Incorporation of an acyclic alkynyl nucleoside analog into siRNA improves silencing activity and nuclease resistance

Article abstract of DOI:10.1016/j.bmcl.2015.04.034

In order to improve the silencing activity and nuclease resistance of small interfering RNA (siRNA), we designed and synthesized an acyclic thymidine analog containing 4-pentyne-1,2-diol instead of d-ribofuranose. The incorporation of this analog into siR

Full text of DOI:10.1016/j.bmcl.2015.04.034

Bioorganic & Medicinal Chemistry Letters 25 (2015) 2574–2578
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Incorporation of an acyclic alkynyl nucleoside analog into siRNA
improves silencing activity and nuclease resistance
Aya Ogata a, Yoshihito Ueno a,b,
⇑
a United Graduate School of Agricultural Science, Gifu University, 1-1 Yanagido, Gifu 501-1193, Japan
b 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:
Received 8 December 2014
Revised 6 April 2015
Accepted 11 April 2015
Available online 17 April 2015
In order to improve the silencing activity and nuclease resistance of small interfering RNA (siRNA), we
designed and synthesized an acyclic thymidine analog containing 4-pentyne-1,2-diol instead of
D
-ribofuranose. The incorporation of this analog into siRNAs at specific positions in the strands was found
to enhance the silencing activity of siRNAs and to increase the resistance of the siRNA to hydrolytic
degradation by a 30 exonuclease.
Ó 2015 Elsevier Ltd. All rights reserved.
This Letter is dedicated to the late Professor
Akio Nomoto
Keywords:
siRNA
Acyclic nucleoside
Alkyne
RNAi
Nuclease resistance
Small interfering RNAs (siRNAs) are key molecules in the cellu-
lar RNA interference (RNAi) pathway of gene expression control.1,2
In RNAi, the guide strand (antisense strand) of an siRNA forms the
RNA-induced silencing complex (RISC) together with the
Argonaute (Ago) protein. In RISC, the siRNA guide strand base-pairs
with a complementary mRNA strand, which is then cleaved by the
RNase activity of the Ago protein, thus inhibiting the mRNA func-
tion. Since siRNAs can be rationally designed and synthesized if
the sequence of the target gene is known, they have considerable
potential as new therapeutic drugs for intractable diseases.3 So
far, several chemically modified siRNAs have been synthesized
for improved nuclease resistance and RNAi-eliciting abilities.4
The synthesis of oligonucleotides (ONs) comprising acyclic
nucleosides has not received much attention because of the low
stability of duplexes containing such nucleoside analogs.5
Recently, however, it was reported that a site-specific incorpora-
tion of unlocked nucleic acid (UNA, 1, Fig. 1), an acyclic analog of
RNA, into siRNAs improves the specificity of gene silencing by
the siRNAs by reducing unintended mRNA silencing called off-tar-
get effects.6 Meggers and co-workers reported that an ON com-
posed of an acyclic nucleoside analog containing glycol (GNA, 2,
with complementary RNA,7 and modification of ONs with alkynyl
residues at the 5 positions of pyrimidine moieties is known to fur-
ther stabilize the duplexes.8
Based on these reports, an acyclic thymidine analog (3, Fig. 1)
containing 4-pentyne-1,2-diol instead of
D-ribofuranose was
designed in this study. In this Letter, we report the synthesis and
thermal stability of siRNAs containing the nucleoside analog 3 as
well as the silencing activity and nuclease resistance of the
siRNA modified with 3.
The synthetic route used to synthesize the thymidine analog 3
is shown in Scheme 1. A 4-pentyne-1,2-diol derivative (4), which
was synthesized according to the reported method,9 was coupled
with 1-methyl-5-iodouracil (5) in the presence of CuI, Pd(PPh3)4,
and Et3N in DMF to produce the uracil analog 6 at a 58% yield.
Subsequently, 6 was de-silylated by tetrabutylammonium fluoride
(TBAF) in THF, and the primary hydroxy group of 310 was protected
with a 4,40-dimethoxytrityl (DMTr) group to give 7 at a 92% yield.
The DMTr derivative 7 was phosphitylated using a standard proce-
dure to give rise to the corresponding phosphoramidite 8 at a 88%
yield. To introduce the analog 3 at the end of an RNA strand, 7 was
converted to the corresponding succinate, which was then linked
to controlled pore glass (CPG) to generate the solid support 9
Fig. 1) instead of D-ribofuranose forms a thermally stable duplex
linked to 7 (39 lmol/g).
All RNAs containing 3 (Tables 1 and 2) were synthesized using
the phosphoramidite 8 and the solid support 9. The RNAs were
⇑
Corresponding author. Tel./fax: +81 58 293 2919.
E-mail address: uenoy@gifu-u.ac.jp (Y. Ueno).
http://dx.doi.org/10.1016/j.bmcl.2015.04.034
0960-894X/Ó 2015 Elsevier Ltd. All rights reserved.

A. Ogata, Y. Ueno / Bioorg. Med. Chem. Lett. 25 (2015) 2574–2578
2575
O
a buffer composed of 10 mM sodium phosphate (pH 7.0) and
100 mM NaCl. Melting temperatures (Tms) and Tms (Tm of each
duplex ꢀ Tm of duplex 1) are listed in Table 1. The Tm of the natural
RNA duplex was found to be 67.0 °C, whereas those of the duplexes
containing 3 ranged from 60.2 °C to 64.3 °C, demonstrating that the
Me
O
N
D
N
H
O
N
NH
N
H
O
O
HO
N
O
HO
HO
O
incorporation of
duplexes. The Tm value of the duplex containing a 3:A matched
base pair was higher than the Tms of the duplexes containing a
3 into siRNAs thermally destabilizes RNA
D
HO
OH
HO
2 (GNA)
Figure 1. Structures of acyclic nucleoside analogs.
HO
D
3:U, 3:C, or 3:G mismatched base pair. This result indicates that
3 exhibits base discrimination ability.
1 (UNA)
3
Next, the ability of the siRNAs containing 3 to suppress gene
expression was investigated using a dual-luciferase reporter assay.
The psiCHECK-2 vector encoding Renilla luciferase and firefly luci-
ferase was used as the reporter4c and the percent Renilla: Firefly
luciferase activities compared to transfection without siRNA are
shown in Table 2. The luciferase activity ratios for an unmodified
siRNA (siRNA 1) were 28.0 and 22.3 at 1 and 10 nM, respectively.
When two molecules of 3 were incorporated into each dangling
end of the sense and antisense strands (siRNA 2), the activity
analyzed by matrix-assisted laser desorption/ionization time-of-
flight mass spectrometry (MALDI-TOF/MS), and the observed
molecular weights from these analyses were in agreement with
the structures of the RNAs.
First, the stabilities of the RNA duplexes containing 3 in the
middle of the strands were evaluated by thermal denaturation in
Me
Me
N
O
H
N
O
H
Si
O
a
+
TBDMSO
TBDPSO
N
N
I
O
O
O
Si
4
5
6
Me
N
Me
N
O
H
O
HO
N
HO
N
c
b
H
DMTrO
HO
O
O
7
3
e
d
Me
N
Me
N
NiPr2
O
O
O
O
O
H
NC
DMTrO
P
CPG
N
H
(CH2)2
DMTrO
O
N
O
N
H
O
O
9
8
Scheme 1. Reagents and conditions: (a) CuI, Pd(PPh3)4, Et3N, DMF, 40 °C, 58%; (b) TBAF, THF, rt, 86%; (c) DMTrCl, pyridine, rt, 92%; (d) chloro(2-cyanoethoxy)(N,N-
diisopropylamino)phosphine, i-Pr2NEt, THF, rt, 88%; (e) (1) succinic anhydride, DMAP, pyridine, rt; (2) CPG, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimideꢁHCl (EDCIꢁHCl),
DMF, rt, 39 lmol/g loading amount.
Table 1
Sequences of ONs and Tm values of RNA/RNA duplexes containing 3
b
Abbreviation of duplex
Duplex 1
Abbreviation of ON
Sequencea
Tm (°C)
D
Tm (°C)
ON 1
ON 2
50-AGAGAUACAUUGACCUUC-30
30-UCUCUAUGUAACUGGAAG-50
67.0
—
Duplex 2
Duplex 3
ON 3
ON 2
50-AGAGAUACA3UGACCUUC-30
64.3
60.2
ꢀ2.7
ꢀ6.8
30-UCUCUAUGUAACUGGAAG-50
ON 3
ON 4
50-AGAGAUACA3UGACCUUC-30
30-UCUCUAUGUUACUGGAAG-50
Duplex 4
Duplex 5
ON 3
ON 5
50-AGAGAUACA3UGACCUUC-30
30-UCUCUAUGUCACUGGAAG-50
61.4
63.6
ꢀ5.6
ꢀ3.4
ON 3
ON 6
50-AGAGAUACA3UGACCUUC-30
30-UCUCUAUGUGACUGGAAG-50
a
Underlined letters indicate mismatched bases.
b
D
Tm = Tm (each duplex) ꢀ Tm (duplex 1).

2576
A. Ogata, Y. Ueno / Bioorg. Med. Chem. Lett. 25 (2015) 2574–2578
Table 2
Sequences of siRNAs and their abilities to suppress gene expression
Abbreviation of siRNA
Abbreviation of RNA
Sequencea
Upper: 1 nMb
lower: 10 nM
Control
siRNA 1
—
—
100 17.7
28.0 8.0
ON 7
Sense (Passenger) strand
50-CUUCUUCGUCGAGACCAUGtt-30
30-ttGAAGAAGCAGCUCUGGUAC-50
Antisense (Guide) strand
ON 8
22.3 3.9
siRNA 2
siRNA 3
siRNA 4
siRNA 5
siRNA 6
siRNA 7
ON 9
ON 10
50-CUUCUUCGUCGAGACCAUG33-30
30-33GAAGAAGCAGCUCUGGUAC-50
23.8 3.1
16.5 4.3
ON 11
ON 8
50-3UUCUUCGUCGAGACCAUGtt-30
30-ttGAAGAAGCAGCUCUGGUAC-50
41.4 7.3
22.4 2.7
ON 7
ON 12
50-CUUCUUCGUCGAGACCAUGtt-30
30-ttGAAGAAGCAGCUCUGGUA3-50
47.1 4.7
36.1 2.5
ON 13
ON 8
50-C3UCUUCGUCGAGACCAUGtt-30
30-ttGAAGAAGCAGCUCUGGUAC-50
29.0 1.3
19.2 2.0
ON 14
ON 8
50-CUUCUUCGUCGAGACCA3Gtt-30
30-ttGAAGAAGCAGCUCUGGUAC-50
8.3 1.0
4.2 0.5
ON 7
ON 15
50-CUUCUUCGUCGAGACCAUGtt-30
30-ttGAAGAAGCAGCUCUGG3AC-50
22.0 2.4
20.1 4.9
a
Small letters indicate 20-deoxyribonucleosides.
Activities of firefly and Renilla luciferases in cell lysates were determined with a dual-luciferase assay system (Promega)
b
according to a manufacturer’s protocol. The results were confirmed by at least three independent transfection experiments with
two cultures each and are expressed as the average from four experiments as mean SD.
ratios were 23.8 and 16.5 at
1
and 10 nM, respectively,
activity of the siRNA. The luciferase activity ratios for siRNA 5
incorporating 3 at the second position from the 50 end of the sense
strand were 29.0 and 19.2 at 1 and 10 nM, respectively, whereas
those for siRNA 6 with 3 at the fourth position from the 30 end of
the sense strand were 8.3 and 4.2 at 1 and 10 nM, respectively.
Modification of the 30 region of the sense strand, therefore, signif-
icantly enhanced the silencing activity of the siRNA. It has been
suggested that thermodynamic features of siRNAs influence silenc-
ing activity; in particular, it has been hypothesized that RISC pref-
erentially selects and incorporates one of the two strands of siRNA,
depending on their thermodynamic features: the strand with its 50
end at the thermodynamically less stable region is preferentially
incorporated into RISC and becomes a mature siRNA.13 In this
study, 3 was found to thermally destabilize the RNA–RNA duplex,
although the base distinguishing ability is conserved. By incorpo-
rating 3 at the fourth position from the 30 end of the sense strand
demonstrating that the modification of the dangling ends by 3
slightly enhances the ability of the siRNA to suppress gene
expression. The activity ratio of siRNA 3 incorporating 3 at the 50
end of the sense strand was 22.4 at 10 nM, while that for siRNA
4 incorporating 3 at the 50 end of the antisense strand was 36.1.
Modification of the 50 end of the antisense strand was therefore
found to slightly decrease the silencing activity of siRNA. Ago
proteins are composed of four domains: N-terminal (N), Piwi/
Argonaute/Zwille (PAZ), Middle (MID), and P-element-induced
wimpy testis (PIWI).13 It is known that the 50 ends of siRNAs are
phosphorylated by kinases in cells and the 50 phosphates of the
antisense (guide) strands of siRNAs are recognized and anchored
by the MID domain in RISC.11,12 Modification of the 50 end of the
antisense strand (siRNA 4) may thus affect the phosphorylation
process of the strand in cells, thereby diminishing the silencing
Table 3
Sequences of siRNAs, their Tm values and their abilities to suppress gene expression
Sequencea
Tm (°C)
D
Tm (°C)
Upper: 1 nMc
lower:0.1 nM
b
Abbreviation of siRNA
Abbreviation of RNA
Control
siRNA 1
—
—
—
—
—
100 1.7
36.2 1.2
ON 7
Sense (Passenger) strand
73.2
50-CUUCUUCGUCGAGACCAUGtt-30
30-ttGAAGAAGCAGCUCUGGUAC-50
Antisense (Guide) strand
ON 8
13.0 1.2
siRNA 6
siRNA 8
ON 14
ON 8
50-CUUCUUCGUCGAGACCA3Gtt-30
72.3
72.6
ꢀ0.9
ꢀ0.6
15.0 1.1
5.7 0.5
30-ttGAAGAAGCAGCUCUGGUAC-50
50-CUUCUUCGUCGAGACCAAGtt-30
30-ttGAAGAAGCAGCUCUGGUAC-50
ON 16
ON 8
21.3 1.6
7.6 0.7
50-CUUCUUCGUCGAGACCAGGtt-30
30-ttGAAGAAGCAGCUCUGGUAC-50
siRNA 9
ON 17
ON 8
72.2
72.1
ꢀ1.0
ꢀ1.1
21.0 1.7
6.8 0.8
50-CUUCUUCGUCGAGACCACGtt-30
30-ttGAAGAAGCAGCUCUGGUAC-50
siRNA 10
ON 18
ON 8
22.1 3.2
7.4 0.4
a
b
c
Small letters indicate 20-deoxyribonucleosides. Underlined letters represent mismatched bases.
Tm = Tm (each siRNA) ꢀ Tm (siRNA 1).
Activities of firefly and Renilla luciferases in cell lysates were determined with a dual-luciferase assay system (Promega) according to a manufacturer’s protocol. The
D
results were confirmed by at least three independent transfection experiments with two cultures each and are expressed as the average from four experiments as mean SD.

A. Ogata, Y. Ueno / Bioorg. Med. Chem. Lett. 25 (2015) 2574–2578
2577
Figure 2. 20% PAGE of RNAs hydrolyzed by SVPD. F denotes a fluorescein.
of the siRNA, the 50 region of the antisense strand thus becomes
less stable than its 30 region (Table 3). As a result, incorporation
of the antisense strand into RISC may be enhanced, increasing
the silencing ability of the siRNA. Incorporation of 3 at the third
position from the 50 end of the antisense strand (siRNA 7) was
found to slightly enhance the silencing activity of the siRNA (22.0
and 20.1 at 1 and 10 nM, respectively); however, the silencing
activity of siRNA 7 was lower than that of siRNA 6. The region from
the second to the eighth positions from the 50 end of the antisense
strand is known as the seed region and initial target base pairing of
siRNA in RISC occurs in this region.11 Analog 3 thermally destabi-
lizes the RNA–RNA duplex and the thermodynamic advantage of
RISC binding the antisense strand by incorporating 3 may therefore
be offset by the disadvantage in initial base pairing of siRNA by
incorporating 3.
To confirm whether the enhanced silencing activity of siRNA 6
is attributed to thermal destabilization of the siRNA by incorporat-
ing 3 at the fourth position from the 30 end of the sense strand, we
examined silencing activities of siRNA 8, 9, and 10 with an A:A,
G:A, or C:A mismatched base pair at the same position, respec-
tively. As shown in Table 3, it was found that the silencing activi-
ties of siRNA 8, 9, and 10 were greater than that of siRNA 1 with
a matched base pair, but siRNA 6 containing 3 was more potent
than siRNA 8, 9, and 10.
Improving the nuclease resistance of siRNA is important for
therapeutic applications of synthetic siRNA, and the susceptibili-
ties of the modified ONs to snake venom phosphodiesterase
(SVPD), a 30 exonuclease, were therefore assessed in this study.
Unmodified ON and ON modified with two molecules of 3 at the
30 end were labeled with fluorescein at the 50 ends and incubated
with SVPD. The reactions were analyzed using polyacrylamide
gel electrophoresis (PAGE) under denaturing conditions.4c As
shown in Figure 2, unmodified ON was randomly hydrolyzed
after 1 min of incubation, whereas ON modified with 3 was
resistant to the enzyme. The half-life (t1/2) of unmodified ON was
<1 min, while that of the modified ON was 15 min. Nuclease
resistance of ON incorporating 3 at the fourth position from the
30 end was also examined. As shown in Figure S3, it turned out
that the phosphodiester bonds between 3 and G, and A and 3
were more resistant to hydrolytic degradation by SVPD than
other phosphodiester bonds. Thus, the enhanced silencing
activity of siRNA 6 might be also attributable to the nuclease-
resistant property of siRNA 6.
exhibit resistance to hydrolytic degradation by a 30 exonuclease.
We conclude from these findings that modification of siRNAs by
the nucleoside analog 3 may hold promise as a means of improving
the silencing activity and nuclease resistance of siRNAs.
Acknowledgements
This research was partially supported by the Ministry of
Education, Culture, Sports, Science, and Technology of Japan
(Grant-in-Aid for Exploratory Research, 24659045) and by a C19
Kiyomi Yoshizaki research grant.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2015.04.
034.
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