Stable triplex formation using the strong stacking effect of consecutive thionucleoside moieties

Article abstract of DOI:10.1039/c1cc14339e

In this study, it was found that the arrangement of consecutive thiocarbonyl groups of s²T and m⁵s²C remarkably stabilized the pre-protonated form of the triplex, and that the stabilization of the pre-protonated form increased the pKa value of a cytosine derivative in the triplex.

Full text of DOI:10.1039/c1cc14339e

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COMMUNICATION
Stable triplex formation using the strong stacking effect of consecutive
thionucleoside moietiesw
Akihiro Ohkubo, Yudai Nishino, Akira Yokouchi, Yu Ito, Yasuhiro Noma, Yuuki Kakishima,
Yoshiaki Masaki, Hirosuke Tsunoda, Kohji Seio and Mitsuo Sekine*
Received 19th July 2011, Accepted 15th September 2011
DOI: 10.1039/c1cc14339e
In this study, it was found that the arrangement of consecutive
thiocarbonyl groups of s2T and m5s2C remarkably stabilized the
pre-protonated form of the triplex, and that the stabilization of
the pre-protonated form increased the pKa value of a cytosine
derivative in the triplex.
strong stacking effect of the thiocarbonyl group at the
2-position of the pyrimidine base. In this study, we focused our
interest on this strong stacking effect of the thiocarbonyl groups
of 2-thiothymidine (s2T), 5-methyl-2-thiocytidine (m5s2C),
8-thioxoadenosine (s8A), and 8-thioxoinosine (s8I) (Fig. 1) and
tried to increase the triplex-forming abilities of TFOs by using
these thionucleosides arranged in a consecutive manner.
Over the last two decades, researchers have reported a large
number of synthetic DNA and RNA oligomers1 having various
functional groups as useful molecules for gene therapies2 such as
RNAi and antisense strategies, the exhaustive analysis of gene
expression,3 the detection of single nucleotide polymorphisms,4
and DNA- (or RNA-) nanostructure.5 Among them, triplex-
forming oligonucleotides (TFOs)6 have long been recognized
as powerful tools for straightforward regulation of gene
expression. This antigene strategy is apparently rational because
it allows a single target of nuclear DNA, but the other methods
require targeting a large number of mRNA copies in the
cytoplasm. Although this advantage is often considered, there
is a disadvantage that the binding affinity of an unmodified
TFO to the corresponding DNA duplex is very weak under
neutral conditions compared with that of an antisense molecule
with the target RNA. To overcome this serious problem, a large
number of research groups have reported various chemical
modifications of TFOs. In particular, it is well known that
the introduction of amino moieties, such as aminopropargyl,
guanidium ethyl, and 2-aminoethyl groups, into the 5-position
of pyrimidine bases and the 20-position of the ribose moiety
can considerably increase the stability of triplexes even under
neutral conditions because of the charge–charge interactions
between cationic amino and anionic phosphate groups of
DNA duplexes.6b–d In addition, Imanishi et al. reported that
20,40-BNA with N-form sugar puckering could strongly enhance
the stability of the triplex structure.6e
To incorporate the thionucleosides into DNA- and RNA-type
TFOs 1–10, we first synthesized their corresponding phosphor-
amidite units 1–5 (Schemes S1–S4, ESIw). The elongation and
deprotection of TFOs containing consecutive thionucleoside
residues was performed using these phosphoramidite units in a
DNA synthesizer by the general procedure. Interestingly, during
deprotection of the 2-(trimethylsilyl)ethylthio (TMSES) group
from the protected s8A and s8I species in 20-OMe RNAs, we
found that the desulfurization of the thiocarbonyl group
occurred when TFOs 11 and 12 were treated with tetrabutyl-
ammonium fluoride (TBAF) for more than 1 h, although such a
thiocarbonyl group in DNA was stable under the same conditions
for 2 h.8 Therefore, the TMSES group was deprotected by
treatment with 1 M TBAF in THF for a short time of 30 min
in the synthesis of TFOs 11 and 12. After TFOs were released
from resins, they were isolated by HPLC and characterized by
MALDI-TOF mass spectrometry.
On the other hand, we previously reported that the replacement
of the thymidine of TFOs with 2-thiothymidine (s2T)7 in a TAT
triad increased their affinity for DNA duplexes because of the
4259 Nagatsuta, Department of Life Science,
Tokyo Institution of Technology, Yokohama 226-8501, Japan.
E-mail: msekine@bio.titech.ac.jp; Fax: +81 45 924 5772;
Tel: +81 45 924 5706
w Electronic supplementary information (ESI) available. See DOI:
10.1039/c1cc14339e
Fig. 1 Base pairs of (a) m5s2C or s8A with G–C and (b) s2T or s8I
with A–T.
c
12556 Chem. Commun., 2011, 47, 12556–12558
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Table 1 Tm values of triplexes and duplexes containing C, m5s2C and
s8A at pH 7.0
Fig. 2 Computer modelling of
a
DNA triplex containing s2T
and m5s2C residues. (a) Top view and (b) side view. The sequence of
TFO is 50-s2Tm5s2Cs2T-30.
those of TFO 3 having Cs. Moreover, we also found that the
base-pair recognition ability of a m5s2C (DTm = 28 1C), which
refers to the difference (DTm) in the Tm values between the
matched and most stable mismatched triplexes, was greater
than those of C (DTm = 22 1C) (Table S1, ESIw).
Next, we measured the Tm values of a triplex formed
between TFO 16 containing a m5s2C residue and HP 8 at
various pHs (pH 6.0–7.2) to examine if protonation could
occur by the consecutive 2-thiocarbonyl groups under mild
conditions near pH 7.0 (Fig. S1, ESIw). As a result, we found
that the pKa value of a m5s2C in the triplex surprisingly
increased to 6.7. This value near 7.0 should be noteworthy
because the pKa value of the protonated C moiety in an
unmodified DNA triplex was reported to be 5.5 according to
Dervan and Singleton.9 We also found that the pKa value of
the protonated C moiety in a triplex formed between TFO 17
and HP 5 increased to 6.3 (Fig. S2, ESIw). The pKa value of a
protonated m5s2C monomer (pKa = 4.24) was almost equal
to that of the protonated C monomer (pKa = 4.3); these
results strongly suggested that the protonation of a cytosine
derivative in the triplexes could be considerably promoted by
the pre-protonated form stabilized by the stacking effect of the
thiocarbonyl groups.
Entry
Oligonucleotides
Tm/1C
DTm/1C
1
2
3
4
5
6
7
8
TFO 1–HP 1
TFO 2–HP 1
TFO 3–HP 1
TFO 4–HP 1
TFO 5–HP 1
TFO 6–HP 2
TFO 7–HP 2
TFO 8–HP 2
TFO 9–HP 3
TFO 10–HP 3
TFO 11–HP 3
TFO 12–HP 3
TFO 10–RNA1
TFO 11–RNA1
16a
17a
37a
44a
52a
14a
13a
55a
29b
55b
57b
19b
77c
28c
—
1
—
7
15
—
ꢀ1
41
—
26
28
ꢀ10
22
ꢀ29
9
10
11
12
13
14
a
The Tm values are accurate within ꢁ0.5 1C. The Tm measurements
were carried out in a buffer containing 10 mM sodium cacodylate
(pH 7.0), 500 mM NaCl, 10 mM MgCl2, and 2 mM triplex. The
Tm values of HP 1–2 themselves were 79 1C and 82 1C, respectively.
Entries 6–8 (Table 1) show the Tm values of triplexes formed
between TFOs 6–8 containing four consecutive C derivatives
and HP 2 under neutral conditions. The hybridization affinity
of TFO 7 containing four m5s2Cs was slightly lower than that
of TFO 6 containing Cs. These results demonstrated that the
destabilizing effect of the internal cation repulsion arising from
the consecutive protonated cytosine bases was much stronger
than the stabilizing effect of the consecutive thiocarbonyl
groups of m5s2Cs. Therefore, we changed the sequence of
four m5s2Cs to an alternate sequence of four s8As as neutral
thionucleobase analogs having two donor protons (see Fig. 1–a)
to avoid internal cation repulsion. As a result, the hybridization
ability dramatically increased to 55 1C (DTm = 41 1C). It
should be emphasized that the alternate arrangement of two
modified bases, s8A and s2T, keeping eight thionucleoside
residues in a consecutive sequence, such as s2T-s2T-s8A-s8
A-s8A-s8A-s2T-s2T, was highly effective in enhancing the
binding affinities of TFOs for double-stranded DNAs.
b
Tm conditions; 10 mM sodium phosphate (pH 7.0), 100 mM NaCl, and
c
2 mM triplex. The Tm value of HP 3 itself was 78 1C. Tm conditions;
10 mM sodium phosphate (pH 7.0), 100 mM NaCl, and 2 mM duplex.
Table 1 shows the Tm values of triplexes formed between
TFOs 1–12 and three hairpin-type duplexes (HPs) 1–3, under
neutral conditions (pH 7.0). When a m5s2C was incorporated
into an unmodified oligonucleotide (TFO 1) in place of C at the
middle position, the hybridization affinity of TFO 2 containing
a m5s2C for HP 1 was almost the same as that of the unmodified
TFO 1 [16 1C (entry1) vs. 17 1C (entry 2)]. However, the
hybridization affinity of TFO 4 containing a m5s2C was greater
by 7 1C than TFO 3 containing a C [44 1C (entry 4) vs. 37 1C
(entry 3)] when four s2T residues were incorporated into TFOs
1–2 to stabilize the triplex structures by the stacking effect of the
thiocarbonyl group. In addition, the hybridization affinity of
TFO 5 containing three discontinuous m5s2C residues surprisingly
increased to 52 1C, as shown in entry 5. These results indicated
that the presence of the consecutive 2-thiocarbamoyl groups
could remarkably result in the stabilization of the triplex structure
by three-dimensionally fixing the positions of m5s2Cs because
of the stacking effect (Fig. 2), so that the hybridization
affinities of TFOs 4 and 5 having m5s2Cs were greater than
Subsequently, we changed the sugar moieties of TFOs
incorporating consecutive thionucleobases from 20-deoxyribose to
20-OMe-ribose to increase their triplex-forming abilities, because it
is well known that RNA-type TFO can strongly bind to the target
DNA duplex compared with the corresponding DNA-type TFO.
The triplex-forming ability of RNA-type TFO 10 having s2Ts and
c
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Chem. Commun., 2011, 47, 12556–12558 12557

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complete at a concentration 500-fold that of TFO under
neutral conditions. These results also suggested that the triplex
forming ability of TFOs incorporating consecutive modified
nucleobases having the thiocarbonyl group was surprisingly
greater (more than 500-fold) than that of unmodified
oligonucleotides.
In summary, we found that the arrangement of consecutive
thiocarbonyl groups of s2T and m5s2C remarkably stabilized
the pre-protonated form of the triplex, and that the stabilization
of the pre-protonated form increased the pKa value of a
cytosine derivative in the triplex. In addition, we could avoid
cation repulsion in a triplex having a series of C+-G-C
sequences arranged in a continuous manner by converting the
sequence of four m5s2Cs into the alternate arrangement of four
s8As. Furthermore, we also observed that 20-OMe TFOs
containing s8As and s2Ts could strongly and selectively bind
to the complementary DNA duplex (but not to the single-
stranded RNA) compared with the unmodified TFOs and
DNA-type modified TFOs. These results indicated that TFOs
having s2Ts and s8As might be useful for the direct regulation
of gene expressions. The excellent properties of TFOs
incorporating thionucleoside moieties could provide new
insight into various fields of nanotechnology. Further studies
in this direction are now in progress.
Fig. 3 Triplex formation of TFOs 13–15 with HP 4 containing the
sequence of c-myc P2 promoter. (a) Sequences of TFOs 13–15 and
HP 4. (b) Electrophoretic mobility shift assay of the triplex formed
between TFO 15 and HP 4 (40 nM) on a 10% nondenaturing
polyacrylamide gel at pH 7.0. At various concentrations, TFO 15
was incubated with HP 4 for 2 h at 37 1C.
discontinuous m5s2Cs was expectedly much greater than the
corresponding DNA-type TFO 9 (entries 9 and 10, Table 1).
In addition, we found that the use of s8As in place of m5s2Cs in
entry 11 slightly enhanced its binding affinity even in the
sequence of TFOs containing discontinuous C derivatives.
We previously reported that the backbone structure of
triplexes was somewhat disturbed by the presence of a bigger
s8A base in place of the protonated C because the distance
between the C10 atoms in the neighboring mononucleotide
units increased only at the position of s8A (Fig. S3, ESIw).8 To
avoid distortion of the triplex structure and reinforce the
structure, we introduced s8I into TFO 11 in place of s2T.
The distortion of the triplex structure might not have occurred
because the size of s8I was very similar to that of s8A. However,
we unexpectedly found that the Tm value of TFO 12 was much
lower than that of TFO 11 (19 1C vs. 57 1C).
Moreover, we examined the hybridization affinity of TFO 10
containing m5s2Cs and TFO 11 containing s8As toward a single-
stranded RNA (entries 13 and 14, Table 1). The Tm value of
TFO 10 toward the complementary RNA was higher than that
toward the complementary DNA duplex (77 1C vs. 55 1C).
These results indicated that TFO containing m5s2Cs could
strongly bind to the complementary single-stranded RNA
compared to the complementary DNA duplex, although the
m5s2C residue of TFO 10 would be expected to interfere with
the duplex formation due to the steric hindrance resulting
from the thiocarbonyl group.7a On the other hand, the
Tm value of TFO 11 toward the complementary RNA was
much lower than that toward the complementary DNA duplex
(28 1C vs. 57 1C). These results suggested that TFO containing
s8As and s2Ts could selectively bind to the complementary
DNA duplex but not to the single-stranded RNA. This
property of TFO containing s8As and s2Ts is very useful for
the direct regulation of gene expression without interference
by a huge number of mRNA copies in the cytoplasm.
Notes and references
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Finally, we examined the triplex formation of TFOs targeting
the model sequence of the c-myc P2 promoter10 by gel mobility
shift experiments at 37 1C and pH 7.0 (Fig. 3). We found that the
triplexes formed by the unmodified parallel- and antiparallel-type
TFOs 13–14 were unstable under neutral conditions and could
not be detected even at a concentration 25 000-fold that of
TFO (Fig. S4 and S5, ESIw). However, the triplex formation
using 20-OMe TFO 15 having s2T and s8A residues was
c
12558 Chem. Commun., 2011, 47, 12556–12558
This journal is The Royal Society of Chemistry 2011