Abstract
Small interfering RNAs (siRNAs) have been widely exploited for sequence-specific gene knockdown, predominantly to investigate gene function in cultured vertebrate cells, and also hold promise as therapeutic agents. Because not all siRNAs that are cognate to a given target mRNA are equally effective, computational tools have been developed based on experimental data to increase the likelihood of selecting effective siRNAs. Furthermore, because target-complementary siRNAs can also target other mRNAs containing sequence segments that are partially complementary to the siRNA, most computational tools include ways to reduce potential off-target effects in the siRNA selection process. Though these methods facilitate selection of functional siRNAs, they do not yet alleviate the need for experimental validation. This perspective provides a practical guide based on current wisdom for selecting siRNAs.
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References
Meister, G. & Tuschl, T. Mechanisms of gene silencing by double-stranded RNA. Nature 431, 343–349 (2004).
Dorsett, Y. & Tuschl, T. siRNAs: applications in functional genomics and potential as therapeutics. Nat. Rev. Drug Discov. 3, 318–329 (2004).
Echeverri, C.J. & Perrimon, N. High-throughput RNAi screening in cultured cells: a user's guide. Nat. Rev. Genet. 7, 373–384 (2006).
Dykxhoorn, D.M., Palliser, D. & Lieberman, J. The silent treatment: siRNAs as small molecule drugs. Gene Ther. 13, 541–552 (2006).
Elbashir, S.M., Harborth, J., Weber, K. & Tuschl, T. Analysis of gene function in somatic mammalian cells using small interfering RNAs. Methods 26, 199–213 (2002).
Reynolds, A. et al. Rational siRNA design for RNA interference. Nat. Biotechnol. 22, 326–330 (2004).
Holen, T., Amarzguioui, M., Wiiger, M.T., Babaie, E. & Prydz, H. Positional effects of short interfering RNAs targeting the human coagulation trigger Tissue Factor. Nucleic Acids Res. 30, 1757–1766 (2002).
Harborth, J. et al. Sequence, chemical, and structural variation of small interfering RNAs and short hairpin RNAs and the effect on mammalian gene silencing. Antisense Nucleic Acid Drug Dev. 13, 83–105 (2003).
Birmingham, A. et al. 3′ UTR seed matches, but not overall identity, are associated with RNAi off-targets. Nat. Methods 3, 199–204 (2006).
Jackson, A.L. & Linsley, P.S. Noise amidst the silence: off-target effects of siRNAs? Trends Genet. 20, 521–524 (2004).
Lin, X. et al. siRNA-mediated off-target gene silencing triggered by a 7 nt complementation. Nucleic Acids Res. 33, 4527–4535 (2005).
Jackson, A.L. et al. Widespread siRNA “off-target” transcript silencing mediated by seed region sequence complementarity. RNA 12, 1179–1187 (2006).
Fedorov, Y. et al. Off-target effects by siRNA can induce toxic phenotype. RNA 12, 1188–1196 (2006).
Huesken, D. et al. Design of a genome-wide siRNA library using an artificial neural network. Nat. Biotechnol. 23, 995–1001 (2005).
Saetrom, P. & Snove, O., Jr. A comparison of siRNA efficacy predictors. Biochem. Biophys. Res. Commun. 321, 247–253 (2004).
Zhao, H.F. et al. High-throughput screening of effective siRNAs from RNAi libraries delivered via bacterial invasion. Nat. Methods 2, 967–973 (2005).
Ito, M., Kawano, K., Miyagishi, M. & Taira, K. Genome-wide application of RNAi to the discovery of potential drug targets. FEBS Lett. 579, 5988–5995 (2005).
Kasim, V., Taira, K. & Miyagishi, M. Screening of siRNA target sequences by using fragmentized DNA. J. Gene Med. 8, 782–791 (2006).
Sontheimer, E.J. Assembly and function of RNA silencing complexes. Nat. Rev. Mol. Cell Biol. 6, 127–138 (2005).
Tomari, Y. & Zamore, P.D. Perspective: machines for RNAi. Genes Dev. 19, 517–529 (2005).
Filipowicz, W., Jaskiewicz, L., Kolb, F.A. & Pillai, R.S. Post-transcriptional gene silencing by siRNAs and miRNAs. Curr. Opin. Struct. Biol. 15, 331–341 (2005).
Valencia-Sanchez, M.A., Liu, J., Hannon, G.J. & Parker, R. Control of translation and mRNA degradation by miRNAs and siRNAs. Genes Dev. 20, 515–524 (2006).
Hsieh, A.C. et al. A library of siRNA duplexes targeting the phosphoinositide 3-kinase pathway: determinants of gene silencing for use in cell-based screens. Nucleic Acids Res. 32, 893–901 (2004).
McManus, M.T. et al. Small interfering RNA-mediated gene silencing in T lymphocytes. J. Immunol. 169, 5754–5760 (2002).
Ren, Y. et al. siRecords: an extensive database of mammalian siRNAs with efficacy ratings. Bioinformatics 22, 1027–1028 (2006).
Chalk, A.M., Warfinge, R.E., Georgii-Hemming, P. & Sonnhammer, E.L. siRNAdb: a database of siRNA sequences. Nucleic Acids Res. 33, D131–D134 (2005).
Truss, M. et al. HuSiDa–the human siRNA database: an open-access database for published functional siRNA sequences and technical details of efficient transfer into recipient cells. Nucleic Acids Res. 33, D108–D111 (2005).
Smith, C. Sharpening the tools of RNA interference. Nat. Methods 3, 475–486 (2006).
Shabalina, S.A., Spiridonov, A.N. & Ogurtsov, A.Y. Computational models with thermodynamic and composition features improve siRNA design. BMC Bioinformatics 7, 65 (2006).
Yuan, B., Latek, R., Hossbach, M., Tuschl, T. & Lewitter, F. siRNA Selection Server: an automated siRNA oligonucleotide prediction server. Nucleic Acids Res. 32, W130–W134 (2004).
Chalk, A.M., Wahlestedt, C. & Sonnhammer, E.L. Improved and automated prediction of effective siRNA. Biochem. Biophys. Res. Commun. 319, 264–274 (2004).
Ui-Tei, K. et al. Guidelines for the selection of highly effective siRNA sequences for mammalian and chick RNA interference. Nucleic Acids Res. 32, 936–948 (2004).
Takasaki, S., Kotani, S. & Konagaya, A. An effective method for selecting siRNA target sequences in mammalian cells. Cell Cycle 3, 790–795 (2004).
Jagla, B. et al. Sequence characteristics of functional siRNAs. RNA 11, 864–872 (2005).
Amarzguioui, M. & Prydz, H. An algorithm for selection of functional siRNA sequences. Biochem. Biophys. Res. Commun. 316, 1050–1058 (2004).
Ding, Y., Chan, C.Y. & Lawrence, C.E. Sfold web server for statistical folding and rational design of nucleic acids. Nucleic Acids Res. 32, W135–W141 (2004).
Luo, K.Q. & Chang, D.C. The gene-silencing efficiency of siRNA is strongly dependent on the local structure of mRNA at the targeted region. Biochem. Biophys. Res. Commun. 318, 303–310 (2004).
Yiu, S.M. et al. Filtering of ineffective siRNAs and improved siRNA design tool. Bioinformatics 21, 144–151 (2005).
Kumar, R., Conklin, D.S. & Mittal, V. High-throughput selection of effective RNAi probes for gene silencing. Genome Res. 13, 2333–2340 (2003).
Malik, I., Garrido, M., Bahr, M., Kugler, S. & Michel, U. Comparison of test systems for RNAinterference. Biochem. Biophys. Res. Commun. 341, 245–253 (2006).
Cullen, B.R. Enhancing and confirming the specificity of RNAi experiments. Nat. Methods 3, 677–681 (2006).
Khvorova, A., Reynolds, A. & Jayasena, S.D. Functional siRNAs and miRNAs exhibit strand bias. Cell 115, 209–216 (2003).
Schwarz, D.S. et al. Asymmetry in the assembly of the RNAi enzyme complex. Cell 115, 199–208 (2003).
Hutvagner, G. Small RNA asymmetry in RNAi: function in RISC assembly and gene regulation. FEBS Lett. 579, 5850–5857 (2005).
Patzel, V. et al. Design of siRNAs producing unstructured guide-RNAs results in improved RNA interference efficiency. Nat. Biotechnol. 23, 1440–1444 (2005).
Taxman, D.J. et al. Criteria for effective design, construction, and gene knockdown by shRNA vectors. BMC Biotechnol. 6, 7 (2006).
Matranga, C., Tomari, Y., Shin, C., Bartel, D.P. & Zamore, P.D. Passenger-strand cleavage facilitates assembly of siRNA into Ago2-containing RNAi enzyme complexes. Cell 123, 607–620 (2005).
Manoharan, M. RNA interference and chemically modified small interfering RNAs. Curr. Opin. Chem. Biol. 8, 570–579 (2004).
Hossbach, M., Gruber, J., Osborn, M., Weber, K. & Tuschl, T. Gene silencing with siRNA duplexes composed of target-mRNA-complementary and partially palindromic or partially complementary single-stranded siRNAs. RNA Biology 3 (2006).
Cullen, B.R. Induction of stable RNA interference in mammalian cells. Gene Ther. 13, 503–508 (2006).
Heale, B.S., Soifer, H.S., Bowers, C. & Rossi, J.J. siRNA target site secondary structure predictions using local stable substructures. Nucleic Acids Res. 33, e30 (2005).
Schubert, S., Grunweller, A., Erdmann, V.A. & Kurreck, J. Local RNA target structure influences siRNA efficacy: systematic analysis of intentionally designed binding regions. J. Mol. Biol. 348, 883–893 (2005).
Overhoff, M. et al. Local RNA target structure influences siRNA efficacy: a systematic global analysis. J. Mol. Biol. 348, 871–881 (2005).
Mittal, V. Improving the efficiency of RNA interference in mammals. Nat. Rev. Genet. 5, 355–365 (2004).
Judge, A.D. et al. Sequence-dependent stimulation of the mammalian innate immune response by synthetic siRNA. Nat. Biotechnol. 23, 457–462 (2005).
Hornung, V. et al. Sequence-specific potent induction of IFN-α by short interfering RNA in plasmacytoid dendritic cells through TLR7. Nat. Med. 11, 263–270 (2005).
Marques, J.T. & Williams, B.R. Activation of the mammalian immune system by siRNAs. Nat. Biotechnol. 23, 1399–1405 (2005).
Judge, A.D., Bola, G., Lee, A.C. & MacLachlan, I. Design of noninflammatory synthetic siRNA mediating potent gene silencing in vivo. Mol. Ther. 13, 494–505 (2006).
Snøve, O. Jr. & Rossi, J.J. Expressing short hairpin RNAs in vivo. Nat. Methods 3, 689–695 (2006).
Elbashir, S.M. et al. Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature 411, 494–498 (2001).
Elbashir, S.M., Lendeckel, W. & Tuschl, T. RNA interference is mediated by 21- and 22-nucleotide RNAs. Genes Dev. 15, 188–200 (2001).
Caplen, N.J., Parrish, S., Imani, F., Fire, A. & Morgan, R.A. Specific inhibition of gene expression by small double-stranded RNAs in invertebrate and vertebrate systems. Proc. Natl. Acad. Sci. USA 98, 9742–9747 (2001).
Paddison, P.J., Caudy, A.A., Bernstein, E., Hannon, G.J. & Conklin, D.S. Short hairpin RNAs (shRNAs) induce sequence-specific silencing in mammalian cells. Genes Dev. 16, 948–958 (2002).
Kim, D.H. et al. Synthetic dsRNA Dicer substrates enhance RNAi potency and efficacy. Nat. Biotechnol. 23, 222–226 (2005).
Amarzguioui, M., Rossi, J.J. & Kim, D. Approaches for chemically synthesized siRNA and vector-mediated RNAi. FEBS Lett. 579, 5974–5981 (2005).
Rose, S.D. et al. Functional polarity is introduced by Dicer processing of short substrate RNAs. Nucleic Acids Res. 33, 4140–4156 (2005).
Reynolds, A. et al. Induction of the interferon response by siRNA is cell type- and duplex length-dependent. RNA 12, 988–993 (2006).
Marques, J.T. et al. A structural basis for discriminating between self and nonself double-stranded RNAs in mammalian cells. Nat. Biotechnol. 24, 559–565 (2006).
Vermeulen, A. et al. The contributions of dsRNA structure to Dicer specificity and efficiency. RNA 11, 674–682 (2005).
Soutschek, J. et al. Therapeutic silencing of an endogenous gene by systemic administration of modified siRNAs. Nature 432, 173–178 (2004).
Jackson, A.L. et al. Expression profiling reveals off-target gene regulation by RNAi. Nat. Biotechnol. 21, 635–637 (2003).
Bartel, D.P. MicroRNAs: genomics, biogenesis, mechanism, and function. Cell 116, 281–297 (2004).
Haley, B. & Zamore, P.D. Kinetic analysis of the RNAi enzyme complex. Nat. Struct. Mol. Biol. 11, 599–606 (2004).
Martinez, J. & Tuschl, T. RISC is a 5′ phosphomonoester-producing RNA endonuclease. Genes Dev. 18, 975–980 (2004).
Holen, T. et al. Tolerated wobble mutations in siRNAs decrease specificity, but can enhance activity in vivo. Nucleic Acids Res. 33, 4704–4710 (2005).
Elbashir, S.M., Martinez, J., Patkaniowska, A., Lendeckel, W. & Tuschl, T. Functional anatomy of siRNAs for mediating efficient RNAi in Drosophila melanogaster embryo lysate. EMBO J. 20, 6877–6888 (2001).
Du, Q., Thonberg, H., Wang, J., Wahlestedt, C. & Liang, Z. A systematic analysis of the silencing effects of an active siRNA at all single-nucleotide mismatched target sites. Nucleic Acids Res. 33, 1671–1677 (2005).
Dykxhoorn, D.M., Schlehuber, L.D., London, I.M. & Lieberman, J. Determinants of specific RNA interference-mediated silencing of human beta-globin alleles differing by a single nucleotide polymorphism. Proc. Natl. Acad. Sci. USA 103, 5953–5958 (2006).
Boese, Q. et al. Mechanistic insights aid computational short interfering RNA design. Methods Enzymol. 392, 73–96 (2005).
Santoyo, J., Vaquerizas, J.M. & Dopazo, J. Highly specific and accurate selection of siRNAs for high-throughput functional assays. Bioinformatics 21, 1376–1382 (2005).
Naito, Y., Yamada, T., Ui-Tei, K., Morishita, S. & Saigo, K. siDirect: highly effective, target-specific siRNA design software for mammalian RNA interference. Nucleic Acids Res. 32, W124–W129 (2004).
Jackson, A.L. et al. Position-specific chemical modification of siRNAs reduces “off-target” transcript silencing. RNA 12, 1197–1205 (2006).
Rodriguez-Lebron, E. & Paulson, H.L. Allele-specific RNA interference for neurological disease. Gene Ther. 13, 576–581 (2006).
Kubodera, T., Yokota, T., Ishikawa, K. & Mizusawa, H. New RNAi strategy for selective suppression of a mutant allele in polyglutamine disease. Oligonucleotides 15, 298–302 (2005).
Samakoglu, S. et al. A genetic strategy to treat sickle cell anemia by coregulating globin transgene expression and RNA interference. Nat. Biotechnol. 24, 89–94 (2006).
Martinez, J., Patkaniowska, A., Urlaub, H., Luhrmann, R. & Tuschl, T. Single-stranded antisense siRNAs guide target RNA cleavage in RNAi. Cell 110, 563–574 (2002).
Kittler, R., Heninger, A.K., Franke, K., Habermann, B. & Buchholz, F. Production of endoribonuclease-prepared short interfering RNAs for gene silencing in mammalian cells. Nat. Methods 2, 779–784 (2005).
Buchholz, F., Kittler, R., Slabicki, M. & Theis, M. Enzymatically prepared RNAi libraries. Nat. Methods 3, 696–700 (2006).
Bernards, R., Brummelkamp, T.R. & Beijersbergen, R.L. shRNA libraries and their use in cancer genetics. Nat. Methods 3, 701–706 (2006).
Chang, K., Elledge, S.J. & Hannon, G.J. Lessons from nature: microRNA-based shRNA libraries. Nat. Methods 3, 707–714 (2006).
Root, D.E., Hacohen, N., Hahn, W.C., Lander, E.S. & Sabatini, D.M. Genome-scale loss-of-function screening with a lentiviral RNAi library. Nat. Methods 3, 715–719 (2006).
Pillai, R.S. MicroRNA function: multiple mechanisms for a tiny RNA? RNA 11, 1753–1761 (2005).
Yekta, S., Shih, I.H. & Bartel, D.P. MicroRNA-directed cleavage of HOXB8 mRNA. Science 304, 594–596 (2004).
Rand, T.A., Petersen, S., Du, F. & Wang, X. Argonaute2 cleaves the anti-guide strand of siRNA during RISC activation. Cell 123, 621–629 (2005).
Leuschner, P.J., Ameres, S.L., Kueng, S. & Martinez, J. Cleavage of the siRNA passenger strand during RISC assembly in human cells. EMBO Rep. 7, 314–320 (2006).
Acknowledgements
We apologize to authors whose works are not cited owing to space limitations. We thank C. Echeverri at Cenix Bioscience for valuable discussion. We also thank M. Landthaler, P. Landgraf, J. Brennecke and C. Rogler for critical reading of the manuscript. Y.P. is supported by the Ruth L. Kirschstein Fellowship from the US National Institutes of Health–National Institute of General Medical Sciences.
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T.T. is a cofounder of Alnylam Therapeuticals and serves on its Scientific Advisory Board.
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Pei, Y., Tuschl, T. On the art of identifying effective and specific siRNAs. Nat Methods 3, 670–676 (2006). https://doi.org/10.1038/nmeth911
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DOI: https://doi.org/10.1038/nmeth911
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