Synthesis, RNA selective hybridization and high nuclease resistance of an oligonucleotide containing novel bridged nucleic acid with cyclic urea structure

Article abstract of DOI:10.1039/c0cc00154f

A novel bridged nucleic acid bearing cyclic urea structure was successfully synthesized and introduced into oligonucleotide, displaying attractive characteristics of highly RNA selective hybridization ability and excellent resistance towards nuclease degradation.

Full text of DOI:10.1039/c0cc00154f

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Synthesis, RNA selective hybridization and high nuclease resistance
of an oligonucleotide containing novel bridged nucleic acid with cyclic
urea structurew
Masaru Nishida, Takeshi Baba, Tetsuya Kodama, Aiko Yahara, Takeshi Imanishi and
Satoshi Obika*
Received 27th February 2010, Accepted 26th May 2010
First published as an Advance Article on the web 22nd June 2010
DOI: 10.1039/c0cc00154f
A novel bridged nucleic acid bearing cyclic urea structure was
successfully synthesized and introduced into oligonucleotide,
displaying attractive characteristics of highly RNA selective
hybridization ability and excellent resistance towards nuclease
degradation.
is still unclear how the bridged moiety itself affects the
hybridization properties of ONs, and further evaluation of
the relationship between the bridged structure and properties
of the modified ONs is ongoing.
Here, we focus on a urea structure, containing both N–H
and CQO groups as a proton donor and acceptor, respectively,
and introduced it into the bridged moiety connecting the
20- and 40-positions of nucleoside to evaluate hybridization
properties and enzymatic stability.14
Since the Human Genome Project was completed, much
attention has been given to genome technologies to regulate
target gene expression or function. The most simple and
promising approach to down-regulate target gene expression
in a living cell is antisense strategy, which prevents translation
by hybridization of an oligonucleotide with its complementary
RNA strand.1 For practical use in antisense strategy, chemical
modification of oligonulcleotides is essential to achieve high
resistance towards nuclease degradation, high affinity to target
mRNA with sequence specificity and RNA selectivity.
20,40-BNA (20,40-Bridged Nucleic Acid)2/LNA3 whereby the
sugar moiety is restricted to North-type (N-type) conformation
was developed by our group and Wengel’s group independently
(Fig. 1). Oligonucleotides (ONs) containing 20,40-BNA confer
moderate resistance against enzymatic degradation3a,4 and
strong affinity with their RNA complements,3,5 but they still
showed high affinity with their DNA complements.3,5 On the
other hand, other bridged nucleic acids bearing a different type
of bridge structure between 20- and 40-positions have been
developed to date (Fig. 1).6–13 Depending on the ring size
and/or the elements which compose the bridged structure,
the modified ONs varied among properties such as
enzymatic stability and RNA selectivity. In general, ONs
containing bridged nucleosides with a large bridged ring
size revealed higher resistance against enzymatic degradation
As shown in Scheme 1, the phosphoramidite derivative of a
novel bridged nucleic acid bearing a cyclic urea moiety
was synthesized from known 40-hydroxymethyl nucleoside
derivative 1.3 At first, 1 was treated with trifluoromethane-
sulfonyl chloride in the presence of DMAP to afford a
2,20-anhydro intermediate, which was then converted to an
arabino-type nucleoside under alkaline conditions. Subsequent
triflation at the 20-hydroxy group afforded ditriflate 2.
Replacement of the two triflate groups by azide groups
successfully proceeded to give diazide 3. Reduction under
Staudinger conditions,15 followed by a ring-closure reaction
with p-nitrophenyl chloroformate gave desired compound 4.
(i.e., 20,40-BNA/LNA vs. ENA,7 aza-ENA,8 20,40-BNANC 9
20,40-BNACOC 10 PrNA,11 etc.6b). In addition, incorporation
of heteroatoms in an appropriate position of the bridged
moiety showed tendency to enhance hybridization
ability (i.e., PrNA11 vs. 20,40-BNACOC 10
or carbocyclic
,
,
a
,
ENA12,13 vs. 20,40-BNANC 9) and in some cases RNA selectivity
(i.e., aza-ENA,8 20,40-BNANC,9 20,40-BNACOC 10). However, it
Graduate School of Pharmaceutical Sciences, Osaka University,
1-6 Yamadaoka, Suita, Osaka, 565-0871, Japan.
E-mail: obika@phs.osaka-u.ac.jp; Fax: +81 6 6879 8204;
Tel: +81 6 6879 8200
w Electronic supplementary information (ESI) available: Full experi-
mental procedures, characterization data of all new compounds, UV
melting profiles, CD spectra, and energy-minimized structures. See
DOI: 10.1039/c0cc00154f
Fig. 1 Structure of 20,40-BNA/LNA and selected derivatives with a
different type of bridge structure. B = nucleobase, T = thymin-1-yl.
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stabilized. Changes in Tm values (DTm) ranged from +1 1C to
+14 1C, which are comparable to those for 20,40-BNACOC and
better than those for PrNA.16 In contrast, the thermal stability
of the hybrids formed by modified ONs 7–10 with DNA
complement was diminished compared with the duplex
involving natural DNA ON 12. In the case of ONs 8, 9 and
10, the differences in DTm values with RNA (DTm (RNA)) and
those with DNA (DTm (DNA)) were +10 1C, +13 1C and
+17 1C, respectively, clearly indicating that modification of
ON with 20,40-BNA bearing cyclic urea structure 5 enhances
RNA selective hybridization ability. The RNA selective binding
abilities of the modified ONs were also revealed in the recently
developed modified ONs8–10,18–21 and would be suitable for
antisense strategy targeting mRNA or miRNA.
Helical structure of the duplexes formed by ONs 7–10
and RNA and DNA complements was evaluated by CD
measurements, and the CD spectra were compared with those
of the corresponding natural duplexes, DNA 12/RNA
complement and DNA 12/DNA complement. As shown in
Fig. S3 (ESIw) duplexes involving ONs 7–10 and their RNA
complement exhibited almost identical CD spectra to that of
the DNA 12/RNA duplex, indicating that 20,40-BNA containing
cyclic urea structure 5 completely adapted to the DNA
12/RNA helical structure when it was incorporated into 12.
In contrast, incorporation of 5 into the DNA 12/DNA duplex
changed its CD profile (Fig. S4, ESIw). As the quantity of 5
increased, the intensity at 220 nm gradually decreased while
that at 260 nm increased. This result suggests that the DNA
12/DNA helix shifted from B to A-like helical structure by
the incorporation of 5, probably due to the rigid N-type
(RNA-type) sugar conformation of 5. The RNA selectivity
of ONs containing the 20,40-BNA with cyclic urea structure 5
could be caused by such differences in the shift of helical
structure revealed by the change of CD profile. In both cases,
no evidence of unexpected distortion of the helical structure
arising from a urea moiety containing hydrogen donor and
acceptor sites was observed.
Scheme 1 Reagents and conditions: (i) TfCl, DMAP, CH2Cl2, 0 1C;
(ii) 1 M NaOH aq., 1,4-dioxane, rt; (iii) Tf2O, pyridine, CH2Cl2, rt,
37% over 3 steps; (iv) NaN3, DMF, rt, quant.; (v) Me3P, THF–H2O,
rt; (vi) p-nitrophenyl chloroformate, Et3N, CH2Cl2, rt, 60% over 2
steps; (vii) H2, 20% Pd(OH)2/C, THF, rt, 94%; (viii) DMTrCl,
pyridine, rt, 76%; (ix) 4,5-dicyanoimidazole, (i-Pr2N)2PO(CH2)2CN,
THF–CH3CN, rt, 45%. X denotes the bridged nucleoside with cyclic
urea structure.
Benzyl protection groups of 4 were removed by hydrogeno-
lysis with 20% Pd(OH)2/C to afford desired diol 5. The sugar
conformation of 516 was confirmed to be N-type by means of
1H-NMR spectroscopy where the H10 signal was observed as a
singlet.2,17 Phosphoramidite 6 was obtained by protection
of the 50-hydroxyl group with 4,40-dimethoxytrityl chloride
and phosphitylation of the 30-hydroxy group with
Enzymatic stability of decathymidylate derivative (ON 11)
involving a single 20,40-BNA with cyclic urea structure 5 was
evaluated by using 30-exonuclease (Crotalus admanteus venom
phosphodiesterase, CAVP) and compared with the corres-
ponding natural (13), 20,40-BNA(LNA)-modified (14) and
20,40-BNANC[NH]-modified (15), ENA-modified (16), 20,40-
BNANC[NMe]-modified (17), phosphorothioate-modified
(18) and 20,40-BNACOC-modified (19) ONs. After incubation
of each ON solution at 37 1C in the presence of CAVP, the
reaction mixture was analyzed at several time points by
reversed-phase HPLC, and the percentage of the remaining
intact ONs was plotted in Fig. 2. Under the conditions used in
this experiment, natural ON 13 was completely degraded
within 2 min, and the 20,40-BNA(LNA) modified congener
14 was digested in 20 min. In contrast, modification by the
20,40-BNA with the cyclic urea structure 5 significantly
enhanced the stability against CAVP; ca. 90% of ON 11
survived after 40 min, which was considerably better than
20,40-BNANC[NH]-modified ON 15, ENA-modified ON 16,
and comparable to 20,40-BNANC[NMe]-modified ON 17,
phosphorothioate-modified ON 18, and 20,40-BNACOC-modified
ON 19. These results clearly demonstrate the enhanced resistance
2-cyanoethyl
N,N,N0,N0-tetraisopropylphosphordiamidite
and 4,5-dicyanoimidazole.
Phosphoramidite 6 was incorporated into ONs on an
automated DNA synthesizer using standard phosphoramidite
chemistry except for a prolonged coupling time of 40 min with
5-ethylthio-1H-tetrazole as an activator. The coupling
efficiency of the phosphoramidite 6 was estimated to be more
than 95% from a trityl monitor. Modified ONs 7–11
(Scheme 1) were purified by reverse phase HPLC and
characterized by MALDI-TOF mass spectra (Table S1, ESIw).
The ability of ONs 7–10 to hybridize to complementary
RNA and DNA strands was evaluated via UV melting
experiments and compared with the corresponding natural
DNA ON 12, 50-d(GCGTTTTTTGCT)-30. The UV melting
profiles are shown in Fig. S1 and S2 (ESIw), and the Tm values
are summarized in Table 1. The Tm values for duplexes formed
by modified ONs 7–10 with RNA complement were higher
than that of the duplex formed by the unmodified DNA 12
and RNA complement. As the number of modifications
increased, the duplex formed with RNA complement was well
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Table 1 Tm values (1C) of duplexes formed by ONs 7–10 and 12 with their complementary strandsa
RNA
DNA
RNA selectivity
Tm
DTm
(DTm/mod.)
Tm
DTm
(DTm/mod.)
DTm (RNA) ꢁ DTm (DNA)
—
+5
+10
+13
+17
ONs
12
7
8
9
10
48
49
51
55
62
—
—
52
48
45
46
49
—
—
+1
+3
+7
+14
(+1.0)
(+1.5)
(+2.3)
(+2.3)
ꢁ4
ꢁ7
ꢁ6
ꢁ3
(ꢁ4.0)
(ꢁ3.5)
(ꢁ2.0)
(ꢁ0.5)
The UV melting experiments were carried out in 10 mM sodium phosphate buffer (pH 7.2) containing 100 mM NaCl at a scan rate of 0.5 1C minꢁ1
a
at 260 nm with target strand, 50-r(AGCAAAAAACGC)-30 or 50-d(AGCAAAAAACGC)-30. Final concentration of each ON was 4 mM.
Notes and references
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Fig.
2
Enzymatic stability of 50-d(TTTTTTTTXT)-30 against
Crotalus admanteus venom phosphodiesterase (CAVP, Pharmacia
Biotech). X = 20,40-BNA with cyclic urea structure-T (open diamond)
(ON 11); natural DNA-T (closed square) (ON 13); 20,40-BNA(LNA)-T
(closed triangle) (ON 14); 20,40-BNANC[NH]-T (closed diamond)
(ON 15); ENA-T (open circle) (ON 16); 20,40-BNANC[NMe]-T
(open square) (ON 17); phosphorothioate-T (closed circle) (ON 18)
and 20,40-BNACOC-T (open triangle) (ON 19). Experiments
were performed at 37 1C in 100 mL of buffer containing 50 mM
Tris-HCl (pH 8.0), 10 mM MgCl2, 0.75 nmol each ON and CAVP
(0.175 mg).
of BNAs can be attributed to steric hindrance around the
phosphodiester linkage rather than the elements composing
the bridge structure.
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In conclusion, we successfully synthesized novel bridged
nucleic acid monomer 5 bearing cyclic urea structure and
incorporated it into ONs. To the best of our knowledge, this
is the first example of a nucleic acid analogue with a bridged
structure between 20- and 40-positions containing a carbonyl
group. Without any distortion of a helical structure brought
about by the urea bridge, ONs containing 5 formed a stable
duplex with RNA complement in a highly RNA selective
manner. Nuclease resistance of this nucleic acid analogue is
abundantly higher than that of natural DNA and 20,40-
BNA(LNA) and is also slightly higher than that of phosphoro-
thioate. The characteristics of this nucleic acid analogue are
essential for application to antisense technology, and research
in this direction is now in progress.
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A. Yahara, M. Nishida, T. Baba, T. Kodama, T. Imanishi and
S. Obika, Nucleic Acids Symp. Ser., 2009, 53, 11.
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16 Energy-minimized structure of 5 was calculated using HF/6-31G*
model (Spartan ’06, Wavefunction Inc) and compared with those
of 20,40-BNACOC and PrNA. We made a brief discussion of the
structure–RNA affinity relationship, see Fig. S5 (ESIw).
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21 S. Kumar, M. H. Hansen, N. Albæk, S. I. Steffansen, M. Petersen
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A part of this work was supported by the Program for
Promotion of Fundamental Studies in Health Sciences of the
National Institute of Biomedical Innovation (NIBIO).
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This journal is The Royal Society of Chemistry 2010
Chem. Commun., 2010, 46, 5283–5285 | 5285