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prerequisite.[17,18] We assume that PDI-modified single strands
are folded in such a way that the PDI caps are shielded and
Table 1. Melting temperatures (Tm) of DNA1-DNA4.
Metal ion
DNA1
DNA2
DNA3
DNA4
PDI dimerization is inhibited. The changes of the ratios A506/
Tm [8C] DTm [8C] Tm [8C] DTm [8C] Tm [8C] DTm [8C] Tm [8C]
A544 from 908C down to 108C support this interpretation. The
ratio 1.05 at 908C decreases to about 0.85 when the Tm value
is reached and increases again up to 1.15 at temperatures
lower than Tm. This indicates that the complexation of the bi-
pyridyl ligands of DNA1 with Ni2+ might be stable even when
the DNA branches of the three way junction are not annealed
(above Tm). Thereby, this Ni2+-mediated preassembly leads an
unfolding or different folding that unshields the PDI caps of
the singles strands and thereby allows PDI dimerization. This
kind of construct first breaks down when the samples reaches
the Tm value and the DNA branches start to anneal. At temper-
atures lower than Tm the full hybridization of the oligonucleo-
tide branches leads to PDI-mediated assembly of DNA1 in the
way how it was previously observed in the presence of Zn2+
/Fe3+ and in the absence of metal ions, as well as with DNA3
(not metal ion-dependent).
No[a]
47.6
54.8
55.9
57.6
–
45.5
51.9
62.2
62.1
–
46.3
46.3
47.0
45.9
–
49.3
Zn2+[b]
Fe3+[c]
Ni2+[b]
+7.2
+8.3
+10.0
+6.4
+16.7
+16.6
ꢀ0.0
+0.7
+0.4
[a] In the presence of EDTA. [b] 1.1 equiv. [c] 6.0 equiv.
only a minor influence on the metal ion lock in the center part.
Interestingly, DNA2 in the presence of Ni2+ yielded a three
way junction with a DTm =16.68C that is significantly more
stable than the metal free construct. This special effect cannot
be explained at the moment.
According to our[17,18,20] and others’ previous studies[14,21–24]
the dimerization of the 5’-terminally attached PDI chromo-
phores in DNA can be followed by significant changes in the
UV/Vis absorption and fluorescence spectroscopy (Figure 2
In order to study the influence of the unbound cytidines in
the middle of the construct (mainly DNA3) DNA4 was synthe-
sized lacking those cytidine spacers. DNA4 revealed an in-
creased Tm value (DTm =38C) compared to DNA3 but it is still
lower than the corresponding Tm values of metal ion-complex-
ated DNA1 (and DNA2). This is an interesting result since it
shows that the stability of PDI-capped three way junctions can
be controlled to a certain extent by the sequential design of
the branching part. Nevertheless, the incorporation of the bi-
pyridyl modified and metal complexating cytidines in the
middle lead to the most stable constructs (DNA1).
To obtain additional information on the PDI-mediated as-
sembly of the DNA three way junctions we wanted to apply
atomic force microscopy (AFM) on mica surfaces. The sequen-
ces of DNA1-DNA4 contain arms with 10 base pairs that corre-
spond to a length of 3.4 nm. These three way junctions clearly
are below the resolution limit of AFM application in air. Hence,
DNA5 was synthesized as a bigger three way construct. Each
branch of DNA5 bears three times the sequence of the corre-
sponding branches of DNA1 (see Supporting Information) and
is equipped with the PDI capped at the terminus. Hence DNA5
contains 30 base pair per arm which corresponds to a length
of 10.2 nm. DNA6 serves as a non-aggregating reference since
it bears the same sequence as DNA5 but lacks the PDI modifi-
cations. The corresponding AFM images of samples with DNA5
(Figure 3) immobilized by NiCl2 on freshly cleaved mica[26] clear-
ly show the formation of higher-ordered structures as the
result of DNA assemblies using concentrations of 1 and
0.02 mm. AFM images of oligomeric (tetrameric) assemblies of
DNA5 (see Supporting Information) help to interpret and visu-
alize the formation of the ordered polymeric aggregates
(Figure 3, top). These structures must be PDI-mediated since
they are not observed in similar experiments with the control
construct DNA6 that lacks the PDI caps. In contrast to our pre-
vious results, where we obtained primarily PDI-mediated
dimers,[18] the increased size of DNA5 obviously allows the for-
mation and observation of higher ordered structure on the
mica surface.
top). The A506/A544 as well as F553
/
ratios show the formation
680
of excitonically interacting PDI dimers. It is established and
does not need further comments that both ratios proof the as-
sembly and finally aggregation of the constructs DNA1, DNA3
and DNA4 mediated by the PDI caps.[14,17,18,20–24] Accordingly,
the PDI-mediated assembly of DNA1 and DNA3 in the pres-
ence of different metal ions (Fe3+, Ni2+, Zn2+) and in the pres-
ence of EDTA (to exclude any metal ion complexation) has
been investigated by both UV/Vis absorption and fluorescence
spectroscopy at different temperatures (90!108C) (Figure 2
bottom and Supporting Information). In the presence of Fe3+
and Zn2+, the absorption ratios A506/A544 of DNA1 at tempera-
tures higher than Tm are around 0.7–0.8 (typical for PDI mono-
mers) and increase to values of >1 for temperatures below Tm.
The latter ratios can be assigned to PDI dimers and thereby to
formation of assemblies of DNA1. Similar experiments with
DNA3 (see Supporting Information) showed that there is only
a minor influence of the bipyridyl modification (in the center
part of DNA1) on the assembly of the PDI dyes. The fluores-
cence measurements support these results. When excited at
505 nm the fluorescence of DNA1 and DNA3 at temperatures
below Tm is dominated by a red-shifted, structureless and exci-
mer-type band with a broad maximum at ~680 nm. The char-
acteristic fluorescence at this wavelength confirms an aggre-
gated PDI dimer that undergoes a structural relaxation process
after photoexcitation.[25] Moreover, the spectra of DNA1 in the
presence of Fe3+ and Zn2+ at temperatures higher than Tm ex-
hibit fluorescence at 553 nm that is typical for PDI monomers.
Hence, the ratios F553/F680 give information about the relative
amount of monomeric and dimeric PDI ensembles in the
sample.
The presence of Ni2+ plays a special role. DNA1 in the pres-
ence of Ni2+ at temperatures above Tm exhibits ratios A506/A544
of 1.05 and ratios F553/F680 of 7 indicating a partial PDI dimeriza-
tion of this three way junction. This DNA assembly must be
driven by Ni2+ since it is known that the dimerization of the
PDI caps needs the double helical DNA construct as a structural
Chem. Eur. J. 2014, 20, 12009 – 12014
12011
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