Molecules and Cells

Korean Society for Molecular and Cellular Biology (한국분자세포생물학회)
권호 표지
DOI QR코드

DOI QR Code

Dual-Target Gene Silencing by Using Long, Synthetic siRNA Duplexes without Triggering Antiviral Responses

  • Chang, Chan Il (Global Research Laboratory for RNAi Medicine, Department of Chemistry and Brain Korea 21 School of Chemical Materials Science, Sungkyunkwan University) ;
  • Kang, Hye Suk (Global Research Laboratory for RNAi Medicine, Department of Chemistry and Brain Korea 21 School of Chemical Materials Science, Sungkyunkwan University) ;
  • Ban, Changill (Department of Chemistry, Pohang University of Science and Technology) ;
  • Kim, Soyoun (Department of Biomedical Engineeering, Dongguk University) ;
  • Lee, Dong-ki (Global Research Laboratory for RNAi Medicine, Department of Chemistry and Brain Korea 21 School of Chemical Materials Science, Sungkyunkwan University)
  • Received : 2009.05.06
  • Accepted : 2009.05.20
  • Published : 2009.06.30
⟨ Previous Next ⟩

Abstract

Chemically synthesized small interfering RNAs (siRNAs) can specifically knock-down expression of target genes via RNA interference (RNAi) pathway. To date, the length of synthetic siRNA duplex has been strictly maintained less than 30 bp, because an early study suggested that double-stranded RNAs (dsRNAs) longer than 30 bp could not trigger specific gene silencing due to the induction of non-specific antiviral interferon responses. Contrary to the current belief, here we show that synthetic dsRNA as long as 38 bp can result in specific target gene silencing without non-specific antiviral responses. Using this longer duplex structure, we have generated dsRNAs, which can simultaneously knock-down expression of two target genes (termed as dual-target siRNAs or dsiRNAs). Our results thus demonstrate the structural flexibility of gene silencing siRNAs, and provide a starting point to construct multifunctional RNA structures. The dsiRNAs could be utilized to develop a novel therapeutic gene silencing strategy against diseases with multiple gene alternations such as viral infection and cancer.

Keywords

Acknowledgement

Supported by : Korea Foundation for International Cooperation of Science and Technology

References

  1. Birmingham, A., Anderson, E.M., Reynolds, A., Ilsley-Tyree, D., Leake, D., Fedorov, Y., Baskerville, S., Maksimova, E., Robinson, K., Karpilow, J., et al. (2006). 3' UTR seed matches, but not overall identity, are associated with RNAi off-targets. Nat. Methods P, 199-204
  2. Boden, D., Pusch, O., Lee, F., Tucker, L., and Ramratnam, B. (2003). Human immunodeficiency virus type 1 escape from RNA interference. J. Virol. 77, 11531-11535 https://doi.org/10.1128/JVI.77.21.11531-11535.2003
  3. Caplen, N.J., Fleenor, J., Fire, A., and Morgan, R.A. (2000). dsRNA-mediated gene silencing in cultured Drosophila cells: a tissue culture model for the analysis of RNA interference. Gene 252, 95-105 https://doi.org/10.1016/S0378-1119(00)00224-9
  4. Chang, C.I., Hong, S.W., Kim, S., and Lee, D.K. (2007). A structureactivity relationship study of siRNAs with structural variations. Biochem. Biophys. Res. Commun. 359, 997-1003 https://doi.org/10.1016/j.bbrc.2007.06.004
  5. Chang, C.I., Yoo, J.W., Hong, S.W., Lee, S.E., Kang, H.S., Sun, X., Rogoff, H.A., Ban, C., Kim, S., Li, C.J., et al. (2009). Asymmetric shorter-duplex siRNA structures trigger efficient gene silsilencing with reduced nonspecific effects. Mol. Ther. 17, 725-732 https://doi.org/10.1038/mt.2008.298
  6. Clark, P.R., Pober, J.S., and Kluger, M.S. (2008). Knockdown of TNFR1 by the sense strand of an ICAM-1 siRNA: dissection of an off-target effect. Nucleic Acids Res. 36, 1081-1097 https://doi.org/10.1093/nar/gkm630
  7. Das, A.T., Brummelkamp, T.R., Westerhout, E.M., Vink, M., Madiredjo, M., Bernards, R., and Berkhout, B. (2004). Human immunodeficiency virus type 1 escapes from RNA interferencemediated inhibition. J. Virol.78, 2601-2605 https://doi.org/10.1128/JVI.78.5.2601-2605.2004
  8. Elbashir, S.M., Lendeckel, W., and Tuschl, T. (2001a). RNA interference is mediated by 21- and 22-nucleotide RNAs. Genes Dev. 15, 188-200 https://doi.org/10.1101/gad.862301
  9. Elbashir, S.M., Harborth, J., Lendeckel, W., Yalcin, A., Weber, K., and Tuschl, T. (2001b). Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature 411, 494-498 https://doi.org/10.1038/35078107
  10. Elbashir, S.M., Martinez, J., Patkaniowska, A., Lendeckel, W., and Tuschl, T. (2001c). Functional anatomy of siRNAs for mediating efficient RNAi in Drosophila melanogaster embryo lysate. EMBO J. 20, 6877-6888 https://doi.org/10.1093/emboj/20.23.6877
  11. Fire, A., Xu, S., Montgomery, M.K., Kostas, S.A., Driver, S.E., and Mello, C.C. (1998). Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature 391, 806-811 https://doi.org/10.1038/35888
  12. Grimm, D., and Kay, M.A. (2007). Combinatorial RNAi: a winning strategy for the race against evolving targets? Mol. Ther. 15, 878-888 https://doi.org/10.1038/sj.mt.6300116
  13. Hossbach, M., Gruber, J., Osborn, M., Weber, K., and Tuschl, T. (2006). Gene silencing with siRNA duplexes composed of target-mRNA-complementary and partially palindromic or partially complementary single-stranded siRNAs. RNA Biol. 3, 82-89 https://doi.org/10.4161/rna.3.2.3110
  14. Jackson, A.L., Bartz, S.R., Schelter, J., Kobayashi, S.V., Burchard, J., Mao, M., Li, B., Cavet, G., and Linsley, P.S. (2003). Expression profiling reveals off-target gene regulation by RNAi. Nat. Biotechnol. 21, 635-637 https://doi.org/10.1038/nbt831
  15. Jackson, A.L., Burchard, J., Leake, D., Reynolds, A., Schelter, J., Guo, J., Johnson, J. M., Lim, L., Karpilow, J., Nichols, K., et al. (2006). Position-specific chemical modification of siRNAs reduces 'off-target' transcript silencing. RNA 12, 1197-1205 https://doi.org/10.1261/rna.30706
  16. Jacque, J.M., Triques, K., and Stevenson, M. (2002). Modulation of HIV-1 replication by RNA interference. Nature 418, 435-438 https://doi.org/10.1038/nature00896
  17. Kennerdell, J.R., and Carthew, R.W. (1998). Use of dsRNAmediated genetic interference to demonstrate that frizzled and frizzled 2 act in the wingless pathway. Cell 95, 1017-1026 https://doi.org/10.1016/S0092-8674(00)81725-0
  18. Khaled, A., Guo, S., Li, F., and Guo, P. (2005). Controllable selfassembly of nanoparticles for specific delivery of multiple therapeutic molecules to cancer cells using RNA nanotechnology. Nano Lett. 5, 1797-1808 https://doi.org/10.1021/nl051264s
  19. Khvorova, A., Reynolds, A., and Jayasena, S.D. (2003). Functional siRNAs and miRNAs exhibit strand bias. Cell 115, 209-216 https://doi.org/10.1016/S0092-8674(03)00801-8
  20. Kim, D.H., Behlke, M.A., Rose, S.D., Chang, M.S., Choi, S., and Rossi, J.J. (2005). Synthetic dsRNA Dicer substrates enhance RNAi potency and efficacy. Nat. Biotechnol. 23, 222-226 https://doi.org/10.1038/nbt1051
  21. Kim, J.Y., Choung, S., Lee, E.J., Kim, Y.J., and Choi, Y.C. (2007). Immune activation by siRNA/liposome complexes in mice is sequence-independent: lack of a role for Toll-like receptor 3 signaling. Mol. Cells 24, 247-254
  22. Konstantinova, P., de Vries, W., Haasnoot, J., ter Brake, O., de Haan, P., and Berkhout, B. (2006). Inhibition of human immunodeficiency virus type 1 by RNA interference using long-hairpin RNA. Gene Ther. 13, 1403-1413 https://doi.org/10.1038/sj.gt.3302786
  23. Liu, Y.P., Haasnoot, J., and Berkhout, B. (2007). Design of extended short hairpin RNAs for HIV-1 inhibition. Nucleic Acids Res. 35, 5683-5693 https://doi.org/10.1093/nar/gkm596
  24. Manche, L., Green, S.R., Schmedt, C., and Mathews, M.B. (1992). Interactions between double-stranded RNA regulators and the protein kinase DAI. Mol. Cell. Biol. 12, 5238-5248 https://doi.org/10.1128/MCB.12.11.5238
  25. Marques, J.T., Devosse, T., Wang, D., Zamanian-Daryoush, M., Serbinowski, P., Hartmann, R., Fujita, T., Behlke, M.A., and Williams, B.R. (2006). A structural basis for discriminating between self and nonself double-stranded RNAs in mammalian cells. Nat. Biotechnol. 24, 559-565 https://doi.org/10.1038/nbt1205
  26. Menendez, J.A., Vellon, L., Mehmi, I., Oza, B.P., Ropero, S., Colomer, R., and Lupu, R. (2004). Inhibition of fatty acid synthase (FAS) suppresses HER2/neu (erbB-2) oncogene overexpression in cancer cells. Proc. Natl. Acad. Sci. USA 101, 10715-10720 https://doi.org/10.1073/pnas.0403390101
  27. Nishitsuji, H., Ikeda, T., Miyoshi, H., Ohashi, T., Kannagi, M., and Masuda, T. (2004). Expression of small hairpin RNA by lentivirus-based vector confers efficient and stable gene-suppression of HIV-1 on human cells including primary non-dividing cells. Microbes Infect. 6, 76-85 https://doi.org/10.1016/j.micinf.2003.10.009
  28. Novina, C.D., Murray, M.F., Dykxhoorn, D.M., Beresford, P.J., Riess, J., Lee, S.K., Collman, R.G., Lieberman, J., Shankar, P., and Sharp, P.A. (2002). siRNA-directed inhibition of HIV-1 infection. Nat. Med. 8, 681-686 https://doi.org/10.1038/nm725
  29. Reynolds, A., Anderson, E.M., Vermeulen, A., Fedorov, Y., Robinson, K., Leake, D., Karpilow, J., Marshall, W.S., and Khvorova, A. (2006). Induction of the interferon response by siRNA is cell typeand duplex length-dependent. RNA 12, 988-993 https://doi.org/10.1261/rna.2340906
  30. Sano, M., Li, H., Nakanishi, M., and Rossi, J.J. (2008). Expression of long anti-HIV-1 hairpin RNAs for the generation of multiple siRNAs: advantages and limitations. Mol. Ther. 16, 170-177 https://doi.org/10.1038/sj.mt.6300298
  31. 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 https://doi.org/10.1016/S0092-8674(03)00759-1
  32. Soutschek, J., Akinc, A., Bramlage, B., Charisse, K., Constien, R., Donoghue, M., Elbashir, S., Geick, A., Hadwiger, P., Harborth, J., et al. (2004). Therapeutic silencing of an endogenous gene by systemic administration of modified siRNAs. Nature 432, 173-178 https://doi.org/10.1038/nature03121
  33. Stark, G.R., Kerr, I.M., Williams, B.R., Silverman, R.H., and Schreiber, R.D. (1998). How cells respond to interferons. Annu. Rev. Biochem. 67, 227-264 https://doi.org/10.1146/annurev.biochem.67.1.227
  34. ter Brake, O., Konstantinova, P., Ceylan, M., and Berkhout, B. (2006). Silencing of HIV-1 with RNA interference: a multiple shRNA approach. Mol. Ther. 14, 883-892 https://doi.org/10.1016/j.ymthe.2006.07.007
  35. Ui-Tei, K., Zenno, S., Miyata, Y., and Saigo, K. (2000). Sensitive assay of RNA interference in Drosophila and Chinese hamster cultured cells using firefly luciferase gene as target. FEBS Lett. 479, 79-82 https://doi.org/10.1016/S0014-5793(00)01883-4
  36. Vickers, T.A., Lima, W.F., Nichols, J.G., and Crooke, S.T. (2007). Reduced levels of Ago2 expression result in increased siRNA competition in mammalian cells. Nucleic Acids Res. 35, 6598-6610 https://doi.org/10.1093/nar/gkm663
  37. Watanabe, T., Sudoh, M., Miyagishi, M., Akashi, H., Arai, M., Inoue, K., Taira, K., Yoshiba, M., and Kohara, M. (2006). Intracellulardiced dsRNA has enhanced efficacy for silencing HCV RNA and overcomes variation in the viral genotype. Gene Ther. 13, 883-892
  38. Westerhout, E.M., Ooms, M., Vink, M., Das, A.T., and Berkhout, B. (2005). HIV-1 can escape from RNA interference by evolving an alternative structure in its RNA genome. Nucleic Acids Res. 33,796-804 https://doi.org/10.1093/nar/gki220
  39. Yekta, S., Shih, I.H., and Bartel, D.P. (2004). MicroRNA-directed cleavage of HOXB8 mRNA. Science 304, 594-596 https://doi.org/10.1126/science.1097434
  40. Yoo, J.W., Kim, S., and Lee, D.K. (2007). Competition potency of siRNA is specified by the 5′-half sequence of the guide strand. Biochem. Biophys. Res. Commun. 367, 78-83 https://doi.org/10.1016/j.bbrc.2007.12.099
  41. Zamore, P.D., Tuschl, T., Sharp, P.A., and Bartel, D.P. (2000). RNAi: double-stranded RNA directs the ATP-dependent cleavage of mRNA at 21 to 23 nucleotide intervals. Cell 101, 25-33 https://doi.org/10.1016/S0092-8674(00)80620-0

Cited by

  1. RNA interference-based therapeutics for human immunodeficiency virus HIV-1 treatment: synthetic siRNA or vector-based shRNA? vol.10, pp.2, 2009, https://doi.org/10.1517/14712590903448158
  2. Structural Diversity Repertoire of Gene Silencing Small Interfering RNAs vol.21, pp.3, 2011, https://doi.org/10.1089/nat.2011.0286
  3. Long Double-Stranded RNA-Mediated RNA Interference and Immunostimulation: Long Interfering Double-Stranded RNA as a Potent Anticancer Therapeutics vol.21, pp.3, 2009, https://doi.org/10.1089/nat.2011.0296
  4. Small interfering ribonucleic acid design strategies for effective targeting and gene silencing vol.6, pp.3, 2011, https://doi.org/10.1517/17460441.2011.555394
  5. A Boost for the Emerging Field of RNA Nanotechnology : Report on the First International Conference on RNA Nanotechnology vol.5, pp.5, 2009, https://doi.org/10.1021/nn200989r
  6. Branched, Tripartite-Interfering RNAs Silence Multiple Target Genes with Long Guide Strands vol.22, pp.1, 2012, https://doi.org/10.1089/nat.2011.0315
  7. Long Double-Stranded Multiplex siRNAs for Dual Genes Silencing vol.23, pp.4, 2009, https://doi.org/10.1089/nat.2013.0416
  8. Development of RNA Interference-Based Therapeutics and Application of Multi-Target Small Interfering RNAs vol.24, pp.4, 2009, https://doi.org/10.1089/nat.2014.0480
  9. Type 2 Diabetes Mellitus: Limitations of Conventional Therapies and Intervention with Nucleic Acid-Based Therapeutics vol.115, pp.11, 2009, https://doi.org/10.1021/cr5002832
  10. Multi-target siRNA: Therapeutic Strategy for Hepatocellular Carcinoma vol.7, pp.10, 2009, https://doi.org/10.7150/jca.15157
  11. Inhibition of Respiratory Syncytial Virus Replication by Simultaneous Targeting of mRNA and Genomic RNA Using Dual-Targeting siRNAs vol.58, pp.11, 2016, https://doi.org/10.1007/s12033-016-9976-4
  12. A multifunctional toolkit for target-directed cancer therapy vol.55, pp.6, 2009, https://doi.org/10.1039/c8cc08823c