Download Application of small interfering RNAs modified by unlocked nucleic

FEBS Letters 584 (2010) 591–598
journal homepage: www.FEBSLetters.org
Application of small interfering RNAs modified by unlocked nucleic acid (UNA)
to inhibit the heart-pathogenic coxsackievirus B3
Denise Werk a, Jesper Wengel b, Suzy L. Wengel c, Hans-Peter Grunert a, Heinz Zeichhardt a, Jens Kurreck d,e,*
a
Institute for Virology, Campus Benjamin Franklin, Charité-University Medicine, Berlin, Germany
Nucleic Acid Center, Department of Physics and Chemistry, University of Southern Denmark, Odense M, Denmark
c
RiboTask ApS, Odense C, Denmark
d
Institute of Industrial Genetics, University of Stuttgart, Stuttgart, Germany
e
Institute of Biotechnologie, Technical University Berlin, Germany
b
a r t i c l e
i n f o
Article history:
Received 4 November 2009
Revised 28 November 2009
Accepted 6 December 2009
Edited by Tamas Dalmay
Keywords:
Unlocked nucleic acid
Locked nucleic acid
RNA interference
Coxsackievirus
Small interfering RNAs
a b s t r a c t
This study describes the first application of unlocked nucleic acid (UNA)-modified small interfering
RNAs (siRNAs) directed against a medically relevant target, the coxsackievirus B3. We systematically
analyzed the impact of different siRNA modification patterns and observed good compatibility of
the introduction of UNA with the maintenance of high antiviral activity. Additionally, the polarity
of an siRNA was successfully reversed by modulating the relative stability of the termini with locked
nucleic acid (LNA) and UNA as shown in a reporter assay. The potency of the reversed siRNA against
the full-length target was, however, too low to inhibit the infectious virus. Altogether, combined
modification of siRNAs with LNA und UNA provides a promising approach to alter and improve
properties of an siRNA.
Ó 2009 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
1. Introduction
RNA interference (RNAi) has emerged as one of the most important technologies of biomedical research within just a few years.
This evolutionary conserved mechanism of post-transcriptional
gene silencing is triggered by small double-stranded RNAs, which
induce sequence-specific degradation of a target RNA (for recent
reviews see [1–3]). Double-stranded small interfering RNAs
(siRNAs) of 21 nucleotides in length become incorporated into
a multimeric complex called the RNA-induced silencing complex
(RISC) with a member of the argonaut family nucleases as its main
component. While the sense strand of the siRNA is degraded, the
antisense strand acts as a guide for RISC to its complementary
target mRNA. After binding of activated RISC, cleavage of target
mRNA is initiated. Depending on the thermodynamic parameters
of the siRNA duplex, the strand containing the less stable 50 -end
is preferred to guide RISC [4,5].
The potency and efficacy of siRNAs can be optimized by incorporation of chemically modified RNA analogues. Locked nucleic
acid (LNA) nucleotides are conformationally restricted due to a
methylene bridge connecting the 20 -oxygen with the 40 -carbon of
* Corresponding author. Address: Institute of Biotechnologie, Technical University Berlin, Germany. Fax: +49 30 314 27502.
E-mail address: [email protected] (J. Kurreck).
the ribose ring. Due to their nuclease resistance and low toxicity,
LNAs improve the properties of antisense oligonucleotides (for reviews, see [6,7]) and aptamers [8]. Incorporation of LNA is tolerated in various positions of the siRNA and can improve its
efficacy [9,10]. Similarly, silencing by the modified strand is inhibited when LNA monomers are introduced at specific sites of an
siRNA [9].
Unlocked nucleic acid (UNA), an acyclic analogue of RNA, has an
incomplete ribose ring open between the 20 - and 30 -carbon (Fig. 1).
The structural flexibility of UNA monomers destabilizes duplexes
[11] and thermal denaturation studies demonstrated the potential
of UNA monomers to decrease or increase mismatch discrimination depending on their sites of incorporation [12]. Modification
of siRNA with both UNA and LNA nucleotides showed highly potent silencing activity. Importantly, the high efficacy of UNA-modified siRNAs was accompanied by low cell toxicity [13].
Coxsackievirus B3 (CVB-3) belongs to the picornavirus family
and is one of the most frequent causes of heart muscle infections.
The small, non-enveloped virus possesses a single-stranded RNA
genome in positive orientation that is used directly as mRNA in
infected cells [14]. After entering the host cell, the genomic RNA
is transcribed into a complementary negative RNA strand by the
viral RNA dependent RNA polymerase (RdRP), which is used as a
template for generation of new viral mRNA [15]. Despite improvements in virological and medical research, there is still no specific
0014-5793/$36.00 Ó 2009 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
doi:10.1016/j.febslet.2009.12.007
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D. Werk et al. / FEBS Letters 584 (2010) 591–598
2. Materials and methods
2.1. Oligonucleotides
Fig. 1. Structure of unlocked nucleic acid (UNA), RNA and locked nucleic acid (LNA)
monomers.
clinical therapy available against CVB-3. Among other applications,
RNAi has been found to efficiently inhibit viruses [16]. We and others have described the successful inhibition of CVB-3 by means of
RNAi in cell culture [17–21] and in vivo [22–24]. By using LNAmodified siRNAs, RNAi-silencing was shown to be mediated via
the genomic plus-strand rather than the intermediate minusstrand [25]. Furthermore, LNA-modified siRNAs were successfully
employed to target the highly structured internal ribosome entry
site of CVB-3 [26].
Recently, a large number of modified siRNAs including LNA- and
UNA-modified siRNAs targeting the Green Fluorescent Protein
(GFP) were analysed with respect to their potency and specificity
[13]. Our present study describes the first application of LNAand UNA-modified siRNAs to the medically relevant target CVB-3.
Based on the above mentioned study, we systematically analysed
the impact of different siRNA modification patterns and screened
for the best variant with respect to its inhibitory activity against
CVB-3. In experiments with reporter assays, LNA and UNA monomers were introduced at the ends of an siRNA and succeeded in
reversing its polarity. These experiments add to our knowledge
about the relevance of the thermodynamic features of an siRNA
for its silencing activity.
The LNA- and UNA-modified as well as unmodified RNA oligonucleotides were synthesized by RiboTask (Odense, Denmark).
Both, siRdRP (target sequence CUA AGG ACC UAA CAA AGU U)
and siRev (target sequence UCU CAU AGC AUU UGA UUA C), are directed against the 3D RNA dependent RNA polymerase (RdRP) of
CVB-3 (GenBank Acc. No. M33854; target nucleotides 6315–6333
and 6594–6612, respectively). Both siRNAs were already used in
an earlier study [25] where siRev was dubbed siRev1 and siRdRP
was dubbed siRdRP2.
As a control, we used an siRNA designed by Qiagen (Hilden, Germany) with no known homology in the human and viral genome:
UUC UCC GAA CGU GUC ACG U. The modification patterns of all oligonucleotides are summarized in Tables 1 and 2. DNA oligonucleotides used for cloning procedure were purchased from TibMolBiol
(Berlin, Germany).
2.2. Cells and virus
Vero (ATCC, Manassas, VA) and COS7 (DSMZ, Braunschweig,
Germany) cells were propagated in monolayer culture in MEM
(GIBCO-Invitrogen, USA) containing 5% heat inactivated fetal bovine serum (FBS), 1% antibiotic/antimycotic, gentamicin and nonessential amino acids, at 37 °C in a humidified atmosphere with
5% carbon dioxide. CVB-3 (strain Nancy; ATCC No. VR-30) was
propagated in Vero cells.
2.3. Luciferase reporter assay
The reporter constructs psiCheck2-RdRP(+) and psiCheck2RdRP() containing the cDNA of the viral RNA dependent RNA polymerase (RdRP) in plus- and minus-strand orientation, respectively,
were described previously [25]. To obtain a plasmid producing the
Table 1
Sequences and modification patterns of classical 21-mer siRNAs with 2 nt overhangs.
Name
Pictogram
Sequence and modification pattern
Nucleotides
siRdRP 2_2
21
21
siRev 2_2
21
21
siCtr 2_2
21
21
mod A
21
21
mod B
21
21
mod C
21
21
mod D
21
21
mod E
21
21
mod F
21
21
Top strand depicts the sense strand in 50 –30 direction (complementary to viral minus-strand). Bottom strand depicts the antisense strand in 30 –50 direction (complementary to
viral plus-strand). Pictogram: open circle = UNA; filled square = LNA. Modification pattern: L = LNA; U = UNA.
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D. Werk et al. / FEBS Letters 584 (2010) 591–598
Table 2
Modified siRNAs containing two and three nucleotide 30 -overhangs.
Name
siRdRP 2_2
Pictogram
Sequence and modification pattern
Nucleotides
21
21
siRdRP 2_3
21
22
siRdRP 3_3
22
22
siCtr 2_2
21
21
siCtr 3_3
22
22
mod 1
22
22
mod 2
22
22
mod 3
22
22
mod 4
22
22
mod 5
22
22
mod 6
22
22
mod 7
22
22
mod 8
21
22
mod 9
21
22
mod 10
21
22
mod 11
21
22
mod 12
21
22
(continued on next page)
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D. Werk et al. / FEBS Letters 584 (2010) 591–598
Table 2 (continued)
Name
Pictogram
Sequence and modification pattern
Nucleotides
21
mod 13
22
22
mod 14
22
22
mod 15
22
22
mod 16
22
22
mod 17
22
21
mod 18
22
Top strand depicts the sense strand in 50 –30 direction. Bottom strand depicts the antisense strand in 30 –50 direction. Pictogram: open circle = UNA; filled square = LNA, a short
separate line indicates a three nucleotide 30 overhang. Modification pattern: L = LNA; U = UNA.
Fig. 2. Titre of the virus propagated in Vero cells after transfection with modified (siRdRP mod. A), unmodified (siRdRP 2_2) and non-silencing (siCtr) siRNA. Cells were pretreated with siRNAs in the range of 1–100 nM, followed by virus inoculation. The virus titer was determined by plaque forming assay 19 h after infection. The mean and S.D. of
at least three independent experiments, each performed in duplicate are shown.
19mer plus-strand siRev target site, the corresponding sense and
antisense oligodeoxynucleotides containing overhangs for XhoI
and NotI restriction sites were annealed and cloned into the multiple
cloning site of psiCheck2 (Promega, Mannheim, Germany) downstream of the Renilla luciferase stop codon.
For reporter assays, COS7 cells were seeded in 96-well plates
at a density of 7000 cells per well in medium without antibiotics
and incubated in a humidified atmosphere. The next day, cells
were co-transfected with 0.1 lg vector and 10 nM of the respective siRNAs by using 0.3 ll Lipofectamine™ 2000 (Invitrogen,
Karlsruhe, Germany) per well according to manufacturer’s
protocol. Following an incubation for 24 h, cells were lysed with
20 ll passive lysis buffer (Promega) and luciferase activity was
measured using the Dual Luciferase Reporter Assay System
(Promega) according to the manufacturer’s instructions on a
Centro LB 960 luminometer (BertholdTech, Bad Wildbach,
Germany). The Renilla luciferase signals were normalized to the
firefly luciferase signals.
2.4. SiRNA transfection and virus inoculation
For transfection, Vero cells were seeded at a density of 7500
cells per well in 96-well plates in medium without antibiotics.
The next day, cells were incubated for 4 h with different amounts
D. Werk et al. / FEBS Letters 584 (2010) 591–598
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Fig. 3. Antiviral potency of various modified siRNAs targeting the RdRP of CVB-3. Vero cells were transfected with 10 nM of the indicated siRNA, incubated overnight and
subsequently infected with CVB-3 for 8 h. The modification patterns are represented as pictograms (open circle: UNA; filled square: LNA; a short separate line indicates a
three nucleotide 30 overhang). siCtr: Non-silencing siRNA; virus: without transfection; siRdRP 2_2: 2 nt overhangs; siRdRP 2_3: 2 nt overhang in the sense strand and 3 nt in
the antisense strand; siRdRP 3_3: 3 nt overhangs. The mean and S.D. of at least three independent experiments, each performed in duplicate are shown.
Fig. 4. Analysis of unspecific or modification dependent reduction of virus propagation after RNAi treatment. Cells were transfected with 50 nM siRdRP (dark gray) and siCtr
(non-silencing; light gray) with indicated modifications, respectively. Virus titer was determined after 8 h incubation. The mean and S.D. of at least three independent
experiments, each performed in duplicate are shown.
of siRNA as indicated and 0.3 ll Lipofectamine™ 2000 per well,
following the manufacturer’s instructions. Cells were then inoculated with CVB-3 at a multiplicity of infection (m.o.i.) of 0.1 in
medium without FBS for 30 min and maintained in cell culture
medium for additional 19 h. Further details and variations of the
standard procedure are described in Section 3.
2.5. Determination of virus titer
The amount of infectious virus in lysed cells was determined on
Vero cells by an agar overlaid plaque forming assay according to
the procedure described elsewhere [20]. Briefly, the serially diluted
samples were incubated for 30 min on Vero cells. Subsequently,
cells were overlaid with agar containing Eagle’s MEM. After incubation in a humidified atmosphere for 3 days, cells were stained
with neutral red and virus titers were determined by counting
plaques.
3. Results and discussion
In the present study, we evaluated the impact of incorporation
of UNA monomers into a previously identified, highly efficient
siRNA, dubbed siRdRP, against the heart-pathogenic CVB-3. UNA
monomers are very flexible due to an open structure of the ribose
ring, leading to a gradual decrease in the thermodynamic stability
of duplexes [12]. In contrast, the widely used LNA monomers
enhance the target affinity and nuclease resistance [9,25,26]. As
published by Bramsen et al. [13], the insertion of UNA and LNA
monomers at specific positions of an siRNA against GFP lead to
an improvement of the silencing activity.
Our highly potent antiviral siRdRP 2_2 composed of a 19mer
duplex and 2 nt overhangs in accordance with conventional siRNA
design, was modified by the incorporation of UNA and LNA monomers based on the modification pattern reported by Bramsen et al.
[13] (siRdRP mod A; Table 1). The CVB-3 titer after siRNA transfec-
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D. Werk et al. / FEBS Letters 584 (2010) 591–598
Fig. 5. Reversal of the siRNA polarity by incorporation of UNA and/or LNA monomers. (A and C) Luciferase reporter knockdown by modified siRev. Shown are the mean and
S.D. of three independent experiments determined from measurements of the luminescence of Renilla luciferase normalized to the firefly luciferase expressed from the same
vector. (A) Reporter-construct containing the 19 nt siRev target sequence in plus-strand orientation. (B) Schematic representation of the siRNA modification patterns (open
circle: UNA; filled square: LNA). The top strand depicts the sense strand, complementary to the viral minus-strand; the bottom strand depicts the antisense strand,
complementary to the viral plus-strand. (C) Reporter constructs containing the full length RdRP target sequence in plus-strand (light gray) or minus-strand (dark gray)
orientation. (D) Virus propagation in Vero cells transfected with 100 nM of the indicated siRNA followed by incubation with CVB-3 for 19 h. The mean and S.D. of six
independent experiments, each performed in duplicate are shown. siCtr: Non-silencing siRNA; virus: without transfection.
D. Werk et al. / FEBS Letters 584 (2010) 591–598
tion and subsequent virus infection of cells clearly revealed a potent and concentration-dependent inhibition of virus propagation
of the UNA-modified siRdRP (Fig. 2). Compared to the unmodified
counterpart, the UNA-modified siRNA was only slightly less potent.
This finding demonstrates siRdRP tolerated the introduction of
UNA without significant loss of antiviral activity.
The next aim was to analyse in more detail the impact of the
introduction of LNA and UNA at different positions in the siRNA
on its antiviral activity. To this end, a set of sense and antisense
strands with three nucleotide overhangs and UNA as well as LNA
modifications at different positions was designed. Annealing of
each of the 22 nt antisense strands with each of the 22 nt sense
strands, as well as with 21 nt sense strands resulted in 18 modified
siRNAs (shown in Table 2). To evaluate the antiviral potential of
these modified siRNAs, the generation of new infectious virus during the first viral replication cycle was determined. For this purpose, cells were transfected with 10 nM of each siRNA and
infected with CVB-3 for 8 h on the next day. Virus titer was determined by titration of cell lysates on confluent cells. As shown in
Fig. 3, each of the siRNAs was capable of reducing the virus titer
at least 10-fold. This finding demonstrates that LNA and UNA
monomers can be introduced into certain positions of the siRNA
without detrimental effects on its silencing activity.
Modified control siRNAs were then used to further investigate
the specificity of the approach. Some of the most potent modification patterns were selected and the corresponding control siRNAs
without homology to the host or viral genome were synthesized.
Fig. 4 shows that neither the modified nor the unmodified 22 nt or
21 nt control siRNAs (light grey bars) had any significant effect on
CVB-3, even at a comparably high concentration of 50 nM. This result clearly confirms that the observed reduction of virus
propagation is a specific RNAi effect rather than an unspecific consequence of the transfected siRNAs or the incorporated modifications.
Prominent properties of LNA and UNA monomers are their high
and low affinity towards complementary nucleotides, respectively.
The next aim of our study was to make use of these features to further investigate the relevance of the thermodynamic design of an
siRNA for its activity. It has previously been shown that the relative
stability of the two ends of an siRNA determines which of strands
of the duplex will be incorporated into RISC as the guide strand
[4,5]. The Dicer associated double-stranded RNA binding protein
was identified as the protein sensor for the strand asymmetry
[27]. We now wanted to investigate whether the affinity differences of LNA and UNA can be employed to reverse the polarity of
an siRNA. In a previous study, we purposely designed siRNAs
which were exclusively active against the CVB-3 RNA in either
plus- or minus-strand orientation [25]. The experiments with
infectious virus revealed that only the plus-strand virus RNA is
amendable to silencing by siRNA, while the minus-strand is resistant against RNAi. We now chose one siRNA, which is exclusively
active against the minus-strand orientation of the CVB-3 RNA, referred to as siRev. UNA and LNA monomers were then introduced
into the 30 and 50 ends of the siRev sense strand, respectively (Table
1 and pictograms in Fig. 5B). These modifications can be expected
to destabilize base pairing at the 50 -end of the antisense strand,
while enhancing the strength of base pairing at its 30 -end. As a consequence, polarity of the siRNAs should be reversed.
To test this hypothesis, the target site of the siRNA was cloned
in positive orientation downstream of Renilla luciferase. As can be
seen in Fig. 5A, the siRNA without UNA or LNA monomers at the
termini of the duplex does not reveal any silencing activity against
the target site in positive orientation. This result confirms our previous findings with the unmodified siRNA [25]. In sharp contrast,
siRNAs with the same sequence but with reversed relative stability
of the two ends by the introduction of LNA und UNA into the 50 and 30 -end of the sense strand, respectively, gained significant
597
activity against the target site in plus-strand orientation (mod.
B–F). Interestingly, a single UNA at the 30 -end (mod. E) as well as
a single LNA at the 50 -end (mod. B) of the sense strand was sufficient to reverse the polarity of siRev. The combination of LNA
monomers to stabilize the 30 -end of the antisense strand and
UNA monomers to destabilize its 50 -end at the same time slightly
increased the silencing potency (mod. C).
Next, we wanted to investigate the activity of the modified
siRNAs against the full length RNA encoding RdRP. To this end,
the RdRP sequence was cloned downstream of Renilla luciferase
in either plus- or minus-strand orientation. As reported previously
[25], siRdRP was exclusively active against the plus-strand orientation, whereas siRev 2_2 inhibited expression of the reporter construct with the minus-stranded RdRP-RNA, but not the plus-strand
orientation (Fig. 5C).
In line with data reported by Elmen et al. [9], a single LNA
monomer in the first position completely abrogates silencing activity of so modified strand (mod. B–D). The experiments presented
here revealed that UNA at the 30 -end of the sense strand (mod. E
and F) also impairs the inhibitory potency of this stand. Furthermore, the modified siRNAs which had the relative stability of their
ends reversed showed silencing of the RdRP-RNA plus-strand, as
observed for the isolated target site (Fig. 5A). However, the siRNA
had a lower efficacy against the full-length target as compared to
the efficiency of silencing the isolated target site. A possible reason
for this discrepancy might be the differences in the environment of
the target site, as we and others have previously shown that the
structure of the target RNA has a great influence on siRNA-mediated silencing [28–30].
In experiments with infectious virus (Fig. 5D) the siRdRP 2_2,
which is directed against an accessible target site in the viral
plus-strand RNA, showed high antiviral potency as described previously [20]. Since the modified siRNAs already had a decreased
activity against the full-length target, it was not unexpected that
no pronounced inhibitory effect on virus replication was observed
with any of the siRNAs.
In summary, our study clearly showed that introduction of a
limited number of UNA and LNA monomers into an siRNA is compatible with maintenance of high silencing activity. UNA-modified
siRNAs have been found to have very low toxic side effects on cell
viability [13]. To the best of our knowledge, the data presented
here show for the first time the potential of UNA- and LNA-modified siRNAs to inhibit a medically relevant target, the coxsackievirus B3, a typical representative of the picornavirus family.
Furthermore, UNA nucleotides can be employed to investigate
the mechanism of RNAi in more detail, as for example their introduction at the ends of an siRNA duplex can reverse its polarity.
Note added in proof
After submission of the present manuscript, a study was published, in which UNA monomers were systematically introduced
into each position of an siRNA guide strand [31]. UNA modifications at various positions were found to be detrimental to siRNA
activity. Particularly, UNAs at positions 1 and 2 prevented phosphorylation of the siRNA and thereby abrogated its binding to
Ago2.
Acknowledgements
The authors wish to thank V. Lindig for practical help and Erik
Wade for proofreading and valuable comments. This work was
supported by the Deutsche Forschungsgemeinschaft (KU 1436/61) and the Fonds der Chemischen Industrie and the Danish Research Agency (STVF). H.Z. and D.W. are grateful for support from
598
Otto-Kuhn-Stiftung
Wissenschaft.
D. Werk et al. / FEBS Letters 584 (2010) 591–598
im
Stifterverband
für
die
Deutsche
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