated at 365 nm, followed by piperidine treatment to
reveal oxidative lesions, the duplex showed damage at all 5
′
-G
in 5
′
-GG-3
′
doublets. This experiment is in effect a positive
control for the types of long range charge transport that would
occur in single crossovers if four-way junction stacking
afforded constructs as stable as duplex DNA.
In the absence of Mg
2+
four-way junctions assume an
extended conformation where the arms are not stacked beyond
their canonical base pairing regions (Figs 5 and 6). Thus, long
range charge transport experiments in the absence of magnesium
would be expected only to yield damage at the guanine doublet
in arm I, as radical migration should not occur across the
empty, open junction. Instead, guanine oxidation is found to
occur at 5
′
-G of guanine doublets in all arms. As damage is
Figure 4.
(
A
) Long range oxidation of DNA by covalently tethered
Rh(phi)
2
bpy
′
3+
in four-way junctions as a function of magnesium concentra-
tion. The gels are labeled as in Figure 2. Strand b of the covalently tethered four-
way junction is shown to be cleaved at the 5
′
-end on arm I by the covalently
attached metal complex upon 313 nm irradiation and long range charge trans-
port is evident upon 365 nm photo-irradiation in the absence and presence o
f
magnesium. (
B
) Long range oxidation of DNA by covalently tethered
Rh(phi)
2
bpy
′
3+
in four-way junctions as a function of magnesium concentration.
Strand c of the covalently tethered four-way junction is shown to be unaffected by
313 nm irradiation, but long range charge transport is evident upon 365 n
m
photo-irradiation in the absence and, particularly, in the presence of magnesium.
Nucleic Acids Research, 2001, Vol. 29, No. 10
2031
found in arm III, the four-way junction must be transiently
sampling conformations that permit even the highly disfavored
parallel alignment of arm I over arm III to occur. Clearly,
radical migration is occuring across the junction. The binding
location of the tethered metallointercalator, as revealed by
direct strand scission at 313 nm, is exclusively at the terminus
of arm I, hence, any damage seen in other locations is caused at
long range.
InthepresenceofMg
2+
, hole transport across four-way junc-
tions with a covalently tethered metallointercalator should
cause oxidative lesions in arms I and II and, to a lesser extent,
in arm IV, which in general make up the favored, anti-parallel
stacking partners. The arm I/arm III stacking arrangement
should be strongly disfavored regardless of magnesium ion
concentration and thus the guanine doublet located in arm III
would not be expected to show oxidative damage.
The results summarized in Figure 6 show that addition of
magnesium to this system, which presumably causes folding of
the otherwise separated arms into coaxial base stacks,
increases guanine oxidation overall, but especially in the
guanine doublets that are located in the stacked arms I and II.
Most dramatically, the guanine doublet in arm II is much more
damaged by radical migration when Mg
2+
is present. As
predicted above, this increase is probably a result of the favored
stacking of arm I with arm II, which would electronically couple
these two base stacks much more efficiently and thus allow
radical migration across the junction. Damage also increases,
though more modestly, to the guanine doublet in arm IV, again
consistent with the above analysis of the arm I/arm IV stacking
conformer as the minor species. Furthermore, the differential
increase, where arm II is more intensely damaged than arm IV,
reflects the trend, if not the absolute numbers, of the previously
determined 20:1 partition ratio of the stacking arrangements of
these arms.
Notably, however, guanines are also oxidized on arm III, the
highly disfavored stacking partner for arm I. This result may
reflect transient sampling of this strongly disfavored stacking
arrangement by the four-way junction. This suggestion would
explain the guanine oxidation seen in arm III in the absence of
Mg
2+
, as in the absence of highly charged cations the four-way
junction is extremely fluxional. It is more surprising, however,
when considering the conformation of the four-way junction in
thepresenceofMg
2+
, where the separate arms are generally
presumed to be in a more tightly packed arrangement and the
overall assembly to be much more rigid.
In some cases, especially crossover strand b, damage also
occurs in guanines at the 3
′
position of doublets and single
guanines as well, even ones that would be expected to be
entirely orthogonal to the oxidation caused by the tethered
metallointercalator. As suggested previously, the flexible junc-
tions likely sample conformations that permit long range
charge transport to proceed, but may also sample conforma-
tions that weaken the stacking geometry that concentrates
HOMO localization onto the 5
′
-G of 5
′
-GG-3
′
doublets (17).
The damage seen on single guanines and 3
′
-G in 5
′
-GG-3
′
doublets on strand b, otherwise unexpected, could be the result.
Figure 5.
Schematic illustration of the damage pattern, location and intensity
of direct photocleavage and photolysis-induced oxidative damage in four-way
j
unction assemblies by the non-covalent metal complex derived from Figure
4A. Direct photocleavage damage is shown by black arrows, whereas the long
range charge transport at 5
′
-GG-3
′
doublets is shown with red arrows. The
assemblies are shown with their presumed structural forms, open in the
absence of magnesium, and with stacked arms in the presence of magnesium.
Figure 6.
Schematic illustration of the damage pattern, location and intensity
of direct photocleavage and photolysis-induced oxidative damage in four-way
j
unction assemblies by the covalently tethered metal complex derived from
Figure 4B. The relative intensity of damage here is represented qualitatively by
the size of arrows.
2032
Nucleic Acids Research, 2001, Vol. 29, No. 10
Magnesium ion effects on long range transport
These results suggest that, in general, the addition of Mg
2+
strongly stabilizes tertiary structures with well-stacked arms
and enhances charge transport to guanines by rigidifying the
base stack. This effect is especially notable when metal
complexes are covalently tethered to the end of one of the base
stacks. Probing by direct photoinduced cleavage shows that the
tethered metallointercalator binds to the end of arm I inde-
pendent of the Mg
2+
concentration. In contrast, in the presence
of Mg
2+
the level of guanine damage observed (Fig. 6) is
greatly increased at all positions, despite the difference in
relative amount at each location. However, the largest increases
are at the predicted sites of greatest oxidative sensitivity, the 5
′
-G
of guanine doublets.
Similar increases in guanine oxidation are observed when a
non-covalent rhodium complex is used, reflecting an increased
rigidity of the duplex arms (Fig. 5). This increase occurs on
strand b not only at the 5
′
-G of 5
′
-GG-3
′
doublets, but at all
guanines regardless of location. Remarkably, and in contrast to
the results with tethered metallointercalator, direct strand scis-
sion in the presence of magnesium ion is much lower with non-
covalently bound Rh(phi)
2
DMB
3+
. In other words, it appears
that lower levels of binding are yielding greater amounts of
oxidative damage.
In general, then, while Mg
2+
must rigidify the individual
duplex arms, there is still apparently significant flexibility
between the arms with Mg
2+
. This may be concluded based
upon our finding of significant long range charge transport to
disfavored sites in the assemblies containing tethered rhodium.
While a duplex stack may be more rigid with Mg
2+
,thereare
still significant transient changes in folding among the arms
that must occur.
Comparison of charge transport in four-way junctions to
DX assemblies
DNA single crossover assemblies are known to be more flex-
ible than their DNA DX counterparts. This fact accounts for
why a large body of work exists exploring the nanostructural
architecture of DX assemblies (10–13), but to date few papers
have been published regarding the attempted use of single cross-
overs as architectural elements (46,61). However, considerable
effort has gone into delineating the different folding behaviors
of single crossover junctions (36–38).
Previously investigated DX assemblies showed remarkable
fidelity in radical migration through single stacks, even across
crossover junctions, due to their rigid and inflexible structures
(51). Notably, this rigid structure is only made possible by the
presence of 10 mM Mg(OAc)
2
. Resistance to crosstalk in separate
but adjoining stacks within DX molecules implies that they
may be used successfully in nanotechnological systems that
involve charge migration.
Similar considerations point to single crossover molecules as
far ‘leakier’ systems towards charge migration. The results
described here on single four-way junction assemblies suggest
that there is a transient sampling of conformers that allows
perhaps brief, but significant, base stacking to occur in
normally disfavored alignments and, thus, radical migration
throughout the assembly. These data suggest that single cross-
over assemblies, although interesting from a structural and
architectural standpoint, would not make effective components
in nanoconstruction, which demands controlled charge migra-
tion.
ACKNOWLEDGEMENTS
We are grateful to the NIH (GM49216) for financial support.
We also thank the Parsons Foundation for pre-doctoral support
of D.T.O. and the Caltech SURF office for a summer fellow-
ship to E.A.D.
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