Synthesis of modified siRNA bearing C-5 polyamine-substituted pyrimidine nucleoside in their 3′-overhang regions and its RNAi activity

Article abstract of DOI:10.1016/j.bmcl.2010.11.125

Short interfering RNA (siRNA) induces specific gene silencing by the RNA interference (RNAi) pathway. Nucleosides in the 3′-overhang regions of siRNAs were replaced with 5-bis(aminoethyl)aminoethylcarbamoylmethyl-2′- deoxyuridine or thymidine. siRNA beari

Full text of DOI:10.1016/j.bmcl.2010.11.125

Bioorganic & Medicinal Chemistry Letters 21 (2011) 715–717
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis of modified siRNA bearing C-5 polyamine-substituted pyrimidine
nucleoside in their 30-overhang regions and its RNAi activity
Mohammad Mehedi Masud a,b, Tomokazu Masuda a, Yusuke Inoue a, Masayasu Kuwahara a,
Hiroaki Sawai a, Hiroaki Ozaki a,
⇑
a Department of Chemistry and Chemical Biology, Graduate School of Engineering, Gunma University, Tenjin-cho 1-5-1, Kiryu, Gunma 376-8515, Japan
b Department of Pharmaceutical Chemistry, Faculty of Pharmacy, University of Dhaka, Dhaka 1000, Bangladesh
a r t i c l e i n f o
a b s t r a c t
Article history:
Short interfering RNA (siRNA) induces specific gene silencing by the RNA interference (RNAi) pathway.
Nucleosides in the 30-overhang regions of siRNAs were replaced with 5-bis(aminoethyl)amin-
oethylcarbamoylmethyl-20-deoxyuridine or thymidine. siRNA bearing modified nucleoside was more
Received 28 October 2010
Revised 29 November 2010
Accepted 30 November 2010
Available online 5 December 2010
active in silencing the gene expression of hepatocyte nuclear factor 4
bearing thymidine.
a (HNF4a) compared with siRNA
Ó 2010 Elsevier Ltd. All rights reserved.
Keywords:
RNAi
siRNA
Modified siRNA
HNF4a
Modified CPG support
Targeted gene silencing through RNA interference (RNAi) opens
up exciting possibilities in gene technology.1 In nature, this ancient
and evolutionarily conserved viral defense mechanism consists of
short interfering RNA (siRNA), sometimes known as small interfer-
ing RNA or silencing RNA, which is a class of double-stranded RNA
molecules enzymatically generated by the Dicer protein from long-
er RNAs of viral origin. These siRNAs interact with several proteins
to form an RNA-induced silencing complex (RISC), which recog-
nizes corresponding sequences in mRNA, causes degradation of
the sequences, and interferes with expression of a specific gene.
This silences gene expression without triggering non-specific
RNA degradation and translation inhibition based on the interferon
response. Thus, siRNA empowers researchers with a highly specific
tool to ablate the gene of interest and has proven to be a robust
agent for analysis of biological function in cell culture. siRNAs also
have considerable potential as new therapeutic drugs for intracta-
ble diseases because they can be rationally designed and prepared
on the basis of genes related to disease. Various siRNAs that are
stable against nuclease have been designed for use as therapeutic
candidates. siRNA with a modified nucleoside in its 30-overhang re-
gions is an example of such modified siRNAs.2 The 2-nt 30-over-
hang region is considered the most efficient in an experiment
using 21-nt siRNA, and this 2-nt region of the guide strand of siRNA
is recognized by the PAZ domain, which is found in Argonaute2, a
key component of RISC.3 In addition, most commercial available
siRNA had thymidines in their 30-overhang region.
Previously, we reported the synthesis and properties of an oli-
godeoxyribonucleotide (ODN) bearing polyamine-substituted
pyrimidine nucleoside at the C-5 position.4 Matsukura et al. also
found that ODN with C5-modified pyrimidine bases, particularly
with a polyamine moiety (trisamine), exhibits a higher antisense
activity as well as reduced cytotoxicity in cells infected with
HIV-1.5 Recently, RNA bearing 5-propynyl pyrimidine nucleoside
in the region complementary to target mRNA was found to be det-
rimental to RNAi activity due to the bulkiness.6
Hepatocyte nuclear factor 4a (HNF4a), a member of the nuclear
hormone receptor family, is one of the most abundant nuclear or-
phan receptors expressed in the liver. This receptor is involved in
early liver development.7 It is also expressed in kidney, intestine,
and pancreas and is required for expression of many tissue-specific
traits in all of these organs. Recent reports have demonstrated that
siRNA for HNF4a can decrease expression of HNF4a and the DNA
binding activity to the target promoters in HepG2 cells.8
Here, we synthesized siRNAs having modified nucleosides in
their 30-overhang regions. In in vitro experiments, these modified
siRNAs were found to be more effective against HNF4a suppression
compared with siRNAs without modification. We also report the
nuclease-resistant properties of these modified RNAs.
Synthesis: We synthesized three duplex RNAs for these experi-
ments. All of these RNAs contained modified nucleosides or deoxy-
ribonucleosides in their 30-overhang regions (Table 1). For these
⇑
Corresponding author. Tel./fax: +81 27 220 7562.
E-mail address: h-ozaki@gunma-u.ac.jp (H. Ozaki).
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.11.125

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M. M. Masud et al. / Bioorg. Med. Chem. Lett. 21 (2011) 715–717
Table 1
Then, compound 2 was phosphitylated to obtain the corresponding
phosphoramidite 3 by a standard procedure.9 Phosphoramidite 3
was purified by chromatography on silica gel and eluted with ethyl
acetate, dichloromethane, and triethylamine [4.5:4.5:1 (v/v)]. The
yield of compound 3 was 60%.
Sequences and structures of modified siRNAs
dsRNA
ssRNA
Sequence
siRNA 1
ORN 1
ORN 2
ORN 3
ORN 4
ORN 5
ORN 6
50-ggcagugcgugguggacaaTT-30
50-uuguccaccacgcacugccTT-30
50-ggcagugcgugguggacaaTZ-30
50-uuguccaccacgcacugccTZ-30
50-ggcagugcgugguggacaaZZ-30
50-uuguccaccacgcacugccZZ-30
Solid support was synthesized by a standard procedure.10,11 30-
Succinate 4 was synthesized from compound 2 and succinic anhy-
siRNA 2
siRNA 3
dride. It was then immobilized to
a long-chain alkylamine
controlled-pore glass (LCAA-CPG) resin using 1-(3-dimethyamino-
propyl)ethylcarbodiimide and DMAP in dry pyridine. The amount
of loaded compound 2 was measured by quantifying the DMTr cat-
ion released by a solution of trichloroacetic acid.
T
represent thymidine and
Z
represent 5-bis(aminoethyl)-aminoethyl-20-
deoxyuridine.
RNA synthesis: The RNA sequence was designed on the basis of
the study of HNF4
sized by the phosphoramidite method using a 392 DNA/RNA syn-
thesizer (Applied Biosystems) on 1- mol scale (Table 1).
a
.8 Six oligoribonucleotides (ORNs) were synthe-
a
l
Coupling time for this synthesis was 10 min. Phosphoramidites of
thymidine (T) and CPG-T were used for ORNs 1 and 2, respectively,
to incorporate T in the 30-overhang region. Similarly, phosphorami-
dites of T and modified CPG 5 were used for ORNs 3 and 4 and
those of modified nucleoside 3 and modified CPG 5 were used for
ORNs 5 and 6, respectively. After cleavage from support and depro-
tection, ORNs were purified by HPLC in two steps using an ODS-
80Ts column (Tosoh, 0.46 ꢀ 25 cm), 100 mM TEAA, and CH3CN to
obtain ORNs 1, 2, 3, 4, 5, and 6 at optical densities of 0.1, 0.2, 2.5,
0.4, 36, and 4.8 (260 nm), respectively. ORNs 1, 2, 3, 4, and 6 were
repeatedly purified by HPLC to obtain the high purity sample. This
purification steps caused the loss of ORN and their yields were low.
Extinction coefficients of ORNs were calculated from those of
mononucleotides according to the nearest-neighbor approxima-
tion method. Purified ORNs were verified by denaturing 12% poly-
acrylamide gel electrophoresis (data not shown). The result
showed that RNAs had good purity. In the gel electrophoresis,
the movement of modified RNAs bearing C-5 polyamine-substi-
tuted pyrimidine nucleosides (ORNs 3–6) was slower than that
of others (ORNs 1 and 2) and the order of mobility was ORNs 5
and 6 > ORNs 3 and 4 > ORNs 1 and 2. This indicates that the move-
ment of ORN bearing more cationic amino groups was slow be-
cause the net charge was low.
Nuclease-resistant property: We investigated the nuclease-resis-
tant property of ORNs 1, 3, and 5 using snake venom phosphodies-
terase (Worthington Biochemical Corp., SVPD), a 30-exonuclease.
Each ORN (0.8 nmol) was incubated with SVPD (0.01 U) in a buffer
containing 100 mM MgCl2 and 200 mM Tris–HCl (pH 8.0) at 37 °C
for 5 and 30 min, respectively. The degraded products were ana-
purposes, phosphoramidite 3 and modified solid support 5 were
synthesized according to the routes shown in Scheme 1. First, mod-
ified nucleoside 1 was synthesized according to our previous re-
port.4 Nucleoside 1 was protected with a 4,40-dimethoxytrityl
(DMTr) group to obtain a mono-DMTr derivative 2 in 30% yield.
Scheme 1. Synthesis of modified nucleoside phosphoramidite and modified solid support. Reagents and conditions: (a) DMTr-Cl, pyridine, rt; (b) 2-cya-
noethyldiisopropylchlorophosphoramidite, i-Pr2NEt, pyridine, CH2Cl2, rt; (c) succinic anhydride, DMAP, pyridine, rt; (d) LCAA-CPG, DMAP, 1-(3-dimethylaminopropyl)eth-
ylcarbodiimide, pyridine, rt.

M. M. Masud et al. / Bioorg. Med. Chem. Lett. 21 (2011) 715–717
717
In conclusion, we have prepared siRNAs bearing C-5 polyamine-
substituted pyrimidine nucleosides in their 30-overhang regions.
RNAi activity of these modified siRNAs against the nuclear hor-
mone receptor HNF4
a was evaluated in HepG2 cells. Our results
suggest that 30-terminal modification improves RNAi activity. This
could be due to the increased nuclease resistance caused by the
modification.
Acknowledgments
We thank Professor Kazuo Shinozuka for encouragement and
helpful discussions. We are also grateful to Mr. Yuichi Tsuchida
(Gunma University) for providing support for the RNAi experiment.
This work was supported by grants from the Tokyo Biochemical
Research foundation (TBRF) and Grant-in-Aid for Scientific
Research (C).
Figure 1. Nuclease-resistant activity of ORNs 1, 3, and 5.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2010.11.125.
References and notes
1. Kurreck, J. Angew. Chem., Int. Ed. 2009, 48, 1378.
2. Behlke, M. A. Oligonucleotides 2008, 18, 305.
3. Ueno, Y.; Watanabe, Y.; Shibata, A.; Yoshikawa, K.; Takano, T.; Kohara, M.;
Kitade, Y. Bioorg. Med. Chem. 2009, 17, 1974.
4. Ozaki, H.; Nakamura, A.; Arai, M.; Endo, M.; Sawai, H. Bull. Chem. Soc. Jpn. 1995,
68, 1981.
5. Matsukura, M.; Okamoto, T.; Miike, T.; Sawai, H.; Shinozuka, K. Biochem.
Biophys. Res. Commun. 2002, 293, 1341.
6. Terrazas, M.; Kool, E. T. Nucleic Acids Res. 2009, 37, 346.
7. Li, J.; Ning, G.; Duncan, S. A. Genes Dev. 2000, 14, 464.
8. Sumi, K. et al Mol. Cell Biol. 2007, 27, 4248.
Figure 2. Real-time RT-PCR for measuring HNF4a mRNA. The cell was treated with
9. Sinha, N. D.; Biernat, J.; Köster Tetrahedron Lett. 1983, 24, 5843.
10. Pon, R. T. In Protocols for Oligonucleotides and Analogs (Methods in Molecular
Biology); Agrawel, S., Ed.; Humana Press: NJ, USA, 1993; Vol. 20, pp 465–
496.
11. Solid support synthesis: Succinic anhydride (0.07253 g, 0.72 mmol), DMAP
(30 mg, 0.25 mmol), and compound 2 (0.15 g, 0.17 mmol) were dissolved in
dry pyridine (1 ml), and the obtained mixture was stirred at room temperature
for 20 h. The mixture was then partitioned between CH2Cl2 and 5% aqueous
solution of Na2HPO4, and the organic layer was coevaporated three times with
siRNA (20 nM). NEG is nonsense control siRNA.
lyzed by RP-HPLC. The ratio of the remaining full-length RNA to the
total RNA was calculated from the peak area on a chromatogram.
Modified ORNs 3 and 5 were more resistant compared with
unmodified ORN 1, even after 30 min (Fig. 1). Because both ORNs
3 and 5 with one and two modified nucleosides, respectively, in
the 30-overhang region showed nuclease resistance, it was con-
cluded that only one modification in the 30-overhang region is en-
ough for resistance to exonuclease.
toluene to remove pyridine. The desired corresponding succinate
4 was
purified by column chromatography on silica gel, eluted with 10% MeOH in
CH2Cl2, and finally precipitated in hexane (70 ml). The LCAA-CPG resin (3-
Prime, 0.5 g) was then added to a solution of DMAP (6 mg, 0.05 mmol), 1-(3-
dimethylaminopropyl)ethylcarbodiimide (191 mg, 1 mmol), and succinate 4
(20 mg, 0.04 mmol) in dry pyridine (5 ml). The mixture was shaken slowly for
4 h at room temperature after adding triethylamine (40 ll). After filtration, the
resin was washed with dry pyridine and then with CH2Cl2 and left to dry.
Finally, we obtained the modified solid support 5 on which the amount of
RNAi activity: The RNAi experiment for three siRNAs was con-
ducted using human hepatoblastoma HepG2 cells, which express
endogenous HNF4
a at high level. Solutions of siRNAs 1, 2, and 3
were prepared by annealing ORN 1/ORN 2, ORN 3/ORN 4, and
ORN 4/ORN 5, respectively. These three siRNAs were evaluated
loaded compound
2 was 23.68 lmol/g. The amount was measured by
quantifying the DMTr cation released by a solution of 3% trichloroacetic acid
in CH2Cl2.
for their activity against mRNA level of HNF4
a by real-time RT-
PCR.12 The obtained results are shown in Figure 2 as the relative
12. RNAi activity: HepG2 cells were cultured in Dulbecco’s modified Eagle’s
medium (DMEM) containing 5% FBS at 37 °C. Each siRNA was then transfected
into the cells with transfection reagent (Hily Max, DOJINDO). After incubation at
37 °C for 24 h, medium was changed to DMEM containing 10% FBS. After
additional 24 h incubation, total RNA was extracted from the cells using RNAiso
Plus (TaKaRa). The RNA was reverse-transcribed using ReverTraAce qPCR RT kit
(TOYOBO). Real-time PCR was performed on a MyiQ Real-time PCR detection
mRNA level to the level using control siRNA. siRNA 2 was found
to be more active against HNF4
(Fig. 2). In addition, the expression level of the HNF4
a
compared with siRNAs 1 and 3
protein
a
was confirmed by Western blotting (data not shown). These data
showed that the expression level was decreased by treatment with
siRNA 2 compared with siRNAs 1 and 3.
system (Bio-Rad). Purified cDNAs encoding HNF4
a
and glyceraldehyde
3-phosphate dehydrogenase (GAPDH) were amplified using THUNDERBIRD
These results suggest that only one modification in the 30-over-
hang region is effective enough to increase siRNA activity. In the
experiment conducted to determine nuclease-resistant property,
the 30-terminal modification was found to confer nuclease resis-
tance to siRNA. Modification of two nucleosides in the 30-overhang
region resulted in same or slightly less RNAi activity. Modification
of the second nucleoside in the 30-overhang region might lead to
poor recognition of mRNA.
SYBR qPCR Mix (TOYOBO) with specific primers (HNF4a
: 50-CAGGCTCAAG
AAATGCTTCC-30 and 50-GGCTGCTGTCCTCATAGCTT-30; GAPDH: 50-CAATGACC
CCTTCATTGACC-30 and 50-GACAAGCTTCCCGTTCTCAG-30). PCR was performed at
95 °C for 1 min followed by 40 cycles of 15 s at 95 °C and 1 min at 60 °C. For each
sample, the mean threshold cycle (Ct) from two replicate PCR using RNA isolated
from independent cells was taken. Expression levels of HNF4
a mRNA were
normalized to GAPDH by the DDCt method.