Angewandte
Communications
Chemie
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influenced by the adjacent bases. In contrast, AP-YdC and
AP-YdC showed selective CG base pair recognition regard-
less of the sequence context. Notably, these triplets provide
a triplex-stabilizing effect comparable to the canonical T-AT
base triplet, which is also reported to be similar with A-AT
[4a,c]
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and G-GC triplets.
The selectivity of AP-YdC for the
CG inversion site was also confirmed by the footprinting
assay using DNase I (Figure S2). To the best of our knowl-
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edge, AP-YdC and AP-YdC are the first non-natural
nucleosides that selectively stabilize the antiparallel triplex
DNA at the inverted CG base pairs in all combinations of the
flanking bases. Contrary to the 2-aminopyridinyl-YdCs, AB-
YdC with a 4-aminobenzene unit showed no selective
interaction with any base pair in any of the sequences.
These results strongly support the fact that the selective
recognition of the CG base pair by the YdC derivatives is
Figure 2. a) The hTERT promoter sequence (hTR) containing four CG
inversion sites and the sequences of the corresponding TFOs (TFO-T
and TFO-Z). FAM-labeled hTR (32 bp; 100 nm) was incubated with
increasing concentrations of each TFO (26-mer; 0–1000 nm) in the
attributable to the specific interaction of the 2-aminopyridine
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ring with the CG base pair. The pK values of AP-YdC and
a
buffer containing 20 mm Tris-HCl, 2.5 mm MgCl , and 2.5 mm spermi-
2
AP-YdC were determined to be 6.3 and 6.1, respectively
dine at pH 7.5 and 378C. Electrophoresis was performed with a 10%
(
Figure S3). In our previous study, protonation to the 2-
non-denaturing polyacrylamide gel at 48C. K
s
6
À1
(10 m )=[Triplex]/([TFO][Duplex]). The results of the gel mobility
aminopyridine unit of AP-isodC, a related analogue with
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shift assays are shown for (b) TFO-T and (c) TFO-Z.
AP-YdC, was supported by the fact that the triplex stability
[
6b]
was greater at pH 6 than at pH 7.5.
Accordingly, we
speculated that that the 2-aminopyridine unit is protonated
and interacts with the guanine base by hydrogen bonding
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direction, and the thymidine or APÀYdC was incorporated
(
Figure 1).
The possible formation of hydrogen bonds was checked by
into the positions corresponding to each of the four CG base
pairs in TFO-T or TFO-Z, respectively. The triplex formation
2
+
molecular dynamics (MD) calculations of the triplex DNAs
containing each YdC derivative, in either a protonated or
non-protonated form, and the CG base pair on the counter-
was performed at relatively low Mg concentrations resem-
bling physiological conditions (2.5 mm), and the triplex was
observed as the slow-moving bands by the gel shift assay. The
negative control TFO-T did not form a stable triplex, as
shown by the duplex bands at the high concentrations
(Figure 2b). This is due to the low stability of the TÀCG
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part. In the triplex DNA containing AP-YdC, the most
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stable structure was obtained by the protonated form; AP-
YdC was shown to form a nearly coplanar base triplet with
the CG base pair through the hydrogen bonds (Figure S4a).
triplets. In contrast, TFO-Z formed a stable triplex with the
target duplex even at its low concentration (Figure 2c). It
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When AP-YdC was not protonated, on the other hand, the
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2
-aminopyridine unit was dissociated from the CG pair and
should be noted that APÀYdC is useful for the formation of
significant distortion of the triplex structure was observed.
Similar results were observed with AP-YdC in which the
protonated 2-aminopyridine forms coplanar hydrogen bonds
with the guanine of the CG base pair (Figure S4b). As for AB-
YdC, a significant structural disturbance was observed
around the AB-YdC, clearly depicting the unfavorable
interaction between the 4-aminobenzene group and the
Hoogsteen face of the guanine base (Figure S4c). The
molecular modeling also suggested that the dihedral angle
between the pseudocytosine ring and the 2-aminopyridine
ring is not strictly fixed but is partially flexible, being located
at the appropriate position to interact with the guanine base.
Such partial mobility might allow the 2-aminopyridinyl–YdC
to adjust its conformation to interact with a CG base pair in
a different flanking base context.
the stable triplex even in the presence of multiple and
consecutive CG base pairs. This characteristic feature of
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APÀYdC has been also confirmed by the formation of
stable triplexes with the EGFR gene promoter sequence,
which contains four CG inversion sites (Figure S5).
The stable triplex formation against the hTERT promoter
sequence indicates the potential to inhibit transcription of the
hTERT gene. Thus, TFO-T and TFO-Z were tested for
transcriptional inhibition of the endogenous hTERT gene in
cultured human cancer cells. To prevent the digestion of the
TFOs by exonuclease, which is known as the dominant
nuclease species inside the cells, the TFOs were modified with
am
an aminopropyl group at their 3’ end (Figure 3a; TFO-T
am
[10]
and
TFO-Z).
When treated with exonuclease I, the
nuclease resistance of the aminopropyl-modified TFOs was
enhanced, whereas the non-modified ones were immediately
digested (Figure 3b). APÀYdC in the TFO-Z and the
TFO-Z displayed a slight resistance to S1 endonuclease
(Figure S6). Notably, the higher nuclease resistance was
observed for TFO-Z compared to TFO-T; the incorpo-
ration of the artificial nucleosides might prevent access of
exonuclease I to the oligonucleotides. Meanwhile, the triplex-
forming ability of the aminopropyl-modified TFOs were
The highly general recognition ability of 2-aminopyri-
dinyl–YdC for a CG pair was further demonstrated by the
triplex formation against the promoter sequence of the
hTERT gene, which is known to be associated with human
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am
[
9]
am
am
carcinogenesis. Importantly, the triplex-forming site in the
target duplex DNA (hTR) contains four CG inversion sites
with two of them consecutive (Figure 2a). The TFOs were
designed to bind to this target region in an antiparallel
Angew. Chem. Int. Ed. 2016, 55, 1 – 6
ꢀ 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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Okamura, Hidenori
