RNA interference in mammalian cells by siRNAs modified with morpholino nucleoside analogues

Article abstract of DOI:10.1016/j.bmc.2009.02.001

siRNAs modified with morpholino nucleoside analogues were synthesized and their biological properties were examined in details. The gene silence abilities of modified siRNAs were correlated to the positions of the modifications, some of which appeared to

Full text of DOI:10.1016/j.bmc.2009.02.001

Bioorganic & Medicinal Chemistry 17 (2009) 2441–2446
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
RNA interference in mammalian cells by siRNAs modified with morpholino
nucleoside analogues
Nan Zhang a,b, Chunyan Tan b, Puqin Cai b, Peizhuo Zhang c, Yufen Zhao a, Yuyang Jiang b,d,
*
a Key Laboratory of Bioorganic Phosphorus Chemistry and Chemical Biology, Ministry of Education, Department of Chemistry, Tsinghua University, Beijing 100084, PR China
b Key Laboratory of Chemical Biology, Guangdong Province, Graduate School at Shenzhen, Tsinghua University, Shenzhen 518055, PR China
c Shanghai GenePharma Co., Ltd, 1011 Halley Road, Z.-J. High Tech Park, Shanghai 201203, PR China
d School of Medicine, Tsinghua University, Beijing 100084, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
siRNAs modified with morpholino nucleoside analogues were synthesized and their biological properties
were examined in details. The gene silence abilities of modified siRNAs were correlated to the positions of
the modifications, some of which appeared to be more potent than the native siRNA. The 30-end modifi-
cation improved the stability of siRNAs in serum significantly. Furthermore, the dose–response and time-
course experiments demonstrated that the siRNAs with 30-end modification have potent gene silence
activity at lower concentration and prolonged action time. These favorable properties make the morpho-
lino modified siRNA a potentially useful tool in therapeutic applications.
Received 27 November 2008
Revised 31 January 2009
Accepted 2 February 2009
Available online 8 February 2009
Keywords:
RNA interference
Small interfering RNA (siRNA)
Morpholino nucleoside analogues
Chemical modification
Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction
thesis, bioactivity evaluation and the structure-and-activity rela-
tionship of a series of morpholino modified siRNAs.
Small interfering RNAs (siRNAs) are a class of short double-
stranded RNA molecules involved in the RNA interference (RNAi)
pathway, which leads to the target mRNA degradation.1–7 Although
siRNAs have become powerful tools in biological research,8–11 their
applications as therapeutic agents were partially limited by their
poor stability against nucleases.12 Therefore, chemical modification
of synthetic siRNAs to improve nuclease stability, potency, specific-
ity and in vivo cellular delivery emerged as an active research area.
Many chemical modifications have been reported (reviewed in Refs.
13–15), such as thiophosphate,16 boranophosphate,17 40-thio,12,18
locked nucleic acid (LNA),19 20-alkyl20 and 20-deoxy-20-fluoro.20,21
Morpholino oligonucleotides with morpholino rings in subunits
instead of the ribose have become one of the promising candidates
for biological research, because they inhibit the translation of tar-
get mRNA by steric blocking and have high resistance to enzymatic
degradations.22 In our previous work, we have synthesized and
investigated the properties of chimeric oligonucleotides containing
morpholino thymidine analogues.23 These modified oligonucleo-
tides have shown moderate thermal stability with complementary
RNA and DNA, as well as enhanced resistance towards nucleases.
The favorable properties prompted us to further evaluate the gene
silence activity of such modified siRNAs. Here we report the syn-
2. Result and discussion
2.1. Chemistry
Synthesis of the building block, phosphoramidite uridine nucle-
oside monomer 6 is shown in Scheme 1 following our previous
procedure.23 Briefly, uridine (1) was first treated with DMTr–Cl in
anhydrous pyridine for overnight under argon atmosphere to af-
ford 50-O-DMTr-uridine (2) in 83% yield, which was then subjected
to the construction of the morpholino ring in one step by treating it
with sodium periodate and then ammonium biborate to afford
20,30-dihydroxyl-morpholino-uridine (4). Reduction of 20 and 30 hy-
droxyl groups on compound 4 gave compound 5.24,25 The total
yield of the above 3 steps was 78%. The building block monomer
6 was then obtained by phosphitylation of compound 5 under
the conditions with 2-cyanoethyl-N,N,N0,N0-tetraisopropylphos-
phorodiamidite and 4,5-dicyanoimidazole (DCI) in anhydrous
CH2Cl2 for 4 h under argon atmosphere with a total yield of 86%.
All compounds were characterized by 1H NMR, 13C NMR, 31P
NMR and HRMS.
These modified building blocks were incorporated into siRNAs
in order to test their effects on gene silence and stability. RNA oli-
gonucleotides (sequences of sense and antisense strands are
shown in Scheme 2) were synthesized on a DNA synthesizer via
* Corresponding author. Tel.: +86 755 2603 6017; fax: +86 755 2603 2094.
E-mail address: jiangyy@sz.tsinghua.edu.cn (Y. Jiang).
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.02.001

2442
N. Zhang et al. / Bioorg. Med. Chem. 17 (2009) 2441–2446
O
O
N
O
N
O
NH
NH
NH
NH
N
O
O
O
N
O
DMTrO
HO
DMTrO
(i)
(iii)
HO
DMTrO
(ii)
O
O
O
O
N
H
OH
OH OH
OH OH
O
O
4
1
2
3
O
O
NH
NH
O
(v)
(iv)
N
O
DMTrO
N
O
DMTrO
O
N
H
N
P
CN
iPr2N
O
5
6
Scheme 1. Synthesis of the morpholino uridine building block. Reagents and conditions: (i) DMTr-Cl, NEt3, Py, rt, Ar; (ii) and (iii) NaIO4, (NH4)2B4O7, CH3OH, rt; (iv) NaCNBH3,
CH3OH, rt; (v) 2-cyanoethyl-N,N,N0,N0- tetraisopropylphosphorodiamidite, DCI, CH2Cl2, rt, Ar.
phosphoramidite route, using 5-ethylthio-1H-tetrazole (ETT) as
the activator in 10-min coupling time. After the removal from solid
support, crude oligonucleotides were purified by HPLC and ana-
lyzed by MALDI-TOF mass spectroscopy (Table S1 in Supplemen-
tary data). siRNA duplexes were obtained by annealing of equal
molar of sense and antisense strands, which were then purified
by HPLC.
2.2. Gene silence by modified siRNAs
To evaluate the gene silence effect of modified siRNAs, one na-
tive siRNA7 and 13 modified siRNA8–20 were analyzed for their
ability to inhibit luciferase expression in HeLa cells (Fig. 1). Cells
were co-transfected with luciferase vector and siRNA (25 nM), fol-
lowed by the luciferase activity measurement after 24 h post-
5' CUU ACG CUG AGU ACU UCG Att 3'
3 ' tt GAA UGC GAC UCA UGA AGC U5'
Native
7
5' CUU ACG CUG AGU ACU UCG AUU 3'
3' tt GAA UGC GAC UCA UGA AGC U 5'
3' modification
8
5' CUU ACG CUG AGU ACU UCG Att 3'
3' UU GAA UGC GAC UCA UGA AGC U 5'
9
5' CUU ACG CUG AGU ACU UCG AUU 3'
3' UU GAA UGC GAC UCA UGA AGC U 5
10
11
12
13
14
15
16
17
18
19
20
antisense
modification
5' CUU ACG CUG AGU ACU UCG Att 3'
3' tt GAA UGC GAC UCA UGA AGC U 5'
O
N
5' CUU ACG CUG AGU ACU UCG Att 3'
3' tt GAA UGC GAC UCA UGA AGC U 5'
O
N
NH
NH
5' CUU ACG CUG AGU ACU UCG Att 3'
3' tt GAA UGC GAC UCA UGA AGC U 5'
O
O
O
O
N
O
O
5' CUU ACG CUG AGU ACU UCG Att 3'
3' tt GAA UGC GAC UCA UGA AGC U 5'
O
P
OH
O
P
O
O-
5' CUU ACG CUG AGU ACU UCG Att 3'
3' tt GAA UGC GAC UCA UGA AGC U 5'
sense
modification
O
O-
O
O
5' CUU ACG CUG AGU ACU UCG Att 3'
3' tt GAA UGC GAC UCA UGA AGC U5'
U (Uridine)
U (Modified Uridine)
5' CUU ACG CUG AGU ACU UCG Att 3'
3' tt GAA UGC GAC UCA UGA AGC U 5'
5' CUU ACG CUG AGU ACU UCG Att 3'
3' tt GAA UGC GAC UCA UGA AGC U 5'
5' CUU ACG CUG AGU ACU UCG Att 3'
3' tt GAA UGC GAC UCA UGA AGC U 5'
5' CUU ACG CUG AGU ACU UCG Att 3'
3' tt GAA UGC GAC UCA UGA AGC U 5'
Scheme 2. Sequences and the chemical structure of morpholino modified siRNAs. siRNAs are shown with the sense strand above (50–30) and the antisense strand below (30–
50). RNA is in uppercase letter, DNA is in lowercase letter, and modification is in uppercase letter with underline.

N. Zhang et al. / Bioorg. Med. Chem. 17 (2009) 2441–2446
2443
100
80
60
40
20
0
92.5
siRNA7
87.3
siRNA9
32.8
22.8
siRNA10
17.7
16.3
15.2
14.7
14.3
8
11.8
12.5
7
11.6
10.5
9
8.9
100
80
60
40
20
0
10 11 12 13 14 15 16 17 18 19 20 21
siRNA
Figure 1. Effects of the morpholino modification on silencing activity. Luciferase
activity was assessed according to the Dual-Luciferase Reporter Assay protocol.
transfection. The native siRNA7 inhibited the activity of luciferase
to about 12.5%, while as a comparison, some modified siRNAs ap-
peared to have comparable or better inhibition effect and others
showed decreased activity. The results suggest that the silencing
activities of siRNAs correlated with the positions of the modifica-
tions as described in the following structure-and-relationships:
(1) siRNAs with the sense strand modification at different positions
(siRNA15–20) demonstrated good inhibition effect with the lucif-
erase activity in the range of 11.6–17.7%, which is similar as the na-
tive siRNA7. In particular, siRNA15 which has the morpholino
uridine modified at the 50 end appeared to be the most potent,
whereas siRNA17 and 18 which have the modification in the mid-
dle of the strands were a little less potent than the native siRNA7.
This result is consistent with other reports.19–21 (2) Modification on
the 30-overhangs in either antisense or both sense and antisense
strands revealed better inhibitory activity compared to siRNA7.
Specifically, siRNA9 and siRNA10 showed more potent effect than
siRNA7. (3) Modification on the antisense strand led to a significant
decrease of inhibitory activity, especially for siRNA11 and 13. It
was reported that the cleavage site of the antisense strand is be-
tween position 10 and 11 starting from 50-terminal.26 siRNA13
with modification at the cleavage site showed significant loss of
gene silencing activity. Phosphorylation at antisense strand 50
end is crucial for siRNA function.27 Our finding showed that siR-
NA11 with modification at antisense strand 50 end significantly re-
duced the inhibitory activity, due to a lack of 50 phosphorylation.
Overall speaking, the sense strand was shown to have better toler-
ance of chemical modification, which is in agreement with other
reports.19–21
0
200
400
600
Time / min
Figure 2. Stability of siRNA7 (j), siRNA9 (d) and siRNA10 (N) in 10% fetal bovine
serum at 37 °C. siRNAs were taken at indicated time points, separated by PAGE and
stained with GelRed.
Table 1
Half-livesa and IC50 values of siRNA7–10
Compound
Half-life (min)
IC50 (nM)
siRNA7
siRNA8
siRNA9
siRNA10
45
—
200
300
8.1
8.3
5.1
4.6
a
Duplexes of siRNA (2 nmol) were incubated at 37 °C in 10% fetal bovine serum
(Hyclone) diluted in phosphate buffered saline (PBS) for the corresponding time.
of siRNA9 than siRNA10 indicate that the 30 end modification on
both sense and antisense strand could increase the stability against
exonucleases in serum.
2.3. Bio-stability in serum
2.4. Dose–response and time–response
Bio-stability is crucial for therapeutic application of siRNAs. The
stability of modified siRNAs was evaluated using siRNA7 as a con-
trol in serum. Based on the fact that native siRNAs degrade primar-
ily via 30 exonuclease in serum,21 it is reasonable to propose that
the protection at 30 end would prevent siRNA from degradation.
siRNA9 and 10 with morpholino modification at 30 end were cho-
sen, because they showed improving gene inhibition effect com-
pared to the native siRNA. As shown in Figure 2a and b, native
siRNA7 degraded rapidly with a half-life time at about 45 min,
while on the contrary, both modified siRNA9 and 10 showed signif-
icantly enhanced resistance to degradation with half lives at about
200 and 300 min, respectively (Table 1). The longer half-life times
Dose–response of modified siRNAs 8–10 was compared with
the native siRNA7 (Fig. 3). Generally speaking, the dose–response
curves of siRNAs 8–10 are similar to that of the native siRNA7.
All siRNAs demonstrated good inhibition effect at the concentra-
tion of 10 nM, and all siRNAs showed enhanced inhibition effect
as the concentration increased and reached to the maximum activ-
ity when the concentration is about 50 nM. This result indicated
that the modified siRNAs inhibited the luciferase activity through
RNAi mechanism instead of other non-specific effects. Further-
more, IC50 values were obtained from the dose–response experi-

2444
N. Zhang et al. / Bioorg. Med. Chem. 17 (2009) 2441–2446
exonucleases in serum. The results suggest that morpholino mod-
ified siRNAs might find further applications in the development of
synthetic siRNAs in gene therapy.
100
80
60
40
20
0
4. Experimental
4.1. Chemistry
1H NMR and 13C NMR were recorded in the indicated deuter-
ated solvent at 300 MHz JEOL NMR spectrometers. Chemical shifts
are expressed in ppm (d) and coupling constants J in Hertz (Hz).
High-resolution mass spectra were obtained with Waters micro-
mass Q-Tof Premier mass spectrometer. Reaction progress was
monitored using analytical thin-layer chromatography (TLC) on
pre-coated silica gel plates and the spots were detected under
UV light (254 nm). All other reagents and analytical grade solvents
were bought from commercial sources and used without further
purification unless indicated. Methylene chloride, pyridine and tri-
ethylamine were distilled from calcium hydride (CaH2) prior to
use.
0
20
40
60
80
100
[siRNA] / nM
Figure 3. siRNA dose–response as measured by the decrease in luciferase activity.
The native siRNA7 (j) and morpholino modified siRNA8 (d), siRNA9 (N) and
siRNA10 (.). Data was from three independent experiments.
4.1.1. Synthesis of 50-O-DMTr-uridine (2)
Uridine (1) (2.44 g, 10 mmol) was co-evaporated three times
with anhydrous pyridine. Then it was dissolved in anhydrous pyr-
idine (50 ml), and 4,40-dimethoxytrityl chloride (5.0 g, 15 mmol),
anhydrous triethylamine (2.1 ml, 15 mmol) were added. The mix-
ture was stirred overnight at room temperature under argon atmo-
sphere. 5% NaHCO3 solution (50 ml) was added and extracted with
CH2Cl2 (3 ꢀ 50 ml). The organic phase was washed with brine
(30 ml), dried over anhydrous Na2SO4, filtered and evaporated.
The residue was purified by column chromatography (CHCl3–
CH3OH = 15:1) to afford 2 (4.43 g, 8.1 mmol, 81%).
blank
siRNA 7
siRNA10
120
100
80
60
40
20
0
1H NMR(300 MHz, CDCl3): 3.49 (m, 2H, 50-CH2), 3.75 (s, 6H,
OCH3), 3.85 (s, 1H, OH), 4.17 (m, 1H, 40-CH), 4.37 (m, 1H, 30-CH),
4.42 (m, 1H, 20-CH), 5.34 (d, 1H, 5=CH), 5.50 (s, 1H, –OH), 5.92
(d, 1H, 10-CH), 6.79–6.82 (m, 4H, ArH), 7.16–7.39 (m, 9H, ArH),
7.97 (d, 1H, 6=CH), 10.5 (s, 1H, 3-NH).
13C NMR (75 MHz, CDCl3): 55.3 (OCH3), 62.1 (C50), 69.9 (C20),
75.4 (C30), 83.6 (C40), 87.1 (DMTr), 90.2 (C10), 13.4, 127.2–130.3,
135.3, 135.5, 144.5, 158.7 (DMTr), 102.4 (C5), 140.7 (C6), 151.4
(C2), 164.2 (C1).
1
3
5
7
9
Time / day
ESI-HRMS [M+Na]+ m/z Calcd for C30H30N2O8Na: 569.1900,
Found: 569.1896.
Figure 4. Time-course experiment of siRNA7 and siRNA10. Luciferase activity was
measured after 1, 3, 5, and 9 days post-transfection. Data was from three
independent experiments.
7
4.1.2. Synthesis of 50-O-DMTr-morpholino uridine (5)
50-O-DMTr-uridine (2) (2.73 g, 5 mmol) was dissolved in 90 ml
methanol. Sodium periodate (1.1 g, 5.1 mmol) and ammonium bib-
orate (1.3 g, 5.7 mmol) were added with stirring. After stirring for
2.5 h at room temperature, the mixture was filtered and sodium
cyanoborohydride (0.43 g, 6.8 mmol) was added to the filtrate.
After 15 min, another portion of sodium cyanoborohydride
(0.11 g, 1.7 mmol) was added. The reaction mixture was stirred
for 6 h at room temperature, followed by evaporation. The residue
was dissolved in ethyl acetate (100 ml), washed with brine
(3 ꢀ 50 ml). The organic phase was dried over Na2SO4, filtered
and evaporated. The residue was purified by column chromatogra-
phy (CHCl3–CH3OH = 20:1) to afford 5 (2.01 g, 3.8 mmol, 76% in
three steps).
ments (Table 1). As a result, siRNA9 and siRNA10 have IC50 values
at 5.1 and 4.6 nM, respectively, which are lower than that of siR-
NA7 (8.1 nM).
Time-course experiments to measure luciferase activity at dif-
ferent days after transfection were carried out (Fig. 4). During the
complete time-course, the most potent siRNA10 showed better
inhibition effect compared to the native siRNA7. Even at day 9
when the luciferase activity was not inhibited at all for the native
siRNA7 treated sample, the modified siRNA10 treated sample still
showed a 15% luciferase inhibition. The longer effective time of
the modified siRNA might be due to its better stability.
1H NMR (300 MHz, CDCl3): 2.56–2.62 (m, 2H, 20-CHax, 30-CHax),
3.00–3.09 (m, 2H, 30-CHeq, 50-CH), 3.22 (m, 2H, 20-CHeq, 70-CH), 3.78
(s, 6H, OCH3), 3.97 (m, 1H, 40-CH), 5.71 (d, 1H, 10-CH), 5.75 (d,
J = 8.25 Hz, 1H, 5=CH), 6.79-6.84 (m, 4H, ArH), 7.26–7.45 (m, 9H,
ArH), 7.48 (d, J = 8.25 Hz, 1H, 6=CH).
3. Conclusions
In conclusion, we synthesized a series of siRNAs containing
morpholino modified uridine analogues. It was found that the siR-
NA with morpholino modification at 30 end in both sense and anti-
sense strand appeared to be most potent and most stable against
13C NMR (300 MHz, CDCl3): 47.1 (C30), 49.4 (C20), 55.3 (OCH3),
64.5 (C50), 78.2 (C40), 80.9 (C10), 86.2 (DMTr), 102.5 (C5), 113.2,

N. Zhang et al. / Bioorg. Med. Chem. 17 (2009) 2441–2446
2445
127.0–130.1, 135.8, 135.9, 144.8, 158.6 (DMTr), 139.8 (C6), 150.1
(C2), 163.2 (C4).
All transient transfections were performed using Lipofectamine
2000 (Invitrogen) with a 20:1 (pmol: l) ratio of siRNA and Lipo-
l
ESI-HRMS [M+Na]+ m/z Calcd for C30H31N3O6Na: 552.2111.
Found: 552.2134.
fectamine 2000. 2 ꢀ 105 cells per well were seeded in 12-well
plates for 24 h before transfection. Each well of cells were transfec-
ted with 100 ng of pRL-SV40 (as transfection control plasmid
which contains Renilla luciferase gene) and pGL3-control (as re-
porter vector which contains Firefly luciferase gene) and different
quantity of siRNA duplexes. Twenty-four hours or longer times
post-transfection, the media were aspirated and cells were washed
4.1.3. Synthesis of 50-O-DMTr-morpholino uridine-N-[(2-cyanoethyl)
N,N-diisopropyl] phosphoramidite (6)
50-O-DMTr-morpholino uridine (5) (1.6 g, 3 mmol) was dried in
vacuum overnight. It was dissolved in anhydrous CH2Cl2 (30 ml)
and
2-cyanoethyl-N,N,N0,N0-tetraisopropyl-phosphorodiamidite
twice with 1 ꢀ PBS. The PBS was aspirated and 250
ll of passive ly-
(1.1 ml), 4,5-dicyanoimidazole (DCI, 11 mg, 1.5 mmol) were added
to the solution. The reaction was allowed to stir for 4 h under argon
atmosphere, then diluted with CH2Cl2 (20 ml), washed with 5%
NaHCO3 solution (30 ml) and brine (3 ꢀ 30 ml). The organic phase
was dried over Na2SO4, filtered and evaporated. The residue was
purified by column chromatography (n-hexane–ethyl ace-
tate = 1:1, with 1% triethylamine) to afford 6 (2.0 g, 2.7 mmol, 90%).
31P NMR (300 MHz, CDCl3): d = 126.1, 127.9 (two isomers, 1:1).
ESI-HRMS [M+Na]+ m/z Calcd for C30H31N5O7PNa: 752.3189.
Found: 752.3209; [M+H+NEt3]+ m/z Calcd for C45H64N6O7P:
831.4574. Found: 831.4571.
sis buffer (Promega) was added and left for 15 min. Lysed cells
were transferred to a 96-well assay plate for luciferase activity
assay.
4.3.3. Luciferase activity assay
Luciferase activity was assessed according to the Dual-Lucifer-
ase Reporter Assay protocol (Promega) using a NovoSTAR 96-well
format luminometer with substrate dispenser. A 10
ll sample were
placed in each well of a 96 well plate subsequent to which 50
ll
Luciferase Assay Reagent II (substrate for firefly luciferase) was
added to each well by the luminometer and the firefly luciferase
activity was measured. Then 50 ll Stop and Glow reagents (stop
4.2. Synthesis, deprotection and purification of siRNA
solution for firefly luciferase and substrate for Renilla luciferase)
were added and Renilla luciferase activity was measured. The
mean values of the luciferase activities measured for 10 s (100
reading) each were used to calculate ratios between firefly and
Renilla luciferase.
Single strand RNAs were synthesized on ABI 3900 DNA Syn-
thesizer at 0.1 lmol scale, using phosphoramidite approach.
0.1 M solution of phosphoramidate 6 in anhydrous acetonitrile
was used for synthesis of modified oligonucleotides, and 5-eth-
ylthio-1H-tetrazole (ETT) was used as an activator. When
Acknowledgments
phosphoramidate
6 was incorporated into oligonucleotides,
two portions of phosphoramidate solutions were delivered fol-
lowed by 15 min coupling time. Oligonucleotides with modifi-
cations in the 30-terminal ends were synthesized using
universal CPG.
The authors would like to thank the financial supports from the
Ministry of Science and Technology of China (2007AA02Z160), the
Chinese National Natural Science Foundation (20672068,
20872077, 90813013), the Ministry of Education (20070003077).
Oligonucleotides were removed from CPG using aqueous
ammonia at 55 °C for 12 h, and the protecting groups was removed
at the same time. Crude oligonucleotides were purified by HPLC
(LiChrosphere 100, RP-18, 4.6 ꢀ 250 mm, flow 1 ml/min, eluent
A: water, eluent B: acetonitrile, gradient started from
A:B = 100:0, ended with A:B = 30:70 over 40 min). Oligonucleo-
tides were analyzed by HPLC and mass spectroscopy.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2009.02.001.
References and notes
siRNA duplexes were obtained by annealing of equimolar
amounts of sense and antisense strands. The sense and antisense
strands were mixed in 10 mM sodium phosphate and 100 mM
1. Matranga, C.; Tomari, Y.; Shin, C.; Bartel, D. P.; Zamore, P. D. Cell 2005, 123, 607.
2. Rand, T. A.; Petersen, S.; Du, F. H.; Wang, X. D. Cell 2005, 123, 621.
3. Elbashir, S. M.; Harborth, J.; Lendeckel, W.; Yalcin, A.; Weber, K.; Tuschl, T.
Nature 2001, 411, 494.
4. Meister, G.; Tuschl, T. Nature 2004, 431, 343.
5. Martinez, J.; Patkaniowska, A.; Urlaub, H.; Luhrmann, R.; Tuschl, T. Cell 2002,
110, 563.
NaCl buffer (pH 7.2), with a concentration of 2 lM for each strand.
The mixture was heated to 90 °C for 15 min and then cooled slowly
to room temperature. The annealed duplex samples were stored at
4 °C.
6. Hammond, S. M.; Bernstein, E.; Beach, D.; Hannon, G. J. Nature 2000, 404, 293.
7. Mello, C. C.; Conte, D. Nature 2004, 431, 338.
8. Pushparaj, P. N.; Melendez, A. J. Clin. Exp. Pharmacol. Physiol. 2006, 33, 504.
9. Ryther, R. C. C.; Flynt, A. S.; Phillips, J. A.; Patton, J. G. Gene Ther. 2005, 12, 5.
10. Jones, S. W.; de Souza, P. M.; Lindsay, M. A. Curr. Opin. Pharmacol. 2004, 4, 522.
11. Soutschek, J.; Akinc, A.; Bramlage, B.; Charisse, K.; Constien, R.; Donoghue, M.;
Elbashir, S.; Geick, A.; Hadwiger, P.; Harborth, J.; John, M.; Kesavan, V.; Lavine,
G.; Pandey, R. K.; Racie, T.; Rajeev, K. G.; Rohl, I.; Toudjarska, I.; Wang, G.;
Wuschko, S.; Bumcrot, D.; Koteliansky, V.; Limmer, S.; Manoharan, M.;
Vornlocher, H. P. Nature 2004, 432, 173.
12. Dande, P.; Prakash, T. P.; Sioufi, N.; Gaus, H.; Jarres, R.; Berdeja, A.; Swayze, E.
E.; Griffey, R. H.; Bhat, B. J. Med. Chem. 2006, 49, 1624.
13. Nawrot, B.; Sipa, K. Curr. Top. Med. Chem. 2006, 6, 913.
14. Prakash, T. P.; Bhat, B. Curr. Top. Med. Chem. 2007, 7, 641.
15. Koizumi, M. Curr. Top. Med. Chem. 2007, 7, 661.
4.3. Biology
4.3.1. Serum stability
Duplexes of siRNA (2 nmol) were incubated at 37 °C in 10% fetal
bovine serum (Hyclone) diluted in phosphate buffered saline (PBS)
for the corresponding time. After incubation, samples were imme-
diately frozen in 1.5 ꢀ TBE-loading buffer. Samples were subjected
to electrophoresis in 15% polyacrylamide-TBE under non-denatur-
ing conditions and visualized by staining with Gel Red (Biotium)
and quantified by density measurement using the software of GEL-
WORKS 4.0.
16. Li, Z. Y.; Mao, H. B.; Kallick, D. A.; Gorenstein, D. G. Biochem. Biophys. Res.
Commun. 2005, 329, 1026.
17. Hall, A. H. S.; Wan, J.; Shaughnessy, E. E.; Shaw, B. R.; Alexander, K. A. Nucleic
Acids Res. 2004, 32, 5991.
18. Hoshika, S.; Minakawa, N.; Kamiya, H.; Harashima, H.; Matsuda, A. Febs Lett.
2005, 579, 3115.
19. Elmen, J.; Thonberg, H.; Ljungberg, K.; Frieden, M.; Westergaard, M.; Xu, Y. H.;
Wahren, B.; Liang, Z. C.; Urum, H.; Koch, T.; Wahlestedt, C. Nucleic Acids Res.
2005, 33, 439.
4.3.2. Cell culture and transfection
Hela cells were cultured in DMEM (Dulbecco0s modified Eagle0s
medium; pH 7.2, Invitrogen) supplemented with 10% (v/v) fetal
calf serum (Hyclone) and 0.5% penicillin–streptomycin. Cells were
grown at 37 °C with 5% CO2.

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N. Zhang et al. / Bioorg. Med. Chem. 17 (2009) 2441–2446
20. Prakash, T. P.; Allerson, C. R.; Dande, P.; Vickers, T. A.; Sioufi, N.; Jarres, R.;
Baker, B. F.; Swayze, E. E.; Griffey, R. H.; Bhat, B. J. Med. Chem. 2005, 48,
4247.
21. Dowler, T.; Bergeron, D.; Tedeschi, A. L.; Paquet, L.; Ferrari, N.; Damha, M. J.
Nucleic Acids Res. 2006, 34, 1669.
23. Zhang, N.; Tan, C.; Cai, P.; Jiang, Y.; Zhang, P.; Zhao, Y. Tetrahedron Lett. 2008, 49,
3570.
24. Jones, A. S.; Walker, R. T. Carbohydr. Res. 1973, 26, 255.
25. Hansske, F.; Sprinzl, M.; Cramer, F. Bioorg. Chem. 1974, 3, 367.
26. Schwarz, D. S.; Tomari, Y.; Zamore, P. D. Curr. Bio. 2004, 14, 787.
27. Nykanen, A.; Haley, B.; Zamore, P. D. Cell 2001, 107, 309.
22. Summerton, J. Biochim. Biophys. Acta Gene Struct. Expr. 1999, 1489, 141.