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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.2009.12.007 592 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. 593 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) 594 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) 591–598 595 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- 596 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 References [1] Castanotto, D. and Rossi, J.J. (2009) The promises and pitfalls of RNAinterference-based therapeutics. Nature 457, 426–433. [2] Kurreck, J. (2009) RNA interference: from basic research to therapeutic applications. Angew. Chem., Int. Ed. Engl. 48, 1378–1398. [3] Grimm, D. (2009) Small silencing RNAs: state-of-the-art. Adv. Drug Deliv. Rev. 61, 672–703. [4] Khvorova, A., Reynolds, A. and Jayasena, S.D. (2003) Functional siRNAs and miRNAs exhibit strand bias. Cell 115, 209–216. [5] Schwarz, D.S., Hutvagner, G., Du, T., Xu, Z., Aronin, N. and Zamore, P.D. (2003) Asymmetry in the assembly of the RNAi enzyme complex. Cell 115, 199–208. [6] Grunweller, A. and Hartmann, R.K. (2007) Locked nucleic acid oligonucleotides: the next generation of antisense agents? BioDrugs 21, 235–243. [7] Veedu, R.N. and Wengel, J. (2009) Locked nucleic acid as a novel class of therapeutic agents. RNA Biol. 6, 321–323. [8] Di Primo, C., Rudloff, I., Reigadas, S., Arzumanov, A.A., Gait, M.J. and Toulme, J.J. (2007) Systematic screening of LNA/20 -O-methyl chimeric derivatives of a TAR RNA aptamer. FEBS Lett. 581, 771–774. [9] Elmen, J., Thonberg, H., Ljungberg, K., Frieden, M., Westergaard, M., Xu, Y., Wahren, B., Liang, Z., Orum, H., Koch, T. and Wahlestedt, C. (2005) Locked nucleic acid (LNA) mediated improvements in siRNA stability and functionality. Nucleic Acids Res. 33, 439–447. [10] Braasch, D.A., Jensen, S., Liu, Y., Kaur, K., Arar, K., White, M.A. and Corey, D.R. (2003) RNA interference in mammalian cells by chemically-modified RNA. Biochemistry (Moscow) 42, 7967–7975. [11] Jensen, T.B., Langkjaer, N. and Wengel, J. (2008) Unlocked nucleic acid (UNA) and UNA derivatives: thermal denaturation studies. Nucleic Acids Symp. Ser., 133–134. [12] Langkjaer, N., Pasternak, A. and Wengel, J. (2009) UNA (unlocked nucleic acid): a flexible RNA mimic that allows engineering of nucleic acid duplex stability. Bioorg. Med. Chem. 17, 5420–5425. [13] Bramsen, J.B., Laursen, M.B., Nielsen, A.F., Hansen, T.B., Bus, C., Langkjaer, N., Babu, B.R., Hojland, T., Abramov, M., Van Aerschot, A., Odadzic, D., Smicius, R., Haas, J., Andree, C., Barman, J., Wenska, M., Srivastava, P., Zhou, C., Honcharenko, D., Hess, S., Muller, E., Bobkov, G.V., Mikhailov, S.N., Fava, E., Meyer, T.F., Chattopadhyaya, J., Zerial, M., Engels, J.W., Herdewijn, P., Wengel, J. and Kjems, J. (2009) A large-scale chemical modification screen identifies design rules to generate siRNAs with high activity, high stability and low toxicity. Nucleic Acids Res. 37, 2867–2881. [14] Racaniello, V.R. (2007) Picornaviridae: the viruses and their replication in: Fields Virology (Knipe, D.M. and Howley, P.M., Eds.), fifth ed, pp. 795–838, Lippincott–Williams–Wilkins, Philadelphia. [15] Cornell, C.T., Perera, R., Brunner, J.E. and Semler, B.L. (2004) Strand-specific RNA synthesis determinants in the RNA-dependent RNA polymerase of poliovirus. J. Virol. 78, 4397–4407. [16] Haasnoot, J., Westerhout, E.M. and Berkhout, B. (2007) RNA interference against viruses: strike and counterstrike. Nat. Biotechnol. 25, 1435– 1443. [17] Ahn, J., Jun, E.S., Lee, H.S., Yoon, S.Y., Kim, D., Joo, C.H., Kim, Y.K. and Lee, H. (2005) A small interfering RNA targeting coxsackievirus B3 protects permissive HeLa cells from viral challenge. J. Virol. 79, 8620–8624. [18] Lee, H.S., Ahn, J., Jee, Y., Seo, I.S., Jeon, E.J., Jeon, E.S., Joo, C.H., Kim, Y.K. and Lee, H. (2007) Universal and mutation-resistant anti-enteroviral activity: potency of small interfering RNA complementary to the conserved cis-acting replication element within the enterovirus coding region. J. Gen. Virol. 88, 2003–2012. [19] Schubert, S., Grunert, H.P., Zeichhardt, H., Werk, D., Erdmann, V.A. and Kurreck, J. (2005) Maintaining inhibition: siRNA double expression vectors against coxsackieviral RNAs. J. Mol. Biol. 346, 457–465. [20] Werk, D., Schubert, S., Lindig, V., Grunert, H.P., Zeichhardt, H., Erdmann, V.A. and Kurreck, J. (2005) Developing an effective RNA interference strategy against a plus-strand RNA virus: silencing of coxsackievirus B3 and its cognate coxsackievirus–adenovirus receptor. Biol. Chem. 386, 857–863. [21] Yuan, J., Cheung, P.K., Zhang, H.M., Chau, D. and Yang, D. (2005) Inhibition of coxsackievirus B3 replication by small interfering RNAs requires perfect sequence match in the central region of the viral positive strand. J. Virol. 79, 2151–2159. [22] Fechner, H., Sipo, I., Westermann, D., Pinkert, S., Wang, X., Suckau, L., Kurreck, J., Zeichhardt, H., Muller, O., Vetter, R., Erdmann, V., Tschope, C. and Poller, W. (2008) Cardiac-targeted RNA interference mediated by an AAV9 vector improves cardiac function in coxsackievirus B3 cardiomyopathy. J. Mol. Med. 86, 987–997. [23] Kim, J.Y., Chung, S.K., Hwang, H.Y., Kim, H., Kim, J.H., Nam, J.H. and Park, S.I. (2007) Expression of short hairpin RNAs against the coxsackievirus B3 exerts potential antiviral effects in Cos-7 cells and in mice. Virus Res. 125, 9–13. [24] Merl, S., Michaelis, C., Jaschke, B., Vorpahl, M., Seidl, S. and Wessely, R. (2005) Targeting 2A protease by RNA interference attenuates coxsackieviral cytopathogenicity and promotes survival in highly susceptible mice. Circulation 111, 1583–1592. [25] Schubert, S., Rothe, D., Werk, D., Grunert, H.P., Zeichhardt, H., Erdmann, V.A. and Kurreck, J. (2007) Strand-specific silencing of a picornavirus by RNA interference. Evidence for the superiority of plus-strand specific siRNAs. Antiviral Res. 73, 197–205. [26] Dutkiewicz, M., Grunert, H.P., Zeichhardt, H., Lena, S.W., Wengel, J. and Kurreck, J. (2008) Design of LNA-modified siRNAs against the highly structured 50 UTR of coxsackievirus B3. FEBS Lett. 582, 3061–3066. [27] Tomari, Y., Matranga, C., Haley, B., Martinez, N. and Zamore, P.D. (2004) A protein sensor for siRNA asymmetry. Science 306, 1377–1380. [28] Schubert, S., Grunweller, A., Erdmann, V.A. and Kurreck, J. (2005) Local RNA target structure influences siRNA efficacy: systematic analysis of intentionally designed binding regions. J. Mol. Biol. 348, 883–893. [29] Overhoff, M., Alken, M., Far, R.K., Lemaitre, M., Lebleu, B., Sczakiel, G. and Robbins, I. (2005) Local RNA target structure influences siRNA efficacy: a systematic global analysis. J. Mol. Biol. 348, 871–881. [30] Westerhout, E.M. and Berkhout, B. (2007) A systematic analysis of the effect of target RNA structure on RNA interference. Nucleic Acids Res. 35, 4322– 4330. [31] Kenski, D.M., Cooper, A.J., Li, J.J., Willingham, A.T., Haringsma, H.J., Young, T.A., Kuklin, N.A., Jones, J.J., Cancilla, M.T., McMasters, D.R., Mathur, M., Sachs, A.B. and Flanagan, W.M. (in press) Analysis of acyclic nucleoside modifications in siRNAs finds sensitivity at position 1 that is restored by 50 -terminal phosphorylation both in vitro and in vivo. Nucleic Acids Res.