Practical and Reliable Synthesis of 1,2-Dideoxy-D-ribofuranose and its Application in RNAi Studies

Article abstract of DOI:10.1080/15257770.2015.1114128

We developed a practical and reliable method for synthesizing an abasic deoxyribonucleoside, 1,2-dideoxy-D-ribofuranose (dR^H) via elimination of nucleobase from thymidine. To synthesize oligonucleotides bearing dR^H by the standard ph

Full text of DOI:10.1080/15257770.2015.1114128

Nucleosides, Nucleotides and Nucleic Acids
ISSN: 1525-7770 (Print) 1532-2335 (Online) Journal homepage: http://www.tandfonline.com/loi/lncn20
Practical and Reliable Synthesis of 1,2-Dideoxy-d-
ribofuranose and its Application in RNAi Studies
Yuki Nagaya, Yoshiaki Kitamura, Remi Nakashima, Aya Shibata, Masato
Ikeda & Yukio Kitade
To cite this article: Yuki Nagaya, Yoshiaki Kitamura, Remi Nakashima, Aya Shibata, Masato
Ikeda & Yukio Kitade (2016): Practical and Reliable Synthesis of 1,2-Dideoxy-d-ribofuranose
and its Application in RNAi Studies, Nucleosides, Nucleotides and Nucleic Acids, DOI:
10.1080/15257770.2015.1114128
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Date: 31 January 2016, At: 08:09

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
, VOL. , NO. , –
http://dx.doi.org/./..
Practical and Reliable Synthesis of
,-Dideoxy-D-ribofuranose and its Application in RNAi
Studies
Yuki Nagayaa, Yoshiaki Kitamurab,c, Remi Nakashimab, Aya Shibatab,c
Masato Ikedaa,b,c, and Yukio Kitadea,b,c
,
aUnited Graduate School of Drug Discovery and Medical Information Sciences, Gifu University, -
Yanagido, Gifu, Japan; bDepartment of Biomolecular Science, Graduate School of Engineering, Gifu
University, - Yanagido, Gifu, Japan; cDepartment of Chemistry and Biomolecular Science, Faculty of
Engineering, Gifu University, - Yanagido, Gifu, Japan
ABSTRACT
ARTICLE HISTORY
Received  May 
Accepted  October 
We developed a practical and reliable method for synthesizing an
abasic deoxyribonucleoside, 1,2-dideoxy-D-ribofuranose (dRH) via
elimination of nucleobase from thymidine. To synthesize oligonu-
cleotides bearing dRH by the standard phosphoramidite solid-
phase method, dRH was converted to the corresponding phos-
phoramidite derivative and linked to a solid support (controlled
pore glass resin). Chemically modified small interfering RNAs (siR-
NAs) possessing dRH at their 3ꢀ-overhang regions were synthe-
sized. Introducing dRH to the 3ꢀ-end of the antisense strand of
siRNA reduced its knockdown effect.
KEYWORDS
Abasic nucleoside; glycal;
oligonucleotide; siRNA; RNAi
Introduction
The abasic deoxyribonucleoside 1,2-dideoxy-d-ribofuranose (dRH; 1) is a struc-
tural analogue of sugar residue at natural apurinic/apyrimidinic (AP) sites, which
results from the hydrolysis of the N-glycosidic bond of nucleotides in DNA. Sev-
eral oligonucleotides (ONs) containing dRH have been synthesized to study DNA
repair mechanisms[1–5], catalytic activity of ribozymes[6–9], and RNA interference-
based therapy.[10,11] An AP site containing dRH provides a unique hydrophobic
binding pocket in DNA duplex.[12] Several researchers have developed functional
probes bearing dRH by exploiting its chemical properties.[12–14] In addition, Kool
et al. reported various oligodeoxy-fluoroside sensors incorporating dRH as a non-
fluorescent spacer,[15,16] and dRH is a well-known building block for synthesizing
carbapenem antibiotics.[17,18]
Several synthetic approaches to dRH have been developed (see Figure 1), includ-
ing the reduction of 2-deoxy-d-ribose (a)[2,19] or its furanosyl derivatives, such as
furanosyl chloride (b),[1,20,21] methyl furanoside (b),[1,22,23] thiofuranoside (c),[24]
CONTACT Yukio Kitade
ykkitade@gifu-u.ac.jp .
Engineering, Gifu University, - Yanagido, Gifu -, Japan.
Department of Chemistry and Biomolecular Science, Faculty of
©  Taylor & Francis Group, LLC

2
Y. NAGAYA ET AL.
Figure . Approaches for the synthesis of dRH.
and furanose erythro-glycals (furanoid glycals) (d).[25,26] However, most of the
existing protocols are not practical. Chenault et al. synthesized dRH from 2-deoxy-
d-ribose (a) in high yield, although an excess of ion exchange resin is required to
obtain dRH.[23] Beigelman and co-workers reported a simple synthetic method for
dRH precursors via a furanoid glycal, but preparation of the furanoid glycal was
challenging and derivation into dRH was not reported.[27] Ferrero et al. described
a biocatalytic procedure for preparing dRH precursors from readily available mate-
rials that is useful in ON synthesis, but the protocol requires multiple steps.[28] To
study several ONs containing dRH for ON-based therapeutics, a practical and reli-
able synthesis of dRH is needed.
Here we describe a practical and reliable procedure for synthesizing dRH (1)
from 5ꢀ-O-silylated thymidine via the corresponding furanoid glycal by elimina-
tion of nucleobase (Scheme 1). We also studied the biological properties of small
interfering RNAs (siRNAs) bearing dRH dimer instead of the natural nucleotides at
the 3ꢀ-overhang region of siRNAs.
Results and discussion
Synthesis of 1,2-dideoxy-D-ribofuranose and its derivatives
First, 5ꢀ-O-(tert-butyldiphenylsilyl)thymidine (2),[25] which can be easily prepared
from thymidine, was reacted with ammonium sulfate ((NH4)2SO4) in 1,1,1,3,3,3
-hexamethyldisilazane (HMDS) under reflux to give the corresponding 3,5-O-
disilylglycal 3. Although Pedersen’s protocol[25] gave the corresponding furan
derivative 3ꢀ[29] as a major product rather than the desired glycal 3, optimization
of concentration, amount of (NH4)2SO4, and reaction time provided the desired
glycal 3 almost quantitatively. Subsequently, the trimethylsilyl (TMS) group at the
3-position was removed with potassium carbonate to produce 5-mono-silylated gly-
cal 4. Han and Lowary reported obtaining 5 in 87% yield from 4 with Pd/C-catalyzed
hydrogenation in MeOH;[30] in contrast, we found that hydrogenation of olefin in
4 under the above conditions gave the desired 5-O-TBDPS-dRH 5 in 36% yield,
along with a tetrahydrofuran derivative 5ꢀ[29] (31%) produced from the Ferrier
rearrangement[31] followed by hydrogenation of resulting olefin. We could

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
3
completely suppress the side reaction by using i-PrOH, a bulkier alcohol as sol-
vent, and could isolate 5 (95% yield from 2) as a sole product. Finally, desi-
lylation by treatment with tetra-n-butylammonium fluoride (TBAF) provided 1
quantitatively.
For incorporation of 1 into ONs by the standard phosphoramidite solid-
phase method, 1 was converted to the corresponding phosphoramidite deriva-
tive 7 and the solid support 8 on controlled pore glass (CPG). Treatment of
1 with 4,4ꢀ-dimethoxytrityl (DMTr) chloride in DMF/pyridine gave the corre-
sponding 5-DMTr derivative 6, which was phosphorylated with 2-cyanoethyl N,N-
diisopropylchlorophosphoramidite to produce 7 quantitatively. Compound 6 was
succinated, and the resulting succinate was linked with CPG to generate solid sup-
port 8 (54.5 μmol/g).
RNA interfering (RNAi) study
Small interfering RNAs inhibit gene suppression by RNAi and thus hold great
promise as nucleic acid drugs. We have reported the synthesis and biological prop-
erties of siRNAs chemically modified at their 3ꢀ-overhang regions.[32–37] In an RNAi
study, an overhang of two thymidine residues (dTdT) at the 3ꢀ-end of siRNA is gener-
ally used for nuclease protection. Here we synthesized siRNA incorporating an aba-
sic deoxyribonucleoside (dRH) dimer at this position to investigate the effects of lack
of nucleobase moiety at the two nucleotides of the 3ꢀ-overhang region (Figure 2).
Scheme . Reagents and conditions: (a) (NH)SO, HMDS, reflux; (b) KCO, MeOH, rt; (c) H, Pd/C, i-
PrOH, rt, %; (d) TBAF, THF, rt, %; (e) DMTrCl, pyridine, rt, quant; (f) (i-PrN)P(Cl)O(CH)CN, i-PrNEt,
THF, rt, quant; (g) (i) succinic anhydride, DMAP, pyridine, rt; (ii) CPG, EDC·HCl, DMF, rt, . μmol/g.

4
Y. NAGAYA ET AL.
Figure . Structures of modified siRNAs.
Oligonucleotide synthesis
Using 7 and 8, ONs containing dRH were synthesized using a DNA/RNA
synthesizer (Table 1). The fully protected ONs linked to 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 puri-
fied by denaturing 20% polyacrylamide gel electrophoresis (PAGE) to isolate the
deprotected ONs bearing abasic deoxyribonucleoside dRH. These ONs were ana-
lyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrom-
etry (MALDI-TOF/MS), and the observed molecular weights were in good agree-
ment with their structures.[38]
Table . Sequences of ONs and siRNAs used in this study.
No. of siRNA
No. of ON
Sequencea,b
siRNA 9
ON 13
ON 14
ꢀ-GGCCUUUCACUACUCCUACdTdT-ꢀ
ꢀ-dTdTCCGGAAAGUGAUGAGGAUG-ꢀ
siRNA 10
siRNA 11
siRNA 12
ON 15
ON 16
ꢀꢀ-GGCCUUUCACUACUCCUACdRHdRH-ꢀ
 -dRHdRHCCGGAAAGUGAUGAGGAUG-ꢀ
ON 13
ON 16
ꢀꢀ-GGCCUUUCACUACUCCUACdTdT-ꢀ
 -dRHdRHCCGGAAAGUGAUGAGGAUG-ꢀ
ON 15
ON 14
ꢀ-GGCCUUUCACUACUCCUACdRHdRH-ꢀ
ꢀ-dTdTCCGGAAAGUGAUGAGGAUG-ꢀ
ON 17
ON 18
F-ꢀꢀ-GUAGGAGUAGUGAAAGGCCdTdT-ꢀ
F- -GUAGGAGUAGUGAAAGGCCdRHdRH-ꢀ
aCapital letters indicate ribonucleosides.
bF denotes fluorescein.

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
5
Figure . Dual-luciferase assay.
Dual-luciferase assay
The silencing activity of siRNAs 9–12 was examined by a dual-reporter assay
using a psiCHECK-2 vector in HeLa cells. This vector contains the Renilla and
firefly luciferase genes, and the siRNA sequences were designed to target the Renilla
luciferase gene. HeLa cells were co-transfected with the vector and the indicated
amount of siRNAs. The signal levels of Renilla luciferase were normalized by the
signal levels of firefly luciferase. All siRNAs effectively reduced luciferase activity in
a dose-dependent manner (Figure 3). The silencing activity of siRNA 10 with dRH
dimers was slightly less than that of siRNA 9, which possesses natural thymidines,
at 0.1 and 10 nM. Of siRNAs 9–12, the lowest knockdown activity was exhibited by
scrambled siRNA 11, which possesses dRHat the 3ꢀ-end of the antisense strand, and
the highest was exhibited by scrambled siRNA 12, which bears dRHat the 3ꢀ-end of
the sense strand.
Nuclease resistance
We next investigated the enzymatic stability of ONs against 3ꢀ-exonucleases, which
are dominant in human serum. The susceptibility of ONs to snake venom phospho-
diesterase (SVPD), which degrades ONs from their 3ꢀ-end, was examined. Unmod-
ified ON 17 and modified ON 18, labeled with fluorescein at their 5ꢀ-ends, were
incubated with SVPD, and the reactions were analyzed by PAGE under denatur-
ing conditions. The half-life (t1/2) of unmodified ON 17 was <5 min, and that of
modified ON 18 was 15 min, indicating that ON 18 carrying an dRH dimer at
its 3ꢀ-end was significantly more resistant to SVPD than was unmodified ON 17
(Figure 4).

6
Y. NAGAYA ET AL.
Figure . Nuclease resistance of ON 17 and ON 18 against SVPD.
Experimental
General methods
All reactions were carried out under an argon atmosphere, unless otherwise noted.
All reagents and solvents were purchased from commercial vendors and used
without further purification unless indicated otherwise. Pyridine was distilled over
1
CaH2 and stored over activated molecular sieves 4 A˚. H and 13C NMR spectra
were recorded on a JEOL JNM AL-400 spectrometer or JNM ECS-400 spectrom-
eter (400 MHz for 1H NMR, 100 MHz for 13C NMR, and 162 MHz for 31P NMR).
Chemical shifts (δ) were expressed in parts per million (ppm) and internally refer-
enced (7.26 ppm for CDCl3 or 3.31 for CD3OD for 1H NMR, 77.0 ppm for CDCl3 or
49.0 ppm for CD3OD for 13C NMR and 0.00 ppm for H3PO4/CDCl3 for 31P NMR).
Direct analysis in real time (DART) coupled with time-of-flight (TOF) mass spectra
were taken on a JMS T100TD instrument. Electron impact (EI) mass spectra were
taken on a Shimadzu GCMS-QP2010A instrument. Matrix-assisted laser desorp-
tion/ionization (MALDI) coupled with TOF mass spectra were taken on a Shimadzu
AXIMA-CFR plus instrument. Flash column chromatography was performed using
silica gel 60 N (spherical neutral [63–210 µm]) from Kanto Chemical Co. Inc.
5ꢀ-O-(tert-Butyldiphenylsilyl)thymidine (2)[25]
TBDPSCl (600 μL, 2.34 mmol) was added to a solution of thymidine (486 mg,
2.01 mmol) and DMAP (49.2 mg, 403 μmol) in pyridine (10 mL), and the mixture
was stirred at room temperature for 15 h. The mixture was partitioned as EtOAc and
H2O. The organic layer was washed with saturated aqueous NH4Cl solution and
brine, dried over Na2SO4, and concentrated under reduced pressure. The residue
was purified by column chromatography on silica gel (n-hexane/EtOAc, 3:1˜0:1) to
give 2 as a white solid (882 mg, 91%); 1H NMR (CDCl3) δ 9.25 (s, 1H, NH), 7.67–
7.64 (m, 4H, H-Ph), 7.50 (d, J = 0.9 Hz, 1H, H-6), 7.47–7.37 (m, 6H, H-Ph), 6.43 (dd,
J = 8.4 Hz, 5.8 Hz, 1H, H-1ꢀ), 4.58–4.56 (m, 1H, H-3ꢀ), 4.05–4.03 (m, 1H, H-4ꢀ), 3.98
(dd, J = 11.8 Hz, 2.7 Hz, 1H, H-5ꢀα), 3.85 (dd, J = 11.8 Hz, 2.7 Hz, 1H, H-5ꢀβ), 2.43

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
7
(ddd, J = 13.8 Hz, 5.8 Hz, 2.3 Hz, 1H, H-2ꢀα), 2.20 (ddd, J = 13.8 Hz, 8.4 Hz, 5.9 Hz,
1H, H-2ꢀβ), 1.62 (d, J = 0.9 Hz, 3H, CH3 of thymine), 1.08 (s, 9H, C(CH3)3); 13
C
NMR (CDCl3) δ 163.8, 150.5, 135.5, 135.3, 132.8, 132.3, 130.1, 130.0, 128.0, 127.9,
111.2, 87.1, 84.7, 72.3, 64.1, 41.0, 27.0, 19.3, 12.1; MS (DART) m/z 504 [M+Na]+,
HRMS (DART) Calcd. for C26H32N2NaO5Si [M+Na]+: 503.6180. Found: 503.6163.
1,4-Anhydro-5-O-(tert-butyldiphenyl)-2-deoxy-D-erythro-pent-1-enitol (5)[30]
A mixture of 2 (482 mg, 1.00 mmol) and (NH4)2SO4 (265 mg, 2.01 mmol) in HMDS
(8 mL) was refluxed for 1.5 h. The solvent was removed under reduced pressure, and
the residue was diluted with EtOAc and H2O. The organic layer was washed with
brine and dried over Na2SO4, and concentrated under reduced pressure. A suspen-
sion of K2CO3 (28.5 mg, 206 μmol) in MeOH (15 mL) was added to the residue
containing 3 and stirred at room temperature for 2 h. The reaction mixture was
partitioned as EtOAc and H2O. The organic layer was washed with brine, dried over
Na2SO4, and concentrated under reduced pressure. The residue was filtered through
column chromatography on silica gel (n-hexane/EtOAc, 3:1˜2:1) to give 4 as a yel-
low oil. A mixture of 4 and 10% Pd/C (15 wt%) in i-PrOH (10 mL) was vigorously
stirred at room temperature under ambient pressure of hydrogen for 24 h. The reac-
tion mixture was filtered through a celite pad, and the filtrate was evaporated under
reduced pressure. The residue was purified by column chromatography on silica gel
(n-hexane/EtOAc, 7:1˜2:1) to give 5 as a yellow oil (338 mg, 94%); 1H NMR (CDCl3)
δ 7.70–7.66 (m, 4H, H-Ph), 7.46–7.37 (m, 6H, H-Ph), 4.43–4.40 (m, 1H, H-3), 3.96
(dd, J = 8.4 Hz, 5.7 Hz, 2H, H-5), 3.85–3.82 (m, 1H, H-4), 3.78 (dd, J = 10.7 Hz,
4.3 Hz, 1H, H-1α), 3.60 (dd, J = 10.7 Hz, 6.4 Hz, 1H, H-1β), 2.20–2.10 (m, 1H,
H-2α), 1.93–1.86 (m, 1H, H-2β), 1.07 (s, 9H, C(CH3)3); 13C NMR (CDCl3) δ 135.6,
135.5, 133.2, 133.1, 129.8, 129.8, 127.7, 86.1, 77.2, 74.3, 67.1, 64.8, 34.8, 26.8, 19.2; MS
(DART) m/z 379 [M+Na]+, HRMS (DART) Calcd. for C21H28NaO3Si [M+Na]+:
379.1705. Found: 379.1750.
1,2-Dideoxy-D-ribofuranose (1)[28]
Tetra-n-butylammonium fluoride (1.0 M in THF, 660 μL) was added to a solution of
5 (215 mg, 603 μmol) in THF (2.7 mL), and the mixture was stirred at room temper-
ature for 12 h. The mixture was concentrated under reduced pressure. The residue
was purified by column chromatography on silica gel (CH2Cl2/acetone, 5:1˜2:1) to
give 1 as a yellow oil (68.8 mg, 97%); 1H NMR (CD3OD) δ 4.21–4.18 (m, 1H, H-3),
3.97–3.87 (m, 2H, H-1), 3.75–3.72 (m, 1H, H-4), 3.57–3.49 (m, 2H, H-5), 2.18–2.03
(m, 1H, H-2α), 1.88–1.81 (m, 1H, H-2β); 13C NMR (CD3OD) δ 88.1, 73.8, 68.0,
63.6, 35.9; MS (DART) m/z 141 [M+Na]+, HRMS (DART) Calcd. for C5H10NaO3
[M+Na]+: 141.1209. Found: 141.1250.

8
Y. NAGAYA ET AL.
1,2-Dideoxy-5-O-(4,4ꢀ-dimethoxytrityl)-D-ribofuranose (6)[24]
A mixture of 1 (101 mg, 853 μmol) and DMTrCl (376 mg, 1.11 mmol) in pyridine
(3.3 mL) was stirred at room temperature for 12 h. The reaction mixture was par-
titioned as CH2Cl2 and H2O. The organic layer was washed with brine, dried over
Na2SO4, and concentrated under reduced pressure. The residue was purified by col-
umn chromatography on silica gel (CH2Cl2/acetone, 20:1˜10:1) to give 7 as a yellow
oil (265 mg, quant); 1H NMR (400 MHz, CDCl3) δ 7.43 (d, J = 7.8 Hz, 2H, H-Ph),
7.36–7.16 (m, 7H, H-Ph), 6.82 (d, J = 9.2 Hz, 4H, H-Ph), 4.30–4.27 (m, 1H, H-3),
3.99–3.96 (m, 2H, H-1), 3.90–3.86 (m, 1H, H-4), 3.79 (s, 6H, OCH3), 3.23 (dd, J =
9.8 Hz, 4.8 Hz, 1H, H-5α), 3.07 (dd, J = 9.8 Hz, 6.2 Hz, 1H, H-5β), 2.19–2.10 (m,
1H, H-2α), 1.92–1.84 (m, 1H, H-2β); 13C NMR (CDCl3) δ 158.4, 144.8, 136.0, 136.0,
130.0, 128.1, 127.8, 126.8, 113.1, 86.2, 85.0, 74.6, 67.0, 64.5, 55.2, 34.8; MS (EI) m/z
420 (M+)
3-O-[(2-Cyanoethoxy)(N,N-diisopropylamino)phosphanyl]-1,2-dideoxy-5-O-
(4,4ꢀ-dimethoxytrityl)-D-ribofuranose (7)
i-Pr2NEt (730 μL, 4.18 mmol) and 2-cyanoethyl N,N-diisopropyl
chlorophosphoramidite (250 μL, 1.12 mmol) were added to a solution of 6
(292 mg, 694 mmol) in THF, and stirred at room temperature for 2 h. The reaction
mixture was partitioned as CHCl3 and saturated NaHCO3 aqueous solution. The
organic layer was washed with brine, dried over Na2SO4, and concentrated under
reduced pressure. The residue was purified by column chromatography on silica
gel (n-hexane/EtOAc, 4:1) to give 7 as a yellow oil; 31P NMR (162 MHz, CDCl3) δ
148.3, 148.6
Solid support synthesis
A mixture of 7 (102 mg, 243 μmol), succinic anhydride (112 mg, 1.12 mmol), and
DMAP (30.4 mg, 249 μmol) in pyridine (2.4 mL) was stirred for 23 h at room tem-
perature. The reaction mixture was partitioned as EtOAc and saturated NaHCO3
aqueous solution. The organic layer was washed with brine, dried over Na2SO4, and
concentrated under reduced pressure to give the corresponding succinate. Amino-
propyl controlled pore glass (546 mg, 53.5 μmol) was added to a solution of suc-
cinate and EDC·HCl (41.6 mg, 217 μmol) in DMF (5.3 mL), and the mixture was
kept for 72 h at room temperature. After the resin was washed with pyridine, a cap-
ping solution (30 mL, 0.1 M DMAP in pyridine/Ac2O, 9:1, v/v) was added and the
whole mixture was kept for 72 h at room temperature. The resin was washed with
pyridine, EtOH, MeCN, and dried in vacuo. The amount of loaded 6 to the solid
support was 54.5 μmol/g from calculation of released dimethoxytrityl cation by a
solution of 70% HClO4/EtOH, 3:2 (v/v).

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
9
Oligonucleotide synthesis
Synthesis was carried out with a DNA/RNA synthesizer (ABI Expedite 3400) by the
phosphoramidite method. Deprotection of bases and phosphates was performed in
concentrated NH4OH/EtOH, 3:1 (v/v) at room temperature for 12 h. 2ꢀ-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 a highly purified ON 15 (8), ON 16 (7), and ON 18
(12). The yields are indicated in parentheses as OD units at 260 nm starting from
1.0-μmol scale. The extinction coefficients of ONs were calculated from those of the
mononucleotides and dinucleotides according to the nearest neighbor approxima-
tion method.[38]
Mass spectrometric analyses of ONs
Spectra were obtained by MALDI-TOF/MS (negative mode). ON 15: calculated
mass, 6250.8; observed mass, 6251.0. ON 16: calculated mass, 6559.9; observed
mass, 6559.8. ON 18: calculated mass, 7098.0; observed mass, 7099.4.
Dual-luciferase assay
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). HeLa cells (4 ×
104/mL) were transferred to 96-well plates (100 μL per well) 24 h before transfec-
tion. 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 μL) of 20 ng of psi-CHECK-2 vector (Promega), the indicated amounts of
siRNAs, and 0.3 μg of TransFast in Opti-MEM I reduced-serum medium (Invit-
rogen), and incubated at 37°C. Transfection without siRNA was used as a control.
After 1 h, D-MEM (100 μL) containing 10% bovine serum 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 confirmed by at least
three independent transfection experiments with two cultures each and expressed
as an average of mean SD from three experiments.
Nuclease resistance
Each ON (300 pmol) labeled with fluorescein at the 5ꢀ-end was incubated with SVPD
(1 unit/mL) in PBS(–) (100 μL) at 37°C. At appropriate periods, aliquots (5 μL) of
the reaction mixture were separated and added to a solution of 7.0 M urea (15 μL).
The resulting mixtures were heated at 90°C for 10 min and analyzed by electrophore-
sis on 20% PAGE containing 7.0 M urea. The labeled ONs in the gel were visualized
with a lumino image analyzer.

10
Y. NAGAYA ET AL.
Conclusions
We demonstrated a practical and reliable method for preparing dRH. To synthe-
size ONs bearing dRH by the standard phosphoramidite solid phase method, the
corresponding phosphoramidite derivative and a solid support were prepared. The
silencing activity of siRNAs containing dRH in their 3ꢀ-overhang region was exam-
ined using a dual-luciferase assay. Introducing dRH to the 3ꢀ-end of the guide strand
of siRNA was found to attenuate its gene silencing effect in an in vitro dual-luciferase
experiment. It was also found that incorporating dRH into 3ꢀ-end enhances the
nuclease resistance of ONs. Further study of dRH will contribute to the development
of ON-based therapeutics.
Acknowledgment
We acknowledge the Division of Instrumental Analysis, Life Science Research Center, Gifu Uni-
versity, for the maintenance of instruments.
Funding
This work was supported by Grants-in-Aid for Scientific Research from the Japan Society for the
Promotion of Science (JSPS).
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