Double-stranded oligonucleotides containing 5-aminomethyl-2′- deoxyuridine form thermostable anti-parallel triplexes with single-stranded DNA or RNA

Article abstract of DOI:10.1016/j.bmcl.2012.03.024

This Letter describes the synthesis and properties of double-stranded antisense oligonucleotides connected with a pentaerythritol linker. We found that double-stranded antisense oligonucleotides with aminomethyl residues have high affinity for single-stra

Full text of DOI:10.1016/j.bmcl.2012.03.024

Bioorganic & Medicinal Chemistry Letters 22 (2012) 2681–2683
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Double-stranded oligonucleotides containing
5-aminomethyl-20-deoxyuridine form thermostable anti-parallel triplexes
with single-stranded DNA or RNA
Aya Shibata a, Yoshihito Ueno b, , Mari Iwata a, Haruka Wakita a, Akira Matsuda c, Yukio Kitade a
⇑
a Department of Biomolecular Science, Faculty of Engineering, Gifu University, Yanagido, Gifu 501-1193, Japan
b Department of Applied Life Science, Faculty of Applied Biological Sciences, Gifu University, Yanagido, Gifu 501-1193, Japan
c Graduate School of Pharmaceutical Sciences, Hokkaido University, Kita-12, Nishi-6, Kita-ku, Sapporo 060-0812, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Available online 14 March 2012
This Letter describes the synthesis and properties of double-stranded antisense oligonucleotides con-
nected with a pentaerythritol linker. We found that double-stranded antisense oligonucleotides with
aminomethyl residues have high affinity for single-stranded DNA or RNA in buffer solutions with and
without MgCl2. Thus, these oligonucleotides would be useful as antisense oligonucleotides for targeting
single-stranded RNA through triplex formation.
Keywords:
DNA
RNA
Ó 2012 Elsevier Ltd. All rights reserved.
Triplex
Antisense
Aminoalkyl linker
Thermal stability
Thanks to the discovery of a large number of non-coding RNAs,
gene suppression by using antisense oligonucleotides has once
again attracted much attention.1–3 An antisense oligonucleotide
binds to the target mRNA via Watson–Crick hydrogen bonds,
whereas an antigene oligonucleotide binds to the major groove
of double-stranded DNA by Hoogsteen or reverse Hoogsteen
hydrogen bonds and forms a local triple helix (triplex).4 Recently,
we reported an approach whereby single-stranded DNA or RNA
is targeted through triplex formation by using a branched oligonu-
cleotide.5–7
Approaches using branched oligonucleotides can be classified
into 2 categories based on the orientation of the triplexes: one
method utilizes parallel triplexes, in which the third strand binds
to the second strand via Hoogsteen hydrogen bonds (Fig. 1a),
whereas the other method utilizes antiparallel triplexes, where
the third strand binds to the second strand via reverse Hoogsteen
hydrogen bonds (Fig. 1b). In a previous report, we described the
synthesis of double-stranded antisense oligonucleotides connected
with a pentaerythritol linker that could target single-stranded DNA
or RNA by forming parallel triplexes.5–7 It was found that the dou-
ble-stranded oligonucleotides formed more thermally stable tri-
a
Hoogsteen
5'
5'
hydrogen bonds
Linker
Watson-Crick
hydrogen bonds
5'
3'
3'
Hoogsteen
b
5'
3'
hydrogen bonds
Linker
Watson-Crick
hydrogen bonds
5'
Figure 1. Schematic representation of branched oligonucleotides binding to single-
stranded nucleic acids.
Letter, we describe the synthesis and properties of double-
stranded antisense oligonucleotides that can target single-
stranded DNA or RNA by forming antiparallel triplexes.
Thus far, oligonucleotide analogs carrying various polyamines
have been synthesized,8–14 and some of them have been shown
to increase the thermal stability of duplexes and parallel triplexes.
However, to the best of our knowledge, the stabilization effects of
aminoalkyl modifications on antiparallel triplex formation have
not been examined. X-ray crystal structure analysis of antiparallel
triplexes showed that the 5-position of the thymidine residues of
the third strand is positioned very close to the phosphate backbone
plexes than the corresponding Watson–Crick duplex with
a
single-stranded RNA.6,7 This suggests that the double-stranded oli-
gonucleotides are useful as an antisense oligonucleotide. In this
⇑
Corresponding author. Tel./fax: +81 58 293 2919.
E-mail address: uenoy@gifu-u.ac.jp (Y. Ueno).
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.03.024

2682
A. Shibata et al. / Bioorg. Med. Chem. Lett. 22 (2012) 2681–2683
O
N
I
5'-d(CTCCTCCCTCCT)-3'
5'-r(CUCCUCCCUCCU)-3'
3'-d(GAGGAGGGAGGA)-5'
target duplex
3'-d(GAGGAGGGAGGA)-5'
5'-d(CTCCTCCCTCCT)-3'
VIII
II
H2N
HO
NH
II
I
O
O
5'-d(GTGGTGGGTGGT)
3'-d(GAGGAGGGAGGA)
OH
OH
OH
OH
IX
X
Z =
HO
5'-d(GZGGZGGGZGGT)
3'-d(GAGGAGGGAGGA)
Kd (nM)
3rd strand
0.12
0.10
0.08
0.06
0.04
0.02
0
_
+
_
+
_
+
_
+
III 5'-d(GTGGTGGGTGGT)-3'
115
9
(oC)
DNA
Tm
duplex (I:II)
55.3
IV 5'-d(GTGGTGGGZGGT)-3'
128 19
V
5'-d(GTGGZGGGZGGT)-3'
82
48
68
4
6
4
VI 5'-d(GZGGZGGGZGGT)-3'
VII 5'-d(GZGGZGGGZGGZ)-3'
triplex (I:IX)
triplex (I:X)
69.8
71.5
_
+
A
Δ
Figure 2. Binding abilities of the oligonucleotides for the target duplex.
of the second strand.15 Thus, we hypothesized that introduction of
an aminomethyl residue into the 5-position of the 20-deoxyuridine
in the third strand would increase the thermal stability of the anti-
parallel triplex because the ammonium cations of the amino-
methyl residues would reduce the anionic electrostatic repulsion
between the phosphate moieties.
10
50
(oC)
70
20
30 40
60
80
90
0.12
0.10
0.08
0.06
0.04
0.02
0
RNA
Tm
Oligonucleotides containing 5-aminomethyl-20-deoxyuridine
(Z) were synthesized using the phosphoramidite method (Fig. 2).
The synthetic route of a Z phosphoramidite is shown in Figure
S1. All Z-containing oligonucleotides were synthesized using a
DNA/RNA synthesizer. The obtained oligonucleotides were ana-
lyzed using matrix-assisted laser desorption/ionization time-of-
flight mass spectrometry (MALDI-TOF/MS), and the observed
molecular weights were in agreement with their structures.
First, binding of the aminomethyl-modified oligonucleotides to
target DNA was examined using an electrophoretic mobility shift
assay (EMSA). Figure 2 summarizes the dissociation constants
(Kd) of the third strands bound to the duplex DNA target. The Kd
of the unmodified third strand, III, to the target DNA was
115 9 nM, whereas those of the third strands V, VI, and VII con-
taining 2, 3, and 4 Z residues were 82 4, 48 6, and 68 4 nM,
respectively. Thus, this proved that the aminomethyl modification
at the 5-position of the 20-deoxyuridine in the third strand in-
creases the stability of the antiparallel triplex. The Kd
(128 19 nM) of the third strand, IV, containing 1 Z residue was
slightly lower than that of the unmodified third strand, III. Thus,
duplex (VIII:II)
triplex (VIII:IX)
triplex (VIII:X)
54.5
67.6
70.3
A
Δ
10
50
70
20
30 40
60
80
90
Temperature (oC)
Figure 3. UV melting profiles. Melting experiments were performed in Tris–HCl
buffer solution (15 mM, pH 7.0) containing 25 mM NaCl and 5.0 mM MgCl2. The
concentrations of the complexes were 2.0 lM.
match (Fig. S3). The
DTm (Tm [a complex between an oligonucleo-
tide and a complementary target] ꢀ Tm [a complex between an
oligonucleotide and a target containing a mismatched base]) ran-
ged from ꢀ7.2 to ꢀ12.4 °C. Thus, it was found that the branched
oligonucleotide IX has sufficient base discrimination ability as an
antisense oligonucleotide. Incorporation of 3 aminomethyl resi-
dues into the branched oligonucleotide slightly stabilized the tri-
plural
Z residues seemed to be required for the triplex
stabilization.
Branched oligonucleotides IX and X were synthesized using an
appropriately protected pentaerythritol linker (Fig. 3). The stability
of triplexes formed with these oligonucleotides was studied by
thermal denaturation in Tris–HCl buffer solution (15 mM, pH 7.0)
containing 25 mM NaCl and 5 mM MgCl2 (Figs. 3 and S2). One tran-
sition was observed in the melting profile when branched oligonu-
cleotide IX and single-stranded RNA VIII were used, and the
melting temperature (Tm) was 67.6 °C. In contrast, the Tm of the du-
plex between single-stranded DNA II and RNA VIII was 54.5 °C. The
increment in the absorbance melting curve of the VIII:IX complex
was greater than that of the VIII:II duplex. This means that thermal
denaturation from triplex to complete random coil for the VIII:IX
complex occurred cooperatively in a single transition. On the other
hand, the VIII:II:III triplex did not show a clear Tm transition, that
might be due to aggregation of the oligonucleotide strands con-
taining many guanine residues. Thus, it turned out that the oligo-
nucleotide IX connected with the pentaerythritol linker forms a
more thermostable triplex with the single-stranded RNA VIII than
the corresponding VIII:II duplex. We also performed a thermal
denaturation study with target RNA that contained one base mis-
plex in the buffer solution containing MgCl2. The
DTm between
the triplexes VIII:X and VIII:IX was +2.7 °C. Similar phenomena
were observed when single-stranded DNA I was used as the target
(Fig. 3, top graph).
It was reported that divalent cations, such as Mg2+, are neces-
sary for the formation of an antiparallel triplex.16,17 Next, we dem-
onstrated thermal denaturation in a buffer solution that did not
contain MgCl2. As shown in Fig. S2, the Tm of the complexes I:IX,
I:X, VIII:IX, and VIII:X were significantly decreased. The Tm of
the complexes were almost equal to those of the corresponding
I:II and VIII:II duplexes. However, intriguingly, the increment in
the absorbance melting curves of the VIII:X complex was greater
than that of the VIII:II duplex. This suggested that the amino-
methyl residue-containing branched oligonucleotide X formed an
antiparallel triplex with the single-stranded RNA VIII in the ab-
sence of Mg2+. The circular dichroism spectrum demonstrated
not only triplex formation between the branched oligonucleotide
X and the target RNA VIII but also triplex formation between the

A. Shibata et al. / Bioorg. Med. Chem. Lett. 22 (2012) 2681–2683
2683
presence of Mg2+. This indicates that the branched oligonucleotide
5'-Flu-d(GTGGTGGGTGGT)
3'-Dab-d(GAGGAGGGAGGA)
OH
OH
XI forms a triplex with the target RNA VIII in the presence of Mg2+
XI
but not in the absence of Mg2+
.
5'-Flu-d(GZGGZGGGZGGT)
3'-Dab-d(GAGGAGGGAGGA)
OH
OH
XII
When the branched oligonucleotide XII containing the amino-
methyl residues was used, the fluorescence intensities of the solu-
tions decreased as the concentrations of the first strand VIII
increased even in the absence of Mg2+, and plateaued near a 1:1
branched oligonucleotide XII and first strand VIII ratio (Fig. 4, bot-
tom graph). This proved that the branched oligonucleotide XII,
which is modified with the aminomethyl residues, could form an
antiparallel triplex even in the absence of a divalent cation such
VIII
5'-r(CUCCUCCCUCCU)-3'
XI + VIII
100
80
60
40
20
0
as Mg2+
.
In conclusion, we have demonstrated the synthesis of branched
oligonucleotides containing aminomethyl residues, and found that
these oligonucleotides have a high affinity for single-stranded DNA
or RNA in buffer solutions with and without MgCl2. Thus, these oli-
gonucleotides would be useful as antisense oligonucleotides for
targeting single-stranded RNA through triplex formation.
2+
without Mg
2+
with Mg
0
0.1
0.2 0.3 0.4 0.5 0.6
Acknowledgments
XII + VIII
100
80
60
40
20
0
This study was supported in part by a Grant-in-Aid from the
Koshiyama Research Grand and a Grant-in-Aid for Scientific Re-
search (C) from the Japan Society for the Promotion of Science to Y.U.
Supplementary data
2+
without Mg
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2012.03.024.
2+
with Mg
0
0.1
concentration of 1st strand (μM)
0.2 0.3 0.4 0.5 0.6
References and notes
1. Carninci, P.; Kasukawa, T.; Katayama, S.; Gough, J.; Frith, M. C., et al Science
2005, 309, 1559.
2. Katayama, S.; Tomaru, Y.; Kasukawa, T.; Waki, K.; Nakanishi, M., et al Science
2005, 309, 1564.
Figure 4. Profiles of the fluorescence intensities versus the first strand concentra-
tions. Experiments were performed in Tris–HCl buffer solution (15 mM, pH 7.0)
containing 25 mM NaCl in the presence or absence of 5.0 mM MgCl2. The
3. Krützfeldt, J.; Rajewsky, N.; Braich, R.; Rajeev, K. G.; Tuschl, T.; Manoharan, M.;
Stoffel, M. Nature 2005, 438, 685.
concentrations of the labeled oligonucleotides (XI and XII) were 0.30 lM. Excitation
wavelength: 485 nm. Emission wavelength: 535 nm.
4. Thuong, N. T.; Hélène, C. Angew. Chem., Int. Ed. Engl. 1993, 32, 666.
5. Ueno, Y.; Takeba, M.; Mikawa, M.; Matsuda, A. J. Org. Chem. 1999, 64, 1211.
6. Ueno, Y.; Mikawa, M.; Hoshika, S.; Matsuda, A. Bioconjugate Chem. 2001, 12,
635.
7. Ueno, Y.; Shibata, A.; Matsuda, A.; Kitade, Y. Bioconjugate Chem. 2003, 14, 684.
8. Rajeev, K. G.; Jadhav, V. R.; Ganesh, K. N. Nucleic Acids Res. 1997, 25, 4187.
9. Cuenoud; Casset, F.; Hüsken, D.; Natt, F.; Wolf, R. M.; Altmann, K. H.; Martin, P.;
Moser, H. E. Angew. Chem., Int. Ed. 1998, 37, 1288.
10. Bijapur, J.; Keppler, M. D.; Bergqvist, S.; Brown, T.; Fox, K. R. Nucleic Acids Res.
1999, 27, 1802.
11. Ueno, Y.; Tomino, K.; Sugimoto, I.; Matsuda, A. Tetrahedron 2000, 56, 7903.
12. Atsumi, N.; Ueno, Y.; Kanzaki, M.; Shuto, S.; Matsuda, A. Bioorg. Med. Chem.
2001, 10, 2933.
13. Puri, N.; Majumdar, A.; Cuenoud, B.; Natt, F.; Matin, P.; Boyd, A.; Miller, P. S.;
Seidman, M. M. Biochemistry 2001, 41, 7716.
14. Brazier, J. A.; Shibata, T.; Townsley, J.; Taylor, B. F.; Frary, E.; Williams, N. H.;
Williams, D. M. Nucleic Acids Res. 2005, 33, 1362.
15. Radhakrishnan, I.; Patel, D. J. Structure 1993, 1, 135.
16. Giovannangeli, C.; Rougée, M.; Garestier, T.; Thuong, N. T.; Hélène, C. Proc. Natl.
Acad. Sci. U.S.A. 1992, 89, 8631.
17. Malkov, V. A.; Voloshin, O. N.; Soyfer, V. N.; Frank-Kamenetskii, M. D. Nucleic
Acids Res. 1993, 21, 585.
branched oligonucleotide X and the target DNA I in the absence of
Mg2+ (Fig. S4).
In order to confirm the formation of the triplex, we next per-
formed fluorescence resonance energy transfer (FRET) analysis.
For this, we synthesized the branched oligonucleotides XI and
XII, which were modified with fluorescein (Flu) at the 50-end and
with dabcyl (Dab) at the 30-end as a quencher (Fig. 4). When the
branched oligonucleotide XI was mixed with the target RNA VIII
in the absence of Mg2+, the fluorescence intensities of the solutions
were almost the same irrespective of the concentrations of the first
strand VIII (Fig. 4, top graph). On the other hand, the fluorescence
intensities of the solutions decreased as the concentrations of the
first strand VIII increased, and plateaued at a 1:1 branched oligo-
nucleotide XI and first strand VIII ratio when the branched oligo-
nucleotide XI was mixed with the target RNA VIII in the