Synthesis of covalently linked parallel and antiparallel DNA duplexes containing the metal-mediated base pairs T-Hg(ii)-T and C-Ag(i)-C

Article abstract of DOI:10.1039/c0cc02028a

DNA duplexes fixed in anti-parallel and parallel orientations by introducing covalent linkages have been synthesized and metal ions, Hg(ii) and Ag(i), were incorporated into pyrimidine-pyrimidine base pairs.

Full text of DOI:10.1039/c0cc02028a

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Synthesis of covalently linked parallel and antiparallel DNA duplexes
containing the metal-mediated base pairs T–Hg(II)–T and C–Ag(I)–Cw
Takashi Ono, Kyohei Yoshida, Yuko Saotome, Rei Sakabe, Itaru Okamoto and Akira Ono*
Received 23rd June 2010, Accepted 4th November 2010
DOI: 10.1039/c0cc02028a
DNA duplexes fixed in anti-parallel and parallel orientations by
introducing covalent linkages have been synthesized and metal
ions, Hg(II) and Ag(I), were incorporated into pyrimidine–
pyrimidine base pairs.
duplexes. In antiparallel-strand (aps) duplexes, adenine (A)
residues and thymine (T) residues form A–T pairs of
Watson–Crick (W–C) geometry. On the other hand, in
parallel-strand (ps) duplexes, A–T pairs are formed in reverse
W–C geometry (Fig. 1). Since aps duplexes are more stable
than ps duplexes, dAn and Tn form aps duplexes under
physiological conditions. However, ps duplexes can be formed
with meaningful stability if the strand orientation is fixed in
parallel using a hairpin structure.7 The previously reported
hairpin ps-duplex contained a 50–50 phosphodiester linkage at
a hairpin–stem junction, and the effects of this unique
structure on the stability of the duplex were not examined.
Here, we have developed a more sophisticated method for
synthesizing aps and ps duplexes in which the strand orientations
are fixed by attaching the ethylene-linked pair T–(Et)–T at the
duplex ends. The introduction of T–(Et)–T inside an aps
duplex has been reported to efficiently stabilize it.5g
We expected that rotation of the thymine residues around
the ethylene linker would stabilize the reverse W–C geometry,
efficiently stabilizing ps duplexes.
We report a method for the synthesis of highly stabilized, short
DNA duplexes in which the DNA strands are held in an
antiparallel or parallel orientation by inter-strand covalent
linkages. Previously developed methods for the production of
covalently linked nucleotides have led to investigations of the
potential of linked nucleotides to act as decoy DNAs1 and
antisense and anti-gene oligonucleotides.2 As structural
mimics of the damaged sites induced by bi-functional mutagenic
chemicals, inter-strand cross-linked duplexes have also been
used to investigate the process of DNA damage repair.3
One approach for preparing covalently linked DNA
duplexes consists of several steps: first, oligonucleotides
containing artificial reactive residues are synthesized; the
synthesized oligonucleotides are then hybridized to form a
duplex, and finally the reactive residues in close proximity are
chemically conjugated. This approach has been used to prepare
duplexes with various covalent linkages, including disulfide,
psoralen, malondialdehyde, and alkyl groups.4 An alternative
approach is the direct synthesis of covalently linked oligonucleo-
tides on an automatic DNA/RNA synthesizer using amidite
units of a nucleoside dimer linking the base residues of two
nucleosides.5 In pioneering studies carried out by the Hopkins,5a
Miller,5d–i and Kishi5b groups, amidite units of linked nucleosides
were used to synthesize duplexes with covalent linkages in the
middle of the duplexes using sophisticated protecting groups and
the multi-step synthetic procedures. In many of these pioneering
studies, linkages were introduced inside the duplexes,6 and none
have reported the preparation of covalently linked duplexes with
parallel strands.7 In this report, we describe efficient methods for
the synthesis of duplexes that are fixed in an antiparallel or
parallel orientation by covalent linkages placed at the duplex
ends. We also describe the utility of this method through the
application of linked duplexes, allowing the demonstration of
metal ion-mediated base pair formation8 in antiparallel and
parallel duplexes.
Structures of novel phosphoramidite units of thymidine
dimers are shown in Fig. 2. In these phosphoramidite units,
the 3N positions of two thymidines are linked with
ethylene. The phosphoramidites 1 and 2, which are used for
the preparation of aps and ps duplexes, respectively, have
similar structures; they differ only in the positions of the
protecting groups on the sugar residues of the nucleosides
shown at left.
When mixed, homothymidylic acid (Tn) and homodeoxy-
adenylic acid (dAn) intrinsically form antiparallel and parallel
Department of Material & Life Chemistry, Faculty of Engineering,
Kanagawa University, 3-27-1 Rokkakubashi, Kanagawa-ku,
Yokohama, Kanagawa 221-8686, Japan.
E-mail: akiraono@kanagawa-u.ac.jp; Fax: +81 454917915;
Tel: +81 454815661
w Electronic supplementary information (ESI) available: Synthesis of
amidite units of thymidine dimers, oligonucleotides, thermal denatura-
tion studies. See DOI: 10.1039/c0cc02028a
Fig. 1 Base-pairing geometry of A–T pairs and covalently linked
T–(Et)–T pairs in aps and ps duplexes.
c
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Fig. 3 Thermally induced transition profiles of ODNs (5 mM) in 10 mM
sodium cacodylate buffer (pH = 7.0) containing 100 mM NaCl.
a Tm higher than that of the hairpin ps duplex as previously
demonstrated.7a Consequently, as a good fit into the ps duplex
structure was observed, we speculate that the T–(Et)–T linkage
efficiently stabilizes the ps duplex.
The linked duplexes were applied to the determination of
possible geometries of metal-mediated base pairs.8 T–T and
C–C mismatches in DNA duplexes selectively capture Hg(II)
ions and Ag(I) ions, respectively, forming metal-mediated
T–Hg(II)–T and C–Ag(I)–C base pairs (Fig. 4). Using
15N-NMR spectroscopy, we verified the formation of the
3N-Hg(II) bonding in the T–Hg(II)–T structure. As shown in
Fig. 5 right, the metal ion mediated base pairs can be formed
in reverse W–C geometry, probably in ps duplexes.
The thermally induced transition profiles of the linked
duplexes containing pyrimidine–pyrimidine (T–T and C–C)
mismatches in the absence and presence of metal ions are
shown in Fig. 5. As previously reported, aps duplexes were
stabilized by the presence of metal ions. The ps-duplexes were
also stabilized by the presence of metal ions, indicating the
formation of T–Hg(II)–T and C–Ag(I)–C in reverse W–C
geometry.9 The formation of the metal ion mediated base
pairs was also detected by mass spectrometry (ESIw).
In conclusion, we have developed a method for synthesizing
covalently linked, stable aps and ps duplexes. These duplexes
are anticipated as being useful for developing biologically
active nucleic acids, and for structural studies. We demonstrated
the utility of the linked duplexes by showing formation of the
metal ion mediated pyrimidine base pairs in reverse W–C
geometry in duplexes for the first time.
Fig. 2 Upper: structures of the linked thymidine dimer phosphor-
amidite unit 1 (for synthesizing aps duplex) and 2 (for ps duplex).
Lower: schematic representations of the synthetic strategies for aps
(left) and ps (right) linked duplexes.
Covalently linked oligodeoxyribonucleotides (ODNs) were
synthesized on a DNA/RNA synthesizer using a general
protocol recommended by the manufacturer, with slight
modifications. Schemes for the synthesis of covalently linked
duplexes in the antiparallel and the parallel orientations are
shown schematically in Fig. 2. The procedure for synthesizing
linked aps duplexes is as follows: starting with 50-O-DMTr-
nucleoside attached to the solid support, the ODN is elongated
using commercially available nucleoside-30-O-phosphoramidite
units; then, phosphoramidite 1 is attached, and a second
strand is elongated using nucleoside-30-O-phosphoramidite
units. For synthesizing linked ps duplexes, after 2 is attached,
50-O-phosphoramidite units are used in the final elongation
step, in which a second strand is elongated from the 50-end to
the 30-end. The fully protected ODNs are deprotected
and purified by the same procedures as those used for
unmodified ODNs.
This study was partly supported by a Grant-in-Aid for
Scientific Research (B) (21350105) and the Science Frontier
The thermal stabilities of the linked duplexes were
compared to those of hairpin duplexes. The thermally induced
transition profiles of the linked and hairpin duplexes are
shown in Fig. 3. The linked aps and ps duplexes were of
similar stability, and the Tms of the linked duplexes (46 1C for
aps and 47 1C for ps) were higher than those of the hairpin
duplexes (29 1C for aps and 24 1C for ps). Thus, the covalent
linkage attached at the duplex end efficiently stabilized
both the aps and ps duplexes. The hairpin aps duplex displayed
Fig. 4 Proposed structures of the metal ion-mediated base pairs
T–Hg(II)–T and C–Ag(I)–C in W–C (left) and reverse W–C (right)
geometry.
c
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