Chemically modified siRNAs and miRNAs bearing urea/thiourea-bridged aromatic compounds at their 3′-end for RNAi therapy

Article abstract of DOI:10.1016/j.bmc.2013.05.046

We have developed chemically modified siRNAs and miRNAs bearing urea/thiourea-bridged aromatic compounds at their 3′-end for RNAi therapy. Chemically modified RNAs possessing urea/thiourea-bridged aromatic compounds instead of naturally occurring dinucleo

Full text of DOI:10.1016/j.bmc.2013.05.046

Bioorganic & Medicinal Chemistry 21 (2013) 4494–4501
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Chemically modified siRNAs and miRNAs bearing urea/thiourea-
bridged aromatic compounds at their 30-end for RNAi therapy q
Yoshiaki Kitamura a,b, Yuki Masegi a, Shunsuke Ogawa a, Remi Nakashima c, Yukihiro Akao c,
Yoshihito Ueno d, Yukio Kitade a,b,c,
⇑
a Department of Biomolecular Science, Graduate School of Engineering, Gifu University, 1-1 Yanagido, Gifu 501-1193, Japan
b Department of Chemistry and Biomolecular Science, Faculty of Engineering, Gifu University, 1-1 Yanagido, Gifu 501-1193, Japan
c United Graduate School of Drug Discovery and Medical Information Sciences, Gifu University, 1-1 Yanagido, Gifu 501-1193, Japan
d Department of Applied Life Sciences, Faculty of Applied Biological Sciences, Gifu University, 1-1 Yanagido, Gifu 501-1193, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
We have developed chemically modified siRNAs and miRNAs bearing urea/thiourea-bridged aromatic
compounds at their 30-end for RNAi therapy. Chemically modified RNAs possessing urea/thiourea-bridged
aromatic compounds instead of naturally occurring dinucleotides at the 30-overhang region were easily
prepared in good yields and were more resistant to nucleolytic hydrolysis than unmodified RNA. siRNAs
containing urea or thiourea derivatives showed the desired knockdown effect. Furthermore, modified
miR-143 duplexes carrying the urea/thiourea compounds in the 30-end of each strand were able to inhibit
the growth of human bladder cancer T24 cells.
Received 16 April 2013
Revised 20 May 2013
Accepted 21 May 2013
Available online 4 June 2013
Keywords:
RNAi
siRNA
Ó 2013 The Authors. Published by Elsevier Ltd. All rights reserved.
miRNA
Urea/thiourea
Aromatic compound
1. Introduction
loops are excised from the stem by Dicer to create miRNA du-
plexes.8–10 The miRNA duplexes usually have a 30-overhang, simi-
Small interfering RNAs (siRNAs) and microRNAs (miRNAs) are
considered to be attractive candidates as nucleic acid drugs, owing
to their powerful regulation of gene expression in a sequence-
specific manner.1 siRNAs are produced from long double-stranded
RNAs (dsRNAs), which are processed by a Dicer enzyme, a member
of the RNase III family of dsRNA-specific endonuclease, into the 21-
to 26-nucleotide (nt) duplexes containing a 2-nt overhang at the
30-end of each strand.2 miRNAs, in contrast, are produced endoge-
nously in the cells of miRNA genes. The miRNA genes are tran-
scribed by RNA polymerase II to produce primary miRNA
transcripts (pri-miRNAs), which are further processed into precur-
sor stem-loop molecules (pre-miRNA) by a complex consisting of
at least two components: the RNaseIII enzyme Drosha and a pro-
tein called Pasha in Drosophila, or DGCR8 in mammals.3–7 The
resulting pre-miRNAs are exported from the nucleus to the cyto-
plasm by exportin-5-mediated transport, and then their terminal
lar to siRNA. Although siRNA and miRNA are distinguished by
their mode of biogenesis, they regulate the expression of target
genes via a similar process.11,12 One of the strands from the siRNA
or miRNA duplex is loaded onto the RNA-induced silencing com-
plex (RISC), which contains Argonaute proteins as core compo-
nents, to become a functional single-stranded RNA called a guide
strand (antisense strand), which suppresses the expression of tar-
get genes.13–16
Argonaute proteins are composed of PAZ, MID, and PIWI do-
mains. X-ray structural analysis and a nuclear magnetic resonance
(NMR) study have revealed that the 30-overhang region of a guide
strand of siRNA or miRNA duplex is recognized by the PAZ domain,
and the region is accommodated by a hydrophobic pocket com-
posed of aromatic amino acids.13,16–18
We recently designed and synthesized chemically modified
siRNA and miRNA bearing hydrophobic 1,3-bis(hydroxy-
methyl)benzene (B) and/or 1,3-bis(hydroxymethyl)pyridine (P) in
the 30-overhang regions and assessed their biological proper-
ties.19–23 We found these modified siRNAs effectively suppressed
target gene expression in a dose-dependent manner, and their
silencing activities were comparable to or greater than those of
normal siRNA containing natural nucleotides in this region.19,20
In addition, miRNAs with the aryl units at the 30-end showed a
q
This is an open-access article distributed under the terms of the Creative
Commons Attribution-NonCommercial-No Derivative Works License, which per-
mits non-commercial use, distribution, and reproduction in any medium, provided
the original author and source are credited.
⇑
Corresponding author. Tel./fax: +81 58 293 2640.
E-mail address: ykkitade@gifu-u.ac.jp (Y. Kitade).
0968-0896/$ - see front matter Ó 2013 The Authors. Published by Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2013.05.046

Y. Kitamura et al. / Bioorg. Med. Chem. 21 (2013) 4494–4501
4495
significant tumor-suppressive effect on cancer cells, such as colon
and bladder cancer cells.21–23 We have also synthesized siRNAs hav-
ing thymidine dimers consisting of a urea or a carbamate linkage at
their 30-overhang regions and found that these siRNAs inhibited tar-
get protein (RNase L) expression more efficiently than the natural
phosphodiester linkage.24 All of these chemically modified dsRNAs
were more resistant to nucleolytic degradation by snake venom
phosphodiesterase (30-exonuclease) than the corresponding normal
dsRNAs.
On the basis of these findings, we designed and synthesized siR-
NAs and miRNAs possessing urea- or thiourea-bridged aromatic
compounds 1–4 (Fig. 1) instead of the naturally occurring dinucle-
otide at the 30-end. We also studied the biological properties of siR-
NAs and miRNAs bearing urea- or thiourea-bridged aromatic units
instead of the natural nucleotides at the 30-overhang region.
reacted with 8 to give urea 13. The reaction of 8 and 12 furnished
the desired thiourea 15. Deprotection of the silyl group of 13 and
15 using tetra-n-butylammonium fluoride (TBAF) afforded 14
(98%) and 16(90%), respectively (Scheme 1c and d). CDI on treat-
ment with a solution of 8 in THF at room temperature yielded
the symmetric urea 17; subsequent desilylation by treatment with
TBAF followed by mono-DMTr protection gave 18 in moderate
yield (Scheme 1e). Unfortunately, the reaction of 8 with carbon
disulfide gave the intermolecular cyclization compound as the sole
isolable product.26 Therefore, we selected thiophosgeneas the cou-
pling reagent. Picolylic amine 8 was treated with thiophosgene to
give the corresponding thiourea 19, then desilylated to produce 4
(43% from 8). One of the two hydroxyl groups of 4 was protected
with a DMTr group to give 20 (Scheme 1f). To enable attachment
to the solid support, 14, 16, 18, and 20 were succinated to yield
the corresponding succinates, which were linked to controlled pore
glass (CPG) to afford the solid supports 21–24 possessing 1–4,
respectively (Scheme 1g).
2. Results and discussion
2.1. Synthesis of solid support
2.2. Synthesis of oligonucleotides
To synthesize the desired RNAs via the conventional phospho-
siRNA and miRNA sequences used in this study are depicted in
Table 1. All the oligonucleotides (ONs) were synthesized using a
DNA/RNA synthesizer. ONs containing 1–4 were synthesized by
using 21–24. The fully protected ONs immobilized on a solid
support were treated with concentrated NH4OH/EtOH (3:1, v/v)
at room temperature for 12 h and with TBAF (1.0 M solution in
THF) at room temperature for 12 h. The ONs were purified by
denaturing 20% polyacrylamide gel electrophoresis (PAGE) to
isolate deprotected ONs. These ONs were analyzed by matrix-
assisted laser desorption/ionization (MALDI) coupled with time-
of-flight (TOF) mass spectrometry (MALDI-TOF/MS), and the
observed molecular weights were in good agreement with their
structures.
ramidite method using
a DNA/RNA synthesizer, we prepared
controlled pore glass (CPG) solid supports linked to urea/thiourea-
bridged aromatic dimers 1–4 (Scheme 1). To obtain the desired
CPG solid supports, we initially prepared pyridine analogue 8 and
benzene analogues 11 and 12 (Scheme 1a and b). 2,6-Pyridinedi-
methanol (5) was treated with 1 equiv of sodium hydride, and the
resulting mono-anion reacted with a slight excess of tert-butyldi-
methylsilyl chloride (TBDMSCl) to give monoprotected diol 6 in mod-
erate yield. Compound
6 was treated with sodium azide,
triphenylphosphine, carbon tetrabromide, and triethylamine in
DMF to afford the corresponding azide compound 7 in 93% yield.
Reduction of the azide group of 7 using triphenylphosphine gave pic-
olylicamine 8 in good yield (Scheme 1a). Compounds 11 and 12 could
be prepared from commercially available 3-cyanobenzylalcohol (9).
The hydroxyl group was protected with a 4,40-dimethoxytrityl
(DMTr) group to give the corresponding DMTr derivative,10, quanti-
tatively. The cyano group in 10 easily underwent reduction with lith-
ium aluminium hydride, and benzylic amine 11 was obtained in
reasonable yield. Amine 11 was converted to the corresponding iso-
thiocyanate 12 via the dithiocarbamatein 96% yield using di-tert-bu-
tyl dicarbonate (Boc2O) and DMAP as a catalyst (Scheme 1b).25
Benzylic amine 11 was converted into the corresponding imid-
azole-N-carboxamide (1,10-carbonyldiimidazole; CDI), which then
2.3. Thermal denaturation study (siRNA)
We examined the thermal stability of siRNAs bearing urea/thio-
urea-bridged aromatic compounds at their 30-end by thermal
denaturation in a buffer of 10 mM sodium phosphate (pH 7.0) con-
taining 100 mM NaCl (Table 2). The melting temperature (Tms) of
unmodified siRNA 25 was 79.5 °C, while those of siRNAs 26, 27,
28, 29, and 30 were 78.5, 78.9, 77.8, 78.8, and 78.5 °C, respectively.
This result suggests that the substitution of urea/thiourea-bridged
HO
OH
HO
OH
N
B
P
H
N
H
N
H
N
H
N
HO
HO
OH
OH
HO
HO
OH
OH
N
N
O
S
1; BuP
2; BtuP
H
N
H
N
H
N
H
N
N
N
N
N
O
S
3; PuP
4; PtuP
Figure 1. Structures of the urea- or thiourea-bridged aromatic compound synthesized in this study.

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Y. Kitamura et al. / Bioorg. Med. Chem. 21 (2013) 4494–4501
(a)
a
a
b
c
c
HO
OH
TBDMSO
DMTrO
OH
TBDMSO
DMTrO
N3
TBDMSO
DMTrO
NH2
N
5
N
6
N
7
N
8
(b)
b
HO
NH2
NCS
OH
CN
CN
9
10
11
12
(c)
(d)
a
a
a
a
b
b
c
c
H
N
H
N
H
N
H
11
DMTrO
DMTrO
RO
OTBDMS
DMTrO
N
N
N
N
N
O
O
13
14
H
N
H
N
H
N
H
N
12
OTBDMS
DMTrO
DMTrO
DMTrO
OH
OH
OH
N
N
N
S
S
15
16
(e)
(f)
8
H
N
H
N
H
N
H
N
OR
N
N
N
O
O
17: R = TBDMS
18
b
3
: R = H
H
N
H
N
H
N
H
N
8
RO
OR
N
N
S
S
19
4
20
: R = TBDMS
: R = H
b
(g)
a
O
H
N
H
N
H
N
H
N
DMTrO
O
DMTrO
OH
N
H
X
N
X
N
CPG
Y
Y
O
14: X = CH, Y = O
21: X = CH, Y = O
16
18
22
23
: X = CH, Y = S
: X = N, Y = O
: X = CH, Y = S
: X = N, Y = O
20: X = N, Y = S
24: X = N, Y = S
Scheme 1. Reagents and conditions: (a) (a) NaH, DMF, rt, then TBDMSCl, DMF, rt, 49%; (b) CBr4, PPh3, NaN3, Et3N, DMF, rt, 93%; (c) PPh3, THF, H2O, rt, 93%. (b) (a) DMTrCl,
pyridine, DMF, rt, 91%; (b) LiAlH4, THF, rt, 94%; (c) CS2, Et3N, EtOH, rt, then Boc2O, DMAP, EtOH, rt, 96%. (c) (a) CDI, THF, rt, then 8, THF, rt, 54%; (b) TBAF, THF, rt, 98%. (d) (a)
Compound 8, CHCl3, reflux, 75%; (b) TBAF, THF, rt, 90%. (e) (a) CDI, THF, rt, 96%; (b) TBAF, THF, rt; (c) DMTrCl, pyridine, DMSO, rt, 43%. (f) (a) CSCl2, Et3N, CH2Cl2, rt; (b) TBAF,
THF, rt, 43%; (c) DMTrCl, pyridine, DMF, rt, 18%. (g) (a) (i) Succinic anhydride, DMAP, pyridine, rt; (ii) CPG, EDCI, DMF, rt.
aromatic compounds for naturally occurring dinucleotides at the
30-overhang regions did not have a crucial effect on the thermal
stability of the duplex.
quences were designed to target the Renilla luciferase gene. The
levels of Renilla luciferase were normalized to those of firefly lucif-
erase. Figure 2 shows the silencing activity of the siRNAs. All siR-
NAs effectively reduced luciferase activity in a dose-dependent
manner. At low concentrations, the silencing activity of siRNAs
27–30, which carried 1–4 containing urea/thiourea-bridged aro-
matic residues, was greater than that of siRNA 25 possessing natu-
ral thymidines and siRNA 26 containing a phosphodiester-bridged
B-P analogue (BP) in the 30-overhang regions.
2.4. Dual-luciferase assay (siRNA)
We examined the silencing activity of siRNAs 25–30 by a dual-
reporter assay using a psiCHECK-2 vector in HeLa cells. The vector
contains the Renilla and firefly luciferase genes, and the siRNA se-

Y. Kitamura et al. / Bioorg. Med. Chem. 21 (2013) 4494–4501
4497
Table 1
Sequences of ONs and siRNAs/miRNAs used in this study
No. of dsRNA
No. of ON
Sequence
siRNA 25
ON 36
ON 37
50-GGCCUUUCACUACUCCUACtt-30
50-GUAGGAGUAGUGAAAGGCCtt-30
siRNA 26
siRNA 27
siRNA 28
siRNA 29
siRNA 30
ON 38
ON 39
50-GGCCUUUCACUACUCCUACBP-30
50-GUAGGAGUAGUGAAAGGCCBP-30
ON 40
ON 41
50-GGCCUUUCACUACUCCUACBuP-30
50-GUAGGAGUAGUGAAAGGCCBuP-30
ON 42
ON 43
50-GGCCUUUCACUACUCCUACBtuP-30
50-GUAGGAGUAGUGAAAGGCCBtuP-30
ON 44
ON 45
50-GGCCUUUCACUACUCCUACPuP-30
50-GUAGGAGUAGUGAAAGGCCPuP-30
Figure 2. Dual-luciferase assay.
ON 46
ON 47
50-GGCCUUUCACUACUCCUACPtuP-30
50-GUAGGAGUAGUGAAAGGCCPtuP-30
ON 48
ON 49
ON 50
ON 51
ON 52
ON 53
F-50-GUAGGAGUAGUGAAAGGCCtt-30
F-50-GUAGGAGUAGUGAAAGGCCBP-30
F-50-GUAGGAGUAGUGAAAGGCCBuP-30
F-50-GUAGGAGUAGUGAAAGGCCBtuP-30
F-50-GUAGGAGUAGUGAAAGGCCPuP-30
F-50-GUAGGAGUAGUGAAAGGCCPtuP-30
50-UGAGCUACAGUGCUGCAUCUCUBP-30
50-UGAGAUGAAGCACUGUAGCUCABP-30
miRNA 31
miRNA 32
miRNA 33
miRNA 34
miRNA 35
ON 54
ON 55
50-UGAGCUACAGUGCUGCAUCUCUBuP-30
50-UGAGAUGAAGCACUGUAGCUCABuP-30
ON 56
ON 57
50-UGAGCUACAGUGCUGCAUCUCUBtuP-30
50-UGAGAUGAAGCACUGUAGCUCABtuP-30
ON 58
ON 59
50-UGAGCUACAGUGCUGCAUCUCUPuP-30
50-UGAGAUGAAGCACUGUAGCUCAPuP-30
ON 60
ON 61
Figure 3. Nuclease resistance of ONs against SVPD.
50-UGAGCUACAGUGCUGCAUCUCUPtuP-30
50-UGAGAUGAAGCACUGUAGCUCAPtuP-30
ON 62
ON 63
most potent effects on growth inhibition.21,22 On the basis of these
results, we designed miRNAs 32–35 (see Table 1). The sequences of
these miRNA are the same as miRNA 31, except for the 30-overhang
regions. Figure 4 shows the growth inhibitory effect of miRNAs on
human bladder cancer T24 cells. miRNAs 32–35, which carry the
urea/thiourea compounds 1–4 in their 30-overhang region of each
strand, inhibited cell viability, but were less potent than
BP-modified miRNA 31.
a
Capital letters indicate ribonucleosides and small letters show 20-
deoxyribonucleosides.
b
F denotes fluorescein.
Underline indicates mismatched position.
c
Table 2
Melting temperatures (Tms) of siRNAs
No. of siRNA
Tm (°C)
3. Conclusions
siRNA 25
siRNA 26
siRNA 27
siRNA 28
siRNA 29
siRNA 30
79.5
78.5
79.5
77.8
78.8
78.5
In this study, we synthesized chemically modified siRNAs and
miRNAs bearing urea/thiourea-bridged aromatic compounds at
their 30-overhang region for RNAi therapy. In an in vitro dual-
luciferase experiment, these modified siRNAs were more effective
than siRNA possessing the natural nucleosides at the 30-end, espe-
cially at low concentrations. Furthermore, these modified miR-143
duplexes inhibited the growth of human bladder cancer T24 cells.
We found that incorporation of these urea/thiourea compounds
into the 30-end enhances the nuclease resistance of ONs. These
results should help to advance the development of RNAi-based
medicine.
2.5. Nuclease resistance
The susceptibility of the ONs to snake venom phosphodiesterase
(SVPD), a 30-exonuclease, was examined. ONs 48, 49, 50, 51, 52, and
53, which were labeled with fluorescein at their 50-ends, were incu-
bated with SVPD. The reactions were analyzed by PAGE under dena-
turing conditions. Unmodified ON 48 was hydrolyzed after 5 min of
incubation, while all modified ONs were resistant to SVPD (Fig. 3).
The half-lives (t1/2s) of ONs 48, 49, 50, 51, 52, and 53 were 0.64,
1.56, 2.85, 2.63, 2.90, and 2.16 min, respectively. ONs 50, 51, 52,
and 53, which carried 1–4, were approximately 1.4–1.9 times more
resistant to the enzyme than the BP-modified ON 49.
4. Experimental
4.1. General remarks
All reactions were carried out under an argon atmosphere, un-
less otherwise noted. All reagents and solvents were purchased
from commercial vendors and used without further purification,
unless indicated otherwise. Pyridine was distilled over CaH2 and
stored over activated molecular sieves 4 Å. 1H and 13C NMR spectra
were recorded on a JEOL JNMAL-400 spectrometer or JNM ECS 400
spectrometer (400 MHz for 1H NMR and 100 MHz for 13C NMR).
2.6. Cell viability assay (miRNA)
We previously reported that miRNA 31, the BP-modified miR-
143 duplex with the mismatch sequence that was changed to
match the 30-region of the passenger strand, demonstrated the

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Y. Kitamura et al. / Bioorg. Med. Chem. 21 (2013) 4494–4501
J = 7.6 Hz, 1H), 4.83 (s, 2H), 4.44 (s, 2H), 0.96 (s, 9H), 0.12 (s, 6H);
13C NMR (CDCl3) d = 161.6, 154.4, 137.6, 120.0, 119.2, 65.9, 55.6,
25.9, 18.3, ꢀ5.4; MS (FAB, NBA) m/z 279 [M+H]+, HRMS (FAB,
NBA) Calcd for C13H23N4OSi [M+H]+: 279.1641. Found: 279.1634.
4.1.3. 2-Aminomethyl-6-tert-butyldimethylsilyloxymethyl-
pyridine (8)
A mixture of 7 (3.97 g, 14.3 mmol) and Ph3P (7.50 g, 28.6 mmol)
in THF (130 mL) and H2O (5.2 mL) was stirred at room temperature
for 24 h. The mixture was partitioned between EtOAc and H2O. The
organic layer was wash with brine, dried over Na2SO4, and concen-
trated under reduced pressure. The residue was purified by column
chromatography on silica gel (EtOAc/MeOH, 1:0–2:1) to give 3 as a
pale yellow oil (3.35 g, 93%). 1H NMR (CDCl3) d 7.66 (t, J = 7.8 Hz,
1H), 7.37 (d, J = 7.8 Hz, 1H), 7.13 (d, J = 7.6 Hz, 1H), 4.82 (s, 2H),
3.94 (s, 2H), 0.96 (s, 9H), 0.12 (s, 6H); 13C NMR (CDCl3) d 160.8,
160.7, 137.0, 119.0, 117.8, 66.0, 47.6, 25.8, 18.2, ꢀ5.4; MS (FAB,
NBA) m/z 253 [M+H]+, HRMS (FAB, NBA) Calcd for C13H25N2OSi
[M+H]+: 253.1736. Found: 253.1722.
Figure 4. Cell viability assay.
Chemical shifts (d) were expressed in parts per million and inter-
nally referenced (7.26 ppm for CDCl3 or 2.49 ppm for DMSO-d6
for 1H NMR and 77.0 ppm for CDCl3 or 39.5 ppm for DMSO-d6 for
13C NMR). Fast-atom-bombardment (FAB) mass spectra were taken
on a JEOL JMS SX102A instrument. Direct analysis in real time
(DART) coupled with TOF mass spectra were taken on a JMS
T100TD instrument. MALDI-TOF mass spectra were taken on a
Shimazu AXIMA-CFR plus instrument. Elemental analyses were
performed by YANACO MT-5 instrument. Flash column chromatog-
raphy was performed using silica gel 60 N [spherical neutral (63–
4.1.4. 3-(4,40-Dimethoxytrityloxymethyl)benzonitrile (10)
A
mixture of 3-hydroxymethylbenzonitrile (9) (1.12 g,
8.38 mmol) and 4,40-dimethoxytritylchloride (3.41 g, 10.1 mmol)
in pyridine (23 mL) and DMF (23 mL) was stirred at room temper-
ature for 12 h. The reaction was quenched by the addition of cold
H2O (20 mL) and the mixture was partitioned between EtOAc
and H2O.The organic layer was wash with brine, dried over Na2SO4,
and concentrated under reduced pressure. The residue was puri-
fied by column chromatography on silica gel (n-hexane/EtOAc,
10:1) to give 10 as a colourless oil (3.31 g, 91%).This was used in
the next step without further purification.
210 lm)] from Kanto Chemical Co., Inc. ONs were synthesized
using an ABI 3400 DNA/RNA synthesizer or a NTS H-6 DNA/RNA
synthesizer. Melting temperatures (Tms) of dsRNAs were measured
on a Shimadzu UV-2400.
4.1.1. 2-tert-Butyldimethylsilyloxymethyl-6-
hydroxymethylpyridine (6)
4.1.5. 3-(4,40-Dimethoxytrityloxymethyl)benzylamine (11)
A suspension of LiAlH4 (1.12 g, 30.0 mmol) in THF (150 mL)
was cooled to 0 °C and a solution of 10 (4.14 g, 9.82 mmol) in
THF (450 mL) was added dropwise at a rate that maintained a
temperature below 0 °C. When the addition was complete, the
reaction mixture was stirred at room temperature for 15 h.
The reaction was quenched by the addition of H2O (10 mL)
and MeOH (50 mL), and filtered through a Celite pad. The fil-
trate was concentrated under reduced pressure. The residue
was purified by column chromatography on silica gel (CHCl3/
MeOH, 20:1–10:1) to give 11 as a colourless oil (4.04 g, 94%).
1H NMR (CDCl3) d 7.52–7.21 (m, 13H), 6.84 (d, J = 8.8 Hz, 4H),
4.17 (s, 2H), 3.86 (s, 2H), 3.79 (s, 6H); 13C NMR (CDCl3) d
158.4, 145.0, 143.2, 139.6, 136.2, 130.0, 128.4, 128.2, 127.8,
126.7, 125.7, 125.6, 125.4, 113.1, 86.3, 65.5, 55.1, 46.5; MS
(FAB, NBA) m/z 440 [M+H]+, HRMS (FAB, NBA) Calcd for
A suspension of NaH (60% dispersion in paraffin oil, 1.45 g,
35.9 mmol) in DMF (60 mL) was cooled to 0 °C and a solution of
2,6-pyridinedimethanol (5) (5.0 g, 35.9 mmol) in DMF (30 mL)
was added dropwise at a rate that maintained a temperature below
0 °C. When the addition was complete, the reaction mixture was
stirred at room temperature for 1 h. A solution of TBDMSCl
(6.54 g, 43.3 mmol) in DMF (40 mL) was added dropwise, and then
the mixture was stirred at room temperature for 12 h. The mixture
was partitioned between EtOAc and 10% aqueous NaHCO3 solution.
The organic layer was wash with brine, dried over Na2SO4, and
concentrated under reduced pressure. The residue was purified
by column chromatography on silica gel (n-hexane/EtOAc, 5:1) to
give 6 as a colourless oil (4.46 g, 49%). 1H NMR (CDCl3) d = 7.66
(t, J = 7.6 Hz, 1H), 7.39 (d, J = 7.6 Hz, 1H), 7.10 (d, J = 7.6 Hz, 1H),
4.80 (s, 2H), 4.71 (s, 2H), 0.95 (s, 9H), 0.11 (s, 6H); 13C NMR (CDCl3)
d = 160.3, 157.9, 137.3, 118.5, 118.5, 65.8, 63.9, 25.9, 18.3, ꢀ5.4; MS
(FAB, NBA) m/z 254 [M+H]+, HRMS (FAB, NBA) Calcd for C13H24NO2-
Si [M+H]+: 254.1576. Found: 254.1581.
C
29H30NO3[M+H]+: 440.2226. Found: 440.2220.
4.1.6. 3-(4,40-Dimethoxytrityloxymethyl)benzylisothiocyanate
(12)
4.1.2. 2-Azidomethyl-6-tert-
butyldimethylsilyloxymethylpyridine (7)
The reactions were performed in three 200 mL round-bottom
A mixture of 11 (613 mg, 1.39 mmol), CS2 (850
and Et3N (200 L, 1.39 mmol) in EtOH (2 mL) was stirred at room
temperature for 2 h. A solution of Boc2O (3.06 g, 1.40 mmol) in
EtOH (600 L) and a solution of DMAP (6.12 mg, 60 mol) in EtOH
(600 L) were added dropwise at 0 °C, and then the mixture was
stirred at room temperature for 4 h. The mixture was concentrated
under reduced pressure, and the resulting residue was purified by
column chromatography on silica gel (n-hexane/EtOAc, 10:1) to
give 12 as a pale yellow oil (640 g, 96%). 1H NMR (CDCl3) d 7.52–
7.22 (m, 13H), 6.85 (d, J = 9.2 Hz, 4H), 4.71 (s, 2H), 4.19 (s, 2H),
3.79 (s, 6H); 13C NMR (CDCl3) d 158.4, 144.9, 140.2, 136.0, 134.1,
130.0, 129.1, 128.8, 128.1, 127.8, 126.8, 126.7, 125.4, 125.1,
113.1, 86.5, 65.1, 55.1, 48.6.
lL, 13.9 mmol)
l
flasks. To each them
6 (1.32 g, 5.20 mmol), CBr4 (1.90 g,
5.72 mmol), Ph3P (1.64 g, 6.02 mmol) and NaN3 (1.69 g,
26.0 mmol) were added. The each mixture was dissolved in DMF
(40 mL) and Et3N (1.6 mL, 11.5 mmol) was added, and then the
each mixture was stirred at room temperature for 24 h, respec-
tively. The mixtures were combined and partitioned between
EtOAc and H2O. The organic layer was wash with brine, dried over
Na2SO4, and concentrated under reduced pressure. The residue was
purified by column chromatography on silica gel (n-hexane/EtOAc,
50:1) to give 7 as a pale yellow oil (3.97 g, 93%). 1H NMR (CDCl3)
d = 7.73 (t, J = 7.6 Hz, 1H), 7.47 (d, J = 7.6 Hz, 1H), 7.19 (d,
l
l
l

Y. Kitamura et al. / Bioorg. Med. Chem. 21 (2013) 4494–4501
4499
4.1.7. N-[3-(4,40-Dimethoxytrityloxymethyl)benzyl]-N0-[(6-tert-
129.0, 128.7, 128.0, 127.8, 126.7, 126.5, 126.3, 126.1, 120.9,
119.5, 113.1, 86.4, 65.3, 64.3, 55.1, 49.3.
butyldimethylsilyloxymethylpyridine-2-yl)methyl]urea (13)
A mixture of 11 (160 mg, 370
ole (61.5 mg, 380 mol) in THF (18.5 mL) was stirred at room tem-
perature for 24 h. A solution of 8 (230 mg, 920 mol) in THF (5 mL)
l
mol) and 1,10-carbonyldiimidaz-
l
4.1.11. N,N0-Bis[(6-tert-butyldimethylsilyloxymethylpyridin-2-
yl)methyl]urea (17)
l
was added dropwise, and then the mixture was stirred at room
temperature for 48 h. The mixture was concentrated under re-
duced pressure, and the resulting residue was purified by column
chromatography on silica gel (CHCl3/MeOH, 100:1) to give 13 as
a colourless solid (113 mg, 54%). Mp 62–64 °C; 1H NMR (CDCl3) d
7.66–7.10 (m, 16H), 6.83 (d, J = 8.8 Hz, 4H), 4.71 (s, 2H), 4.47 (d,
J = 4.8 Hz, 2H), 4.11 (d, J = 5.6 Hz, 2H), 4.16 (s, 2H), 3.79 (s, 6H),
0.95 (s, 9H), 0.10 (s, 6H); 13C NMR (CDCl3) d 160.6, 158.4, 158.2,
156.4, 145.0, 139.7, 139.1, 137.4, 136.2, 130.0, 128.5, 128.1,
127.8, 126.7, 126.2, 126.0, 125.9, 119.9, 118.3, 113.1, 86.4, 65.8,
65.4, 55.2, 45.6, 44.6, 25.9, 18.3, ꢀ5.4; MS (FAB, NBA) m/z 718
[M+H]+, HRMS (FAB, NBA) Calcd for C43H52N3O5Si [M+H]+:
718.3676. Found: 718.3670; Anal. Calcd for C43H51N3O5Si: C,
71.93; H, 7.16; N, 5.85. Found: C, 71.78; H, 7.15; N, 5.78.
A mixture of 8 (380 mg, 1.50 mmol) and 1,10-carbonyldiimidaz-
ole (150 mg, 920 lmol) in THF (10 mL)was stirred at room temper-
ature for 24 h. The mixture was concentrated under reduced
pressure, and the resulting residue was purified by column chro-
matography on silica gel (CHCl3/MeOH, 3:1) to give 17 as a colour-
less solid (384 mg, 96%). Mp 110–112 °C; 1H NMR (CDCl3) d 7.66 (t,
J = 7.6 Hz, 2H), 7.38 (d, J = 7.6 Hz, 2H), 7.14 (d, J = 7.6 Hz, 2H), 4.79
(s, 4H), 4.50 (d, J = 5.2 Hz, 4H), 0.96 (s, 9H), 0.12 (s, 6H); 13C NMR
(CDCl3) d 160.7, 158.3, 156.7, 137.3, 119.8, 118.3, 65.8, 45.6, 25.9,
18.3, -5.4; MS (FAB, NBA) m/z 531 [M+H]+, HRMS (FAB, NBA) Calcd
for C27H47N4O3Si2 [M+H]+: 531.3187. Found: 531.3178; Anal. Calcd
for C27H46N4O3Si2: C, 61.09; H, 8.73; N, 10.55. Found: C, 61.30; H,
8.90; N, 10.33.
4.1.12. N-{[3-(4,40-Dimethoxytrityloxymethyl)pyridin-2-
yl]methyl}-N0-[(6-hydroxymethylpyridin-2-yl)methyl]urea (18)
TBAF (1.0 M in THF, 3.70 mL) was added to a solution of 17
(855 mg, 1.61 mmol) in THF (8.1 mL), and then the mixture was
stirred at room temperature for 5 h. The mixture was concentrated
under reduced pressure, and the resulting intermediate alcohol (3)
was used in the next step without further purification.
4.1.8. N-[3-(4,40-Dimethoxytrityloxymethyl)benzyl]-
N0-[(6-hydroxymethylpyridine-2-yl)methyl]urea (14)
TBAF (1.0 M in THF, 640
lL) was added to a solution of 13
(410 mg, 570 mol) in THF (2.2 mL), and then the mixture was stir-
l
red at room temperature for 4 h. The mixture was concentrated
under reduced pressure, and the resulting residue was purified
by column chromatography on silica gel (CHCl3/MeOH, 20:1) to
give 14 as a colourless solid (336 mg, 98%). Mp 64–66 °C; 1H
NMR (CDCl3) d 7.62–7.09 (m, 16H), 6.83 (d, J = 8.8 Hz, 4H), 4.66
(s, 2H), 4.50 (d, J = 5.4 Hz, 2H), 4.40 (d, J = 5.6 Hz, 2H), 4.16 (s,
2H), 3.79 (s, 6H); 13C NMR (CDCl3) d 158.6, 158.4, 157.1, 144.9,
139.5, 139.2, 137.3, 136.1, 130.0, 128.5, 128.1, 127.8, 126.7,
126.0, 125.9, 125.7, 120.2, 118.9, 113.1, 86.4, 65.4, 64.1, 55.1,
45.3, 44.2; MS (FAB, NBA) m/z 604 [M+H]+, HRMS (FAB, NBA) Calcd
for C37H38N3O5 [M+H]+: 604.2811. Found: 604.2803.
DMTrCl (271 mg, 800 lmol) was added to a solution of the
crude alcohol in pyridine (6 mL) and DMSO (1.2 mL), and the mix-
ture was heated at 40 °C. The resulting suspension was stirred at
room temperature for 12 h. An additional portion of DMTrCl
(405 mg, 1.2 mmol) was added and the mixture was stirred for
additional 12 h. Then DMTrCl (405 mg, 1.2 mmol) was further
added and stirring was continued for 26 h. The mixture was parti-
tioned between EtOAc and 10% aqueous NaHCO3 solution. The or-
ganic layer was wash with brine, dried over Na2SO4, and
concentrated under reduced pressure. The residue was purified
by column chromatography on silica gel (CHCl3/MeOH, 20:1) to
give 18 as a pale yellow solid (424 mg, 43%). Mp 62–64 °C; 1H
NMR (CDCl3) d 7.70–7.04 (m, 15H), 6.82 (d, J = 9.0 Hz, 4H), 4.60
(s, 2H), 4.40 (d, J = 5.6 Hz, 4H), 4.28 (s, 2H), 3.78 (s, 6H); 13C NMR
(CDCl3) d 158.8, 158.6, 158.5, 157.1, 144.7, 137.4, 137.2, 135.9,
129.9, 128.0, 127.8, 126.8, 120.2, 119.0, 113.1, 86.6, 66.4, 64.1,
55.1, 45.4.
4.1.9. N-[3-(4,40-Dimethoxytrityloxymethyl)benzyl]-N0-[(6-tert-
butyldimethylsilyloxymethylpyridine-2-yl)methyl]thiourea
(15)
A solution of 8 (910 mg, 3.60 mmol) in CHCl3 (10 mL) was
added to a solution of 12 (3.67 g, 7.62 mmol) in CHCl3 (80 mL),
and then the mixture was refluxed for 7 h. The mixture was con-
centrated under reduced pressure, and the resulting residue was
purified by column chromatography on silica gel (n-hexane/EtOAc,
20:1–3:1) to give 15 as a colourless solid (1.98 g, 75%). Mp 62–
64 °C; 1H NMR (CDCl3) d 7.69–7.09 (m, 16H), 6.83 (d, J = 9.2 Hz,
4H), 4.70 (s, 4H), 4.51 (s, 2H), 4.16 (s, 2H), 3.79 (s, 6H), 0.92 (s,
9H), 0.06 (s, 6H); 13C NMR (CDCl3) d 182.1, 160.6, 158.4, 154.4,
144.9, 140.0, 137.8, 136.0, 130.0, 128.6, 128.1, 127.8, 126.7,
126.3, 126.2, 126.1, 120.4, 118.8, 113.1, 86.3, 65.3, 65.3, 55.1,
49.4, 25.8, 18.2, ꢀ5.4; MS (FAB, NBA) m/z 734 [M+H]+, HRMS
(FAB, NBA) Calcd for C43H52N3O4SiS [M+H]+: 734.3448. Found:
734.3443; Anal. Calcd for C43H51N3O5Si: C, 70.36; H, 7.00; N,
5.72. Found: C, 70.39; H, 7.21; N, 5.67.
4.1.13. N,N0-Bis[(hydroxymethylpyridin-2-yl)methyl]thiourea
(4)
The reactions were performed in two 500 mL round-bottom
flasks. Compound 8 (1.79 g, 7.09 mmol) was added to one flask
and dissolved in CH2Cl2 (142 mL). Et3N (1.0 mL, 7.09 mmol) was
added to the solution, and the mixture was cooled to 0 °C. Thio-
phosgene (360 lL, 4.73 mmol) was added dropwise and the reac-
tion mixture was stirred at room temperature. In a similar
manner, 8 (1.56 g, 6.18 mmol) was added to another flask and dis-
solved in CH2Cl2 (124 mL). Et3N (900
the solution, and the mixture was cooled to 0 °C. Thiophosgene
(314 L, 4.12 mmol) was added dropwise and the reaction mixture
lL, 6.18 mmol) was added to
4.1.10. N-[3-(4,40-Dimethoxytrityloxymethyl)benzyl]-
l
N0-[(6-hydroxymethylpyridine-2-yl)methyl]thiourea (16)
TBAF (1.0 M in THF, 2.90 mL) was added to a solution of 15
(1.94 g, 2.65 mmol) in THF (12 mL), and then the mixture was stir-
red at room temperature for 20 h. The mixture was concentrated
under reduced pressure, and the resulting residue was purified
by column chromatography on silica gel (CHCl3/MeOH, 50:1–
40:1) to give 16 as a colourless solid (1.48 g, 90%). Mp 74–76 °C;
1H NMR (CDCl3) d 7.62–7.12 (m, 16H), 6.83 (d, J = 9.0 Hz, 4H),
4.67 (s, 4H), 4.46 (s, 2H), 4.16 (s, 2H), 3.78 (s, 6H); 13C NMR (CDCl3)
d 182.2, 158.8, 158.4, 155.1, 144.8, 139.8, 137.7, 136.0, 130.0,
was stirred at room temperature. After 5 h, the mixtures were
combined and partitioned betweenCH2Cl2 and H2O.The organic
layer was wash with brine, dried over Na2SO4, and concentrated
under reduced pressure. The residue was passed through a column
of silica (CHCl3/MeOH, 50:1) to give 19 as a brown oil. This was
used in the next step without further purification.
TBAF (1.0 M in THF, 13.3 mL) was added to a solution of the
crude thiourea in THF (33 mL), and then the mixture was stirred
at room temperature for 24 h. The mixture was concentrated under
reduced pressure, and the resulting residue was purified by

4500
Y. Kitamura et al. / Bioorg. Med. Chem. 21 (2013) 4494–4501
column chromatography on silica gel (CHCl3/MeOH, 20:1–10:1) to
(6), ON 58 (11), ON 59 (7), ON 60 (8), ON 61 (6), ON 62 (3) and
ON 63 (1). The yields are indicated in parentheses as OD units at
give 4 as a pale brown solid (909 mg, 43%). 125–127 °C; 1H NMR
(DMSO-d6)
d
8.27 (s, 2H), 7.75 (t, J = 7.6 Hz, 2H), 7.34 (d,
260 nm starting from 1.0 lmol scale. The extinction coefficients
J = 7.6 Hz, 2H), 7.16 (d, J = 7.6 Hz, 2H), 5.39 (t, J = 5.8 Hz, 2H), 4.73
(s, 4H), 4.55 (d, J = 5.6 Hz, 4H); 13C NMR (DMSO-d6) d 183.3,
161.3, 157.0, 137.2, 119.3, 118.4, 64.1, 48.9; MS (DART) m/z 319
[M+H]+, HRMS (DART) Calcd for C15H19N4O2S [M+H]+: 319.1229.
Found: 319.1244.
of the ONs were calculated from those of the mononucleotides
and dinucleotides according to the nearest-neighbor approxima-
tion method.27
7. Mass spectrometric analyses of ONs
4.1.14. N-{[3-(4,40-Dimethoxytrityloxymethyl)pyridin-2-
yl]methyl}-N’-[(6-hydroxymethylpyridin-2-yl)methyl]thiourea
(20)
Spectra were obtained by MALDI-TOF/MS (negative mode). ON
36: calculated mass, 6498.9; observed mass, 6502.6. ON 37: calcu-
lated mass, 6808.0; observed mass, 6807.6. ON 38: calculated
mass, 6291.8; observed mass, 6290.4. ON 49: calculated mass,
6600.9; observed mass, 6599.3. ON 40: calculated mass, 6253.9;
observed mass, 6254.0. ON 41: calculated mass, 6563.0; observed
mass, 6568.4. ON 42: calculated mass, 6269.9; observed mass,
6273.1. ON 43: calculated mass, 6579.0; observed mass, 6581.7.
ON 44: calculated mass, 6254.9; observed mass, 6258.9. ON 45:
calculated mass, 6564.0; observed mass, 6567.3. ON 46: calculated
mass, 6270.9; observed mass, 6272.6. ON 47: calculated mass,
6580.0; observed mass, 6583.7. ON 48: calculated mass, 7375.1;
observed mass, 7378.0. ON 49: calculated mass, 7168.1; observed
mass, 7170.4. ON 50: calculated mass, 7130.1; observed mass,
7132.8. ON 51: calculated mass, 7146.1; observed mass, 7147.9.
ON 52: calculated mass, 7131.1; observed mass, 7135.1. ON 53:
calculated mass, 7118.1; observed mass, 7118.8. ON 54: calculated
mass, 7352.0; observed mass, 7347.0. ON 55: calculated mass,
7462.0; observed mass, 7454.9. ON 56: calculated mass, 7314.0;
observed mass, 7315.6. ON 57: calculated mass, 7424.1; observed
mass, 7425.9. ON 58: calculated mass, 7330.0; observed mass,
7330.5. ON 59: calculated mass, 7440.1; observed mass, 7439.4.
ON 60: calculated mass, 7315.0; observed mass, 7315.9. ON 61:
calculated mass, 7425.1; observed mass, 7424.7. ON 62: calculated
mass, 7331.0; observed mass, 7331.4. ON 63: calculated mass,
7441.1; observed mass, 7442.0.
A solution of DMTrCl (420 mg, 1.24 mmol) in pyridine (5 mL)
was added to a solution of 4 (650 mg, 2.04 mmol) in DMF (5 mL),
and then the mixture was stirred at room temperature for 2 h.
DMTrCl (420 mg, 1.24 mmol) in pyridine (5 mL) was further added
and stirring was continued for 20 h. The mixture was partitioned
between EtOAc and H2O. The organic layer was wash with brine,
dried over Na2SO4, and concentrated under reduced pressure. The
residue was purified by column chromatography on silica gel (n-
hexane/EtOAc, 1:1) to give 20 as a yellow solid (227 mg, 18%).
66–68 °C; 1H NMR (CDCl3)
d 7.75–7.04 (m, 15H), 6.81 (d,
J = 8.8 Hz, 4H), 4.70 (s, 4H), 4.53 (s, 2H), 4.26 (s, 2H), 3.78 (s, 6H);
13C NMR (CDCl3) d 182.4, 158.8, 158.5, 155.3, 144.6, 137.7, 137.4,
135.7, 129.9, 127.9, 127.8, 126.8, 121.1, 120.4, 119.6, 119.1,
113.3, 86.6, 66.3, 63.9, 55.1, 53.4, 49.4; Anal. Calcd for
C
36H36N4O4Sꢁ2/5H2O: C, 68.85; H, 5.91; N, 8.92. Found: C, 68.77;
H, 5.89; N, 8.54.
5. Solid support synthesis
A
mixture of 14 (290 mg, 470
(141 mg, 1.41 mmol), and 4-dimethylaminopyridine (DMAP;
1.24 mg, 10.2 mol) in pyridine (4.7 mL) was stirred for 72 h at
lmol), succinic anhydride
l
room temperature. The reaction mixture was partitioned between
CHCl3 and saturated NaHCO3 aqueous solution. The organic layer
was wash with brine, dried over Na2SO4, and concentrated under
reduced pressure to give the corresponding succinate. Aminopro-
8. Thermal denaturation study
pyl controlled pore glass (CPG) (979 mg, 118
a solution of the succinate and 1-ethyl-3-(3-dimethylaminopro-
pyl)carbodiimide hydrochloride (90 mg, 470 mol) in DMF
(12 mL), and the mixture was kept for 48 h at room temperature.
After the resin was washed with pyridine, a capping solution
(15 mL, 0.1 M DMAP in pyridine/Ac2O, 9:1, v/v) was added and
the whole mixture was kept for 12 h at room temperature. The re-
sin was washed with pyridine, EtOH, MeCN, and dried in vacuo.
The amount of loaded compound 14 to the solid support was
lmol) was added to
Each solution containing each siRNA (3.0 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.
l
9. Dual-luciferase assay
38.5 lmol/g from calculation of released dimethoxytritylcation
by a solution of 70% HClO4/EtOH, 3:2 (v/v). In a similar manner, so-
lid supports with 16, 18 and19 were obtained in 48.3, 79.9,
39.2 lmol/g loading amounts, respectively.
HeLa cells were grown at 37 °C in a humidified atmosphere of
5% CO2 in air in D-MEM (Wako) supplemented with 10% bovine
serum (Sigma). 24 h before transfection, HeLa cells (4 ꢂ 104/mL)
were transferred to 96-well plates (100 lL per well). They were
transfected, using TransFast (Promega), according to instructions
6. Oligonucleotide synthesis
for transfection of adherent cell lines. Cells in each well were trans-
fected with a solution (35
mega), the indicated amounts of siRNAs, and 0.3
Opti-MEM I Reduced-Serum Medium (Invitrogen), and incubated
at 37 °C. Transfection without siRNA was used as a control. After
l
L) of 20 ng of psi-CHECK-2 vector (Pro-
Synthesis was carried out with a DNA/RNA synthesizer (ABI
Expedite 3400) by phosphoramidite method. Deprotection of the
bases and phosphates was performed in concentrated NH4OH/
EtOH, 3:1 (v/v) at room temperature for 12 h. 20-TBDMS groups
were removed by TBAF (1.0 M in THF) at room temperature for
12 h. The reaction was quenched with 0.1 M triethylammonium
acetate buffer (pH 7.0) and desalted on a Sep-Pak C18 cartridge.
Deprotected ONs were purified by 20% PAGE containing 7.0 M urea
to give the highly purified ON 36 (10), ON 37 (8), ON 38 (7), ON 39
(6), ON 40 (4), ON 41 (3), ON 42 (6), ON 43 (4), ON 44 (2), ON 45 (4),
ON 46 (8), ON 47 (2), ON 48 (8), ON 49 (10), ON 50 (22), ON 51 (8),
ON 52 (3), ON 53 (2), ON 54 (14), ON 55 (0.9), ON 56 (10), ON 57
lg of TransFast in
1 h, D-MEM (100 lL) containing 10% bovine serum and antibiotics
was added to each well, and the whole was further incubated at
37 °C. After 24 h, cell extracts were frozen at ꢀ80 °C. Activities of
firefly and Renilla luciferases in cell lysates were determined with
a dual-luciferase assay system (Promega). The results were con-
firmed by at least three independent transfection experiments
with four cultures each and are expressed as the average from
three experiments as mean SD.

Y. Kitamura et al. / Bioorg. Med. Chem. 21 (2013) 4494–4501
4501
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with 10% heat-inactivated fetal bovine serum (Sigma). 24 h before
transfection, T24 cells (0.5 ꢂ 105/mL) were transferred to 6-well plates
(1 mL per well). They were transfected, using cationic liposomes, that
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1.5 lL of Lipofectamine in Opti-MEM I Reduced-Serum Medium (Invit-
rogen), and incubated at 37 °C. A 19-mer RNA duplex targeted to the
Renilla luciferase, 50-GGCCUUUCACUACUCCUAC-30/30-CCGGAAAGU-
GAUGAGGAUG-50 (Hokkaido System Science), was used as a control
for nonspecific effects. After 48 h, the number of viable cells was deter-
mined by the Trypan blue dye exclusion test. The results are expressed
as the average from three experiments as mean SD.
Acknowledgement
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This work was supported by Grants-in-Aid for Scientific Re-
search from the Japan Society for the Promotion of Science (JSPS).
References and notes
1. For a review on RNA-based drug, see: (a) Pappas, T. C.; Bader, A. G.; Andreas, B.
F.; Brown, D.; Ford, L. P. Expert Opin. Ther. Targets 2008, 12, 115; (b) Hemida, M.