Volume 50 Number 11 9 February 2014 Pages 1273–1386
ChemComm Chemical Communications
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ISSN 1359-7345
COMMUNICATION Yoshihiro Ito, Hiroshi Abe et al. An intracellular buildup reaction of active siRNA species from short RNA fragments
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Cite this: Chem. Commun., 2014, 50, 1284 Received 1st October 2013, Accepted 28th November 2013
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An intracellular buildup reaction of active siRNA species from short RNA fragments† Hideto Maruyama,ab Yuko Nakashima,a Satoshi Shuto,b Akira Matsuda,b Yoshihiro Ito*ac and Hiroshi Abe*abcd
DOI: 10.1039/c3cc47529h www.rsc.org/chemcomm
Here we report a new strategy for the buildup reaction of active siRNA species from short RNA fragments in living cells using a chemical ligation reaction. This strategy could decrease undesired immune responses and provide more latitude for RNAi technology in the design and concentration of introduced RNA compared to traditional siRNA methods.
RNA interference (RNAi) is a potent and highly specific gene silencing phenomenon that was first reported in the nematode Caenorhabditis elegans by Fire and Mello in 1998.1 They discovered that genes could be silenced by introducing long, double-stranded RNAs (dsRNAs) complementary to mRNA sequences. To silence genes in mammalian cells, short dsRNAs of 20–23 nucleotides, referred to as small interfering RNA (siRNA), have been used as an RNAi trigger to avoid nonspecific interferon responses induced by long dsRNAs.2 However, soon after the application of siRNAs in functional genomic studies and the development of therapeutics, it was found that siRNAs activate innate immune responses via pattern recognition receptors (PRRs) (e.g. Toll-like receptors (TLR) and retinoic acid inducible gene I (RIG-I)-like cytoplasmic helicases).3 In addition, naked siRNAs have several drawbacks, including short-term RNAi effects, due to their unstable nature in serum, non-target specificity, and off-target effects induced by unintended incorporation of the passenger strand into the RNAi-induced silencing complex (RISC). To overcome these issues, many types of chemically modified RNA4–6 and RNAi triggering strategies were developed,7 including short-hairpin RNA (shRNA) expressing vectors,8 synthetic shRNA,9 Dicer substrate dsRNA,10 short shRNA (sshRNA),11–13 branched RNA,14,15 small
internally segmented interfering RNA (sisiRNA),16–18 asymmetric interfering RNA (aiRNA),19–22 dumbbell RNA,23,24 and caged siRNA.25 Significantly, sisiRNA and aiRNA oligonucleotides are shorter than canonical siRNA (21–23 bases). sisiRNA consists of two short sense strands (10 + 11 bases) and an antisense strand of 21 bases. aiRNAs consist of a sense strand of 13–19 bases and an antisense strand of 19–21 bases. These strategies suppress unintended off-target effects16,21 caused by canonical siRNA, and shorter siRNAs (e.g. 19 bases) cause less cellular stress than long siRNAs (>23 bases).26 In principle, shorter RNAs suppress immune stimulation, however, the desired RNAi effect is decreased. Thus, there is a dilemma in optimizing the length of siRNA. Here, we report a new strategy for the buildup reaction of biologically active siRNA species from short RNA fragments that are inactive in living cells, which is called intracellular buildup reaction (IBR) of siRNA (Fig. 1). Short RNA fragments for IBR (IBR-RNA) that have chemically reactive groups are introduced into living cells, and then hybridized and activated by glutathione (GSH), and finally ligated by an SN2 reaction to give active siRNA species
a
Nano Medical Engineering Laboratory, RIKEN, 2-1, Hirosawa, Wako-Shi, Saitama 351-0198, Japan. E-mail: [email protected], [email protected] b Faculty of Pharmaceutical Sciences, Hokkaido University, Kita-12, Nishi-6, Kita-ku, Sapporo 060-0812, Japan c Emergent Bioengineering Materials Research Team, RIKEN Center for Emergent Matter Science, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan d PRESTO, Japan Science and Technology Agency, 4-1-8, Honcho, Kawaguchi, Saitama 332-0012, Japan † Electronic supplementary information (ESI) available. See DOI: 10.1039/ c3cc47529h
1284 | Chem. Commun., 2014, 50, 1284--1287
Fig. 1 Proposed mechanism of the intracellular buildup reaction of active siRNA (IBR).
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Fig. 3 Buildup reaction of ligated siRNA from IBR-RNA triggered by GSH in vitro. Lane 1, 25 mer RNA; lanes 2 and 3, each PS RNA (1 and 2); lanes 4 and 5, reaction mixture (1 + 2 + 3 + 4) with (lane 4) or without (lane 5) GSH; lanes 6 and 7, reaction mixture (lane 6, 1 + 3 (sense strand) or lane 7, 2 + 4 (antisense strand)) without the complementary strand. The reaction mixture was incubated at 37 1C for 30 min and then samples were analyzed by 20% denaturing PAGE. The gel was stained with SYBR Green II. Fig. 2 Structures and sequences of IBR-RNA. (A) Structure of siRNA-n-1 with arrows showing segmented positions. (B–D) Segmented RNA sequences and structures of CPS-RNA (B), IAc-RNA (C), and Ac-RNA (D). (E, F) Sequences and structures of siRNA-lig-1 (used in Fig. 4 and 5) and segmented siRNA (used in Fig. 5), respectively.
(ligated siRNA, Fig. 1). To the best of our knowledge, this is the first report of such an IBR strategy to induce RNAi. The benefit of this strategy is that it bypasses the immune stimulation that is normally induced by native siRNA, and can therefore be applied to the development of new classes of RNAi therapeutics. To circumvent innate immune responses, we designed four short RNA fragments, segmented from 25 bases of active siRNA, targeting the luciferase gene, as shown in Fig. 2A. Each RNA strand was segmented into 19 and 6 bases (sense strand) or 18 and 7 bases (antisense strand). In contrast to full-length siRNA, which causes a strong immune response, short RNA fragments were expected to cause a significantly lower immune response. Thus, we designed the IBR strategy based on the sequences of the short RNA strands. This strategy requires RNA strands that are activated inside living cells, without activation outside the cells, and which are automatically ligated to become the full-length active siRNA species. RNA-templated SN2 reactions, between an electrophilic iodoacetyl (IAc) group and a nucleophilic phosphorothioate (PS) group, were used because of their high ligation rate, template dependency, and efficiency.27,28 Nucleophilic PS-RNA is deactivated to caged PS-RNA (CPS-RNA), by introducing the phenyl disulfide group, to avoid chemical ligation prior to internalization into living cells. Following cellular entry, the CPS-RNA is uncaged and activated by the abundant intracellular GSH (B1–10 mM) to PS-RNA. The uncaging reaction in cells was predicted based on the similar chemistry of pyridyl disulfides.29 Finally, PS-RNA and IAc-RNA are ligated inside the cells by chemical ligation to produce active siRNA species. CPS-RNAs (1, 2) and IAc-RNAs (3, 4) were synthesized as shown in Fig. S1 (ESI†). RNA with 20 -deoxynucleoside 30 -phosphorothioate (PS-RNA) was prepared on a DNA synthesizer using standard phosphoramidite chemistry. PS-RNA was treated with diphenyl disulfide and 2,4-dinitrofluorobenzene (to trap extra thiophenol) to produce the desired CPS-RNA. Next, RNA with 50 -amino dT was prepared on a DNA synthesizer using standard phosphoramidite chemistry and treated with N-succinimidyl iodoacetate to obtain IAc-RNA. Non-nucleophilic acetylated RNA (Ac-RNA (5, 6)) was also synthesized as a control.
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To test whether the buildup reaction required the presence of GSH, reactions between CPS-RNAs (1, 2) and IAc-RNAs (3, 4) were carried out under various conditions and analyzed by denaturing polyacrylamide gel electrophoresis (PAGE) (Fig. 3). In Fig. 3, lane 1 shows the full-length 25-base RNA strand. A ligation product was observed when four RNA fragments (fragments 1, 2, 3, and 4, as defined in Fig. 2) were combined in the presence of GSH (lane 4). In contrast, no ligation product was observed in the absence of GSH (lane 5). Thus, we confirmed that GSH triggered the ligation reaction. When only the sense (fragments 1 and 3) or antisense (fragments 2 and 4) fragments were mixed in the presence of GSH, no ligation product was generated (lanes 6 and 7). Therefore, we concluded that the ligation reaction took place only in the presence of GSH with the full set of four RNA fragments. Dicer substrate dsRNA dramatically increases gene silencing activity through enhanced efficiency of RISC (RNA induced silencing complex) formation.10 To determine whether the ligated siRNA was a substrate for Dicer, we carried out RNA cleavage reactions using human Dicer, and analyzed them by PAGE (Fig. S2, ESI†). The results showed that both native siRNA (siRNA-n-1) and ligated siRNA (siRNA-lig-1), with their unnatural backbone, were good substrates. To investigate whether IBR-RNA has an RNAi effect in living cells, pairs of CPS-RNAs and IAc-RNAs, as well as a series of control RNAs, were tested (Fig. 4). Ligated siRNA constructed from the pairs of PS-RNAs and IAc-RNAs in vitro was designed to target the firefly luciferase gene. RNAs were transfected into HeLa cells stably expressing the firefly luciferase gene, and the RNAi effect was evaluated by measuring luciferase activity. Ligated siRNA, prepared in vitro, and a pair of CPS-RNAs and IAc-RNAs reduced luciferase expression to 60% and 70% at 25 nM, respectively. This was less potent than native siRNA, which reduced luciferase expression to 40% at an RNA concentration of 25 nM. Increasing the concentration of IBR-RNA provided more potent RNAi activity; 100 nM of IBR-RNA knocked down luciferase expression to 40%. A combination of CPS-RNAs (fragments 1 and 2) and nonelectrophilic Ac-RNAs (fragments 5 and 6) did not have an RNAi effect at any concentration tested. No significant suppression was observed by treating with 100 nM CPS-RNAs, IAc-RNAs, or Ac-RNAs alone. These results indicated that short RNA fragments of nucleophilic PS-RNA and electrophilic IAc-RNA were activated and assembled to form active siRNA species in living cells. Furthermore, we analyzed cell extracts 24 h post-transfection of
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Fig. 4 Gene silencing by siRNA-lig-1 and IBR-RNA, a pair of CPS RNAs (1, 2), and IAc-RNAs (3, 4) in HeLa cells stably expressing luciferase. Luciferase expression was monitored at 24 h post-transfection. The relative expression of luciferase in scramble siRNA (siRNA-scr-1)-transfected cells was defined as 100%. The plotted data are the means standard deviation of three independent experiments.
RNAs to confirm that PS-RNAs and IAc-RNAs had indeed ligated and formed full-length active siRNA species in living cells. Fig. S3 (ESI†) shows PAGE analysis of total RNA extracts from HeLa cells. Native siRNA (siRNA-n-1) and ligated siRNA (siRNAlig-1) prepared in vitro had the same mobility on the gel, which was consistent with that of IBR-RNA treated with GSH. IBR-RNA assembled with two pairs of CPS-RNAs and IAc-RNAs produced full-length siRNA, whereas a pair of CPS-RNAs and Ac-RNAs did not give a ligated product. These results prove our concept of IBR strategy in which CPS-RNA is activated to become nucleophilic, and the ligation reaction between PS-RNAs and IAc-RNAs generates active siRNA species in living cells. Innate immune stimulation by dsRNA is a serious problem preventing its clinical application. Toll-like receptor 3 recognizes dsRNAs of 21 bases or longer and elicits an immune response by inducing an interferon. For example, expression of interferon-b (IFN-b) increases during an immune response. As shorter RNAs cause less of an immune response,26 we expected that IBR-RNA may also reduce the immune response. To evaluate the immune response caused by IBR-RNA, or a series of control RNAs, RNAs were transfected into human glioblastoma cells (T98G) and the expression level of IFN-b mRNA was analyzed by quantitative reverse transcription-polymerase chain reaction (qRT-PCR, Fig. 5). Full-length siRNA (25-base dsRNA) at 100 nM induced significant up-regulation of IFN-b expression, which was 3.7 times higher than that of poly I:C, a positive control for TLR-3 induction. In contrast, segmented siRNA (Fig. 2F) did not cause immune stimulation. Similarly, IBR-RNA showed no significant up-regulation of IFN-b at 100 nM or even 500 nM, which was comparable with the level of IFN-b expression of the mock transfection. Thus, we concluded that our intracellular buildup reaction strategy could potentially decrease undesired immune responses. The design of IBR-RNAs is important to avoid an immune response, maintain a potent RNAi effect, and allow an efficient buildup reaction. The critical length of RNA for TLR3 activation is between 19 and 21 bases, where RNA with 19 bases does not stimulate an immune response.30 With regard to length, we reported the optimal design of IBR-RNA targeting the luciferase
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Fig. 5 Immunostimulatory effect of IBR-RNA. The expression level of IFN-b mRNA was measured by semi-quantitative RT-PCR at 24 h posttransfection of RNA (100 or 500 nM of either IBR-RNA, segmented siRNA or siRNA-n-1) into T98G cells. The expression level of IFN-b mRNA was normalized to that of b-actin. The relative expression of IFN-b mRNA in mock-transfected cells was set as 1. The plotted data are the means standard deviation of three independent experiments.
gene shown in Fig. 2. However, prior to finding this design, we first designed IBR-RNA from 21 bases of siRNA-n-2, as shown in Fig. S4A (Fig. S4–S7, ESI†). However, chemical ligation reactions were inefficient (Fig. S6, lane 4, ESI†) because the RNA complex was unstable where the double-stranded region of RNA was only a minimum of four bases in length. Consequently, this provided no significant RNAi effect upon transfection with any amount of short RNA fragments (Fig. S7, ESI†). The second design was 15 bases and 10 bases, segmented from 25 bases of siRNA, as shown in Fig. 2A (Fig. S8–S11, ESI†). In this design, the chemical ligation proceeded well (Fig. S10, lane 4, ESI†), but the RNAi effect was weak (Fig. S11, ESI†), presumably because the unnatural backbone was located in a central position in the siRNA-lig-3 (Fig. S8D, ESI†). Finally, we designed IBR-RNA (19 bases + 6 bases) derived from 25 bases of siRNA-n-1, which produced the best RNAi effect (Fig. 4). Generally, introduction of high concentrations of siRNA induces non-specific activation of the immune response. For example, 100 nM of 25-bases siRNA-n-1 caused a strong immune response in our experiment (Fig. 5). In contrast, IBR-RNA, even at a concentration of 500 nM, did not induce an immune response. The length and concentration of introduced RNAs are key factors controlling the immune response. The IBR strategy will allow us to regulate these factors more freely than traditional siRNA approaches. We expect that this will be a major advancement in siRNA technology. However, we need to optimize the chemistry of the ligation reaction to improve the knockdown efficiency. We also expect that a natural RNA backbone on the ligated product will be optimal for potent RNAi activity, and we will incorporate this into future studies. H.A. was financially supported by the Ministry of Education, Culture, Sports, Science, and Technology (MEXT), Precursory Research for Embryonic Science and Technology (PREST) and the New Energy and Industrial Technology Development Organization (NEDO). We are grateful for the support received from the Brain Science Institute (BSI) Research Resource Center for mass spectrum analysis.
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Notes and references 1 A. Fire, S. Xu, M. K. Montgomery, S. A. Kostas, S. E. Driver and C. C. Mello, Nature, 1998, 391, 806. 2 S. M. Elbashir, J. Harborth, W. Lendeckel, A. Yalcin, K. Weber and T. Tuschl, Nature, 2001, 411, 494. 3 K. Tiemann and J. J. Rossi, EMBO Mol. Med., 2009, 1, 142. 4 A. D. Judge, G. Bola, A. C. Lee and I. MacLachlan, Mol. Ther., 2006, 13, 494. 5 H. Peacock, A. Kannan, P. A. Beal and C. J. Burrows, J. Org. Chem., 2011, 76, 7295. 6 S. Shukla, C. S. Sumaria and P. I. Pradeepkumar, ChemMedChem, 2010, 5, 328. 7 C. I. Chang, H. A. Kim, P. Dua, S. Kim, C. J. Li and D. K. Lee, Nucleic Acid Ther., 2011, 21, 125. 8 T. R. Brummelkamp, R. Bernards and R. Agami, Science, 2002, 296, 550. 9 D. Siolas, C. Lerner, J. Burchard, W. Ge, P. S. Linsley, P. J. Paddison, G. J. Hannon and M. A. Cleary, Nat. Biotechnol., 2005, 23, 227. 10 D. H. Kim, M. A. Behlke, S. D. Rose, M. S. Chang, S. Choi and J. J. Rossi, Nat. Biotechnol., 2005, 23, 222. 11 Q. Ge, H. Ilves, A. Dallas, P. Kumar, J. Shorenstein, S. A. Kazakov and B. H. Johnston, RNA, 2010, 16, 106. 12 Q. Ge, A. Dallas, H. Ilves, J. Shorenstein, M. A. Behlke and B. H. Johnston, RNA, 2010, 16, 118. 13 A. Dallas, H. Ilves, Q. Ge, P. Kumar, J. Shorenstein, S. A. Kazakov, T. L. Cuellar, M. T. McManus, M. A. Behlke and B. H. Johnston, Nucleic Acids Res., 2012, 40, 9255. 14 Y. Nakashima, H. Abe, N. Abe, K. Aikawa and Y. Ito, Chem. Commun., 2011, 47, 8367. 15 A. Avino, S. M. Ocampo, J. C. Perales and R. Eritja, J. Nucleic Acids, 2011, 2011, 586935.
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ChemComm 16 J. B. Bramsen, M. B. Laursen, C. K. Damgaard, S. W. Lena, B. Ravindra Babu, J. Wengel and J. Kjems, Nucleic Acids Res., 2007, 35, 5886. 17 O. Mook, J. Vreijling, S. L. Wengel, J. Wengel, C. Zhou, J. Chattopadhyaya, F. Baas and K. Fluiter, Artif. DNA PNA XNA, 2010, 1, 36. 18 B. D. Schyth, J. B. Bramsen, M. M. Pakula, S. Larashati, J. Kjems, J. Wengel and N. Lorenzen, Nucleic Acids Res., 2012, 40, 4653. 19 X. Sun, H. A. Rogoff and C. J. Li, Nat. Biotechnol., 2008, 26, 1379. 20 M. Sano, M. Sierant, M. Miyagishi, M. Nakanishi, Y. Takagi and S. Sutou, Nucleic Acids Res., 2008, 36, 5812. 21 C. I. Chang, J. W. Yoo, S. W. Hong, S. E. Lee, H. S. Kang, X. Sun, H. A. Rogoff, C. Ban, S. Kim, C. J. Li and D.-k. Lee, Mol. Ther., 2009, 17, 725. 22 L. Lv, X.-y. Xiao, Z.-h. Gu, F.-q. Zeng, L.-q. Huang and G.-s. Jiang, Mol. Cell. Biochem., 2010, 346, 11. 23 N. Abe, H. Abe and Y. Ito, J. Am. Chem. Soc., 2007, 129, 15108. 24 N. Abe, H. Abe, C. Nagai, M. Harada, H. Hatakeyama, H. Harashima, T. Ohshiro, M. Nishihara, K. Furukawa, M. Maeda, S. Tsuneda and Y. Ito, Bioconjugate Chem., 2011, 22, 2082. 25 J. P. Casey, R. A. Blidner and W. T. Monroe, Mol. Pharmaceutics, 2009, 6, 669. 26 A. Reynolds, E. M. Anderson, A. Vermeulen, Y. Fedorov, K. Robinson, D. Leake, J. Karpilow, W. S. Marshall and A. Khvorova, RNA, 2006, 12, 988. 27 H. Abe, Y. Kondo, H. Jinmei, N. Abe, K. Furukawa, A. Uchiyama, S. Tsuneda, K. Aikawa, I. Matsumoto and Y. Ito, Bioconjugate Chem., 2008, 19, 32. 28 S. M. Gryaznov and R. L. Letsinger, J. Am. Chem. Soc., 1993, 115, 3808. 29 J. Carlsson, H. Drevin and R. Axen, Biochem. J., 1978, 173, 723. 30 M. E. Kleinman, K. Yamada, A. Takeda, V. Chandrasekaran, M. Nozaki, J. Z. Baffi, R. J. Albuquerque, S. Yamasaki, M. Itaya, Y. Pan, B. Appukuttan, D. Gibbs, Z. Yang, K. Kariko, B. K. Ambati, T. A. Wilgus, L. A. DiPietro, E. Sakurai, K. Zhang, J. R. Smith, E. W. Taylor and J. Ambati, Nature, 2008, 452, 591.
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ChemComm Chemical Communications
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ISSN 1359-7345
COMMUNICATION Yoshihiro Ito, Hiroshi Abe et al. An intracellular buildup reaction of active siRNA species from short RNA fragments
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Published on 29 November 2013. Downloaded by Aston University on 09/01/2014 15:36:39.
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Cite this: Chem. Commun., 2014, 50, 1284 Received 1st October 2013, Accepted 28th November 2013
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An intracellular buildup reaction of active siRNA species from short RNA fragments† Hideto Maruyama,ab Yuko Nakashima,a Satoshi Shuto,b Akira Matsuda,b Yoshihiro Ito*ac and Hiroshi Abe*abcd
DOI: 10.1039/c3cc47529h www.rsc.org/chemcomm
Here we report a new strategy for the buildup reaction of active siRNA species from short RNA fragments in living cells using a chemical ligation reaction. This strategy could decrease undesired immune responses and provide more latitude for RNAi technology in the design and concentration of introduced RNA compared to traditional siRNA methods.
RNA interference (RNAi) is a potent and highly specific gene silencing phenomenon that was first reported in the nematode Caenorhabditis elegans by Fire and Mello in 1998.1 They discovered that genes could be silenced by introducing long, double-stranded RNAs (dsRNAs) complementary to mRNA sequences. To silence genes in mammalian cells, short dsRNAs of 20–23 nucleotides, referred to as small interfering RNA (siRNA), have been used as an RNAi trigger to avoid nonspecific interferon responses induced by long dsRNAs.2 However, soon after the application of siRNAs in functional genomic studies and the development of therapeutics, it was found that siRNAs activate innate immune responses via pattern recognition receptors (PRRs) (e.g. Toll-like receptors (TLR) and retinoic acid inducible gene I (RIG-I)-like cytoplasmic helicases).3 In addition, naked siRNAs have several drawbacks, including short-term RNAi effects, due to their unstable nature in serum, non-target specificity, and off-target effects induced by unintended incorporation of the passenger strand into the RNAi-induced silencing complex (RISC). To overcome these issues, many types of chemically modified RNA4–6 and RNAi triggering strategies were developed,7 including short-hairpin RNA (shRNA) expressing vectors,8 synthetic shRNA,9 Dicer substrate dsRNA,10 short shRNA (sshRNA),11–13 branched RNA,14,15 small
internally segmented interfering RNA (sisiRNA),16–18 asymmetric interfering RNA (aiRNA),19–22 dumbbell RNA,23,24 and caged siRNA.25 Significantly, sisiRNA and aiRNA oligonucleotides are shorter than canonical siRNA (21–23 bases). sisiRNA consists of two short sense strands (10 + 11 bases) and an antisense strand of 21 bases. aiRNAs consist of a sense strand of 13–19 bases and an antisense strand of 19–21 bases. These strategies suppress unintended off-target effects16,21 caused by canonical siRNA, and shorter siRNAs (e.g. 19 bases) cause less cellular stress than long siRNAs (>23 bases).26 In principle, shorter RNAs suppress immune stimulation, however, the desired RNAi effect is decreased. Thus, there is a dilemma in optimizing the length of siRNA. Here, we report a new strategy for the buildup reaction of biologically active siRNA species from short RNA fragments that are inactive in living cells, which is called intracellular buildup reaction (IBR) of siRNA (Fig. 1). Short RNA fragments for IBR (IBR-RNA) that have chemically reactive groups are introduced into living cells, and then hybridized and activated by glutathione (GSH), and finally ligated by an SN2 reaction to give active siRNA species
a
Nano Medical Engineering Laboratory, RIKEN, 2-1, Hirosawa, Wako-Shi, Saitama 351-0198, Japan. E-mail: [email protected], [email protected] b Faculty of Pharmaceutical Sciences, Hokkaido University, Kita-12, Nishi-6, Kita-ku, Sapporo 060-0812, Japan c Emergent Bioengineering Materials Research Team, RIKEN Center for Emergent Matter Science, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan d PRESTO, Japan Science and Technology Agency, 4-1-8, Honcho, Kawaguchi, Saitama 332-0012, Japan † Electronic supplementary information (ESI) available. See DOI: 10.1039/ c3cc47529h
1284 | Chem. Commun., 2014, 50, 1284--1287
Fig. 1 Proposed mechanism of the intracellular buildup reaction of active siRNA (IBR).
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Fig. 3 Buildup reaction of ligated siRNA from IBR-RNA triggered by GSH in vitro. Lane 1, 25 mer RNA; lanes 2 and 3, each PS RNA (1 and 2); lanes 4 and 5, reaction mixture (1 + 2 + 3 + 4) with (lane 4) or without (lane 5) GSH; lanes 6 and 7, reaction mixture (lane 6, 1 + 3 (sense strand) or lane 7, 2 + 4 (antisense strand)) without the complementary strand. The reaction mixture was incubated at 37 1C for 30 min and then samples were analyzed by 20% denaturing PAGE. The gel was stained with SYBR Green II. Fig. 2 Structures and sequences of IBR-RNA. (A) Structure of siRNA-n-1 with arrows showing segmented positions. (B–D) Segmented RNA sequences and structures of CPS-RNA (B), IAc-RNA (C), and Ac-RNA (D). (E, F) Sequences and structures of siRNA-lig-1 (used in Fig. 4 and 5) and segmented siRNA (used in Fig. 5), respectively.
(ligated siRNA, Fig. 1). To the best of our knowledge, this is the first report of such an IBR strategy to induce RNAi. The benefit of this strategy is that it bypasses the immune stimulation that is normally induced by native siRNA, and can therefore be applied to the development of new classes of RNAi therapeutics. To circumvent innate immune responses, we designed four short RNA fragments, segmented from 25 bases of active siRNA, targeting the luciferase gene, as shown in Fig. 2A. Each RNA strand was segmented into 19 and 6 bases (sense strand) or 18 and 7 bases (antisense strand). In contrast to full-length siRNA, which causes a strong immune response, short RNA fragments were expected to cause a significantly lower immune response. Thus, we designed the IBR strategy based on the sequences of the short RNA strands. This strategy requires RNA strands that are activated inside living cells, without activation outside the cells, and which are automatically ligated to become the full-length active siRNA species. RNA-templated SN2 reactions, between an electrophilic iodoacetyl (IAc) group and a nucleophilic phosphorothioate (PS) group, were used because of their high ligation rate, template dependency, and efficiency.27,28 Nucleophilic PS-RNA is deactivated to caged PS-RNA (CPS-RNA), by introducing the phenyl disulfide group, to avoid chemical ligation prior to internalization into living cells. Following cellular entry, the CPS-RNA is uncaged and activated by the abundant intracellular GSH (B1–10 mM) to PS-RNA. The uncaging reaction in cells was predicted based on the similar chemistry of pyridyl disulfides.29 Finally, PS-RNA and IAc-RNA are ligated inside the cells by chemical ligation to produce active siRNA species. CPS-RNAs (1, 2) and IAc-RNAs (3, 4) were synthesized as shown in Fig. S1 (ESI†). RNA with 20 -deoxynucleoside 30 -phosphorothioate (PS-RNA) was prepared on a DNA synthesizer using standard phosphoramidite chemistry. PS-RNA was treated with diphenyl disulfide and 2,4-dinitrofluorobenzene (to trap extra thiophenol) to produce the desired CPS-RNA. Next, RNA with 50 -amino dT was prepared on a DNA synthesizer using standard phosphoramidite chemistry and treated with N-succinimidyl iodoacetate to obtain IAc-RNA. Non-nucleophilic acetylated RNA (Ac-RNA (5, 6)) was also synthesized as a control.
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To test whether the buildup reaction required the presence of GSH, reactions between CPS-RNAs (1, 2) and IAc-RNAs (3, 4) were carried out under various conditions and analyzed by denaturing polyacrylamide gel electrophoresis (PAGE) (Fig. 3). In Fig. 3, lane 1 shows the full-length 25-base RNA strand. A ligation product was observed when four RNA fragments (fragments 1, 2, 3, and 4, as defined in Fig. 2) were combined in the presence of GSH (lane 4). In contrast, no ligation product was observed in the absence of GSH (lane 5). Thus, we confirmed that GSH triggered the ligation reaction. When only the sense (fragments 1 and 3) or antisense (fragments 2 and 4) fragments were mixed in the presence of GSH, no ligation product was generated (lanes 6 and 7). Therefore, we concluded that the ligation reaction took place only in the presence of GSH with the full set of four RNA fragments. Dicer substrate dsRNA dramatically increases gene silencing activity through enhanced efficiency of RISC (RNA induced silencing complex) formation.10 To determine whether the ligated siRNA was a substrate for Dicer, we carried out RNA cleavage reactions using human Dicer, and analyzed them by PAGE (Fig. S2, ESI†). The results showed that both native siRNA (siRNA-n-1) and ligated siRNA (siRNA-lig-1), with their unnatural backbone, were good substrates. To investigate whether IBR-RNA has an RNAi effect in living cells, pairs of CPS-RNAs and IAc-RNAs, as well as a series of control RNAs, were tested (Fig. 4). Ligated siRNA constructed from the pairs of PS-RNAs and IAc-RNAs in vitro was designed to target the firefly luciferase gene. RNAs were transfected into HeLa cells stably expressing the firefly luciferase gene, and the RNAi effect was evaluated by measuring luciferase activity. Ligated siRNA, prepared in vitro, and a pair of CPS-RNAs and IAc-RNAs reduced luciferase expression to 60% and 70% at 25 nM, respectively. This was less potent than native siRNA, which reduced luciferase expression to 40% at an RNA concentration of 25 nM. Increasing the concentration of IBR-RNA provided more potent RNAi activity; 100 nM of IBR-RNA knocked down luciferase expression to 40%. A combination of CPS-RNAs (fragments 1 and 2) and nonelectrophilic Ac-RNAs (fragments 5 and 6) did not have an RNAi effect at any concentration tested. No significant suppression was observed by treating with 100 nM CPS-RNAs, IAc-RNAs, or Ac-RNAs alone. These results indicated that short RNA fragments of nucleophilic PS-RNA and electrophilic IAc-RNA were activated and assembled to form active siRNA species in living cells. Furthermore, we analyzed cell extracts 24 h post-transfection of
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Fig. 4 Gene silencing by siRNA-lig-1 and IBR-RNA, a pair of CPS RNAs (1, 2), and IAc-RNAs (3, 4) in HeLa cells stably expressing luciferase. Luciferase expression was monitored at 24 h post-transfection. The relative expression of luciferase in scramble siRNA (siRNA-scr-1)-transfected cells was defined as 100%. The plotted data are the means standard deviation of three independent experiments.
RNAs to confirm that PS-RNAs and IAc-RNAs had indeed ligated and formed full-length active siRNA species in living cells. Fig. S3 (ESI†) shows PAGE analysis of total RNA extracts from HeLa cells. Native siRNA (siRNA-n-1) and ligated siRNA (siRNAlig-1) prepared in vitro had the same mobility on the gel, which was consistent with that of IBR-RNA treated with GSH. IBR-RNA assembled with two pairs of CPS-RNAs and IAc-RNAs produced full-length siRNA, whereas a pair of CPS-RNAs and Ac-RNAs did not give a ligated product. These results prove our concept of IBR strategy in which CPS-RNA is activated to become nucleophilic, and the ligation reaction between PS-RNAs and IAc-RNAs generates active siRNA species in living cells. Innate immune stimulation by dsRNA is a serious problem preventing its clinical application. Toll-like receptor 3 recognizes dsRNAs of 21 bases or longer and elicits an immune response by inducing an interferon. For example, expression of interferon-b (IFN-b) increases during an immune response. As shorter RNAs cause less of an immune response,26 we expected that IBR-RNA may also reduce the immune response. To evaluate the immune response caused by IBR-RNA, or a series of control RNAs, RNAs were transfected into human glioblastoma cells (T98G) and the expression level of IFN-b mRNA was analyzed by quantitative reverse transcription-polymerase chain reaction (qRT-PCR, Fig. 5). Full-length siRNA (25-base dsRNA) at 100 nM induced significant up-regulation of IFN-b expression, which was 3.7 times higher than that of poly I:C, a positive control for TLR-3 induction. In contrast, segmented siRNA (Fig. 2F) did not cause immune stimulation. Similarly, IBR-RNA showed no significant up-regulation of IFN-b at 100 nM or even 500 nM, which was comparable with the level of IFN-b expression of the mock transfection. Thus, we concluded that our intracellular buildup reaction strategy could potentially decrease undesired immune responses. The design of IBR-RNAs is important to avoid an immune response, maintain a potent RNAi effect, and allow an efficient buildup reaction. The critical length of RNA for TLR3 activation is between 19 and 21 bases, where RNA with 19 bases does not stimulate an immune response.30 With regard to length, we reported the optimal design of IBR-RNA targeting the luciferase
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Fig. 5 Immunostimulatory effect of IBR-RNA. The expression level of IFN-b mRNA was measured by semi-quantitative RT-PCR at 24 h posttransfection of RNA (100 or 500 nM of either IBR-RNA, segmented siRNA or siRNA-n-1) into T98G cells. The expression level of IFN-b mRNA was normalized to that of b-actin. The relative expression of IFN-b mRNA in mock-transfected cells was set as 1. The plotted data are the means standard deviation of three independent experiments.
gene shown in Fig. 2. However, prior to finding this design, we first designed IBR-RNA from 21 bases of siRNA-n-2, as shown in Fig. S4A (Fig. S4–S7, ESI†). However, chemical ligation reactions were inefficient (Fig. S6, lane 4, ESI†) because the RNA complex was unstable where the double-stranded region of RNA was only a minimum of four bases in length. Consequently, this provided no significant RNAi effect upon transfection with any amount of short RNA fragments (Fig. S7, ESI†). The second design was 15 bases and 10 bases, segmented from 25 bases of siRNA, as shown in Fig. 2A (Fig. S8–S11, ESI†). In this design, the chemical ligation proceeded well (Fig. S10, lane 4, ESI†), but the RNAi effect was weak (Fig. S11, ESI†), presumably because the unnatural backbone was located in a central position in the siRNA-lig-3 (Fig. S8D, ESI†). Finally, we designed IBR-RNA (19 bases + 6 bases) derived from 25 bases of siRNA-n-1, which produced the best RNAi effect (Fig. 4). Generally, introduction of high concentrations of siRNA induces non-specific activation of the immune response. For example, 100 nM of 25-bases siRNA-n-1 caused a strong immune response in our experiment (Fig. 5). In contrast, IBR-RNA, even at a concentration of 500 nM, did not induce an immune response. The length and concentration of introduced RNAs are key factors controlling the immune response. The IBR strategy will allow us to regulate these factors more freely than traditional siRNA approaches. We expect that this will be a major advancement in siRNA technology. However, we need to optimize the chemistry of the ligation reaction to improve the knockdown efficiency. We also expect that a natural RNA backbone on the ligated product will be optimal for potent RNAi activity, and we will incorporate this into future studies. H.A. was financially supported by the Ministry of Education, Culture, Sports, Science, and Technology (MEXT), Precursory Research for Embryonic Science and Technology (PREST) and the New Energy and Industrial Technology Development Organization (NEDO). We are grateful for the support received from the Brain Science Institute (BSI) Research Resource Center for mass spectrum analysis.
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