Stabilization of DNA triplexes by dangling aromatic residues

Article abstract of DOI:10.1016/j.tetlet.2003.11.144

Novel nucleoside analogues containing 2′-deoxyinosine and aromatic rings, which are connected by short linker groups, were synthesized and incorporated into oligodeoxyribonucleotides (ODNs). ODNs containing the nucleoside analogues formed stable duplexes

Full text of DOI:10.1016/j.tetlet.2003.11.144

Tetrahedron
Letters
Tetrahedron Letters 45 (2004) 1187–1190
Stabilization of DNA triplexes by dangling aromatic residuesq
Mio Kubota and Akira Ono*
Department of Chemistry, Graduate School of Science, Tokyo Metropolitan University, Minami-ohsawa 1-1, Hachioji,
Tokyo 192-0397, Japan
Received 24 October 2003; revised 27 November 2003; accepted 28 November 2003
Abstract—Novel nucleoside analogues containing 20-deoxyinosine and aromatic rings, which are connected by short linker groups,
were synthesized and incorporated into oligodeoxyribonucleotides (ODNs). ODNs containing the nucleoside analogues formed
stable duplexes and triplexes with target nucleic acids. The stacking interaction between base residues in the nucleoside analogues
appears to be a major cause of stabilization.
Ó 2003 Elsevier Ltd. All rights reserved.
The stacking interaction as well as hydrogen bond for-
mation between nucleobases have been recognized as the
major factors stabilizing duplex formation of nucleic
acids.1 Recently, it has been reported that oligodeoxy-
ribonucleotides (ODNs) containing nucleoside ana-
logues carrying aromatic groups, instead of nucleobases,
form stable duplexes without forming hydrogen
bonds.2–6 These results and the studies of selective
DNA-polymerase-mediated replication of oligodeoxy-
ribonucletides that contain hydrophobic, non-hydrogen-
bonding base pairs7;8 led to a re-evaluation of the
Figure 1. Structures of nucleoside–aromatic ring conjugates.
importance of aromatic stacking interactions in duplex
formation. However, no study examining the effects of
stacking interactions of non-hydrogen-bonding base
analogues on triplex stability has been reported even
though triplexes as well as duplexes are important
motifs for designing biochemical tools.9 In this report,
the synthesis and stabilization of triplex formation as
ring conjugates 6 and 7, which were subsequently con-
verted into the corresponding phosphoramidite building
blocks, 8 and 9. Oligodeoxyribonucleotides (ODNs)
containing 1 and 2 were synthesized by the standard
well as duplex formation of ODNs containing new
nucleoside–aromatic ring conjugates (1 and 2 in Fig. 1)
is described.
protocols on an automated DNA synthesizer and puri-
fied using established procedures. Each ODN prepared
in this study exhibited a sharp major peak by HPLC
analysis. Each ODN was completely hydrolyzed by
The synthesis of the phosphoramidite building blocks of
venom phosphodiesterase and alkaline phosphatese to
the corresponding nucleoside–aromatic ring conjugates
the corresponding nucleosides, which were analyzed by
HPLC (see supporting information).
are shown in Scheme 1. Dinitrophenylation10 of 3 fol-
lowed by hydrazine treatment yielded the N1-amino
derivative 5. Condensation of 5 with aromatic rings by
amide bond formation gave the nucleoside–aromatic
assessed by determination of the melting temperatures
The thermal stability of the duplexes (Fig. 2) was
(Tm) (Fig. 2) using UV spectroscopy in appropriate
buffers. Typical thermal transition profiles are shown in
q Supplementary data associated with this article can be found, in the
online version, at doi:10.1016/j.tetlet.2003.11.144
Figure 3a. The dangling residues were added to the
homopyrimidine strands of the control duplex consist-
ing of homopurine and homopyrimidine strands (D-I in
Fig. 2). Addition of the dAMP residue at the 50-end of
* Corresponding author. Tel.: +81-426-77-2545; fax: +81-426-77-2525;
e-mail: akiraono@comp.metro-u.ac.jp
0040-4039/$ - see front matter Ó 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2003.11.144

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M. Kubota, A. Ono / Tetrahedron Letters 45 (2004) 1187–1190
Scheme 1. A schematic representation of the synthesis of the phosphoramidite building blocks 8 and 9. Reagents and conditions: (a) 2,4-dinitro-
chlorobenzene, K2CO3, DMF, 75%; (b) NH2NH2/H20, K2CO3, CH3CN, 55%; (c) RCOOH, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide/HCl,
73–76%; (d) standard procedures.
Figure 2. Tms for the thermal transition of the duplexes. Thermally
induced transitions for D-I–D-IV were measured at 260 nm in buffer
(10 mM Na cacodylate, 200 mM NaCl, 20 mM MgCl2; pH 6.6) con-
taining duplex (7 lM), and thermally induced transitions for scD-I–
scD-III were measured in buffer (10 mM Na phosphate, 1 M NaCl,
0.1 mM EDTA; pH 7.0) containing duplex (5 lM).
Figure 3. (a) Relative absorbance, A=A0 ¼ ½ðAt °C ꢀ A10 °CÞ=ðA80 °C
ꢀ
the control duplex did not significantly stabilize duplex
formation (D-II). In contrast, addition of conjugates 1
and 2 efficiently stabilized duplex formation as shown by
the increased Tm values (D-III, D-IV).
A10 °CÞꢁ at 260 nm versus temperature. (d) D-I, (M) D-III, (j) D-IV.
(b) Relative absorbance, A=A0 ¼ ½ðAt °C ꢀ A10 °CÞ=ðA80 °C ꢀ A10 °CÞꢁ at
260 nm versus temperature. (d) T-I, (M) T-III, (j) T-IV (c) and (d)
Hypochromic and bathochromic shift of kmax of the aromatic rings
according to the formation of duplexes D-IV (c) and triplexes T-III (d).
The spectra were measured at 70 °C (above Tm) (—) and 10 °C (below
Tm) (- - -) in the same buffers used for thermal transition experiments.
The effects of dangling nucleotides on duplex stability
should be sequence dependent. For example, it has been
reported that the sequence, 50-d(ACGCGCG)-30, which
has the dangling dAMP residue at the 50-end of the self-
complementary sequence was largely stabilized (scD-II in
Fig. 2) compared to the control sequence (scD-I).2 Thus,
the stabilization effect of pyrene conjugate 2, which most
effectively stabilized the homopyrimidine–homopurine
duplex formation, was re-examined using the self-com-
plementary sequence and it was found that the addition
of conjugate 2 to the self-complementary sequence
greatly stabilized the duplex formation (scD-III). Con-
sequently, it was inferred that the addition of the con-
jugate at the dangling position can stabilize a wide range
of duplexes in addition the duplexes studies here.
The duplex-stabilizing mode of dAMP and the conju-
gates should be different. From the typical DNA duplex

M. Kubota, A. Ono / Tetrahedron Letters 45 (2004) 1187–1190
1189
Figure 4. Illustrations of possible 50-end stacking geometry for the
conjugates. The conjugates placed at the 50-ends adjacent to C-G base
pair and Cþ-C-G base triad. (a) Dangling 2 at the 50-end of duplex
D-IV. (b) Dangling 1 at the 50-end of the third strand of triplex T-III.
(c) Dangling 1 at the 50-end of the duplex part of triplex T-IV.
structure1 it has been inferred that dangling nucleotides
such as dAMP stabilize the duplex formation mainly by
the intra-strand-stacking interaction. In contrast, the
conjugates at the dangling position stabilize the duplex
formation by both of intra-strand stacking (for example,
the stacking between hypoxanthine and cytidine in D-
III) and inter-strand stacking (for example, the stacking
between the aromatic ring and guanine in D-III) as
illustrated in Figure 4. The reason that the dangling
dAMP residue did not significantly enhance the stability
of the duplex D-II is probably because the intra-strand-
stacking effect through the homopyrimidine strand was
weak,1 consequently additional intra-strand stacking
does not confer any additional stability. The situation
was totally different for D-IV. The pyrene ring of 2 at
the dangling position may interact with the guanine base
of the homopurine strand. Since the stacking ability of
purine bases is much higher than that of pyrimidine
bases,1 D-IV was significantly stabilized by the addi-
tional stacking interaction.
Figure 5. Tms for the thermal transition of triplexes. Thermally
induced transitions for T-I–T-VIII were measured at 260 nm in buffer
(10 mM Na cacodylate, 200 mM NaCl, 20 mM MgCl2; pH 5.0) con-
taining triplex (7 lM).
strand and duplex ends are synergetic, greatly enhancing
the stability of the triplexes (T-V and T-VIII). The large
aromatic surface formed by the two aromatic residues
(hypoxanthine and acridine or pyrene) of the conjugate
could overlap with the surface of the base triad as
illustrated in Figure 4, and the stacking interaction
between the large aromatic surfaces could significantly
stabilize the triplex formation. The hypochromic11;12 and
bathochromic shift of the absorption kmax of the aro-
matic residue with duplex and the triplex formation
provides evidence of aromatic stacking interactions
between the aromatic residues and base-pairs or base-
triads (Fig. 3c and d).
This inter-strand-stacking ability of conjugates 1 and 2
is likely to be crucial in stabilizing triplex formation
(Fig. 5). Tm values in Figure 5 were corresponding to the
dissociation of the triplexes directly to the single strands,
without passing through the duplexes, since the triplexes
were more stable than the corresponding duplexes in the
solution containing the divalent cation in acidic pH
(20 mM MgCl2; pH 5.0) (see supporting information).
The addition of a dAMP residue at the 50-end of the
third strand showed no detectable effect on the thermal
dissociation profile of the control triplex T-I (Fig. 3b).
However, addition of conjugates 1 and 2 at the dangling
position of the third strand significantly stabilized the
triplex formation (Fig. 5, T-III and T-VI). Interestingly,
addition of the conjugates to the 50-end of the homo-
pyrimidine strand in the duplex part also stabilized the
triplex formation (T-IV and T-VII) and the stabilizing
effects of the conjugates attached at both the third
The results are the first reported example of stabilizing
the triplex formation using dangling residues and indi-
cate that the large aromatic surfaces of the conjugates
consisting of nucleoside and aromatic rings can enhance
the stability of triplexes as well as duplexes in cases
where both intra- and inter-strand-stacking interactions
between the aromatic rings and base-pairs or base-triads
are possible. This rationale may prove useful in the
design of biochemical probes for antisense, antigene,
and decoy13 methodologies.
Acknowledgements
This work was supported in part by the National Project
on Protein Structural and Functional Analyses from the

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M. Kubota, A. Ono / Tetrahedron Letters 45 (2004) 1187–1190
6. Nakano, S.; Uotani, Y.; Nakashina, S.; Anno, Y.; Fujii,
M.; Sugimoto, N. J. Am. Chem. Soc. 2003, 125, 8086–
8087.
Ministry of Education, Science, and Culture. We thank
Prof. W. S. Price for kind discussion.
7. Kool, E. T.; Morales, J. C.; Guckian, K. M. Angew.
Chem., Int. Ed. 2000, 39, 990–1009.
8. Wu, Y.; Ogawa, A. K.; Berger, M.; McMinn, D. L.;
Schultz, P. G.; Romesberg, F. E. J. Am. Chem. Soc. 2000,
122, 7621–7632.
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