NUCLEIC ACID THERAPEUTICS

Volume 27, Number 5, 2017 Brief Communication

ª Mary Ann Liebert, Inc.
DOI: 10.1089/nat.2017.0663

Effective Anti-miRNA Oligonucleotides

Show High Releasing Rate of MicroRNA
from RNA-Induced Silencing Complex

Jumpei Ariyoshi,1,2 Yohei Matsuyama,1 Akio Kobori,1 Akira Murakami,3

Downloaded by Asako Yamayoshi from online.liebertpub.com at 10/05/17. For personal use only.

Hiroshi Sugiyama,2,4 and Asako Yamayoshi2,5

MicroRNAs (miRNAs) regulate gene expression by forming RNA-induced silencing complexes (RISCs) and
have been considered as promising therapeutic targets. MiRNA is an essential component of RISC for the
modulation of gene expression. Therefore, the release of miRNA from RISC is considered as an effective
method for the inhibition of miRNA functions. In our previous study, we reported that anti-miRNA oligonu-
cleotides (AMOs), which are composed of the 2¢-O-methyl (2¢-OMe) RNA, could induce the release of miRNA
from RISC. However, the mechanisms underlying the miRNA-releasing effects of chemically modified AMOs,
which are conventionally used as anti-cancer drugs, are still unclear. In this study, we investigated the rela-
tionship between the miRNA releasing rate from RISC and the inhibitory effect on RISC activity (IC50) using
conventional chemically modified AMOs. We demonstrated that the miRNA-releasing effects of AMOs are
directly proportional to the IC50 values, and AMOs, which have an ability to promote the release of miRNA
from RISC, can effectively inhibit RISC activity in living cells.

Keywords: microRNA, RNA-induced silencing complex, anti-miRNA oligonucleotide

Introduction are 2¢-O-methyl RNA (2¢-OMe) [22–24], 2¢-O-methoxyethyl

RNA (2¢-MOE) [25], and locked nucleic acids (LNAs) [26,27].

M icroRNA (miRNA) is a small noncoding RNA of

about 18–22 nucleotides that plays important roles in
many biological processes by regulating gene expression [1].
A large number of studies demonstrated that miRNAs par-
ticipate in a wide spectrum of physiological functions such as
cellular development, differentiation, and apoptosis [2–7].
Until now, thousand kinds of miRNAs have been identified in
humans. The aberrant expression of miRNAs is associated
with the pathology of many human diseases, particularly in
cancers [8–13]. Therefore, miRNAs are considered as at-
tractive therapeutic targets in cancers [14].
The miRNAs that are highly expressed in cancerous cells
(oncogenic miRNA), such as miR-21, miR-221, and miR-222,
are attractive therapeutic targets for cancer therapy [12, 15–18].
The main approach for the inhibition of oncogenic miRNA
functions is anti-miRNA oligonucleotides (AMOs), which have
the complementary sequence of the oncogenic miRNAs [19–
21]. In general, AMOs are chemically modified for increased
nuclease resistance and binding affinity to the target miRNA.
The commonly used types of chemical modifications in AMOs
Especially, the effectiveness of LNA-containing AMOs as
anticancer drugs has been demonstrated in clinical trials. For
instance, Miravirsen, which contains LNAs and targets a liver-
specific miRNA (miR-122), has demonstrated antiviral activity
in patients with chronic hepatitis C virus infection in clinical
phase II [28,29]. Thus, AMOs have been shown as promising
tools for the inhibition of miRNA activity.
MiRNA can exhibit its functions by forming RNA-induced
silencing complex (RISC); therefore, it is expected that the
release of miRNA from RISC effectively inactivates the RISC
activity [30–32]. In our previous study, we have revealed that
AMOs, which were composed of 2¢-OMe RNA, could induce
the release of miRNA from RISC [33]. Furthermore, we also
successfully enhanced the miRNA-releasing effects of AMOs
by conjugating anionic peptides to the AMOs for inhibition of
the holding ability of miRNA in RISC, resulting in enhance-
ment of inhibitory effects of AMOs on RISC activity [33].
These results indicated that release of miRNA from RISC is a
promising strategy for the inhibition of RISC activity. How-
ever, the mechanisms underlying the miRNA-releasing effects

1
Department of Biomolecular Engineering, Kyoto Institute of Technology, Kyoto, Japan.
2
Department of Chemistry, Graduate School of Science, 4Institute for Integrated Cell-Materials Sciences (iCeMS), and 5The Hakubi
Center for Advanced Research, Kyoto University, Kyoto, Japan.
3
Department of Clinical and Translational Physiology, Kyoto Pharmaceutical University, Kyoto, Japan.

303
 304 ARIYOSHI ET AL.

of chemically modified AMOs, which are conventionally used
as anticancer drugs, are still unclear. Furthermore, the rela-
tionship between the miRNA-releasing effects and the inhibi-
tory effects of AMOs on RISC activity is also still incompletely
understood. Hence, in this study, we evaluated the half-time of
the releasing rate of miRNA from RISC (T1/2) and the half
maximal inhibitory concentration (IC50) of AMOs on RISC
activity. We also investigated the relationships between T1/2
and IC50, as well.
Materials and Methods
All AMOs described in this study were purchased from
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move unbound material, the beads were washed with a
washing buffer (20 mM Tris–HCl (pH 8.0), 150 mM NaCl,
2 mM MgCl2, and 0.5 mM TCEP).
For the unloading assay, 20 mL of RISC-bound beads
were suspended in a 1.5 mL centrifuge tube and was pre-
incubated at 37C for 5 min. The unloading reaction was
initiated by the addition of 1 mM (final concentration) of
AMOs and subsequent incubation at 37C. After the incu-
bation, the beads were isolated, and the supernatant was
carefully removed and named as the ‘‘unloaded miRNA’’
fraction. The beads were washed immediately with 40 mL
washing buffer, and all traces of the supernatant were re-
moved. The washed beads were saved and named as the

Gene Design, Inc. (Osaka, Japan).
UV melting profiles of the duplex between an AMO and
mir-Luc were obtained using a UV-spectrophotometer equip-
ped with a programmed thermal controller at an increase rate
of 1.0C/min. The sample solutions were prepared in 10 mM
phosphate buffer (pH 7.0) containing 0.1 M NaCl, and the
concentration of oligonucleotides was fixed at 2.0 mM.
For the dual luciferase assay, HeLa cells were cultured in
Dulbecco’s modified Eagle’s medium supplemented with
10% (v/v) fetal bovine serum, 100 U/mL penicillin, and
100 mg/mL streptomycin at 37C in 5% CO2. The cells were
seeded in 96-well plates at a density of 4.5 · 104 cells/mL in
an antibiotic-free medium, transfected and harvested for
24 h. The next day, cells were transfected with 20 nM of
mir-Luc using Lipofectamine RNAiMAX (GIBCO, CA)
according to the manufacturer’s protocol. The following
day, the cells were cotransfected with plasmids pGL4.13
(25 ng/well) and pGL4.73 (62.5 ng/well) plasmids and
AMOs using Lipofectamine 2000 (GIBCO). After 24-h
incubation period, the cells were lysed, and the lumines-
cence in the lysate was measured with a Dual Luciferase
Assay Kit (Promega, WI).
To prepare the immobilized RISC on beads, HEK293T cell
lysate expressing hAgo2 was prepared, according to a pub-
lished method [34]. For RISC assembly, 4 mL of the cell lysate
was added to 3 mL of 40· reaction mix and 2 mL of mir-Luc
duplex (32P-labeled, stock concentration 100 nM) and incu-
bated at 37C for 30 min. After incubation, the reaction mix
was added to 2 mL anti-hAgo2 antibody and stirred at 4C for
30 min. Then, 12 mL of the reaction mix was added to 20 mL
Dynabeads Protein G for immunoprecipitation (Thermo
Fisher Scientific, Inc., MA) and stirred at 4C for 2 h. To re-
‘‘RISC (including miRNA)’’ fraction. The radioactivity of
each fraction was measured using a radiation analyzer. The
dissociation ratio of miRNA from RISC was evaluated by
measuring the radioactivity.
Results and Discussion
We prepared six kinds of AMOs with sequences entirely
complementary to the target miRNA sequence. As a target of
AMOs, miRNA targeting firefly luciferase mRNA (mir-Luc)
was selected. The melting temperature (Tm) values of AMOs
duplexed with mir-Luc are summarized in Table 1. From the
results, the Tm values were determined to be as follows:
AMO(DNA) (53C), AMO(DNA/OMe) (54C), AMO(OMe)
(69C), AMO(4LNA) (76C), AMO(DNA/LNA) (79C), and
AMO(7LNA) (88C). These results showed that the binding
affinity of AMOs containing DNA to mir-Luc was lower
compared with AMOs which do not contain DNA. In contrast,
AMOs containing LNA, such as AMO(4LNA), AMO(DNA/
LNA), and AMO(7LNA), showed a higher binding affinity for
miRNA compared with AMOs which did not contain LNA.
First, we evaluated the miRNA-releasing effects of AMOs
using the unloading assay (Fig. 1A). Time course analyses of
the percentage of released miRNA from RISC by AMOs are
shown in Fig. 1B. In the case of AMO(ctrl), in which the
sequence was not complementary to mir-Luc, the release of the
miRNA was not observed. In contrast, in the case of AMOs
that have a fully complementary sequence to the target
miRNA, the release of the miRNA from the RISC was
observed in a time-dependent manner. The half-time of the
releasing rate of miRNA from RISC by AMOs (T1/2) was de-
termined by fitting the releasing rates as pseudo-first-order

Table 1. Sequences of Anti-miRNA Oligonucleotides

Seed region
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 Tm (C)
mir-Luc 5¢- a u u g a a u c u u a u a g u c u u g c a -3¢ —
AMO(OMe) 3¢- U A A C U U A G A A U A U C A G A A C G U -5¢ 69
AMO(DNA) 3¢- t a a c t t a g a a t a t c a g a a c g t -5¢ 53
AMO(DNA/OMe) 3¢- t A a c U t a G a a U a t C a g A a c G t -5¢ 54
AMO(DNA/LNA) 3¢- t A a c U t a G a a U a t C a g A a c G t -5¢ 79
AMO(7LNA) 3¢- U A A C U U A G A A U A U C A G A A C G U -5¢ 88
AMO(4LNA) 3¢- U A A C U U A G A A U A U C A G A A C G U -5¢ 76
AMO(Ctrl) 3¢- U U A U G G A G U C A A U G U U A A A U A -5¢ —
augc, RNA; atcg, DNA; AUGC, 2¢-OMe RNA; AUGC, LNA.
Tm: [mir-Luc] = [AMO]s = 2 mM in 10 mM phosphate buffer (pH 7.0), 100 mM NaCl.
 THE MIRNA-RELEASING EFFECTS OF AMOS 305
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FIG. 1. (A) Schematic illustration of the unloading assay. (B) Time course analysis of the percentage of miRNA released
from RISC (RISC on beads = 10 fmol, [AMO]s = 1 mM). (C) The inhibitory effects of AMOs on RISC function in HeLa cell.
AMO, anti-miRNA oligonucleotide; RISC, RNA-induced silencing complex.

reactions (Figs. 1B and 2A). The kinetic rates of T1/2 increased
in the following order: AMO(7LNA) [14 min], AMO(DNA/
LNA) [20 min], AMO(4LNA) [23 min], AMO(OMe) [25 min],
AMO(DNA) [49 min], and AMO(DNA/OMe) [51 min]
(Fig. 1D). Figure 2B shows the plot of the kinetics rates (T1/2)
versus the Tm values. We calculated the correlation coefficient
and found that there was strong correlation between the Tm
values and the kinetics rate (T1/2) of AMOs (R2 = 0.96). The
result clearly indicates that AMOs, which have a high binding
affinity for the target miRNA, can efficiently induce the release
of miRNA from RISC. We consider that these results are ap-
propriate, because association between the AMO and RISC is
required to induce the release of miRNA from RISC.
Next, we evaluated the inhibitory effect of AMOs on RISC
activity in HeLa cells using the luciferase reporter assay. In this
assay, luminescence intensity of firefly luciferase is already
downregulated by RISC incorporated with mir-Luc. If AMOs
inhibit the RISC activity, it is expected that luminescence in-
tensity would be recovered. The results are shown in Fig. 1C.
In the case of AMO(DNA) and AMO(DNA/OMe), the lumi-
nescence intensity was hardly recovered, indicating that nei-
ther of these AMOs had significant inhibitory effects on RISC
 306
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FIG. 2. (A) The table summarizing about the melting
temperature (Tm) values of AMOs duplexed with mir-Luc,
the half maximal inhibitory concentrations (IC50) of AMOs
on RISC activity, and the half-time of the releasing rate of
miRNA from RISC by AMOs (T1/2). (B) The scatter plot of
T1/2 versus Tm. (C) The scatter plot of T1/2 versus IC50.
functions. In contrast, concentration-dependent inhibitory ef-
fects were observed when using AMO(OMe), AMO(DNA/
LNA), AMO(4LNA), and AMO(7LNA). The half maximal
inhibitory concentrations (IC50) of AMOs are summarized in
Fig. 2A. In order of increasing the inhibitory effect of AMOs
on RISC activity, the IC50 values are determined as follows:
ARIYOSHI ET AL.
AMO(7LNA) [1.1 nM], AMO(4LNA) [1.8 nM], AMO(DNA/
LNA) [2.2 nM], and AMO(OMe) [100 nM].
Figure 2C shows the plot of the kinetics rates (T1/2) versus
the IC50 values. In the case of AMO(DNA) and AMO(OMe/
DNA), the IC50 values were immeasurable (IC50: >100);
therefore, the IC50 values of both AMOs were not plotted in
Fig. 2C. In the case of AMO(7LNA), AMO(4LNA), and
AMO(DNA/LNA), there was a positive correlation between
T1/2 and IC50 (R2 = 0.78); however, the plot of AMO(OMe)
was deviated from the linear approximation. We speculated
that AMO(OMe) hardly associates with RISC in living cells
for the following reasons. In the case of unloading assay, we
evaluated the releasing effect of AMOs at 1 mM (final con-
centration), and RISC complexes (10 fmol) were purified on
magnetic beads. On the contrary, in the case of luciferase
assay, AMOs were transfected into HeLa cells in the con-
centration range of 0.16–100 nM and we could not know
exact amount of RISC in the cells. Therefore, the experi-
mental condition of both assays was not exactly the same.
Furthermore, in the case of AMO(7LNA), AMO(4LNA), and
AMO(DNA/LNA), there was a positive correlation between
Tm and IC50 (R2 = 0.67); however, the plot of AMO(OMe)
deviated from the linear approximation (Supplementary
Fig. S1; Supplementary Data are available online at www
.liebertpub.com/nat). From this result, it was considered that
the miRNA-releasing from RISC by AMO(OMe) was not
adequately induced in living cells. If the binding affinity of an
AMO to RISC is not adequately strong in living cells, the
releasing rate of the AMO could be slow in living cells.
Recently, it was revealed that the transient binding between
RISC and target RNA, such as mRNA and AMO, exists in the
gene-silencing pathway mediated by RISC [35]. Thus, we
inferred that an AMO was dissociated from RISC through the
transient binding pathway when the releasing rate of miRNA
by AMO was slow. From these reasons, we considered
that the inhibitory effect of AMO(OMe) on RISC activity
was lower compared with AMO(4LNA), AMO(7LNA), and
AMO(DNA/LNA). However, there was not a linear rela-
tionship but a positive correlation between T1/2 and IC50. This
result suggests that the AMOs, which can efficiently promote
the release of miRNA from RISC, have high inhibitory ef-
fects on RISC activity.
To investigate the role of inhibitory effects of AMOs tar-
geting endogenous miRNA, we evaluated the inhibitory ef-
fects of AMOs targeting miR-21 using a luciferase reporter
assay and a cell growth inhibition assay. In both cases, we
observed that anti-miR21 AMO(7LNA) significantly in-
hibited RISC activity; however, the other AMOs induced
cytotoxicity (Supplementary Figs. S2 and S3). To investigate
the role of the inhibitory effect of AMOs targeting endoge-
nous miRNA, we will examine the inhibitory effects of
AMOs targeting other endogenous miRNA and investigate
the relationship between the inhibitory effect of AMOs on
RISC activity and the releasing effect of AMOs.
Taking the results into consideration, it is clear that
chemically modified AMOs induced the release of miRNA
from RISC. These results suggest that the release of miRNA
from RISC is directly correlated to the inhibitory effect of
AMOs on RISC activity. Recently, it was reported that the
AMO targeting miR-122 induced the degradation of miR-122
in vivo [36,37]. It was also reported that the inhibition of miR-
21 activity using AMOs reduced the amount of miR-21 in
 THE MIRNA-RELEASING EFFECTS OF AMOS
living cells [20]. Because miRNA released from RISC can be
rapidly degraded by nucleases, the above findings support our
hypothesis that the release of miRNA from RISC is generally
induced by AMOs in living cells.
Most importantly, we also found a proportional relationship
between the kinetic rates (T1/2) and the IC50 values. These
findings suggest that the strategy to release the miRNA
from RISC is a promising method for the inhibition of
miRNA activity in cancerous cells. It is also suggested that the
miRNA-releasing effects of AMOs are valuable information
for the development of AMOs as RISC inhibitors. The details
of the mechanisms underlying the release of miRNA from
RISC are still unclear. Understanding these mechanisms is
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important to develop more effective methods to inhibit RISC
activity using AMOs. For instance, we previously reported
that the chemical structure of wild-type RNA is favorable for
the release of miRNA from RISC and that the cleavage re-
action by RISC is necessary to improve the releasing rate of
miRNA from RISC. These experiments suggest that chemi-
cally modified AMOs, which are partly composed of wild-
type RNA to accept cleavage by RISC, might be beneficial to
improve the release of miRNA from RISC and to the inhibi-
tory effect on RISC activity of AMOs. In near future, based on
our findings, we will try to develop the novel AMOs to pro-
mote the release of miRNA from RISC.
Acknowledgments
The authors would like to thank Prof. Hiroyuki Asanuma
and Associate Prof. Yukiko Kamiya (Nagoya University,
Japan) for providing the luciferase-expressing plasmid
containing miR-21 binding sites and for giving us valuable
suggestions.
Author Disclosure Statement
No competing financial interests exist.
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Address correspondence to:
Asako Yamayoshi, PhD
The Hakubi Center for Advanced Research
Kyoto University
Yoshida-Ushinomiyacho
Sakyo-ku
Kyoto 606-8501
Japan
E-mail: asakoy@kuchem.kyoto-u.ac.jp
Received for publication January 9, 2017; accepted after
revision August 2, 2017.