Synthesis of nuclease-resistant siRNAs possessing universal overhangs

Article abstract of DOI:10.1016/j.bmc.2009.01.033

RNA interference (RNAi) induced by small interfering RNA (siRNA) has emerged as a powerful technique for the silencing of gene expression at the post-transcriptional level. It has been shown that in the RNAi machinery, the 3′-overhang region of a guide strand (an antisense strand) of siRNA is recognized by the PAZ domain in the Argonaute protein, and the 2-nucleotide (nt) 3′-overhang is accommodated into a binding pocket composed of hydrophobic amino acids in the PAZ domain. Based on this background information, we designed and synthesized siRNAs possessing aromatic compounds at their 3′-overhang regions. It was found that the modified siRNAs possessing aromatic compounds are more potent than the siRNAs without the 3′-overhang regions. Further, the silencing activities of the modified siRNAs are almost equal to those of normal siRNAs with natural nucleosides at their 3′-overhang regions. We also found that the siRNAs possessing the aromatic compounds at their 3′-overhang region could be used to inhibit hepatitis C virus (HCV) replication. Moreover, the RNAs with aromatic groups at their 3′-ends were more resistant to nucleolytic degradation by snake venom phosphodiesterase (SVPD) (a 3′-exonuclease) than natural RNAs. The aromatic compounds described in this report do not have functional groups capable of forming hydrogen bonds with nucleobases. Therefore, we expect that they can serve as the universal overhang units that can improve the nuclease resistance of siRNAs.

Full text of DOI:10.1016/j.bmc.2009.01.033

Bioorganic & Medicinal Chemistry 17 (2009) 1974–1981
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis of nuclease-resistant siRNAs possessing universal overhangs
a
a
a
e
Yoshihito Ueno a,b,c,f, , Yuuji Watanabe , Aya Shibata , Kayo Yoshikawa , Takashi Takano ,
*
Michinori Kohara e, 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 Department of Microbiology and Cell Biology, The Tokyo Metropolitan Institute of Medical Science, 3-18-22, Honkomagome, Bunkyo-ku, Tokyo 113-8613, Japan
f 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
Article history:
RNA interference (RNAi) induced by small interfering RNA (siRNA) has emerged as a powerful technique
for the silencing of gene expression at the post-transcriptional level. It has been shown that in the RNAi
machinery, the 30-overhang region of a guide strand (an antisense strand) of siRNA is recognized by the
PAZ domain in the Argonaute protein, and the 2-nucleotide (nt) 30-overhang is accommodated into a
binding pocket composed of hydrophobic amino acids in the PAZ domain. Based on this background
information, we designed and synthesized siRNAs possessing aromatic compounds at their 30-overhang
regions. It was found that the modified siRNAs possessing aromatic compounds are more potent than
the siRNAs without the 30-overhang regions. Further, the silencing activities of the modified siRNAs are
almost equal to those of normal siRNAs with natural nucleosides at their 30-overhang regions. We also
found that the siRNAs possessing the aromatic compounds at their 30-overhang region could be used
to inhibit hepatitis C virus (HCV) replication. Moreover, the RNAs with aromatic groups at their 30-ends
were more resistant to nucleolytic degradation by snake venom phosphodiesterase (SVPD) (a 30-exonu-
clease) than natural RNAs. The aromatic compounds described in this report do not have functional
groups capable of forming hydrogen bonds with nucleobases. Therefore, we expect that they can serve
as the universal overhang units that can improve the nuclease resistance of siRNAs.
Received 28 November 2008
Revised 14 January 2009
Accepted 15 January 2009
Available online 21 January 2009
Keywords:
RNAi
siRNA
Aromatic compound
Nuclease-resistant
HCV
Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction
for the therapeutic application of synthetic siRNAs.4 Thus far, many
types of siRNAs modified at the base, sugar, or phosphate moieties
RNA interference (RNAi) is a process of sequence-specific post-
transcriptional gene silencing that is triggered by double-stranded
RNAs (dsRNAs) homologous to the silenced genes.1 The process is
initiated by the processive cleavage of dsRNA into 21- to 23-nucle-
otide (nt) duplexes by the enzyme Dicer. These duplexes, which
contain a 2-nt overhang at the 30-end of each strand, are termed
as short interfering RNAs (siRNAs). The siRNAs associate with the
RNA-induced silencing complex (RISC), which is then guided to
catalyze the sequence-specific degradation of the target mRNA.2–4
siRNA has considerable potential as a new therapeutic drug for
intractable diseases because siRNAs can be rationally designed and
synthesized if the sequences of the disease-causing genes are
known. Several groups have demonstrated the efficacy of siRNA-
mediated inhibition of clinically relevant genes in vivo as well as
in vitro.4–6 Improving the nuclease resistance of siRNA is important
have been synthesized, and their nuclease-resistant properties and
RNAi-inducing activities have been studied.7–26
Argonaute2, a key component of RISC, is responsible for mRNA
cleavage in the RNAi pathway.27,28 Argonaute2 is composed of PAZ,
Mid, and PIWI domains. X-ray structural analysis and a nuclear
magnetic resonance (NMR) study have revealed that the 2-nt 30-
overhang region of the guide strand (the antisense strand) of siRNA
is recognized by the PAZ domain and is accommodated into a bind-
ing pocket composed of hydrophobic amino acids; this pocket is lo-
cated in the domain.29–32 The length of the 30-overhang regions of
siRNA influences the activities of the siRNAs. It is reported that the
2-nt 30-overhang is the most efficient in an experiment using 21-nt
siRNA in Drosophila embryo lysate; however, the multiple addition
of 20-deoxynucleotide to the 30-end of siRNAs is tolerated.33
Considering this background information, we substituted the
natural nucleosides at 30-overhang regions with the aromatic com-
pounds, 1,3-bis(hydroxymethyl)benzene (1), 1,3-bis(hydroxy-
methyl)pyridine (2), and 1,2-bis(hydroxymethyl)benzene (3)
(Fig. 1). We hypothesized that the introduction of lipophilic groups
* Corresponding authors. Tel.: +81 58 293 2639; fax: +81 58 293 2794 (Y.U.).
E-mail addresses: uenoy@gifu-u.ac.jp (Y. Ueno), ykkitade@gifu-u.ac.jp (Y.
Kitade).
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.01.033

Y. Ueno et al. / Bioorg. Med. Chem. 17 (2009) 1974–1981
1975
Figure 1. Structures of the siRNAs.
at the 30-overhang portions of the siRNAs would improve affinities
of the 30-overhang portions of the siRNAs with the PAZ protein
making the siRNAs more potent than normal siRNAs which possess
natural nucleosides at their 30-overhang portion. In addition, the
modified RNAs would be more nuclease-resistant than the normal
siRNAs.
Scheme 1. Reagents and conditions: (a) LiBH4, THF, rt, 95% for 1 and 28% for 2; (b)
DMTrCl, DMAP, pyridine, rt, 51% for 6 and 51% for 7; (c) chloro(2-cyanoethoxy)(N,N-
diisopropylamino)phosphine, i-Pr2NEt, THF, rt, 94% for 8 and 93% for 9; (d) (1)
In this paper, we report the synthesis and the silencing activi-
ties of the siRNAs with the aromatic groups 1–3 at their 30-over-
hang regions. The nuclease-resistant properties of the siRNAs are
also reported.
succinic anhydride, DMAP, pyridine, rt; (2) CPG, WSCI, DMF, rt, 108
and 74 mol/g for 11.
lmol/g for 10
l
2. Results
2.1. Synthesis
Modified siRNAs were synthesized by the phophoramidite
method. In order to incorporate the aromatic compounds at the
30-overhang regions of siRNAs, solid supports carrying 1, 2, or 3
and phosphoramidites of 1, 2, and 3 were synthesized according
to routes shown in Schemes 1 and 2. Dimethyl isophtalate was
treated with LiBH4 to give 1,3-bis(hydroxymethyl)benzene (1) in
95% yield. One of the two hydroxyl groups of 1 was protected with
a 4,40-dimethoxytrityl (DMTr) group to afford a mono-DMTr deriv-
ative 6 in 51% yield. The mono-DMTr derivative 6 was phosphity-
lated by the standard procedure34 to produce the corresponding
phosphoramidite 8 in 94% yield. In a similar manner, the phospho-
ramidites 9 and 14 were synthesized from dimethyl 2,6-pyridine-
dicarboxylate and dimethyl phthalate with total yields of 13%
and 64%, respectively.
To enable attachment to the solid support, the mono-DMTr
derivative 6 was succinated to yield the corresponding succinate,
that was linked to controlled pore glass (CPG) to afford the solid
support 10 possessing 6 (108
derivatives 7 and 13 were succinated and then linked to the CPGs
to afford the solid supports 11 and 15 possessing 7 (74 mol/g) and
13 (86 mol/g), respectively.
lmol/g). Similarly, the mono-DMTr
l
l
Scheme 2. Reagents and conditions: (a) LiBH4, THF, rt, 72%; (b) DMTrCl, DMAP,
pyridine, rt, 90%; (c) chloro(2-cyanoethoxy)(N,N-diisopropylamino)phosphine, i-
Pr2NEt, THF, rt, 98%; (d) (1) succinic anhydride, DMAP, pyridine, rt; (2) CPG, WSCI,
2.2. Oligoribonucleotide synthesis
DMF, rt, 86 lmol/g.
All oligoribonucleotides (ONs) were synthesized with a DNA/
RNA synthesizer. Fully protected ONs (1.0 mol each) linked to so-
lid supports were treated with concentrated NH4OH:EtOH (3:1, v/v)
l
at room temperature for 12 h and then with 1.0 M tetra-n-butyl-
ammonium fluoride (TBAF)/THF at room temperature for 12 h.

1976
Y. Ueno et al. / Bioorg. Med. Chem. 17 (2009) 1974–1981
Table 2
Tm values
The ONs that were released from the support after the treatment
were purified using denaturing 20% polyacrylamide gel electropho-
resis (PAGE) to afford deprotected ONs 36–77. These ONs were
analyzed by matrix-assisted laser desorption/ionization time-of-
flight mass spectrometry (MALDI-TOF MS), and the observed
molecular weights were in agreement with their structures.
siRNA
Tm (°C)
siRNA18
siRNA21
siRNA24
79.0
77.1
78.2
2.3. Thermal stabilities of siRNAs
The thermal stability of siRNAs was studied by thermal dena-
turation in 0.01 M sodium phosphate buffer (pH 7.0) containing
0.1 M NaCl (Table 2). The melting temperatures (Tms) of siRNAs
18, 21, and 24 were 79.0, 77.1, and 78.2 °C, respectively. These re-
sults showed that the thermal stabilities of siRNA 21 which con-
tained 1 and siRNA 24 which contained 2 were lower than that
of siRNA 18 with a natural dinucleotide at its overhang portion.
However, the Tm values of siRNAs 18, 21, and 24 were not consid-
erably different from each other.
which contained Renilla and firefly luciferase genes. The sequences
of the siRNAs were designed to target the Renilla luciferase gene.
HeLa cells were co-transfected with the vector and indicated the
amount of siRNAs. The signals of Renilla luciferase were normalized
to those of firefly luciferase.
The results are shown in Figure 2. The silencing activities of the
siRNAs possessing overhang moieties were markedly greater than
those of siRNA 16, which lacked an overhang portion. Surprisingly,
the silencing activities of the siRNAs carrying aromatic compounds
1, 2, and 3 at their 30-overhang regions were almost equal to those
of siRNAs 17, 18, and 19, which had natural thymidines at their 30-
overhang portions, at all concentrations. The number of aromatic
derivatives at the overhang moieties seemed to influence the activ-
ities of the siRNAs. At all concentrations, the silencing activities of
siRNAs with two aromatic compounds at their 30-overhang regions
tended to be greater than that of those carrying one or three aro-
matic compounds at their 30-overhang portions. The silencing
activities of the siRNAs carrying the 1,3-substituted compound 1,
the 1,2-substituted compound 2, and the pyridine derivative 3
were not considerably different at each concentration.
2.4. Dual-luciferase assay
The ability of modified siRNAs to suppress gene expression was
determined by a dual-luciferase assay using a psiCHECK-2 vector,
Table 1
Sequences of oligonucleotides (ONs) used in this study
Number of siRNA
No. of ON
Sequence
siRNA16
ON36
ON37
ON38
ON39
ON40
ON41
ON42
ON43
ON44
ON45
ON46
ON47
ON48
ON49
ON50
ON51
ON52
ON53
ON54
ON55
ON56
ON57
ON58
ON59
ON60
ON61
ON62
ON63
ON64
ON65
ON66
ON67
ON68
ON69
ON70
ON71
ON72
ON73
ON74
ON75
ON76
ON77
50-GGCCUUUCACUACUCCUAC-30
30-CCGGAAAGUGAUGAGGAUG-50
50-GGCCUUUCACUACUCCUACt-30
30-tCCGGAAAGUGAUGAGGAUG-50
50-GGCCUUUCACUACUCCUACtt-30
30-ttCCGGAAAGUGAUGAGGAUG-50
50-GGCCUUUCACUACUCCUACttt-30
30-tttCCGGAAAGUGAUGAGGAUG-50
50-GGCCUUUCACUACUCCUAC1-30
30-1CCGGAAAGUGAUGAGGAUG-50
50-GGCCUUUCACUACUCCUAC11-30
30-11CCGGAAAGUGAUGAGGAUG-50
50-GGCCUUUCACUACUCCUAC111-30
30-111CCGGAAAGUGAUGAGGAUG-50
50-GGCCUUUCACUACUCCUAC2-30
30-2CCGGAAAGUGAUGAGGAUG-50
50-GGCCUUUCACUACUCCUAC22-30
30-22CCGGAAAGUGAUGAGGAUG-50
50-GGCCUUUCACUACUCCUAC222-30
30-222CCGGAAAGUGAUGAGGAUG-50
50-GGCCUUUCACUACUCCUAC3-30
30-3CCGGAAAGUGAUGAGGAUG-50
50-GGCCUUUCACUACUCCUAC33-30
30-33CCGGAAAGUGAUGAGGAUG-50
50-GGCCUUUCACUACUCCUAC333-30
30-333CCGGAAAGUGAUGAGGAUG-50
50-AGAUCCAUACGUCCGUGAAtt-30
30-tgUCUAGGUAUGCAGGCACUU-50
50-GCACCGGCAGGAGAUCAUAtt-30
30-gtCGUGGCCGUCCUCUAGUAU-50
F-50-GGCCUUUCACUACUCCUACtt-30
F-50-GGCCUUUCACUACUCCUAC11-30
50-GUCUCGUAGACCGUGCAUCAUU-30
30-UUCAGAGCAUCUGGCACGUAGU-50
50-GUCUCGUAGACCGUGCAUCAtt-30
30-ttCAGAGCAUCUGGCACGUAGU-50
50-GUCUCGUAGACCGUGCAUCA11-30
30-11CAGAGCAUCUGGCACGUAGU-50
50-GUCUCGUAGACCGUGCAUCA12-30
30-21CAGAGCAUCUGGCACGUAGU-50
50-GUCUCAUAGGCCAUGCGUCA11-30
30-11CAGAGUAUCCGGGACGCAGU-50
Argonaute2/eIF2C2 (hAgo2) has been identified as a key protein
with endonuclease activity associated with RISC in the RNAi path-
way.27,28 In order to examine whether the observed silencing activ-
ities could be attributed to RNAi, the activities of the modified
siRNAs were studied after treating HeLa cells with eIF2C2-target-
ing siRNAs. We hypothesized that if the silencing activities of the
modified siRNAs resulted from RNAi, the expression levels of lucif-
erase proteins would recover if HeLa cells were treated with siR-
NAs that targeted eIF2C2. Two kinds of siRNAs targeting eIF2C2
were used in this study: one (siRNA 29) that targeted open reading
frame (ORF) positions 1168–1188 and another (siRNA 30) that tar-
geted ORF positions 1897–1917. HeLa cells were transfected with
siRNA 29 or siRNA 30. After incubation for 1 h, the cells were co-
transfected with psiCHECK-2 vector and siRNAs modified with aro-
matic compounds. After incubation for 24 h, the activity of Renilla
luciferase was measured. As shown in Figure 3, the signals of Renil-
la luciferase were recovered on treatment with siRNAs targeting
eIF2C2. These results indicated that the silencing activities of mod-
ified siRNAs could be attributed to RNAi.
siRNA17
siRNA18
siRNA19
siRNA20
siRNA21
siRNA22
siRNA23
siRNA24
siRNA25
siRNA26
siRNA27
siRNA28
siRNA29
siRNA30
2.5. Nuclease-resistance
Improving the nuclease resistance of siRNA is important for the
therapeutic application of synthetic siRNAs. It was expected that
RNAs carrying the aromatic compounds would be more nuclease
resistant than unmodified RNAs. First, the stability of the RNAs in
PBS containing bovine serum was investigated. ONs 66 and 67 la-
beled at the 50-ends with fluorescein were incubated in PBS con-
taining 5% bovine serum. ON 67 contained the aromatic
compound 1 at the 30-overhang region. The reactions were ana-
lyzed with PAGE under denaturing conditions. Figure 4 shows
the results. After 1 min of incubation, both the ONs were hydro-
lyzed completely. The results implied that ON 67, which carried
1, was hydrolyzed mainly by the endonuclease activity in the bo-
vine serum.
–
–
siRNA31
siRNA32
siRNA33
siRNA34
siRNA35
The capital letters indicate ribonucleosides. The small italic letters represent 20-
deoxyribonucleosides. The underlined letters indicate the mismatch bases. F shows
fluorescein.
Next, the susceptibility of the ONs to snake venom phosphodi-
esterase (SVPD), a 30-exonuclease, was examined. Unmodified ON

Y. Ueno et al. / Bioorg. Med. Chem. 17 (2009) 1974–1981
1977
Figure 3. Dual-luciferase assay (2). HeLa cells were transfected with siRNA 29
(50 nM) or siRNA 30 (50 nM). After incubating for 1 h, the cells were co-transfected
with a psiCHECK-2 vector and the modified siRNAs (10 nM). After incubating for
24 h, the activity of Renilla luciferase was measured. The experimental conditions
are as given under Section 4.
Figure 4. 20% PAGE of ONs incubated in PBS containing 5% bovine serum: (a) ON66;
(b) ON67. ONs were incubated for 0 min (lane 1), 1 min (lane 2), 3 min (lane 3),
15 min (lane 4), 1 h (lane 5), 3 h (lane 6), 6 h (lane 7), and 12 h (lane 8). The
experimental conditions are as given under Section 4.
2.6. Inhibition of HCV replication
It has been reported that hepatitis C virus (HCV) replication is
efficiently suppressed by siRNA 31 that targets an internal ribo-
some entry site (IRES) region of HCV.35 Accordingly, in order to
examine the efficacy of the modified siRNAs, we compared the
abilities of the modified siRNAs to suppress HCV replication with
those of normal siRNAs. The sequences of the siRNAs used in this
study are listed in Table 1. The siRNAs 32, 33, and 34 contained
thymidines, benzene derivatives, and benzene and pyridine deriv-
atives at their 30-overhang regions, respectively. siRNA 35 con-
tained 4 mismatched bases in its sequence.
Figure 6 shows the results. siRNAs 32, 33, and 34 dose-depen-
dently inhibited HCV replication. They almost completely sup-
pressed HCV replication at a concentration of 100 pM, while
the replication ratio of the siRNA35, which contained the mis-
matched bases, was 75% at the same concentration. The siRNAs
exerted no cytotoxic effect at 100 pM concentrations (Fig. 6c
and d). Thus, it was found that modified siRNAs 33 and 34 sup-
press HCV replication in a sequence-specific manner. At each
concentration, the replication ratios of HCV in cells treated with
siRNAs 33 and 34, which carried aromatic compounds at their
30-overhang regions, were almost equal to the ratio in the cells
treated with siRNAs 31 and 32 with natural uridines and thymi-
dines at their 30-overhang portions. These results indicated that
the silencing abilities of the modified siRNAs are almost equal
to those of the normal siRNA, which had natural nucleosides at
their 30-overhang regions.
Figure 2. Dual-luciferase assay (1). The experimental conditions are as given under
Section 4.
40, modified ON 46 carrying benzene derivative 1, and modified
ON 52 that contained pyridine derivative 2 were labeled with
[c-
32P]ATP and incubated with SVPD. The reactions were analyzed
using PAGE under denaturing conditions. As shown in Figure 5,
unmodified ON 40 was hydrolyzed randomly after 30 min of incu-
bation, while modified ONs 46 and 52 were resistant to the en-
zyme. The half-lives (t1/2s) of ONs 40, 46, and 52 were 7, 64, and
72 min, respectively. Therefore, it was apparent that ON 46 carrying
benzene derivative 1 and ON 52 carrying pyridine derivative 2 were
9 and 10 times more resistant to SVPD than unmodified ON 40.

1978
Y. Ueno et al. / Bioorg. Med. Chem. 17 (2009) 1974–1981
Figure 5. 20% PAGE of 50-32P-labeled ONs hydrolyzed by SVPD: (a) ON40, (b) ON46, (c) ON52. ONs were incubated with SVPD for 0 min (lane 1), 1 min (lane 2), 3 min (lane 3),
5 min (lane 4), 10 min (lane 5), 30 min (lane 6), and 60 min (lane 7). The experimental conditions are as described in Section 4.
Figure 6. Effect of siRNAs on HCV replication. (a) and (b): Inhibition of HCV replication by siRNAs in R6FLR-N replicon cells. HCV replication was calculated by measuring the
luminescence ratio with a Bright-Glo luciferase assay system. (c) and (d): Cell viability was determined by a WST-8 assay. Data are represented as mean SD (n = 3). The
experimental conditions are as described in Section 4.
21, which contained 1,3-bis(hydroxymethyl)benzene, and siRNA
24, which contained 1,3-bis(hydroxymethyl)pyridine, is slightly
lower than that of siRNA 18, which had a natural dinucleotide at
the overhang portion, the Tm values of these three siRNA were
not considerably different. Therefore, it was considered that the
change in the thermal stability of the siRNAs, caused by the intro-
duction of aromatic residues, influenced the silencing activities of
the siRNAs to a very small extent.
The silencing activities of the siRNAs were examined by dual-
luciferase assay using the psiCHECK-2 vector. It was revealed that
the modified siRNAs were more potent than the siRNAs without
the 30-overhang regions. Moreover, the silencing activities of the
3. Discussion and conclusions
We designed and synthesized siRNAs possessing the aromatic
compounds, 1,3-bis(hydroxymethyl)benzene, 1,3-bis(hydroxy-
methyl)pyridine, and 1,2-bis(hydroxymethyl)benzene at their 30-
overhang regions. RNAs containing these aromatic compounds at
their 30-ends were successfully synthesized by the phosphorami-
dite method by using a DNA/RNA synthesizer. It has been reported
that the overhang nucleosides of RNA/RNA duplexes influence the
thermal stabilities of the RNA/RNA duplexes.36 Therefore, the ther-
mal stability of the siRNAs was studied using thermal denaturation
analysis. It was found that although the thermal stability of siRNA

Y. Ueno et al. / Bioorg. Med. Chem. 17 (2009) 1974–1981
1979
modified siRNAs were almost equal to those of the normal siRNAs.
It has been reported that in the RNAi machinery, the 30-overhang
region of the guide strand (the antisense strand) of siRNA is recog-
nized by a PAZ domain, and the 2-nt 30-overhang is accommodated
into a binding pocket composed of hydrophobic amino acids; this
pocket is located in the domain.29–32 In this report, we tested the
siRNAs with the monocyclic compounds at their 30-overhang re-
gions. They showed silencing activities similar to those of unmod-
ified siRNAs. Therefore, it is considered that recognition of the 30-
overhang portions of the siRNAs by the PAZ domain of human Arg-
onaute is not so stringent. It has been reported that the 2-nt 30-
overhang was the most efficient in an experiment using 21-nt siR-
NA in Drosophila embryo lysate.33 The silencing activity of the siR-
NAs with two aromatic compounds at their 30-overhang regions
also tended to be greater than that of carrying one or three aro-
matic compounds in this experiment.
It has been reported that pre-treatment of tissue culture cells
with siRNAs can inhibit the replication of a large number of
viruses.37 Therefore, we examined the inhibition activity of the
modified siRNAs using the cells harboring HCV replicons. It was
found that the modified siRNAs suppressed HCV replication in a se-
quence-specific manner. The replication ratios of HCV in cells trea-
ted with siRNAs carrying aromatic compounds at their 30-overhang
regions were almost equal to those treated with siRNAs with nat-
ural nucleosides at their 30-overhang portions. Therefore, siRNAs
possessing aromatic compounds at their 30-overhang region can
be used to inhibit HCV replication.
An improvement in nuclease resistance of siRNA is important
for the therapeutic application of synthetic siRNA.4–6 We examined
the susceptibility of the RNAs possessing aromatic compounds at
their 30-ends. SVPD was used as the model 30-exonuclease, and
the stability of the RNAs in bovine serum was also studied. The
30-modified RNAs were hydrolyzed completely by the endonucle-
ase activity in the bovine serum after 1 min of incubation. In con-
trast, the 30-modification was effective for improving resistance to
SVPD, a 30-exonuclease.
In summary, we synthesized the siRNAs possessing the aro-
matic compounds at their 30-overhang regions. It was revealed that
the modified siRNAs were more potent than the siRNAs without 30-
overhang regions and that the modified siRNAs had silencing activ-
ity similar to those of the siRNAs possessing the natural nucleo-
sides at their 30-overhang portions. Moreover, the modified RNAs
were more resistant to 30-exonuclease than the natural RNAs.
The aromatic compounds described in this report do not have func-
tional groups capable of forming hydrogen bonds with nucleo-
bases. Therefore, they can be used as universal overhang units in
order to improve the nuclease resistance of siRNAs.
perature for 23 h. Aqueous NaHCO3 (10 mL) was added to the mix-
ture at 0 °C, and the whole was stirred at room temperature for 1 h.
The solvent was evaporated in vacuo, and the resulting residue was
purified by column chromatography (SiO2, EtOAc) to give 1 (1.36 g,
9.84 mmol) in 95% yield: 1H NMR (CDCl3) d 7.39–7.26 (m, 4H), 4.71
(s, 4H); 13C NMR (CDCl3) d 141.2, 128.7, 126.2, 125.5, 65.1; LRMS
(EI) m/z 138 (M+); HRMS (EI) calcd for C8H10O2 138.0681, found
138.0677. Anal. Calcd for C8H10O2: C, 69.54; H, 7.30. Found: C,
69.45; H, 7.23.
4.1.2. 1-(4,40-Dimethoxytrityloxymethyl)-3-(hydroxymethyl)
benzene (6)
A mixture of 1 (0.50 g, 3.62 mmol), DMTrCl (1.23 g, 3.62 mmol),
and DMAP (22 mg, 0.18 mmol) in pyridine (18 mL) was stirred at
room temperature for 17 h. The mixture was partitioned between
EtOAc and aqueous NaHCO3 (saturated). The organic layer was
washed with brine, dried (Na2SO4), and concentrated. The residue
was purified by column chromatography (SiO2, 20% EtOAc in hex-
ane) to give 6 (0.82 g, 1.86 mmol) in 51% yield: 1H NMR (CDCl3) d
7.52–6.82 (m, 17H), 4.70 (s, 2H), 4.18 (s, 2H), 3.80 (s, 6H); 13C
NMR (CDCl3) d 158.4, 145.0, 140.8, 139.7, 136.2, 130.1, 128.5,
128.2, 127.8, 126.7, 126.3, 125.7, 125.6, 113.1, 86.4, 65.4, 55.2;
LRMS (EI) m/z 440 (M+); HRMS (EI) calcd for C29H28O4 440.1988,
found 440.1981. Anal. Calcd for C29H28O4ꢀ1/5H2O: C, 78.27; H,
6.45. Found: C, 78.33; H, 6.59.
4.1.3. 1-[(2-Cyanoethyloxy)(N,N-diisopropylamino)phosphinyl-
oxymethyl]-3-(4,40-dimethoxytrityloxymethyl)benzene (8)
A
mixture of 6 (0.35 g, 0.80 mmol), N,N-diisopropylethyl-
amine (0.40 mL, 4.00 mmol), and chloro(2-cyanoethoxy)(N,N-
diisopropylamino)phosphine (0.29 mL, 1.60 mmol) in THF
(8 mL) was stirred at room temperature for 1 h. The mixture
was partitioned between EtOAc and aqueous NaHCO3 (satu-
rated). The organic layer was washed with brine, dried (Na2SO4),
and concentrated. The residue was purified by column chroma-
tography (a neutralized SiO2, 50% EtOAc in hexane) to give 8
(0.48 g, 0.75 mmol) in 94% yield: 31P NMR (CDCl3) d 148.8;
LRMS (FAB) m/z 641 (MH+); HRMS (FAB) calcd for C38H46N2O5P:
641.3144, found: 6410.3129.
4.1.4. 2,6-Bis(hydroxymethyl)pyridine (2)
Dimethyl 2,6-pyridinedicarboxylate (2.00 g, 10.3 mmol) was
treated as described in the preparation of 1 to give 2 (0.40 g,
2.88 mmol) in 28%: 1H NMR (CDCl3) d 7.70 (t, 1H, J = 8.0), 7.19 (d,
2H, J = 8.0), 4.79 (s, 4H); 13C NMR (CDCl3) d 158.4, 137.4, 119.1,
64.3; LRMS (FAB) m/z 140 (MH+); HRMS (FAB) calcd for
C7H10NO2 140.0712, found 140.0705. Anal. Calcd for C7H9NO2: C,
60.42; H, 6.52; N, 10.07. Found: C, 60.28; H, 6.50; N, 9.95.
4.1.5. 2-(4,40-Dimethoxytrityloxymethyl)-6-(hydroxymethyl)
pyridine (7)
4. Experimental
4.1. General remarks
Compound 2 (0.50 g, 3.59 mmol) was treated as described in
the preparation of 6 to give 7 (0.81 g, 1.83 mmol) in 51%: 1H
NMR (CDCl3) d 7.76–6.82 (m, 16H), 4.69 (s, 2H), 4.34 (s, 2H), 3.79
(s, 6H); 13C NMR (CDCl3) d 158.5, 158.4, 157.6, 144.8, 137.3,
135.9, 130.0, 128.1, 127.9, 126.9, 119.4, 118.6, 113.2, 86.7, 66.6,
63.6, 55.20; LRMS (FAB) m/z 442 (MH+); HRMS (FAB) calcd for
C28H28NO4: 442.2018, found: 442.2033.
NMR spectra were recorded at 400 MHz (1H) and at 100 MHz
(
13C), and are reported in ppm downfield from tetramethylsilane.
Coupling constants (J) are expressed in Hertz. Mass spectra were
obtained by electron impact (EI) or fast atom bombardment
(FAB). Thin-layer chromatography was performed using Merck
coated plates 60F254. Silica gel column chromatography was car-
ried out on Wako gel C-300. siRNAs directed against eIF2C2
(hAgo2) were purchased from QIAGEN Inc.
4.1.6. 1-[(2-Cyanoethyloxy)(N,N-diisopropylamino)phosphinyl-
oxymethyl]-3-(4,40-dimethoxytrityloxymethyl)pyridine (9)
Compound 7 (0.20 g, 0.45 mmol) was phosphitylated as de-
scribed in the preparation of 8 to give 9 (0.27 g, 0.42 mmol) in
93%: 31P NMR (CDCl3) d 149.1; LRMS (FAB) m/z 642 (MH+); HRMS
(FAB) calcd for C37H45N3O5P: 642.3097, found: 642.3116.
4.1.1. 1,3-Bis(hydroxymethyl)benzene (1)
A mixture of dimethyl isophtalate (2.00 g, 10.3 mmol) and
LiBH4 (1.12 g, 51.5 mmol) in THF (52 mL) was stirred at room tem-

1980
Y. Ueno et al. / Bioorg. Med. Chem. 17 (2009) 1974–1981
4.1.7. 1,2-Bis(hydroxymethyl)benzene (3)
4.4. MALDI-TOF/MS analysis of RNAs
Dimethyl phthalate (2.00 g, 10.3 mmol) was treated as de-
scribed in the preparation of 1 to give 3 (1.02 g, 7.38 mmol) in
72%: 1H NMR (CDCl3) d 7.30 (s, 4H), 4.65 (s, 4H); 13C NMR (CDCl3)
d 139.3, 129.6, 128.5, 63.9; LRMS (FAB) m/z 139 (MH+); HRMS (FAB)
calcd for C8H11O2 139.0765, found 139.0759. Anal. Calcd for
C8H10O2: C, 69.54; H, 7.30. Found: C, 69.75; H, 7.32.
Spectra were obtained with a time-of-flight mass spectrometer.
ON44: calculated mass, 6094.6; observed mass, 6086.2. ON45: cal-
culated mass, 6403.9; observed mass, 6397.7. ON46: calculated
mass, 6294.8; observed mass, 6294.4. ON47: calculated mass,
6604.0; observed mass, 6605.6. ON48: calculated mass, 6494.9;
observed mass, 6489.2. ON49: calculated mass, 6804.1; observed
mass, 6797.1. ON50: calculated mass, 6095.6; observed mass,
6096.8. ON51: calculated mass, 6404.9; observed mass, 6405.0.
ON52: calculated mass, 6296.6; observed mass, 6296.1. ON53: cal-
culated mass, 6605.8; observed mass, 6603.5. ON54: calculated
mass, 6497.9; observed mass, 6503.5. ON55: calculated mass,
6807.1; observed mass, 6807.2. ON56: calculated mass, 6094.6;
observed mass, 6092.2. ON57: calculated mass, 6403.9; observed
mass, 6399.1. ON58: calculated mass, 6294.8; observed mass,
6290.4. ON59: calculated mass, 6604.0; observed mass, 6603.7.
ON60: calculated mass, 6494.9; observed mass, 6492.7. ON61: cal-
culated mass, 6804.2; observed mass, 6806.8. ON66: calculated
mass, 7070.4; observed mass, 7067.5. ON67: calculated mass,
6862.3; observed mass, 6856.9. ON72: calculated mass, 6739.9;
observed mass, 6731.8. ON73: calculated mass, 6802.9; observed
mass, 6794.6. ON74: calculated mass, 6740.9; observed mass,
6734.9. ON75: calculated mass, 6803.9; observed mass, 6797.9.
ON76: calculated mass, 6739.9; observed mass, 6734.2. ON77: cal-
culated mass, 6802.9; observed mass, 6794.7.
4.1.8. 1-(4,40-Dimethoxytrityloxymethyl)-2-(hydroxymethyl)
benzene (13)
Compound 3 (0.50 g, 3.62 mmol) was treated as described in
the preparation of 6 to give 13 (1.44 g, 3.27 mmol) in 90%: 1H
NMR (CDCl3) d 7.50–6.85 (m, 17H), 4.43 (d, 2H, J = 6.0), 4.18 (s,
2H), 3.79 (s, 6H); 13C NMR (CDCl3) d 158.6, 144.5, 140.5, 136.4,
135.7, 129.9, 129.8, 129.5, 128.6, 128.1, 127.9, 126.9, 113.4, 87.6,
65.2, 63.6, 55.2; LRMS (EI) m/z 440 (M+); HRMS (EI) calcd for
C29H28O4 440.1988, found 440.1991. Anal. Calcd for C29H28O4ꢀ1/
5H2O: C, 78.27; H, 6.45. Found: C, 78.24; H, 6.61.
4.1.9. 1-[(2-Cyanoethyloxy)(N,N-diisopropylamino)phosphinyl-
oxymethyl]-2-(4,40-dimethoxytrityloxymethyl)benzene (14)
Compound 13 (0.35 g, 0.80 mmol) was phosphitylated as de-
scribed in the preparation of 8 to give 14 (0.50 g, 0.78 mmol) in
98%: 31P NMR (CDCl3) d 148.4; LRMS (FAB) m/z 641 (MH+); HRMS
(FAB) calcd for C38H46N2O5P: 641.3144, found: 641.3127.
4.2. Solid support synthesis
4.5. Thermal denaturation study
A mixture of 1 (0.30 g, 0.68 mmol), succinic anhydride (0.20 g,
2.04 mmol), and DMAP (4 mg, 0.03 mmol) in pyridine (6.8 mL)
was stirred at room temperature. After 24 h, the solution was par-
titioned between CHCl3 and H2O, and the organic layer was washed
with H2O and brine. The separated organic phase was dried
(Na2SO4) and concentrated to give a succinate. Aminopropyl con-
trolled pore glass (0.50 g, 0.11 mmol) was added to a solution of
the succinate and WSCI (0.11 g, 0.57 mmol) in DMF (10 mL), and
the mixture was kept for 72 h at room temperature. After the resin
was washed with pyridine, a capping solution (6 mL, 0.1 M DMAP
in pyridine:Ac2O = 9:1, v/v) was added and the whole mixture was
kept for 24 h at room temperature. The resin was washed with
MeOH and acetone, and dried in vacuo. Amount of loaded com-
Each solution containing each siRNA (3
lM) in a buffer com-
posed of 10 mM Na2HPO4/NaH2PO4 (pH 7.0) and 100 mM NaCl
was heated at 95 °C for 3 min, then cooled gradually to an appro-
priate temperature, and used for the thermal denaturation studies.
Thermal-induced transitions of each mixture were monitored at
260 nm with a spectrophotometer.
4.6. 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
pound 1 to solid support was 108 lmol/g from calculation of re-
leased dimethoxytrityl cation by a solution of 70% HClO4:EtOH
(3:2, v/v). In a similar manner, solid supports with 2 and 3 were ob-
tained in 74 and 86 lmol/g loading amounts, respectively.
to 96-well plates (100 lL per well). They were transfected, using
TransFast (Promega), according to instructions for transfection of
adherent cell lines. Cells in each well were transfected with a solu-
tion (35
amounts of siRNAs, and 0.3
l
L) of 20 ng of psiCHECK-2 vector (Promega), the indicated
g of TransFast in Opti-MEM I Re-
l
4.3. RNA synthesis
duced-Serum Medium (Invitrogen), and incubated at 37 °C. Trans-
fection without siRNA was used as a control. After 1 h, MEM
Synthesis was carried out with a DNA/RNA synthesizer by phos-
phoramidite method. Deprotection of bases and phosphates was
performed in concentrated NH4OH:EtOH (3:1, v/v) at room tem-
perature for 12 h. 20-TBDMS groups were removed by 1.0 M tetra-
butylammonium fluoride (TBAF, Aldrich) in THF at room
temperature for 12 h. The reaction was quenched with 0.1 M TEAA
buffer (pH 7.0) and desalted on a Sep-Pak C18 cartridge. Deprotec-
ted ONs were purified by 20% PAGE containing 7 M urea to give the
highly purified ON44 (8), ON45 (8), ON46 (8), ON47 (12), ON48 (5),
ON49 (6), ON50 (28), ON51 (28), ON52 (29), ON53 (14), ON54 (35),
ON55 (20), ON56 (11), ON57 (4), ON58 (5), ON59 (7), ON60 (3),
ON61 (2), ON66 (20), ON67 (11), ON72 (14), ON73 (14), ON74
(33), ON75 (37), ON76 (21), ON77 (19). The yields are indicated
(100 lL) containing 10% FBS and antibiotics was added to each
well, and the whole 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 cell lysates were determined
with a dual-luciferase assay system (Promega) according to a man-
ufacturer’s protocol. The results were confirmed by at least three
independent transfection experiments with two cultures each
and are expressed as the average from four experiments as
mean SD.
4.7. Stability of ON in the PBS containing bovine serum
Each ON (600 pmol) labeled with fluorescein at 50-end was
in parentheses as OD units at 260 nm starting from 1.0 lmol scale.
incubated in PBS (300
At appropriate periods, aliquots (5
separated and added to a solution of 9 M urea (15
tures were analyzed by electrophoresis on 20% polyacrylamide
l
L) containing 5% bovine serum at 37 °C.
L) of the reaction mixture were
L). The mix-
Extinction coefficients of the ONs were calculated from those of
mononucleotides and dinucleotides according to the nearest-
neighbor approximation method.38
l
l

Y. Ueno et al. / Bioorg. Med. Chem. 17 (2009) 1974–1981
1981
gel containing 7 M urea. The labeled ON in the gel was visualized
by a Typhoon system (Amersham Biosciences).
Hirata (Gifu University) and Professor K. Kiuchi (Gifu University)
for providing technical assistance in the dual-luciferase assay.
4.8. Partial hydrolysis of ONs with snake venom
phosphodiesterase
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32
Each ON (100 pmol) labeled with P at the 50-end was incu-
bated with snake venom phosphodiesterase (3 ng) in a buffer con-
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This work was supported by a Grant from Precursory Research
for Embryonic Science and Technology (PRESTO) of the Japan Sci-
ence and Technology Agency (JST) and a Grant-in-Aid for Scientific
Research from the Japan Society for the Promotion of Science
(JSPS). We are grateful to Professor Y. Tomari (the University of To-
kyo) for his helpful advice. We are also grateful to Professor Y.