Incorporation of a novel nucleobase allows stable oligonucleotide-directed triple helix formation at the target sequence containing a purine pyrimidine interruption

Article abstract of DOI:10.1039/b103743a

Thermal denaturation experiments have established that an oligonucleotide incorporating the artificial nucleobase S, does form a stable triplex with a double stranded DNA which exhibits a pyrimidine interruption within the oligopurine sequence.

Full text of DOI:10.1039/b103743a

View Article Online / Journal Homepage / Table of Contents for this issue
Incorporation of a novel nucleobase allows stable
oligonucleotide-directed triple helix formation at the target sequence
containing a purine·pyrimidine interruption
Dominique Guianvarc’h,a Rachid Benhida,a Jean-Louis Fourrey,*a Rosalie Maurisseb and
Jian-Sheng Sun*b
a Institut de Chimie des Substances Naturelles, CNRS, Avenue de la Terrasse 91198. Gif-sur-Yvette
Cédex, France. E-mail: fourrey@icsn.cnrs-gif.fr; Fax: +33-1-69077247; Tel: +33-0169283053
b Laboratoire de Biophysique, UMR 8646 CNRS-Muséum National d’Histoire Naturelle, INSERM U201,
43 rue Cuvier 75231. Paris Cedex 05, France. E-mail: sun@mnhn.fr; Fax: +33-1-69077247;
Tel: +33-01407937
Received (in Cambridge, UK) 25th April 2001, Accepted 21st July 2001
First published as an Advance Article on the web 23rd August 2001
Thermal denaturation experiments have established that an
oligonucleotide incorporating the artificial nucleobase S,
does form a stable triplex with a double stranded DNA
which exhibits a pyrimidine interruption within the oligo-
purine sequence.
dazole (D3) has been shown to equally stabilize the triplex at
both A·T and G·C sites.6a Subsequent NMR studies showed that
the binding mode is intercalation6b which is consistent with the
lack of base pair discrimination.
In this work, a new base analog (S) has been synthesized and
incorporated into a TFO in an attempt to achieve better triplex
stabilization than the D3 nucleobase at the purine·pyrimidine
sites within its cognate dsDNA sequence. Compared to D3 the
S nucleobase consists of two unfused aromatic rings which are
linked to 2A-deoxyribose by an acetamide motif instead of a
three ring construct attached directly to 2A-deoxyribose (Fig. 2).
The synthesis of the required phosphoramidite 5 to serve for the
incorporation of S in TFOs is straightforward. Thus, compound
2, readily obtained by catalytic hydrogenation of 2-acetamido-
4-(3-nitrophenyl)thiazole 1,8 was acylated with 2-(2-deoxy-
5-O-dimethoxytrityldeoxyribosyl)acetic acid 39 using 2-chloro-
1-methylpyridinium iodide. Finally, the resulting derivative 4
was phosphitylated in the usual manner to give the desired
phosphoramidite 5 (Scheme 1).
The capacity of triple helix stabilization of the novel
nucleobase S was assessed in a model system where S was
incorporated in the middle of a 18-mer TFO (at position Z)10
and was screened against all four possible base pairs (X·Y =
T·A, C·G, A·T or G·C) in the target oligopyrimidine·oligopurine
sequence (Table 1). The thermal denaturation experiments11
indicated that the Tm value of the triplex containing an A·T3S
triplet (Tm = 50 °C) was very close to those of perfect triplexes
without any interruption of oligopyrimidine·oligopurine se-
quences (T·A3T or C·G3C+, Tm = 51 or 50 °C, respectively).
It was noted that the use of S base provides a 5–8 °C triplex
stabilization as compared to the best base triplet made of natural
bases (A·T3S vs. A·T3G; G·C3S vs. G·C3T), respectively. In
terms of triplex stability, the novel S nucleobase is at least as
good as the previously reported D3 base. The main difference
For many years, triple helix-forming oligonucleotides (TFOs)
have been known to be able to bind in the major groove of
oligopyrimidine·oligopurine sequences of double-stranded
DNA (dsDNA). TFOs establish specific hydrogen bonds with
the oligopurine strand of dsDNA through the formation of
T·A3T and C·G3C+ base triplets1 in Hoogsteen (pyrimidine-
motif, Fig. 1) or T·A3A and T·A3T, and C·G3G triplets in
reverse Hoogsteen configuration (purine- or mixed-motif).2
Hence, dsDNA sequence recognition by TFOs has potential
applications in gene expression modulation and in gene
targeting technologies.3 Unfortunately, these interesting appli-
cations must be restricted to long oligopyrimidine·oligopurine
sequences only ( > about 15 bp) since any interruption by even
a single A·T or G·C base pair strongly destabilizes triple helix
formation.4 Consequently, during the last decade, considerable
efforts have been devoted, so far with limited success, to
circumvent such a sequence limitation.5 Two approaches have
been undertaken to design and synthesize: (1) new base analogs
featuring an extended heterocyclic ring system for achieving
specific hydrogen bonds with all hydrogen bond-forming sites
available in the major groove side of the inverted A·T and G·C
base pairs;6 (2) nucleobases capable of stabilizing, non-
sequence-specifically, triple helix formation at the purine·pyr-
imidine interruption sites, either by the attachment of an
intercalating agent in conjunction with an appropriate nucleo-
base, or by a nucleobase alone.7 The second strategy (universal
base approach) has been more successful than the first (specific
base approach). In the literature, a 4-(3-benzamidophenyl)imi-
Fig. 1 Canonical T·A3T and C·G3C+ base triplets in Hoogsteen
configuration (pyrimidine-motif). R = 2A-deoxyribosyl.
† Electronic supplementary information (ESI) available: experimental
details. See http://www.rsc.org/suppdata/cc/b1/b103743a/
Fig. 2 Structures of the 2A-deoxynucleosides featuring nucleobases S and
D3.
1814
Chem. Commun., 2001, 1814–1815
This journal is © The Royal Society of Chemistry 2001
DOI: 10.1039/b103743a

View Article Online
between the C5 methyl of T in the oligopurine-rich target strand
and the deoxyribose moiety of S can be anticipated. Indeed,
when T was replaced by U in the target strand, a further
stabilization of the triplex featuring an A·U3S triplet was
observed with the Tm increasing by 3 °C. Such an observation is
consistent with the proposed mode of recognition (Fig. 3).
In conclusion, this work shows that a novel S nucleobase
when incorporated into a pyrimidine-motif TFO can effectively
circumvent a purine·pyrimidine base pair interruption in an
oligopyrimidine·oligopurine sequence. This outstanding prop-
erty of S opens new perspective as it could be exploited as a lead
compound, in both universal base or specific base approaches,
to develop new nucleobases capable of achieving sequence-
specific recognition of further extended dsDNA sequences by
oligonucleotide-directed triple helix formation.
Scheme 1 Reagents and conditions: (a) H2, Pd/C, EtOH–AcOH, 95%. (b)
2-Chloro-1-methylpyridinium iodide, NEt3, CH2Cl2, 60 °C, 90%. (c)
2-Cyanoethyl diisopropylchlorophosphoramidite, N,N-diisopropylethyl-
amine, CH2Cl2, rt, 75%.
between S and D3 is the observation that the S nucleobase can
discriminate, to some extent, an A·T base pair from others
(namely A·T3S vs. G·C3S, T·A3S and C·G3S). It is worth
noting that there is evidence that the aminothiazole moiety of S
is involved in the A·T base pair recognition as the replacement
of the heterocyclic moiety of S by aniline caused a 14 °C
decrease in Tm and abolished base pair discrimination. How-
ever, additional studies12 are needed to obtain the definitive
structural arguments which would validate the recognition
pattern proposed in Fig. 3. In this scheme the triple between the
A·T base pair and S is characterized by the establishment of
three hydrogen bonds to the N7 atom and the 6-amino group of
adenine, and to the 4-oxo group of thymine in a co-planar
arrangement. According to this scheme, steric hindrance
Notes and references
1 The symbols · and 3 stand for Watson–Crick and Hoogsteen-like
hydrogen bonding, respectively.
2 (a) T. Le Doan, L. Perrouault, D. Praseuth, N. Habhoud, J. L. Decout,
N. T. Thuong, J. Lhomme and C. Hélène, Nucleic Acids Res., 1987, 15,
7749; (b) H. E. Moser and P. B. Dervan, Science, 1987, 238, 645. For
reviews see (c)–(e) (c) N. T. Thuong and C. Hélène, Angew. Chem., Int.
Ed. Engl., 1993, 32, 666; (d) M. T. Frank-Kamenetskii and S. M.
Mirkin, Annu. Rev. Biochem., 1995, 64, 65; (e) S. Neidle, Anti-Cancer
Drug Des., 1997, 12, 433.
3 (a) J. L. Maher III, Cancer Invest., 1996, 14, 66; (b) C. Giovannangeli
and C. Hélène, Antisense Nucleic Acid Drug Dev., 1997, 7, 413; (c) K.
M. Vasquez and J. H. Wilson, Trends Biochem. Sci., 1998, 23, 4; (d) D.
Praseuth, A. L. Guieysse-Peugeot and C. Hélène, Biochim. Biophys.
Acta, 1999, 1489, 181.
4 (a) J. L. Mergny, J. S. Sun, M. Rougée, T. Garestier, F. Barcelo, J.
Chomilier and C. Hélène, Biochemistry, 1991, 30, 9791; (b) W. A.
Greenberg and P. B. Dervan, J. Am. Chem. Soc., 1995, 117, 5016; (c) G.
C. Best and P. B. Dervan, J. Am. Chem. Soc., 1995, 117, 1187.
5 (a) Reviews: J. S. Sun and C. Hélène, Curr. Opin. Struct. Biol., 1993, 3,
345; (b) S. O. Doronina and J. P. Behr, Chem. Soc. Rev., 1997, 63; (c)
D. M. Gowers and K. R. Fox, Nucleic Acids Res., 1999, 27, 1569 and
references cited therein.
Table 1 Sequence of the triple helices studied in this work. Melting
temperature (Tm) of all combinations of base triplets at the X·Y3Z site.
Estimated accuracy of the melting temperatures is 1 °C
6 (a) L. C. Griffin, L. L. Kiessling, P. A. Beal, P. Gillespie and P. B.
Dervan, J. Am. Chem. Soc., 1992, 114, 7976; (b) K. M. Koshlap, P.
Gillespie, P. B. Dervan and J. Feigon, J. Am. Chem. Soc., 1993, 115,
7908; (c) C. Y. Huang, C. D. Cushman and P. S. Miller, J. Org. Chem.,
1993, 58, 5048; (d) C. Y. Huang, G. Bi and P. S. Miller, Nucleic Acids
Res., 1996, 24, 2606; (e) T. E. Lehmann, W. A. Greenberg, D. A.
Liberles, C. K. Wada and P. B. Dervan, Helv. Chim. Acta, 1997, 80,
2002; (f) I. Prévot-Halter and C. J. Leumann, Bioorg. Med. Chem. Lett.,
1999, 9, 2657.
7 (a) S. Kukreti, J. S. Sun, T. Garestier and C. Hélène, Nucleic Acids Res.,
1997, 25, 4264; (b) S. Kukreti, J. S. Sun, D. Loakes, D. M. Brown, C.
H. Nguyen, E. Bisagni, T. Garestier and C. Hélène, Nucleic Acids Res.,
1998, 26, 2179; (c) D. A. Gianolio and L. W. McLaughlin, J. Am. Chem.
Soc., 1999, 121, 6334.
Z
X·Y
T
C
G
S
T·A
C·G
A·T
G·C
51
40
33
38
31
50
33
35
31
31
45
35
42
41
50
46
8 C. D. Hurd and N. Karasch, J. Am. Chem. Soc., 1946, 68, 658.
9 J. Hovinen and H. Salo, J. Chem. Soc., Perkin Trans. 1, 1997, 3017.
10 The identity and homogeneity of the 18-mer was confirmed by MALDI-
TOF [M calc. 5502.8; found m/z 5501.5 (M 2 H)2] and RP-HPLC.
11 DNA thermal denaturation and renaturation experiments were carried
out by first mixing I and II strands (1.2 and 1.0 mM, respectively), then
adding 1.5 mM of TFO (III) in a 10 mM cacodylate buffer (pH 5.9)
containing 100 mM NaCl, 10 mM MgCl2 and 0.5 mM spermine.
12 A comprehensive NMR study is under way with an intramolecular
system consisting of a 31-mer in order to establish the recognition mode
either within the A·T3S triple and/or between the adjacent bases.
Results will be reported in due course.
Fig. 3 Model proposed for the specific recognition of an A·T base pair by
nucleobase S. R = 2A-deoxyribosyl. Putative steric interaction in the plane
of the A·T3S triple is indicated (see text).
Chem. Commun., 2001, 1814–1815
1815