Metal-mediated DNA assembly using the ethynyl linked terpyridine ligand

Article abstract of DOI:10.1039/c1ob06421e

The terpyridine ligand directly attached to the 5-position of a uridine allows metal-mediated DNA assembly towards potentially electronically coupled DNA conjugates.

Full text of DOI:10.1039/c1ob06421e

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Metal-mediated DNA assembly using the ethynyl linked terpyridine ligand†
Thomas Ehrenschwender,a Anna Barth,b Holger Puchtab and Hans-Achim Wagenknecht*a
Received 19th August 2011, Accepted 25th October 2011
DOI: 10.1039/c1ob06421e
The terpyridine ligand directly attached to the 5-position
of a uridine allows metal-mediated DNA assembly towards
potentially electronically coupled DNA conjugates.
DNA represents an increasingly important tool for the con-
struction of nanoarchitectures or nanoscaled devices due to the
predictable Watson–Crick base pairing.1–6 In order to enhance
complexity of DNA-based nanostructuring it would be highly
desirable to develop additional binding motifs that behave chem-
ically orthogonal to conventional hydrogen bonding of Watson–
Crick base pairing that is applied typically as so-called sticky ends.
The first alternative, hydrophobic p–p interactions have been used
mainly between perylene bisimides as DNA caps to aggregate
DNA7 and Y-shaped DNA constructs8 in a reversible fashion.
Metal ion–ligand interactions represent the second alternative
motif. The latter idea is not new; conjugates of nucleic acids
and metal-chelating moieties have been investigated intensively.9–15
Especially the 2,2¢:6¢,2¢¢-terpyridine (terpy) ligand is known to
efficiently form stable complexes with a broad variety of metal
ions16–21 and has been used to assemble oligonucleotides.22–24
However, it is important to point out that all of these studies
Scheme 1 Structure of terpy-dU in oligonucleotides and sequences of
single strands DNA1–DNA6. Duplexes between two modified oligonu-
cleotides are called DNA1-2, DNA3-4 and DNA5-6. Duplexes of only
one of the modified oligonucleotides with corresponding unmodified
have been carried out with oligonucleotides that were terminally
modified with terpy using a flexible alkyl linker. In supramolecular
electronics a strong electronic coupling is provided mainly by
acetylene bridges.25 In order to go one step further towards DNA-
counterstrands are called DNA1Y etc. (with Y = base opposite to terpy-dU,
based nanoelectronics, we present the DNA building block terpy-
dU in which the terpy ligand is linked to the 5-position of 2¢-
deoxyuridine via the ethynyl bridge. This building block allows
internal and terminal terpy-dU modification and thereby provides
the basis for metal-mediated DNA assembly.
e.g. A in DNA1A).
sequence and surrounded by T, A, G or C; strands DNA5 and
DNA6 carry a terminal terpy-dU label. DNA1 is complementary
to DNA2, DNA3 to DNA4, and DNA5 to DNA6 (Fig. 1).
The synthesis of 2¢-deoxyuridine carrying the terpy-acetylene
moiety in the 5-position was recently reported.26 Accordingly,
the preparation of the corresponding building block was car-
ried out via Sonogashira coupling between DMT-protected
5-iodo-2¢-deoxyuridine and 4¢-ethynyl-2,2¢:6¢,2¢¢-terpyridine fol-
lowed by standard phosphoramidite formation (see Supporting
Information†). The single strands DNA1–DNA4 (Scheme 1) were
prepared with one terpy-dU modification in the middle of the
aKarlsruhe Institute of Technology, Institute for Organic Chemistry, Fritz-
Haber-Weg 6, 76131 Karlsruhe. E-mail: wagenknecht@kit.edu; Fax: +49-
(0)721-608-44825
Fig. 1 Absorption (left) and fluorescence spectra (right) of terpy-dU–
modified single strands DNA1, DNA3 and DNA5 and double strands
DNA1-2, DNA3-4, DNA5-6; 2.5 mM in Na–Pi buffer at pH 7, 250 mM
NaCl, 100 mM EDTA, 20 ◦C, excitation at 325 nm.
bKarlsruhe Institute of Technology, Botanical Institute II, Fritz-Haber-Weg
4, 76131 Karlsruhe. E-mail: puchta@kit.edu; Fax: +49-(0)721-608-44874
† Electronic supplementary information (ESI) available. See DOI:
10.1039/c1ob06421e
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Table 1 Melting temperatures (Tm) and spectroscopic data of double
strands without metal ions
DNA
labs [nm]
lem [nm]
Tm [◦C]
DTm [◦C]a
DNA1A
DNA1-2
DNA3A
DNA3-4
DNA5A
DNA5-6
316
321
316
316
316
321
378
390
—
—
400
400
56.8
65.9
63.9
71.6
61.4
62.7
-5.7
+2.4
-4.1
+3.6
—
—
a Compared to the unmodified references: Tm = 62.5 ◦C for DNA1A and
Tm = 68.0 ◦C for DNA5A, each with T instead of terpy-dU.
First we studied the influence of a single terpy-dU modification
on the melting temperatures (Tm) of double strands (Table 1).
If DNA1 and DNA3 are hybridized with completely unmodified
counterstrands including A opposite to terpy-dU (yielding double
strands DNA1A and DNA3A) the Tm values reveal a strong
destabilization (-5.7 ◦C and -4.1 ◦C) compared to completely
unmodified duplexes. The destabilization is slightly stronger
with other bases opposite to terpy-dU (double strands DNA3T,
DNA3G and DNA3C) (see Supporting Information†). Obviously,
the terpy-dU unit exhibits a small preference for adenine as the
counterbase although the metal ligand has been attached via the
short ethynyl bridge to the 5-position. In contrast, the duplexes
DNA1-2 and DNA3-4 bearing two terpy-dU moieties opposite to
each other are stabilized quite significantly (+2.4 ◦C and +3.6 ◦C,
respectively) compared to completely unmodified duplexes. This
is a remarkable result and shows that the hydrophobic interaction
of the two terpy unit regains more hybridization energy than the
destabilization introduced by the terpy units. Similar results have
been obtained with bipyridine pairs27 and binaphthyl pairs28 inside
DNA.
The terpy chromophore can be excited selectively at 325 nm
yielding a characteristic fluorescence (Fig. 1). Compared to duplex
DNA1A the fluorescence of double strand DNA1-2 is quenched.
This is the typical result of chromophore aggregation and thereby
supports the idea of a hydrophobically interacting terpy “base
pair” inside DNA1-2. The fluorescence of duplexes DNA3A and
DNA3-4 does not allow this interpretation since it is almost
completely quenched, probably due to photoinduced charge
transfer processes to adjacent guanines. The fluorescence intensity
of DNA5 and DNA5-6 is approximately equal since hydrophobic
terpy pairing enforced by the surrounding DNA architecture (as
in DNA1-2) is unlikely with terminal modifications.
More importantly, the terpy fluorescence and its quenching can
be used to follow and quantify metal ion coordination. We chose
Cu2+, Ni2+, Zn2+and Fe2+ as typical representatives, known to form
stable complexes with the terpy ligands. First, we examined double
strands DNA1A and DNA5A bearing only one terpy-dU in the
middle or at the terminus. It is expected that addition of metal
ions induces dimerization. From the titration experiments (see
Supporting Information†) we calculated the quenched fraction
of fluorescence intensity (Fq) at characteristic emission maxima.
The results show that fluorescence quenching is complete after
addition of 0.5–0.75 equiv. of metal ions (Fig. 2, top). This
observation together with the absorption changes (see Supporting
Information†) indicate approximately the expected stoichiometry.
To further evidence the dimer formation we performed non-
Fig. 2 Top: Fluorescence quenching (Fq) for DNA1A (left) and DNA5A
(right) upon addition of metal ions; bottom: non-denaturing gel elec-
trophoresis (8% TBM-PAGE) of DNA1A and DNA5A in absence and
presence of metal ions after silver staining.
denaturing polyacrylamide gel electrophoresis (Fig. 2, bottom).
The gels show dimerization of DNA5A and DNA1A in the presence
of Ni2+ and Fe2+ by a band of slower mobility. This is a remarkable
result by keeping in mind how short the acetylene linkers are
between the metal chelators and the nucleic acids on both sides of
the complex. On the other hand, dimers of DNA5A and DNA1A in
the presence Cu2+ and Zn2+ which are indicated by the fluorescence
measurements seem to be not stable enough for non-denaturing
gel analysis.
In the second part of this study we performed similar exper-
iments with double strands bearing two terpy-dU units either
opposite to each other in the middle (DNA1-2) or at the termini
(DNA5-6). It is expected that these DNA probes potentially are
forming larger DNA assemblies. The Fq analysis of DNA1-2
(Fig. 3, top) reveals a complete fluorescence quenching after
addition of 1.5 equiv. metal ions which is 0.5 equiv. more than
expected. The gel analysis (Fig. 3, bottom) shows dimers of DNA1-
2 only in the presence of Ni2+ but no larger aggregates. Due to the
fact that optical changes clearly indicate metal coordination, it
looks reasonable to assume that the two terpy-dU moieties of
DNA1-2 are forming a metal-mediated base pair inside the duplex
instead of networking between duplexes.
It is important to note that it is problematic to compare Tm
values of the metal-ion coordinated samples of DNA1-2 directly
with the metal free DNA1-2 since the Tm of the latter duplex
revealed an astonishingly stabilized, hydrophobically interacting
terpy-dU pair (as discussed above). Compared to a completely
unmodified reference double strand, however, DNA1-2 shows
significantly higher melting temperatures in the presence of 1
equiv. of Ni2+, Fe2+ and Cu2+ (Table 2). With Ni2+ or Fe2+ two
different Tm values are obtained, of which one is even higher
than the metal free DNA1-2 (DTm positive). The latter observation
strongly supports the idea of a metal ion-mediated, internal terpy-
dU base pair that interferes with the formation of higher DNA
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complementary strands. Higher structures can be formed with
doubly terminally labeled DNA. In principal, the short acetylene
linkers should provide strong electronic coupling between the
metal–ligand complex and the DNA. Hence it is expected that
these kind of DNA materials29 have a significant potential for
DNA-based nanoelectronics.
Acknowledgements
Financial support by the Deutsche Forschungsgemeinschaft (Wa
1386/12-1), the Center for Functional Nanostructures (CFN) and
KIT is gratefully acknowledged.
Notes and references
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48 | Org. Biomol. Chem., 2012, 10, 46–48
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