New nucleotide pairs for stable DNA triplexes stabilized by stacking interaction

Article abstract of DOI:10.1021/ja800991m

New nucleotide pairs applicable to formation of DNA triplexes were developed. We designed oligonucleotides incorporating 5-aryl deoxycytidine derivatives (dC^5Ars) and cyclic deoxycytidine derivatives, dC^PPP and dC^PPI, having an expanded aromatic area, as the second strand. As pairing partners, two types of abasic residues (C3: propylene linker, φ: abasic base) were chosen. It was concluded that, when the 5-aryl-modified cytosine bases paired with the abasic sites in TFOs in a space-fitting manner, the stability of the resulting triplexes significantly increased. The recognition of C3 toward dC^5Ars was selective because of the stacking interactions between their aromatic part and the nucleobases flanking the abasic site. These results indicate the potential utility of new nucleotide triplets for DNA triplex formation, which might expand the variety of structures and sequences and might be useful for biorelated fields such as DNA nanotechnologies. Copyright

Full text of DOI:10.1021/ja800991m

Published on Web 07/09/2008
New Nucleotide Pairs for Stable DNA Triplexes Stabilized by Stacking
Interaction
Masahiro Mizuta, Jun-ichi Banba, Takashi Kanamori, Ryuya Tawarada, Akihiro Ohkubo,
Mitsuo Sekine,* and Kohji Seio*
Department of Life Science, Tokyo Institute of Technology, Nagatsuta, Midoriku, Yokohama 226-8501, Japan
Received February 8, 2008; E-mail: msekine@bio.titech.ac.jp; kseio@bio.titech.ac.jp
Triple-helical structures, which represent a class of major
supramolecular structures of DNA, have various applications,
including fabrication of DNA nanostructures.1 Recently, many
artificial nucleic acids that can improve the stability of triple-helical
DNAs have been reported.2 In typical parallel DNA triplexes that
comprise natural-type nucleotides, only two types of Hoogsteen
base pairs, T•A and C+•G, can be used as stable base pairs between
triplex-forming oligonucleotides (TFO) and DNA duplexes. Con-
Figure 1. Structure of 5-aryl-dC derivatives, dCPPP and dCPPI
.
sequently, the TFO should have homopyrimidine sequences, and
the DNA duplexes should have homopurine-homopyrimidine
sequences. Therefore, if we can develop new artificial base pairs,
which can replace the Hoogsteen base pairs without the loss of
DNA triplex stability, the variety of nucleotide sequences capable
of forming DNA triplexes would increase. Such an idea was first
proposed by Ts’o et al.;3 they designed various artificial nucleobases
such as deoxypseudouridine that could form hydrogen bonds in
the major groove with cytosine.
In this communication, we describe a possibility of new
nucleotide pairs applicable to DNA triplex. We designed oligo-
nucleotide duplexes incorporating 5-aryl deoxycytidine derivatives
(dC5Ars), 1a-c, dCPPP, and dCPPI (Figure 1). The cyclic derivatives,
dCPPP and dCPPI, having an expanded aromatic area, have recently
Figure 2. Sequences of hairpin oligonucleotides (OL-1, -2, and -3) and
TFOs.
been reported by us.4 The aromatic region at the 5-position of these
cytidine derivatives is expected to protrude into the major groove.
As pairing partners in TFO, which recognize these aromatic groups,
we selected an abasic residue (φ) and a propylene linker (C3) that
has a space (Figure 2). This space can accommodate an aromatic
group due to the absence of the nucleobase. In duplex formation,
the recognition of the abasic site by artificial nucleosides that have
an aromatic ring has been reported by other research groups.5 The
strategy shown in this communication is an example of the
recognition of aromatic groups in the major groove by TFO
incorporating φ or C3.
Table 1. Melting Temperaturea (°C) and Selectivity (in
Parentheses) of Modified Triplexes in 50 mM Sodium Cacodylate,
500 mM NaCl, 10 mM MgCl2 (pH 5.4)
OL-1a
X ) dC
-1b
dC5Ph
-1c
dC5Th
-1d
dC5Fur
-1e
dCPPP
-1f
dCPPI
TFO-1a
Y ) φ
-1b
22
(-)
27
28
32
36
41
52
(3b)
21
(2b)
30
(2b)
34
(2b)
39
(3b)
48
dT
-1c
dA
-1d
dC
-1e
dG
-1f
C3
20
25
22
21
16
23
25
27
30
26
31
34
34
38
39
39
45
49
45
The synthesis of 1a-c, dCppp, and dCPPI, and their phosphor-
amidite derivatives is shown in Scheme S1. Hairpin oligodeoxy-
nucleotides, such as OL-1a (X ) dC), -1b (dC5Ph), -1c (dC5Th),
-1d (dC5Fur), -1e (dCPPP), and -1f (dCPPI) were synthesized, using
these phosphoramidite units. In addition, triplex-forming oligo-
deoxynucleotides, TFO-1a (Y ) φ), TFO-1b (T), TFO-1c (dA),
TFO-1d (dC), TFO-1e (dG), and TFO-1f (C3) were also synthe-
sized, as shown in Figure 2.
First, we studied the triplex formation between OL-1a to -1f
and TFO-1a to -1e to clarify the interaction between 5-aryl-dC
and φ. The thermal stabilities of the triplexes are summarized in
Table 1. In all cases, the UV melting curves showed typical two-
step transitions, as shown in Figure S1 giving lower Tm values at
around 16-56 °C and higher Tm values at around 80-86 °C, which
corresponded to the melting patterns of triplexes and hairpin
duplexes, respectively. As a result of the triplex formation of TFO-
1a (Y ) φ)/OL-1a (X ) dC) and TFO-1a (Y ) φ)/OL-1b (X )
30
35
38
44
56
(5b)
(5b)
(4b)
(5b)
(7b)
a Averages of at least three independent experiments. The standard
deviation is (0.6 °C. b The selectivity of X-φ or X-C3 pair calculated
by Tm of X-φ or X-C3 minus the Tm of the most stable X-N pair
indicated by underlining, where N represents the natural type bases such
as T, dA, dG, and dC.
dC5Ph), it was revealed that the introduction of a phenyl ring of
dC5Ph increased the Tm value from 22 to 28 °C of the triplex with
TFO-1a incorporating φ. This result indicated that φ accommodated
the phenyl ring, resulting in the increased stability of the triplex
probably due to the stacking interactions between the phenyl ring
9
9622 J. AM. CHEM. SOC. 2008, 130, 9622–9623
10.1021/ja800991m CCC: $40.75
2008 American Chemical Society

C O M M U N I C A T I O N S
Table 2. Stacking Surface Areas (Å2) between dC5Ars and
Neighboring Bases
Table 3. Melting Temperature (°C) of the Triplexes Incorporating
Two dCPPI-Y Pairs at pH 5.4
a
X
Y
stacking surface areas
Tm (°C)
TFO-
dC
φ
φ
φ
φ
φ
C3
φ
23.4
42.0
50.9
56.3
67.3
73.0
84.7
92.8
22
28
32
36
41
44
52
56
2a
Y ) T
2b
dA
2c
dC
2d
dG
2e
C3
dC5Ph
dC5Th
dC5Fur
dCPPP
dCPPP
dCPPI
dCPPI
OL-2
52
44
53
46
63
TFO-
3a
Y ) T
3b
dA
3c
dC
3d
dG
3e
C3
C3
OL-3
19
19
20
28
22
a These Tm values are also shown in Table 1.
the C3 site. At present, the selectivity of dCPPI toward C3 is as
large as 7 °C. The results shown in this study indicate the potential
utility of new nucleotide triplets stabilized by stacking interaction.
Such nulceotide triplets might be useful for applications such as
DNA nanotechnologies utilizing DNA triplexes. Norden and co-
workers reported that DNA nanostructures consisting of branched
duplexes recognized the complementary TFOs at specific duplex
sequences to construct the addressable DNA nanostructure system.6
Currently, the sequences of such TFOs are limited to homopyrid-
midine sequences which can form Hoogsteen base pairs with the
homopurine sequences. If the dCPPI-C3 pair was incorporated into
this system, the number of addresses could be increased and the
sequences could be designed more freely. The application of the
new nucleotide triplets to such DNA nanotechnologies is underway
and will be reported elsewhere.
and the bases flanking φ. In addition, comparison of the Tm of the
triplexes of TFO-1a with OL-1b, -1c, -1d, -1e, and -1f revealed
that the stability decreased in the order of OL-1f (X ) dCPPI, 52 °C)
> OL-1e (dCPPP, 41 °C) > OL-1d (dC5Fur, 36 °C) > OL-1c
(dC5Th, 32 °C) > OL-1b (dC5Ph, 28 °C). As shown in Table 2, the
stability trend was in correlation with the stacking surface area
between the aromatic ring and the nucleobases.
Next, we evaluated the selectivity of the dC5Ars toward φ over
the canonical bases. As shown in the column of OL-1a in Table 1,
the unmodified dC bound most strongly to T with a Tm value of 27
°C. This stability might be explained by the hydrogen bond between
the amino group of the cytosine and the O4 or O2 of the carbonyl
group of the thymine. In contrast, the modified triplexes incorporat-
ing OL-1b to -1f gave the highest Tm values unexceptionally when
the modified dC bound to φ, but the selectivities were at most 3 °C.
Because OL-1e (X ) dCPPP) and -1f (dCPPI) showed rather high
Tm values toward TFO-1a (Y ) φ), we tried to change the structure
of φ to the one that had higher affinity for dC5Ars. As shown in the
bottom row of Table 1, TFO-1f incorporating C3 showed higher
affinity toward OL-1b, 1c, 1d, 1e, 1f of 30, 35, 38, 44, and 56 °C,
respectively, compared with φ probably due to the reduced steric
crush between the ribose moiety and the aromatic ring. As a result,
the selectivity increased significantly to 4-7 °C when paired with
C3 instead of φ. The stability of the triplexes shown in Table 1
was in correlation with the stacking surface areas between the
dC5Ars and the adjacent natural bases derived from the MD
simulated triplex structures, as shown in Table 2 and Figure S2.
When the melting temperatures of the triplexes consisting of OL-
1f were measured in the absence of Mg2+, the Tm of the triplexes
decreased by 5-8 °C. Even under this condition, triplex OL-1f/
TFO-1f showed the highest stability and selectivity of 6 °C toward
OL-1f/TFO-1d (Table S2).
Acknowledgment. This study was supported by a Grant-in-
Aid from the Ministry of Education, Science, Sports and Culture,
and a Grant-in-Aid from the Scientific Research and Industrial
Technology Research Grant Program ‘05 from New Energy and
Industrial Technology Development Organization (NEDO) of Japan.
Supporting Information Available: The procedures for the syn-
thesis, Tm analyses and the computation, and the NMR charts of new
compounds. This material is available free of charge via the Internet
at http://pubs.acs.org.
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Finally, we measured the Tm of the triplexes using the hairpin
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