Biaryl modification of the 5′-terminus of one strand of a microRNA duplex induces strand specificity

Article abstract of DOI:10.1016/j.bmcl.2010.10.077

MicroRNAs (miRNAs) are single-stranded non-coding RNAs composed of 20-23 nucleotides. They are initially transcribed in the nucleus as pri-miRNAs. After processing, one strand from the miRNA duplex (miR-5p/miR-3p duplex) is loaded onto the RNA-induced sil

Full text of DOI:10.1016/j.bmcl.2010.10.077

Bioorganic & Medicinal Chemistry Letters 20 (2010) 7299–7302
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Biaryl modification of the 50-terminus of one strand of a microRNA duplex
induces strand specificity
Aya Ogata a, , Chihiro Furukawa c, , Kouhei Sakurai c, , Hideo Iba c, Yukio Kitade a,b, Yoshihito Ueno a,b,
⇑
a Department of Biomolecular Science, Faculty of Engineering, Gifu University, 1-1 Yanagido, Gifu 501-1193, Japan
b United Graduate School of Drug Discovery and Medical Information Sciences, Gifu University, 1-1 Yanagido, Gifu 501-1193, Japan
c Division of Host–Parasite Interaction, Department of Microbiology and Immunology, Institute of Medical Science, The University of Tokyo, 4-6-1 Shirokanedai,
Minato-ku Tokyo 108-8639, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 7 July 2010
Revised 13 October 2010
Accepted 15 October 2010
Available online 8 November 2010
MicroRNAs (miRNAs) are single-stranded non-coding RNAs composed of 20–23 nucleotides. They are
initially transcribed in the nucleus as pri-miRNAs. After processing, one strand from the miRNA duplex
(miR-5p/miR-3p duplex) is loaded onto the RNA-induced silencing complex (RISC) to produce a func-
tional, mature miRNA that inhibits the expression of multiple target genes. In the case of some miRNAs,
both strands can be equally incorporated into the RISC as single strands, and both strands can function as
mature miRNAs. Thus, a technique for selective expression of miR-5p and miR-3p strands is required to
identify distinct targets of miRNAs. In this Letter, we report the synthesis and properties of miRNA
duplexes carrying biaryl units at the 50-terminus of one strand. We found that incorporation of biaryl
units at the 50-terminus of one strand of miRNA duplexes induced strand specificity in these duplexes.
Further, we succeeded in identifying endogenous mRNA targets for each strand of the duplex by using
the biaryl-modified miRNA duplexes.
Keywords:
RNAi
miRNA
Biaryl compound
miR-199a-3p
miR-199a-5p
Ó 2010 Elsevier Ltd. All rights reserved.
MicroRNAs (miRNAs) are short non-coding RNAs that suppress
gene expression at the post-transcriptional level in animals and
plants.1 In mammals, miRNAs target mRNAs through translational
repression and degradation. miRNA genes are mainly transcribed
by RNA polymerase II to produce primary miRNA transcripts (pri-
miRNAs) containing a 50-cap structure and a poly(A) tail. The pri-
miRNA is processed into a precursor stem-loop structure called
pre-miRNA by the microprocessor complex Drosha-DGCR8. The
pre-miRNA is then exported from the nucleus to the cytoplasm
where the terminal loop is excised from the stem by the Dicer en-
zyme to create an miRNA duplex of 20–23 bp. One of the strands
from the miRNA duplex is loaded onto the RNA-induced silencing
complex (RISC) to become a functional mature miRNA strand that
inhibits the expression of multiple target genes.2 Over 700 miRNAs
have been identified in humans, and some have been found to play
important roles in development, differentiation, and proliferation.3
Among them, miR-199a has been known to exhibit aberrant
expression patterns in human cancer and infection. It has been re-
ported that miR-199a expression is impaired in different types of
cancer such as hepatic cancer, ovarian cancer, and cervical can-
cer.4–6 Interesting insights about the role of miR-199a in skeletal
development and hepatitis C virus (HCV) replication have also
been published.7,8
It is believed that RISC preferentially selects and incorporates one
of the strands of the miRNA duplex, depending on its thermody-
namic features. The strand with lower thermodynamic stability at
its 50-terminus (the guide strand) preferentially binds to the RISC
and becomes functional, whereas the other strand (the passenger
strand) is degraded.9,10 However, recent studies show that strand
selection does not always follow this rule. In the case of some miRNA
duplexes, both strands can be incorporated into the RISC as single
strands, and each strand can function as a mature miRNA, despite
the thermodynamic asymmetry between the strands.11 Among
human miRNAs registered in the online microRNA database miR-
Base <http://www.mirbase.org/>, 36% originate from the 50-strand
(miRNA-5p), 37% originate from the 30-strand (miRNA-3p), and
27% are miRNA duplexes, each producing two miRNAs from the
50- and 30-strands.12,13 miR-199a is a type of miRNA duplex that
produces two mature miRNAs, miR-199a-5p and miR-199a-3p. It
is believed that the members of an miRNA duplex can target two dis-
tinct sets of genes according to the nucleotide sequence.
A technique for selective expression of miR-199a-5p and miR-
199a-3p is required to identify the distinct targets of miRNAs gen-
erated from pre-miRNA generated in this manner. Pre-miRNA
expression vectors or double-stranded miRNAs are commonly used
for over-expression, but these methods do not specify which
strand is expressed. It was recently reported in siRNA studies that
⇑
Corresponding author. Tel.: +81 58 293 2639; fax: +81 58 293 2794.
E-mail address: uenoy@gifu-u.ac.jp (Y. Ueno).
These authors contributed equally to this work.
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.10.077

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A. Ogata et al. / Bioorg. Med. Chem. Lett. 20 (2010) 7299–7302
Figure 1. Structures of biaryl units and nucleoside analogs.
50-O-methyl-modification of the 50-terminus of one strand
efficiently allows its antisense strand to be loaded onto the RISC,
despite thermodynamic instability.13 This is because phosphoryla-
tion of the 50-terminus—an important factor for RISC loading—is
inhibited by the 50-O-methyl group. However, this modification
can be applied to only the sequence with uridine at the 50-terminus
of the passenger strand, since only 50-O-methylthymidine (3)
(Fig. 1) is now commercially available. Recently, we also found that
the incorporation of naphthalene-type biaryl unit 1 (Nap) into the
50-termini of the passenger strands of siRNAs can suppress the
silencing activities induced by the passenger strands (off-target
effect) without reducing the silencing activities of the guide
strands.14,15 The Nap-modification was more effective than the
50-O-methyl-modification in inhibition of function of a passenger
strand. From these results and existing information, we deduced
that the addition of biaryl units to the 50-terminus of one strand
of an miRNA duplex could induce better strand specificity of the
miRNA duplexes than with the addition of a methyl group, since
biaryl units are sterically bulkier than methyl groups, and increase
the thermal stability of duplexes by hydrophobic interactions.
Further, the Nap-modification can be applied to all sequences
including the sequence without uridine at the 50-terminus of a
passenger strand.
In this Letter, we report the synthesis and properties of miR-
199a duplexes carrying biaryl units at the 50-terminus of one
strand of each duplex (Figs. 1 and 2). We examined gene silencing
activities of the modified miRNA duplexes by the dual-luciferase
reporter assay. In addition, we tried to identify endogenous mRNA
targets for miR-199a-5p and miR-199a-3p strands by using the
modified miR-199a duplexes.
Figure 2. Sequences of oligonucleotides (ONs) and miRNAs used in this study.
Structures of 1 (Nap) and 2 (DANap) are indicated in Fig. 1. Mutated bases are
shown in bold letters.
We selected two types of biaryl compounds, naphthalene-type
1 (Nap) and dimethylnaphthalene type 2 (DANap), as the mono-
mer units to be incorporated in the miRNA duplexes. DANap is ste-
rically bulkier than Nap. The synthesis of the phosphoramidite unit
of DANap is depicted in Scheme 1. An arylboronic acid derivative 4
was coupled with 1-bromo-4-(dimethylamino)naphthalene (5) in
the presence of PdCl2(dppf) (dppf = 1,10-bis(diphenylphosphino)-
ferrocene) at 65 °C to afford the biaryl derivative 6 with a yield
of 81%. Subsequently, 6 was desilylated by tetra-n-butylammo-
nium fluoride (TBAF) treatment to afford biaryl unit 2 with a yield
of 66%. One of the two hydroxy groups of 2 was protected by a 4,40-
dimethoxytrityl (DMTr) group to give a mono-DMTr derivative 7,
with 44% yield. Compound 7 was phosphitylated by the standard
procedure to afford the corresponding phosphoramidite 9 with
76% yield. All oligonucleotides (ONs) were synthesized using the
phosphoramidites of Nap and DANap with a DNA/RNA synthesizer.
The ability of modified miRNAs to suppress gene expression
was determined by the dual-luciferase reporter assay using a psi-
CHECK-2 vector (Promega) with Renilla and firefly luciferase genes
as reporter genes. We constructed psi-199a-5p and psi-199a-3p
vectors by inserting sequences complementary to miR-199a-5p
and miR-199a-3p strands in the Luc 30-UTR region of the
psiCHECK-2 vectors. Reporter vectors and synthesized miRNA
duplexes were co-transfected into HeLa or HeLa S3 cells with
TransFast (Promega) or Lipofectamine2000 (Invitrogen) as
transfection reagents, and luciferase activities were measured after
24 h. The signals of Renilla luciferase were normalized to those of
firefly luciferase (Fig. 3).
An unmodified miR-199a duplex, miRNA1, effectively reduced
luciferase activity in both vectors in a dose-dependent manner;
luciferase activity of the psi-199a-5p vector was reduced from
72% to 41% and that of the psi-199a-3p vector, from 61% to 22%.
Differences in the reduction levels between vectors may reflect
the asymmetry of RISC loading between natural miR-199a-5p
and miR-199a-3p. When miRNA2 and miRNA4—modified with
Nap or DANap at the 50-termini of miR-199a-5p strands—were
transfected, luciferase activity of the psi-199a-5p vector was high-
er than that of the psi-199a-3p vector with both the modifications
(Fig. 3a). Thus, it was found that the Nap and DANap modifications
at the 50-termini of miR-199a-5p strands could induce miR-199a-
3p strand specificity of miR-199a-5p/miR-199a-3p duplexes.
On the other hand, when miRNA3 and miRNA5—modified with
Nap or DANap at the 50-termini of miR-199a-3p strands—were
transfected, luciferase activity of the psi-199a-3p vector was higher
than that of the psi-199a-5p vector with the DANap modification

A. Ogata et al. / Bioorg. Med. Chem. Lett. 20 (2010) 7299–7302
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Scheme 1. Reagents and conditions: (a) PdCl2(dppf)ꢀCH2Cl2, NaOH, THF/H2O (5:1 v/v), 65 °C, 24 h, yield of biaryl derivative 6: 81%; (b) TBAF, THF, room temperature, 12 h,
yield of biaryl unit 2: 66%; (c) DMTrCl, pyridine, room temperature, 12 h, yield of mono-DMTr derivative 7: 44%; (d) chloro(2-cyanoethoxy)(N,N-diisopropylamino)phos-
phane, i-Pr2NEt, THF, room temperature, 1 h, yield of corresponding phosphoramidite 9: 76%.
while that of the psi-199a-3p vector with the Nap-modification was
similar to that of the psi-199a-5p vector at concentrations of 0.3 nM
and 1.0 nM (Fig. 3b). These results indicate that the DANap
modification is the most promising in eliciting miR-199a-5p and
miR-199a-3p strand specificities of miR-199a-5p/miR-199a-3p
duplexes.
We succeeded in inducing the expression of the miR-199a-5p
and miR-199a-3p strands individually, using the DANap-modified
miR-199a duplexes. However, small amounts of unanticipated
changes in expression can still occur in these modified miRNAs
when injected into cells. In order to eliminate this off-target effect,
we further artificially changed the sequence of the opposite
strands to form complete double-stranded RNAs as depicted in
Fig. 2. We evaluated the strand specificity of these duplexes by
Figure 3. Dual-luciferase reporter assay (1). Reporter vectors and synthesized
miRNA duplexes (final concentrations: 0.1, 0.3, or 1.0 nM) were transfected into the
cells (HeLa cells) using TransFast (Promega). Firefly and Renilla lu-ciferase activities
were measured consecutively using Dual-Luciferase Reporter Assay System (Pro-
mega). Renilla lu-ciferase expression was normalized to that of Firefly lu-ciferase.
Normalized expression in mock-transfected cells was set as 1.0. The results were
confirmedby at leastfour independent transfection experimentsand are expressed as
the average from four experiments as mean SD.
Figure 4. Dual-luciferase reporter assay (2). Reporter vectors and synthesized
miRNA duplexes (final concentrations: 0.1, 0.3, or 1.0 nM) were transfected into the
cells (HeLa S3 cells) using Lipofectamine2000 (Invitrogen). Firefly and Renilla lu-
ciferase activities were measured consecutively using Dual-Luciferase Reporter
Assay System (Promega). Renilla lu-ciferase expression was normalized to that of
Firefly lu-ciferase. Normalized expression in mock-transfected cells was set as 1.0.
The results were confirmed by at least four independent transfection experiments
and are expressed as the average from four experiments as mean SD.

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A. Ogata et al. / Bioorg. Med. Chem. Lett. 20 (2010) 7299–7302
miR-199a-3p strands by using the DANap-modified miR-199a-5p/
miR-199a-3p duplex. Thus, DANap modification would be useful
to identify endogenous mRNA targets for other miRNAs.
Acknowledgments
This study was supported in part by a Grant-in-Aid from Precur-
sory Research for Embryonic Science and Technology (PRESTO) of
Japan Science and Technology (JST) and a Grant-in-Aid for Scien-
tific Research (C) from Japan Society for the Promotion of Science
(JSPS) to Y.U., and a Grant-in-Aid for Scientific Research on Priority
Areas from the Ministry of Education, Culture, Sports, Science, and
Technology (MEXT) of Japan (17016015) to H.I. We are also grate-
ful to Dr. Y. Kitamura (Gifu University) for providing technical
assistance.
Figure 5. Detection of target proteins by high-level expression of miR-199a-5p and
miR-199a-3p. MDA-MB-435 cells were transfected with 30 nM DANap-modified
miRNA duplexes, and after 48 h, protein extracts were prepared. IKKb, fibronectin,
and b-actin proteins were analyzed by western blotting.
Supplementary data
using the same luciferase assay system (Fig. 4). For DANap-modi-
fied duplexes, miRNA6 modified at the 50-terminus of the miR-
199a-5p strand reduced luciferase activity of psi-199a-3p from
18% to 5%, but not of psi-199a-5p. In contrast, miRNA7 modified
at the 50-terminus of the miR-199a-3p strand reduced luciferase
activity of psi-199a-5p from 38% to 9%, but not of psi-199a-3p. It
is noteworthy that the 5p and 3p strands of miRNA6 and miRNA7
did not suppress luciferase activity efficiently due to the applied
mutations, and the anticipated effects of the synthesized duplexes
were enhanced as compared to duplexes with the natural miRNA
sequence, thus resulting in better strand selectivity.
It was previously reported that IKKb is a target of miR-199a-5p
and fibronectin, a target of miR-199a-3p.16,17 Therefore, we tried to
determine endogenous miR-199a target genes by using the strand-
specific over-expression method that we devised. MDA-MB-435
cells (expressing low levels of miR-199a) were transfected with
DANap-modified miR-199a duplexes for over-expression. Western
blotting showed that in MDA-MB-435 cells, IKKb expression was
suppressed by selective expression of miR-199a-5p originating
from miRNA7 duplexes, and not miRNA6 (Fig. 5). It was also shown
that fibronectin expression was specifically suppressed by miR-
NA6, and not miRNA7. Our method involving selective over-
expression of miR-199a-5p and miR-199a-3p could be used for
the regulation of endogenous target gene expression and that it
could be applied for the identification of new target genes.
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2010.10.077.
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In conclusion, we have demonstrated the synthesis of miRNA du-
plexes modified with biaryl compounds. We found that the DANap
modification was the most effective in eliciting strand specificity
of miR-199a-5p/miR-199a-3p duplexes. Further, we succeeded in
identifying endogenous mRNA targets for miR-199a-5p and