Remarkable stabilization of antiparallel DNA triplexes by strong stacking effects of consecutively modified nucleobases containing thiocarbonyl groups

Article abstract of DOI:10.1016/j.bmcl.2012.11.079

The consecutive arrangement of 2′-deoxy-6-thioguanosines (s ⁶Gs) and 4-thiothymidines (s⁴Ts) in antiparallel triplex-forming oligonucleotides (TFOs) considerably stabilized the resulting antiparallel triplexes with high base recognit

Full text of DOI:10.1016/j.bmcl.2012.11.079

Bioorganic & Medicinal Chemistry Letters 23 (2013) 776–778
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Bioorganic & Medicinal Chemistry Letters
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Remarkable stabilization of antiparallel DNA triplexes by strong stacking
effects of consecutively modified nucleobases containing thiocarbonyl groups
Kenji Yamada a, Yusaku Hattori a, Takeshi Inde a, Takashi Kanamori b, Akihiro Ohkubo a, Kohji Seio a,
Mitsuo Sekine a,
⇑
a Department of Life Science, Tokyo Institution of Technology, 4259 Nagatsuta, Yokohama 226-8501, Japan
b Education Academy of Computational Life Science, Tokyo Institution of Technology, 4259 Nagatsuta, Yokohama 226-8501, Japan
a r t i c l e i n f o
a b s t r a c t
The consecutive arrangement of 20-deoxy-6-thioguanosines (s6Gs) and 4-thiothymidines (s4Ts) in
antiparallel triplex-forming oligonucleotides (TFOs) considerably stabilized the resulting antiparallel tri-
plexes with high base recognition ability by the strong stacking effects of thiocarbonyl groups. This result
was remarkable because chemical modifications of the sugar moieties and nucleobases of antiparallel
TFOs generally destabilize triplex structures. Moreover, in comparison with unmodified TFOs, it was
found that TFOs containing s6Gs and s4Ts could selectively bind to the complementary DNA duplex
but not to mismatched DNA duplexes or single-stranded RNA.
Article history:
Received 4 October 2012
Revised 31 October 2012
Accepted 20 November 2012
Available online 1 December 2012
Keywords:
Antiparallel triplex-forming oligonucleotide
6-Thioguanosine
Ó 2012 Elsevier Ltd. All rights reserved.
4-Thiothymidine
Stacking effect
Gene therapy
The formation of naturally occurring DNA triplexes between
DNA duplexes and external DNA single strands is well known,
and for more than 2 decades, a large number of chemists have syn-
thesized triplex-forming oligonucleotides (TFOs) that have been
modified in various ways to increase the stabilities of the tri-
plexes.1 Because TFOs can strongly bind to the major grooves of
complementary DNA duplexes, they can be very useful for the sup-
pression of gene expression or for the creation of various artificial
nanostructures. This is particularly important in gene therapy
where the strong affinity of a TFO is one of the most important
requirements because the length of the recognition sequence,
which is a homopurine–homopyrimidine site, can be shortened
in a gene.
increased their affinity, except for studies of the attachment of
intercalators5 or minor groove binders6 to TFOs and the expansion
of recognition sequences.7 It is known that the introduction of
chemical modifications into the sugar moieties and nucleobases
of antiparallel TFOs generally destabilize triplex structures.4b Gee
et al. synthesized modified antiparallel TFOs that contained
20-deoxy-6-thioguanosine (s6G) residues to avoid the self-aggrega-
tion of G-rich TFOs, such as the G-quadruplex, that occurs in the
presence of physical concentrations of potassium ions.4a They
found that the introduction of discontinuous s6G residues into
TFOs destabilized the triplexes while preventing the undesired
self-aggregation that occurs in the presence of potassium ions.
There are 2 kinds of triplex formation: parallel- and antiparal-
lel-oriented triplexes.2 In a parallel-oriented triplex, 2 sets of
planar triads that are composed of T–A–T and protonated C (C+)–
G–C and that involve the Hoogsteen hydrogen bonds contribute
to the stabilization of the triplexes. By contrast, an antiparallel-
oriented triplex contains 2 reverse-Hoogsteen base pairs [T (or
A)–A–T and G–G–C], as shown in Figure 1. Many chemical modifi-
cations of nucleobases or sugar moieties have been developed to
improve the hybridization affinity of parallel TFOs.3 However,
there have been few studies4 on the chemical modifications of
the nucleobases or the sugar moieties of antiparallel TFOs that
(a)
(b)
dR
N
dR
O
N
H
N
N
H3C
N
H
N
N
H
H
N
H
X
X
H
H
CH3
N
H
O
N
O
N
N
N
N
N
H
H
N
N
dR
N
O
N
N
N
dR
O
H
dR
X = O (unmodified) or S (modified)
N
H
dR
⇑
Corresponding author. Tel.: +81 45 924 5706; fax: +81 45 924 5772.
E-mail address: msekine@bio.titech.ac.jp (M. Sekine).
Figure 1. Base pairing of (a) G or s6G with G–C and (b) T or s4T with A–T.
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.11.079

K. Yamada et al. / Bioorg. Med. Chem. Lett. 23 (2013) 776–778
777
(a)
(b)
thiocarbonyl groups
thiocarbonyl groups
Figure 2. Computer model of a DNA triplex containing s6G and s4T residues. The sequences of the TFOs are (a) 50 -s6G s6G T-30 and (b) 50 -s6G s4T G-30.
Table 1
CN
O
CN
S
N
S
N
Binding assaya of TFOs 1, 2, and 3 to HP duplex DNA 1 containing the P2 promoter
sequence
N
N
N
N
OPh
DMTrO
O
N
H
DMTrO
O
O
O
O
O
P
O
P
CN
CN
N
N
Figure 3. Chemical structures of the s6G and s4T phosphoramidite derivatives.
a
The conditions of binding assay: 50 mM HEPES buffer (pH 7.2) containing
10 mM MgCl2, 100 mM NaCl, and 10% sucrose for 12 h at 37 °C.
However, we recently reported that the consecutive arrangement
of the thiocarbonyl groups, 2-thiothymidine (s2T) and 20-deoxy-
5-methyl-2-thiocytidine (m5s2C), considerably stabilized the par-
allel-oriented triplex by the strong stacking effects of thiocarbonyl
groups.8 These results encouraged us to develop new antiparallel
TFOs that contain consecutive thionucleobases. We expected that
the thiocarbonyl groups of s6G and 4-thiothymidine (s4T) would
interact with the 50-upstream nucleobases in TFOs without inter-
rupting the formation of the Hoogsteen base pairs, as shown in
Figure 2. In this Letter, we focused on the triplex-forming and base
recognition abilities of oligonucleotides containing s6G and s4T res-
idues that were arranged consecutively.
Table 2
Binding assaya of TFOs 4 and 5 to HP duplex DNA 2 containing the sequence of the
HIV2 nef gene at 37 °C
In the synthesis of oligonucleotides containing consecutive s6G
and s4T residues, the corresponding monomer building blocks
were used in the general phosphoramidite approach, as shown in
Figure 3. The cyanoethyl groups of the nucleobases and the phos-
phate linkages were deprotected by treatment with 1 M DBU in
CH3CN before the release of the oligonucleotides from the resins.
First, we synthesized oligo DNA 1 d[T s6G s6G s6G s6G T] to examine
the quadruplex formation of the oligomer containing consecutive
s6G residues because the self-aggregation of an oligonucleotide
containing consecutive s6G residues has not been previously re-
ported; however, it was reported that the discontinuous introduc-
tion of s6G destabilized the quadruplex.4a The G-quadruplex of the
unmodified oligomer, d[TGGGGT] was observed by matrix-assisted
laser desorption/ionization-time of flight mass spectrometry, as
shown in Figure S1. However, the oligo DNA 1 could not form
the corresponding quadruplex. These results showed that the
consecutive introduction of s6G disturbed the formation of quadru-
plexes, which supported the results of the discontinuous introduc-
tion that were reported in the previous paper.4a
a The conditions of binding assay: 50 mM HEPES buffer (pH 7.2) containing 10 mM
MgCl2, 100 mM NaCl, and 10% sucrose for 12 h at 37 °C.
consecutive s6G residues was significantly higher. In addition, we
found that the consecutive introduction of s6G and s4T into the
antiparallel TFO surprisingly increased the triplex-forming ability
by the strong stacking effects of thiocarbonyl groups. The binding
affinity of TFO 3 was more than 1200 times higher than that of
the unmodified TFO. This was the first promising observation sug-
gesting that a chemical modification of a nucleoside moiety could
enhance the triplex stability of an antiparallel TFO. Table 2 shows
the binding affinity of the 13-nucleotide TFOs 4 and 5 to the HP
DNA 2 containing a sequence of the HIV2 nef gene.10 The binding
affinity of TFO 5 containing consecutive s6G and s4T residues was
43 times higher than that of the unmodified TFO 4 in the presence
of 100 mM NaCl. A similar result was also observed in the presence
of 100 mM KCl. These results indicated that TFOs containing con-
secutive s6G and s4T residues could strongly bind to the comple-
mentary DNA duplexes without sequence dependency and the
formation of G-quadruplexes.
Subsequently, we examined the binding affinity of TFOs 1, 2,
and 3 to the complementary HP DNA duplex 1, which contained
a sequence of the c-Myc P2 promoter,9 at 37 °C, as shown in Table
1. Although the binding affinity of unmodified TFO 1 was very low
Moreover, we examined the base recognition of s6G and s4T in
the triplexes. Figure 4 shows the results of a gel mobility shift assay
(Kd >800 lM), the affinity of modified TFO 2 containing 2 sets of 3

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K. Yamada et al. / Bioorg. Med. Chem. Lett. 23 (2013) 776–778
served that TFOs containing s6Gs and s4Ts could selectively bind
to the complementary DNA duplex and not to the mismatched
DNA duplexes or single-stranded RNA, compared with unmodified
TFOs. These results indicated that TFOs containing s6Gs and s4Ts
may be useful for the direct regulation of gene expression. The
excellent properties of TFOs incorporating thionucleoside moieties
could provide new insights into various fields of nanotechnology.
Further studies on these issues are now in progress.
Acknowledgment
This study was supported by a Grant-in-Aid for Scientific Re-
search from the Ministry of Education, Culture, Sports, Science
and Technology of Japan.
Figure 4. Electrophoretic mobility shift assay of the triplex formed between TFO 5
(100 lM) and fluorescently labeled HPs 2–8 (40 nM) on a 15% nondenaturing
polyacrylamide gel after the incubation of the triplexes. The conditions of binding
assay: 50 mM HEPES buffer (pH 7.2) containing 10 mM MgCl2, 100 mM NaCl, and
10% sucrose for 12 h at 37 °C.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2012.
11.079.
of the binding of TFO 5 (100 lM) to HP DNAs 2–8 (40 nM).
Although triplex formation was observed in lane 2 with matched
HP DNA 2, TFO 5 could not form stable triplexes with mismatched
HP DNAs 3–7, even with the high concentrations of TFOs. Although
we observed a smeared band in lane 8, the Kd value of the triplex
References and notes
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formed between TFO
>100
2 and HP DNA 8 was very low (Kd
l
M). As a result, the base recognition ability of the TFO con-
taining consecutive s6G and s4T residues was sufficiently high to
distinguish the matched base pair from the other mismatched base
pairs. There were more than 250-fold differences in the Kd values
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between matched (Kd = 0.4 lM) and mismatched triplexes.
Finally, the additional binding assay of TFO 4 or 5 to HP DNA 2
was conducted in the presence of the complementary RNA 1, 50-
CACCACCACCACC-30 (Fig. 5). Although the triplexes were observed
with 40
was formed between TFO 4 and HP DNA 2 disappeared, even in the
presence of an equivalent RNA 1 (40 M, lane 3). This might have
lM of TFO 4 or 5, as shown in lanes 2 and 5, the triplex that
l
resulted from duplex formation between TFO 4 and RNA 1. How-
ever, a large amount of RNA 1 (40 equiv RNA) could not destabilize
the triplex that was formed by TFO 5 and HP DNA 2. These results
suggested that TFO containing s6Gs and s4Ts could selectively bind
to the complementary DNA duplex but not to the single-stranded
RNA. This property of TFOs containing s6Gs and s4Ts is very useful
for the direct regulation of gene expression without the interfer-
ence of a huge number of mRNA copies containing similar se-
quences in the cytoplasm.
In summary, we found that the consecutive arrangement of the
thiocarbonyl groups of s6Gs and s4Ts remarkably stabilized anti-
parallel triplexes by the strong stacking effects of thiocarbonyl
groups. This is the first report of the enhancement of the structural
stability of antiparallel triplexes by the introduction of chemical
modifications into nucleoside moieties. Furthermore, it was ob-