Overview of Gene Silencing by RNA Interference

Daniel H. Kim1, John J. Rossi2

1 Howard Hughes Medical Institute, Department of Molecular Biology, Massachusetts General Hospital, and Department of Genetics, Harvard Medical School, Boston, Massachusetts, 2 Graduate School of Biological Sciences and Division of Molecular Biology, Beckman Research Institute of the City of Hope, Duarte, California
Publication Name:  Current Protocols in Nucleic Acid Chemistry
Unit Number:  Unit 16.1
DOI:  10.1002/0471142700.nc1601s36
Online Posting Date:  March, 2009
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library


The potential of harnessing RNA interference (RNAi) for sequence‐specific gene silencing has generated much excitement and progress in the field. Recent advances in RNAi technology suggest that RNAi‐based approaches may soon become an effective therapeutic strategy against a myriad of diseases. This overview provides a brief description of important considerations when designing an RNAi‐based method for gene silencing and therapeutic development: (a) mechanistic aspects of RNAi‐mediated gene silencing in mammalian cells; (b) structural requirements for potent siRNA duplexes; (c) off‐target effects and interferon responses; and (d) effective delivery of RNAi‐inducing agents. Promising therapeutic applications of RNAi that are currently in the developmental pipeline are also described. Curr. Protoc. Nucleic Acid Chem. 36:16.1.1‐16.1.10. © 2009 by John Wiley & Sons, Inc.

Keywords: RNAi; siRNA; microRNA; shRNA; transcriptional gene silencing

PDF or HTML at Wiley Online Library

Table of Contents

  • Introduction
  • RNAi Mechanisms
  • Enhancing RNAi Potency
  • Improving RNAi Specificity
  • Delivering RNA
  • Therapeutic RNAi Applications
  • Conclusions
  • Acknowledgements
  • Literature Cited
  • Figures
PDF or HTML at Wiley Online Library


PDF or HTML at Wiley Online Library



Literature Cited

Literature Cited
   Amarzguioui, M., Rossi, J.J., and Kim, D. 2005. Approaches for chemically synthesized siRNA and vector‐mediated RNAi. FEBS Lett. 579:5974‐5981.
   Amarzguioui, M., Lundberg, P., Cantin, E., Hagstrom, J., Behlke, M.A., and Rossi, J.J. 2006. Rational design and in vitro and in vivo delivery of Dicer substrate siRNA. Nat. Protoc. 1:508‐517.
   Bartel, D.P. 2004. MicroRNAs: Genomics, biogenesis, mechanism, and function. Cell 116:281‐297.
   Behlke, M.A. 2006. Progress towards in vivo use of siRNAs. Mol. Ther. 13:644‐670.
   Bernstein, E., Caudy, A.A., Hammond, S.M., and Hannon, G.J. 2001. Role for a bidentate ribonuclease in the initiation step of RNA interference. Nature 409:363‐366.
   Bitko, V., Musiyenko, A., Shulyayeva, O., and Barik, S. 2005. Inhibition of respiratory viruses by nasally administered siRNA. Nat. Med. 11:50‐55.
   Brummelkamp, T.R., Bernards, R., and Agami, R. 2002. A system for stable expression of short interfering RNAs in mammalian cells. Science 296:550‐553.
   Cao, R., Wang, L., Wang, H., Xia, L., Erdjument‐Bromage, H., Tempst, P., Jones, R.S., and Zhang, Y. 2002. Role of histone H3 lysine 27 methylation in Polycomb‐group silencing. Science 298:1039‐1043.
   Carmell, M.A., Xuan, Z., Zhang, M.Q., and Hannon, G.J. 2002. The Argonaute family: Tentacles that reach into RNAi, developmental control, stem cell maintenance, and tumorigenesis. Genes Dev. 16:2733‐2742.
   Castanotto, D., Tommasi, S., Li, M., Li, H., Yanow, S., Pfeifer, G.P., and Rossi, J.J. 2005. Short hairpin RNA‐directed cytosine (CpG) methylation of the RASSF1A gene promoter in HeLa cells. Mol. Ther. 12:179‐183.
   Check, E. 2005. A crucial test. Nat. Med. 11:243‐244.
   Chiu, Y.L. and Rana, T.M. 2003. siRNA function in RNAi: A chemical modification analysis. RNA 9:1034‐1048.
   Chu, T.C., Twu, K.Y., Ellington, A.D., and Levy, M. 2006. Aptamer mediated siRNA delivery. Nucleic Acids Res. 34:e73.
   Coburn, G.A., and Cullen, B.R. 2002. Potent and specific inhibition of human immunodeficiency virus type 1 replication by RNA interference. J. Virol. 76:9225‐9231.
   Czauderna, F., Fechtner, M., Dames, S., Aygun, H., Klippel, A., Pronk, G.J., Giese, K., and Kaufman, J. 2003. Structural variations and stabilising modifications of synthetic siRNAs in mammalian cells. Nucleic Acids Res. 31:2705‐2716.
   de Veer, M.J., Sledz, C.A., and Williams, B.R. 2005. Detection of foreign RNA: Implications for RNAi. Immunol. Cell Biol 83:224‐228.
   Dykxhoorn, D.M. and Lieberman, J. 2006. Silencing viral infection. PLoS Med 3:e242.
   Elbashir, S.M., Harborth, J., Lendeckel, W., Yalcin, A., Weber, K., and Tuschl, T. 2001a. Duplexes of 21‐nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature 411:494‐498.
   Elbashir, S.M., Lendeckel, W., and Tuschl, T. 2001b. RNA interference is mediated by 21‐ and 22‐nucleotide RNAs. Genes Dev. 15:188‐200.
   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.
   Grimm, D. and Kay, M.A. 2006. Therapeutic short hairpin RNA expression in the liver: Viral targets and vectors. Gene Ther. 13:563‐575.
   Grimm, D., Streetz, K.L., Jopling, C.L., Storm, T.A., Pandey, K., Davis, C.R., Marion, P., Salazar, F., and Kay, M.A. 2006. Fatality in mice due to oversaturation of cellular microRNA/short hairpin RNA pathways. Nature 441:537‐541.
   Han, J., Kim, D., and Morris, K.V. 2007. Promoter‐associated RNA is required for RNA‐directed transcriptional gene silencing in human cells. Proc. Natl. Acad. Sci. U.S.A. 104:12422‐12427.
   Hornung, V., Guenthner‐Biller, M., Bourquin, C., Ablasser, A., Schlee, M., Uematsu, S., Noronha, A., Manoharan, M., Akira, S., de Fougerolles, A., Endres, S., and Hartmann, G. 2005. Sequence‐specific potent induction of IFN‐alpha by short interfering RNA in plasmacytoid dendritic cells through TLR7. Nat. Med. 11:263‐270.
   Hu‐Lieskovan, S., Heidel, J.D., Bartlett, D.W., Davis, M.E., and Triche, T.J. 2005. Sequence‐specific knockdown of EWS‐FLI1 by targeted, nonviral delivery of small interfering RNA inhibits tumor growth in a murine model of metastatic Ewing's sarcoma. Cancer Res. 65:8984‐8992.
   Hutvagner, G. and Zamore, P.D. 2002. A microRNA in a multiple‐turnover RNAi enzyme complex. Science 297:2056‐2060.
   Jackson, A.L., Burchard, J., Leake, D., Reynolds, A., Schelter, J., Guo, J., Johnson, J.M., Lim, L., Karpilow, J., Nichols, K., Marshall, W., Khvorova, A., and Linsley, P.S. 2006a. Position‐specific chemical modification of siRNAs reduces “off‐target” transcript silencing. RNA 12:1197‐1205.
   Jackson, A.L., Burchard, J., Schelter, J., Chau, B.N., Cleary, M., Lim, L., and Linsley, P.S. 2006b. Widespread siRNA “off‐target” transcript silencing mediated by seed region sequence complementarity. RNA 12:1179‐1187.
   Jacque, J., Triques, K., and Stevenson, M. 2002. Modulation of HIV‐1 replication by RNA interference. Nature 418:435‐438.
   Janowski, B.A., Huffman, K.E., Schwartz, J.C., Ram, R., Hardy, D., Shames, D.S., Minna, J.D., and Corey, D.R. 2005. Inhibiting gene expression at transcription start sites in chromosomal DNA with antigene RNAs. Nat. Chem. Biol. 1:216‐222.
   Janowski, B.A., Huffman, K.E., Schwartz, J.C., Ram, R., Nordsell, R., Shames, D.S., Minna, J.D., and Corey, D.R. 2006. Involvement of AGO1 and AGO2 in mammalian transcriptional silencing. Nat. Struct. Mol. Biol. 13:787‐792.
   Jeffery, L. and Nakielny, S. 2004. Components of the DNA methylation system of chromatin control are RNA‐binding proteins. J. Biol. Chem. 279:49479‐49487.
   Jenuwein, T. and Allis, C.D. 2001. Translating the histone code. Science 293:1074‐1080.
   John, M., Constien, R., Akinc, A., Goldberg, M., Moon, Y.A., Spranger, M., Hadwiger, P., Soutschek, J., Vornlocher, H.P., Manoharan, M., Stoffel, M., Langer, R., Anderson, D.G., Horton, J.D., Koteliansky, V., and Bumcrot, D. 2007. Effective RNAi‐mediated gene silencing without interruption of the endogenous microRNA pathway. Nature 449:745‐747.
   Judge, A.D., Sood, V., Shaw, J.R., Fang, D., McClintock, K., and MacLachlan, I. 2005. Sequence‐dependent stimulation of the mammalian innate immune response by synthetic siRNA. Nat. Biotechnol. 23:457‐462.
   Kim, D.H. and Rossi, J.J. 2007. Strategies for silencing human disease using RNA interference. Nat. Rev. Genet. 8:173‐184.
   Kim, D.H., Longo, M., Han, Y., Lundberg, P., Cantin, E., and Rossi, J.J. 2004. Interferon induction by siRNAs and ssRNAs synthesized by phage polymerase. Nat. Biotechnol. 22:321‐325.
   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.
   Kim, D.H., Villeneuve, L.M., Morris, K.V., and Rossi, J.J. 2006. Argonaute‐1 directs siRNA‐mediated transcriptional gene silencing in human cells. Nat. Struct. Mol. Biol. 13:793‐797.
   Kim, J.W., Zhang, Y.H., Zern, M.A., Rossi, J.J, and Wu, J. 2007. Short hairpin RNA causes the methylation of transforming growth factor‐beta receptor II promoter and silencing of the target gene in rat hepatic stellate cells. Biochem. Biophys. Res. Commun. 359:292‐297.
   Kumar, P., Wu, H., McBride, J.L., Jung, K.E., Kim, M.H., Davidson, B.L., Lee, S.K., Shankar, P., and Manjunath, N. 2007. Transvascular delivery of small interfering RNA to the central nervous system. Nature 448:39‐43.
   Landen, C.N., Jr., Chavez‐Reyes, A., Bucana, C., Schmandt, R., Deavers, M.T., Lopez‐Berestein, G., and Sood, A.K. 2005. Therapeutic EphA2 gene targeting in vivo using neutral liposomal small interfering RNA delivery. Cancer Res. 65:6910‐6918.
   Lee, N.S., Dohjima, T., Bauer, G., Li, H., Li, M., Ehsani, A., Salvaterra, P., and Rossi, J. 2002. Expression of small interfering RNAs targeted against HIV‐1 rev transcripts in human cells. Nature Biotechnol. 19:500‐505.
   Lee, Y., Ahn, C., Han, J., Choi, H., Kim, J., Yim, J., Lee, J., Provost, P., Radmark, O., Kim, S., and Kim, V.N. 2003. The nuclear RNase III Drosha initiates microRNA processing. Nature 425:415‐419.
   Lee, Y., Kim, M., Han, J., Yeom, K.H., Lee, S., Baek, S.H., and Kim, V.N. 2004. MicroRNA genes are transcribed by RNA polymerase II. EMBO. J. 23:4051‐4060.
   Lewis, B.P., Burge, C.B., and Bartel, D.P. 2005. Conserved seed pairing, often flanked by adenosines, indicates that thousands of human genes are microRNA targets. Cell 120:15‐20.
   Liu, J., Carmell, M.A., Rivas, F.V., Marsden, C.G., Thomson, J.M., Song, J.J., Hammond, S.M., Joshua‐Tor, L., and Hannon, G.J. 2004. Argonaute2 is the catalytic engine of mammalian RNAi. Science 305:1437‐1441.
   Liu, J., Valencia‐Sanchez, M.A., Hannon, G.J., and Parker, R. 2005. MicroRNA‐dependent localization of targeted mRNAs to mammalian P‐bodies. Nat. Cell Biol. 7:719‐723.
   Lund, E., Guttinger, S., Calado, A., Dahlberg, J.E., and Kutay, U. 2004. Nuclear export of microRNA precursors. Science 303:95‐98.
   Ma, J.B., Ye, K., and Patel, D.J. 2004. Structural basis for overhang‐specific small interfering RNA recognition by the PAZ domain. Nature 429:318‐322.
   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.
   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.
   Martinez, J. and Tuschl, T., 2004. RISC is a 5′ phosphomonoester‐producing RNA endonuclease. Genes Dev. 18:975‐980.
   Martinez, J., Patkaniowska, A., Urlaub, H., Luhrmann, R., and Tuschl, T. 2002. Single‐stranded antisense siRNAs guide target RNA cleavage in RNAi. Cell 110:563‐574.
   Matranga, C., Tomari, Y., Shin, C., Bartel, D.P., and Zamore, P.D. 2005. Passenger‐strand cleavage facilitates assembly of siRNA into Ago2‐containing RNAi enzyme complexes. Cell 123:607‐620.
   McFarland, T.J., Zhang, Y., Appukuttan, B., and Stout, J.T. 2004. Gene therapy for proliferative ocular diseases. Expert Opin. Biol. Ther. 4:1053‐1058.
   McNamara, J.O., 2nd, Andrechek, E.R., Wang, Y., Viles, K.D., Rempel, R.E., Gilboa, E., Sullenger, B.A., and Giangrande, P.H. 2006. Cell type‐specific delivery of siRNAs with aptamer‐siRNA chimeras. Nat. Biotechnol. 24:1005‐1015.
   Morris, K.V. and Rossi, J.J. 2006. Lentiviral‐mediated delivery of siRNAs for antiviral therapy. Gene Ther. 13:553‐558.
   Morris, K.V., Chan, S.W., Jacobsen, S.E., and Looney, D.J. 2004. Small interfering RNA‐induced transcriptional gene silencing in human cells. Science 305:1289‐1292.
   Morrissey, D.V., Blanchard, K., Shaw, L., Jensen, K., Lockridge, J.A., Dickinson, B., McSwiggen, J.A., Vargeese, C., Bowman, K., Shaffer, C.S., Polisky, B.A., and Zinnen, S. 2005a. Activity of stabilized short interfering RNA in a mouse model of hepatitis B virus replication. Hepatology 41:1349‐1356.
   Morrissey, D.V., Lockridge, J.A., Shaw, L., Blanchard, K., Jensen, K., Breen, W., Hartsough, K., Machemer, L., Radka, S., Jadhav, V., Vaish, N., Zinne, S., Vargeese, C., Bowman, K., Shaffer, C.S., Jeffs, L.B., Judge, A., MacLachlan, I., and Palisky, B. 2005b. Potent and persistent in vivo anti‐HBV activity of chemically modified siRNAs. Nat. Biotechnol. 23:1002‐1007.
   Novina, C.D., Murray, M.F., Dykxhoorn, D.M., Beresford, P.J., Riess, J., Lee, S., Collman, R.G., Leiberman, J., Shankar, P., and Sharp, P.A. 2002. siRNA‐directed inhibtion of HIV‐1 infection. Nature Medicine 8:681‐686.
   Paddison, P.J., Caudy, A.A., and Hannon, G.J. 2002. Stable suppression of gene expression by RNAi in mammalian cells. PNAS 99:1443‐1448.
   Pai, S.I., Lin, Y.Y., Macaes, B., Meneshian, A., Hung, C.F., and Wu, T.C. 2006. Prospects of RNA interference therapy for cancer. Gene Ther. 13:464‐477.
   Palliser, D., Chowdhury, D., Wang, Q.Y., Lee, S.J., Bronson, R.T., Knipe, D.M., and Lieberman, J. 2006. An siRNA‐based microbicide protects mice from lethal herpes simplex virus 2 infection. Nature 439:89‐94.
   Peer, D., Park, E.J., Morishita, Y., Carman, C.V., and Shimaoka, M. 2008. Systemic leukocyte‐directed siRNA delivery revealing cyclin D1 as an anti‐inflammatory target. Science 319:627‐630.
   Preall, J.B. and Sontheimer, E.J. 2005. RNAi: RISC gets loaded. Cell 123:543‐545.
   Ralph, G.S., Radcliffe, P.A., Day, D.M., Carthy, J.M., Leroux, M.A., Lee, D.C., Wong, L.F., Bilsland, L.G., Greensmith, L., Kingsman, S.M., Mitrophanous, K.A., Mazarakis, N.D., and Azzouz, M. 2005. Silencing mutant SOD1 using RNAi protects against neurodegeneration and extends survival in an ALS model. Nat. Med. 11:429‐433.
   Rand, T.A., Petersen, S., Du, F., and Wang, X. 2005. Argonaute2 cleaves the anti‐guide strand of siRNA during RISC activation. Cell 123:621‐629.
   Raoul, C., Abbas‐Terki, T., Bensadoun, J.C., Guillot, S., Haase, G., Szulc, J., Henderson, C.E., and Aebischer, P. 2005. Lentiviral‐mediated silencing of SOD1 through RNA interference retards disease onset and progression in a mouse model of ALS. Nat. Med. 11:423‐428.
   Raoul, C., Barker, S.D., and Aebischer, P. 2006. Viral‐based modelling and correction of neurodegenerative diseases by RNA interference. Gene Ther. 13:487‐495.
   Robbins, M.A., Li, M., Leung, I., Li, H., Boyer, D.V., Song, Y., Behlke, M.A., and Rossi, J.J. 2006. Stable expression of shRNAs in human CD34+ progenitor cells can avoid induction of interferon responses to siRNAs in vitro. Nat. Biotechnol. 24:566‐571.
   Rossi, J.J., 2006. RNAi as a treatment for HIV‐1 infection. Biotechniques Suppl:25‐29.
   Sarnow, P., Jopling, C.L., Norman, K.L., Schutz, S., and Wehner, K.A. 2006. MicroRNAs: Expression, avoidance and subversion by vertebrate viruses. Nat. Rev. Microbiol. 4:651‐659.
   Seth, R.B., Sun, L., and Chen, Z.J. 2006. Antiviral innate immunity pathways. Cell Res. 16:141‐147.
   Song, E., Zhu, P., Lee, S.K., Chowdhury, D., Kussman, S., Dykxhoorn, D.M., Feng, Y., Palliser, D., Weiner, D.B., Shankar, P., Marasco, W.A., and Lieberman, J. 2005. Antibody mediated in vivo delivery of small interfering RNAs via cell‐surface receptors. Nat. Biotechnol. 23:709‐717.
   Soutschek, J., Akinc, A., Bramlage, B., Charisse, K., Constien, R., Donoghue, M., Elbashir, S., Geick, A., Hadwiger, P., Harborth, J., John, M., Kesavan, V., Lavine, G., Pandey, R.K., Racie, T., Rajeev, K.G., Rohl, I., Toudjarska, I., Want, G., Wuschko, S., Bumcrot, D., Koteliansky, V., Limmer, S., Manoharan, M., and Vornlorcher, H.‐P. 2004. Therapeutic silencing of an endogenous gene by systemic administration of modified siRNAs. Nature 432:173‐178.
   Ting, A.H., Schuebel, K.E., Herman, J.G., and Baylin, S.B. 2005. Short double‐stranded RNA induces transcriptional gene silencing in human cancer cells in the absence of DNA methylation. Nat. Genet. 37:906‐910.
   Vire, E., Brenner, C., Deplus, R., Blanchon, L., Fraga, M., Didelot, C., Morey, L., Van Eynde, A., Bernard, D., Vanderwinden, J.M., Bollen, M., Esteller, M., Di Croce, L., deLaunoit, Y., and Fuks, F. 2005. The Polycomb group protein EZH2 directly controls DNA methylation. Nature 439:794‐795.
   Weinberg, M.S., Villeneuve, L.M., Ehsani, A., Amarzguioui, M., Aagaard, L., Chen, Z.X., Riggs, A.D., Rossi, J.J., and Morris, K.V. 2006. The antisense strand of small interfering RNAs directs histone methylation and transcriptional gene silencing in human cells. RNA 12:256‐262.
   Xia, H., Mao, Q., Eliason, S.L., Harper, S.Q., Martins, I.H., Orr, H.T., Paulson, H.L., Yang, L., Kotin, R.M., and Davidson, B.L. 2004. RNAi suppresses polyglutamine‐induced neurodegeneration in a model of spinocerebellar ataxia. Nat. Med. 10:816‐820.
   Yi, R., Qin, Y., Macaran, I.G., and Cullen, B.R. 2003. Exportin‐5 mediates the nuclear export of pre‐microRNAs and short hairpin RNAs. Genes Dev. 17:3011‐3016.
   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.
   Zaratiegui, M., Irvine, D.V., and Martienssen, R.A. 2007. Noncoding RNAs and gene silencing. Cell 128:763‐776.
   Zeng, Y., Wagner, E.J., and Cullen, B.R. 2002. Both natural and designed micro RNAs can inhibit the expression of cognate mRNAs when expressed in human cells. Mol. Cell 9:1327‐1333.
   Zhang, W., Yang, H., Kong, X., Mohapatra, S., San Juan‐Vergara, H., Hellerman, G., Behera, S., Singam, R., Lockey, R.F., and Mohapatra, S.S. 2005. Inhibition of respiratory syncytial virus infection with intranasal siRNA nanoparticles targeting the viral NS1 gene. Nat. Med. 11:56‐62.
   Zilberman, D., Cao, X., and Jacobsen, S.E. 2003. ARGONAUTE4 control of locus‐specific siRNA accumulation and DNA and histone methylation. Science 299:716‐719.
   Zimmermann, T.S., Lee, A.C., Akinc, A., Bramlage, B., Bumcrot, D., Fedoruk, M.N., Harborth, J., Heyes, J.A., Jeffs, L.B., John, M., Judge, A.D., Lam, K., McClintock, K., Nechev, L.V., Palmer, L.R., Racie, T., Rohl, I., Seiffert, S., Shanmugam, S., Sood, V., Soutschek, J., Toudjarska, I., Wheat, A.J., Yaworski, E., Zedalis, W., Koteliansky, V., Manoharan, M., Vornlocher, H.P., and MacLachlan, I. 2006. RNAi‐mediated gene silencing in non‐human primates. Nature 441:111‐114.
PDF or HTML at Wiley Online Library