Synthesis of nuclease-resistant siRNAs possessing benzene-phosphate backbones in their 3′-overhang regions

Article abstract of DOI:10.1016/j.bmcl.2008.08.086

We describe the synthesis and silencing activities of siRNA possessing N¹-[3,5-bis(hydroxymethyl)phenyl]thymine (b^t) in their 3′-overhang regions. We found that an siRNA possessing b^t in the 3′-overhang region was more effective than an siRNA with natural nucleosides and the siRNA possessing 1,3-bis(hydroxymethyl)benzene (b) without a nucleobase at the 3′-overhang region in in vitro experiment using HeLa cells system. Furthermore, the RNA possessing b^t at its 3′-end was more resistant to nucleolytic hydrolysis by snake venom phosphodiesterase (a 3′-exonuclease) than the RNA possessing the natural nucleoside 2′-deoxythymidine at the 3′-end. Thus, the compound b^t will be a novel 3′-overhang moiety that can enhance the silencing activity and nuclease-resistant property of siRNAs.

Full text of DOI:10.1016/j.bmcl.2008.08.086

Bioorganic & Medicinal Chemistry Letters 18 (2008) 5194–5196
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis of nuclease-resistant siRNAs possessing benzene-phosphate
backbones in their 30-overhang regions
a
a
a
a
Yoshihito Ueno a,b,c,e, , Takumi Inoue , Mahito Yoshida , Kayo Yoshikawa , Aya Shibata ,
*
Yoshiaki Kitamura a, Yukio Kitade a,b,c,d,
*
a Department of Biomolecular Science, Faculty of Engineering, Gifu University, 1-1 Yanagido, Gifu 501-1193, Japan
b United Graduate School of Drug Discovery and Medical Information Sciences, Gifu University, 1-1 Yanagido, Gifu 501-1193, Japan
c Center for Emerging Infectious Diseases, Gifu University, 1-1 Yanagido, Gifu 501-1193, Japan
d Center for Advanced Drug Research, Gifu University, 1-1 Yanagido, Gifu 501-1193, Japan
e PRESTO, JST (Japan Science and Technology Agency), 4-1-8 Honcho Kawaguchi, Saitama 332-0012, Japan
a r t i c l e i n f o
a b s t r a c t
We describe the synthesis and silencing activities of siRNA possessing N1-[3,5-bis(hydroxy-
methyl)phenyl]thymine (bt) in their 30-overhang regions. We found that an siRNA possessing bt in the
30-overhang region was more effective than an siRNA with natural nucleosides and the siRNA possessing
1,3-bis(hydroxymethyl)benzene (b) without a nucleobase at the 30-overhang region in in vitro experi-
ment using HeLa cells system. Furthermore, the RNA possessing bt at its 30-end was more resistant to
nucleolytic hydrolysis by snake venom phosphodiesterase (a 30-exonuclease) than the RNA possessing
the natural nucleoside 20-deoxythymidine at the 30-end. Thus, the compound bt will be a novel 30-over-
hang moiety that can enhance the silencing activity and nuclease-resistant property of siRNAs.
Ó 2008 Elsevier Ltd. All rights reserved.
Article history:
Received 23 July 2008
Revised 21 August 2008
Accepted 25 August 2008
Available online 28 August 2008
Keywords:
RNA
siRNA
RNAi
Aromatic compound
Nucleobase
Introduction. Short interfering RNA (siRNA) molecules have
drawn considerable attention since it was demonstrated that they
mediate potent gene knock-down in a variety of mammalian cells
without triggering non-specific RNA degradation and translation
inhibition based on interferon response.1,2 siRNA has considerable
potential as a new therapeutic drug for intractable diseases be-
cause siRNAs can be rationally designed and synthesized if the se-
quences of the disease-causing genes are known.2 Improved
nuclease stability of siRNAs is of prime importance for the efficient
therapeutic application of synthetic siRNAs. Thus far, many types
of siRNAs modified at the base, sugar, or phosphate moieties have
been synthesized, and their nuclease-resistant properties and
RNAi-inducing activities have been studied.3,4
Recently, we have designed and synthesized siRNAs possessing
the aromatic compound 1,3-bis(hydroxymethyl)benzene (b) in
their 30-overhang regions (Fig. 1).11,12 We found that these mod-
ified siRNAs are more effective than the siRNAs without the 30-
overhang regions in in vitro experiment using HeLa cells system.
Argonaute2, a key component of the RNA-induced silencing
complex (RISC), is responsible for mRNA cleavage in the RNAi path-
way.5,6 It is composed of PAZ, Mid, and PIWI domains. X-ray struc-
tural analysis and NMR studies have revealed that the 2-nucleotide
30-overhang region of the guide strand (antisense strand) of siRNA
is recognized by the PAZ domain and is accommodated into its
hydrophobic binding pocket.7–10
* Corresponding authors. Tel.: +81 58 293 2639; fax: +81 58 293 2749 (Y.U).
E-mail addresses: uenoy@gifu-u.ac.jp (Y. Ueno), ykkitade@gifu-u.ac.jp (Y.
Kitade).
Figure 1. Structures of modified siRNAs.
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.08.086

Y. Ueno et al. / Bioorg. Med. Chem. Lett. 18 (2008) 5194–5196
5195
Further, the silencing activities of the modified siRNAs are very
similar to those of normal siRNAs with natural nucleosides in
their 30-overhang regions. Moreover, RNAs possessing the aro-
matic groups at their 30-ends were more resistant to nucleolytic
degradation by snake venom phosphodiesterase (SVPD; a 30-exo-
nuclease) than normal RNAs with natural nucleosides at their 30-
ends.
These results prompted us to investigate the silencing activities
of the modified siRNAs possessing the aromatic compound N1-[3,5-
bis(hydroxymethyl)phenyl]thymine (bt), which has a nucleobase
capable of forming hydrogen bonds. We predicted that the silenc-
ing activities of the siRNAs possessing b in their 30-regions would
be enhanced by introducing the nucleobase capable of forming
hydrogen bonds in their 30-overhang regions. In this paper, we re-
port the synthesis and silencing properties of siRNAs possessing bt
in their 30-overhang regions (Table 1).
Results and discussion. Synthesis. Modified siRNAs were syn-
thesized by the standard phosphoramidite method. In order to
incorporate the aromatic compound in the 30-overhang regions of
the siRNAs, a solid support carrying the aromatic compound 6
and a phosphoramidite of 6 were synthesized according to the
route shown in Scheme 1. Trimesic acid (1) was treated with LiAlH4
to give tris(hydroxymethyl)benzene (2) in a 91% yield. The 3 hy-
droxyl groups of 2 were protected with tert-butyldiphenylsilyl
(TBDPS) groups to afford a mono-TBDPS derivative 3a, a di-TBDPS
derivative 3b, and a tri-TBDPS derivative 3c, in 10%, 34%, and 44%
yields, respectively. The 3b derivative was coupled with N3-ben-
zoylthymine under the Mitsunobu conditions to give the N1-substi-
tuted thymine derivative 4 in a 92% yield. The silyl groups of 4
were removed by treatment with tetra-n-butylammonium fluoride
(TBAF). One of the 2 hydroxyl groups of 5 was protected with a
4,40-dimethoxytrityl (DMTr) group to afford a mono-DMTr deriva-
tive 6 in a 69% yield. The mono-DMTr derivative 6 was phosphity-
lated by the standard procedure13 to produce the corresponding
phosphoramidite 7 in an 80% yield. In order to incorporate 6 at
the 30-ends of RNAs, 6 was further modified to afford the corre-
sponding 30-succinate, which was then reacted with controlled
Table 1
Sequences of ONs and siRNAs used in this study
No. of siRNA
No. of ON
Sequence
pore glass (CPG) to afford a solid support containing 6 (42
All the oligoribonucleotides (ONs) were synthesized using a
DNA/RNA synthesizer. Fully protected ONs (1.0 mol each) linked
lmol/g).
siRNA 9
ON 13
ON 14
50-GGCCUUUCACUACUCCUAC-30
30-CCGGAAAGUGAUGAGGAUG-50
l
50-GGCCUUUCACUACUCCUACtt-30
30-ttCCGGAAAGUGAUGAGGAUG-50
50-GGCCUUUCACUACUCCUACbtbt-30
30-btbtCCGGAAAGUGAUGAGGAUG-50
to solid supports were treated with concentrated NH4OH:EtOH
(3:1, v/v) at room temperature for 12 h and then with 1.0 M
TBAF/THF at room temperature for 12 h. The ONs released after
the treatment were purified by denaturing 20% polyacrylamide
gel electrophoresis (20% PAGE) to afford deprotected ONs 17 and
18 in 12 and 9 OD260 absorbance units, respectively.14 These ONs
were analyzed by matrix-assisted laser desorption/ionization
time-of-flight mass spectrometry (MALDI-TOF/MS), and observed
molecular weights were in agreement with their structures.16
siRNA 10
siRNA 11
siRNA 12
ON 15
ON 16
ON 17
ON 18
ON 19
ON 20
50-GGCCUUUCACUACUCCUACbb-30
30-bbCCGGAAAGUGAUGAGGAUG-50
Capital letters represent ribonucleosides and small letters represent 20-deoxyribo-
nucleosides. The compounds b and bt are 1,3-bis(hydroxymethyl)benzene and N1-
[3,5-bis(hydroxymethyl)phenyl]thymine, respectively.
Scheme 1. Reagents and conditions: (a) LiAlH4, THF, rt, 91%; (b) TBDPSCl, imidazole, DMF, rt, 10% for 3a, 34% for 3b, and 44% for 3c; (c) N3-benzoylthymine, PPh3,
diethylazodicarboxylate, THF, rt, 92%; (d) TBAF, THF, rt, 87%; (e) DMTrCl, pyridine, rt, 69%; (f) chloro(2-cyanoethoxy)(N,N-diisopropylamino)phosphine, i-Pr2NEt, CH2Cl2, rt,
80%; (g) (1) succinic anhydride, DMAP, pyridine, rt; (2) CPG, WSCI, DMF, rt, 42 lmol/g.

5196
Y. Ueno et al. / Bioorg. Med. Chem. Lett. 18 (2008) 5194–5196
Dual-luciferase assay. The ability of modified siRNAs to suppress
resistant to SVPD than the unmodified ON 16. The half-lives
(t1/2s) of the ONs 16 and 18 were 3.7 min and 11.4 min, respec-
tively. Thus, it was found that the ON 18 possessing bt was 3 times
more resistant to SVPD than the unmodified ON 16.
gene expression was studied by a dual-luciferase assay using a psi-
CHECK-2 vector (Promega), which contained the Renilla and firefly
luciferase genes. The siRNA sequences were designed to target the
Renilla luciferase gene. HeLa cells were co-transfected with the
vector and indicated amounts of siRNAs, and the signals of Renilla
luciferase were normalized to those of firefly luciferase.17
As shown in Figure 2, the silencing activities of siRNAs 10, 11,
and 12, which possessed overhang moieties, were markedly great-
er than that of siRNA 9, which had no overhang moiety. The silenc-
ing activity of the siRNA 12, which had b in its overhang region,
was almost equal to that of the unmodified siRNA 10, which had
thymidine in its overhang region, at each concentration,11 whereas
the activity of the siRNA 11, which possessed bt in its overhang re-
gion, was greater than that of the siRNAs 10 and 12 at each concen-
tration. Thus, it was found that the siRNA 11, which had thymine
base in the overhang region, was more potent than the siRNA 12,
which had no base in the overhang region.
In conclusion, we have demonstrated the synthesis of the siR-
NAs possessing N1-[3,5-bis(hydroxymethyl)phenyl]thymine (bt) in
their 30-overhang regions. The silencing activities of the siRNAs
were examined by the dual-luciferase assay. It was found that
the siRNA possessing bt in the 30-overhang region was more effec-
tive than the siRNA with 20-deoxythymidine as the natural
nucleosides in the 30-overhang region and the siRNA possessing
b without the nucleobase in in vitro experiment using HeLa cells
system. Furthermore, the RNA possessing bt was more resistant to
nucleolytic hydrolysis by SVPD than the RNA possessing the nat-
ural 20-deoxythymidine. Thus, bt can be a novel overhang unit
that enhances the silencing activity and nuclease-resistant prop-
erties of the siRNAs.
Nuclease-resistant property. Next, the susceptibility of the ONs
to snake venom phosphodiesterase (SVPD), a 30-exonuclease, was
examined. The unmodified ON 16 and the modified ON 18 possess-
ing the benzene derivative bt at its 30-end, were labeled with
Acknowledgments
This work was supported by a grant from PRESTO of the Japan
Science and Technology Agency (JST) and a Grant-in-Aid for Scien-
tific Research from the Japan Society for the Promotion of Science
(JSPS). We are grateful to Professors Y. Hirata (Gifu University)
and Professor K. Kiuchi (Gifu University) for providing technical
assistance in the dual-luciferase assay.
[c-
32P]ATP and incubated with SVPD. The reactions were analyzed
by PAGE under denaturing conditions (data not shown). Densities
of radioactivity of the gel were visualized by a Bio-imaging ana-
lyzer. As shown in Figure 3, the ON 18 possessing bt was more
References and notes
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Figure 2. Dual-luciferase assay.17
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14. The yields are indicated as OD units at 260 nm starting from 1.0 lmol scale.
Extinction coefficients of the ONs were calculated from those of
mononucleotides and dinucleotides according to the nearest-neighbor
approximation method.15
.
15. Puglisi, J. D.; Tinoco, I., Jr.. In Methods Enzymol.; Dahlberg, J. E., Abelson, J. N.,
Eds.; Academic press, Inc.: San Diego, 1989; Vol. 180, pp 304–325.
16. MALDI-TOF/MS analyses of RNAs. The following spectra were obtained using a
time-of-flight mass spectrometer. ON 17: calculated mass, 6571.0; observed
mass, 6567.9. ON 18: calculated mass, 6880.3; observed mass, 6877.3.
17. Dual-luciferase assay. HeLa cells were grown at 37 °C in
a humidified
atmosphere of 5% CO2 in air in Minimum Essential Medium (MEM)
(Invitrogen) supplemented with 10% fetal bovine serum (FBS). Twenty-four
hours before transfection, HeLa cells (4 ꢀ 104/mL) were transferred to 96-well
plates (100 lL per well). They were transfected using TransFast (Promega)
according to the manufacturer’s instructions, for transfection of adherent cell
lines. Cells in each well were transfected with a solution (35 L) of 20 ng of
psiCHECK-2 vector (Promega), the indicated amounts of siRNAs, and 0.3 g of
TransFast in Opti-MEM I Reduced-Serum Medium (Invitrogen), and incubated
at 37 °C. After 1 h, MEM (100 L) containing 10% FBS and antibiotics was added
to each well, and the mixture was further incubated at 37 °C. After 24 h, cell
extracts were prepared in Passive Lysis Buffer (Promega). Activities of firefly
and Renilla luciferases in the cell lysates were determined using a dual-
luciferase assay system (Promega) according to the manufacturer’s protocol.
The results were confirmed by performing at least 3 independent transfection
experiments with 2 cultures each and are expressed as the average from 4
experiments as mean SD.
l
l
Figure 3. Nuclease resistance of ON 16 and ON 18 against SVPD. Each ON
(100 pmol) labeled with 32P at the 50-end was incubated with snake venom
phosphodiesterase (8 ꢀ 10ꢁ3 U) in a buffer containing 37.5 mM Tris–HCl (pH 7.0)
l
and 7.5 mM MgCl2 (total 40
l
L) at 37 °C. At appropriate periods, aliquots (5
lL) of
the reaction mixture were separated and added to a solution of 9 M urea (10
lL).
The mixtures were analyzed by electrophoresis on 20% PAGE containing 7 M urea.
Densities of radioactivity of the gel were visualized by a Bio-imaging analyzer (Bas
2000, Fuji Co., Ltd).