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Recommended Reading in siRNA Research

Frequently cited siRNA-related articles from peer-reviewed journals and scientific texts.

GETTING STARTED

The following articles describe important insights into the history and uses of RNAi.

RNAi in review - Many excellent reviews on RNAi can be found in the literature. Here are a few favorites.

Zamore, P. D. (2001) RNA interference: listening to the sound of silence. Nat Struct Biol 8, 746750.

Tuschl, T. (2001). RNA Interference and Small Interfering RNAs. Chembiochem Europ J Chem Biol 2, 239-245.

Hannon, G. J. (2002). RNA interference. Nature 418, 244-251. A thorough review of the underlying biologyand mechanics of RNA interference.

Agrawal, N., Dasaradhi, P. V. N., Mohmmed, A., Malhotra, P., Bhatnagar, R. K., and Mukherjee, S. K. (2003).

RNA Interference: Biology, Mechanism, and Applications. Microbiol Mol Biol Rev 67, 657-685. Acomprehensive review of the biology and proposed mechanism of RNA interference with over 200 references.

Bonetta, L. (2004) RNAi: Silencing never sounded better. Nature Methods. 1:79-86.

Gilmore, I., S. Fox, A. Hollins, M. Sohail and S. Akhtar. (2004) The design and exogenous delivery of siRNAfor post-transcriptional gene silencing. J Drug Target. 12:315-40.

Sandy, P. V., Andrea. Jacks, Tyler. (2005) Mammalian RNAi: a practical guide. Bio Techniques. 39:1-10.

Dykxhoorn, D. and J. Lieberman. (2005) The silent revolution: RNA interference as basic biology, researchtool, and therapeutic. Annu Rev Med. 56:401-23.

MILESTONES IN siRNA RESEARCH

Early siRNA Research across the phyla (Petunia, Neurospora, and C. elegans)

Napoli, C., Lemieux, C., and Jorgensen, R. (1990). Introduction of a Chimeric Chalcone Synthase Gene intoPetunia Results in Reversible Co-Suppression of Homologous Genes in trans. Plant Cell 2, 279-289.

Romano, N., and Macino, G. (1992). Quelling: transient inactivation of gene expression in Neurospora crassa bytransformation with homologous sequences. Mol Microbiol 6, 3343-3353.

Fire, A., Xu, S., Montgomery, M. K., Kostas, S. A., Driver, S. E., and Mello, C. C. (1998). Potent and specificgenetic interference by double-stranded RNA in Caenorhabditis elegans. Nature 391, 806-811. The first reportshowing that double-stranded RNA (dsRNA)comprising both sense and antisense strandsis far more potentthan the individual antisense or sense strands alone in silencing gene targets.

Demonstrations of siRNA knockdown in mammalian cells

Elbashir, S. M., Harborth, J., Lendeckel, W., Yalcin, A., Weber, K., and Tuschl, T. (2001). Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature 411, 494-498.In this landmark publication, these authors demonstrate the utility of chemically synthesized siRNAs for targetedgene knockdown in vertebrates, effectively bypassing the interferon response.

Caplen, N. J., Parrish, S., Imani, F., Fire, A., and Morgan, R. A. (2001). Specific inhibition of gene expression bysmall double-stranded RNAs in invertebrate and vertebrate systems. PNAS 98, 9742-9747. This work providedindependent support for the ability to achieve siRNA-mediated silencing in vertebrates cell culture.

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Recommended Reading insiRNA Research

Bantounas, I., L. A. Phylactou and J. B. Uney. (2004) RNA interference and the use of small interfering RNA tostudy gene function in mammalian systems. J. Mol. Endocrinol. 33:545-557.

Demonstration of siRNA silencing in vertebratesProof-of-principle studies demonstrating the utility of siRNA or shRNA-mediated silencing in animal models.

McCaffrey, A. P., Meuse, L., Pham, T. T., Conklin, D. S., Hannon, G. J., and Kay, M. A. (2002). Geneexpression: RNA interference in adult mice. Nature 418, 38-39.This report demonstrates the utility of siRNAs and shRNAs as potential therapeutic agents whether delivered aschemical entities or expressed from vectors.

Lewis, D. L., Hagstrom, J. E., Loomis, A. G., Wolff, J. A., and Herweijer, H. (2002). Efficient delivery of siRNA forinhibition of gene expression in postnatal mice. Nat Genet 32, 107.This paper describes efficient delivery of siRNAs via hydrodynamic tail injections and subsequent silencing of co-injected reporter plasmids in a dose dependent manner.

Demonstration of siRNA silencing in Plants, Pathogens, and Virus

Susi, P., M. Hohkuri, T. Wahlroos and N. J. Kilby. (2004) Characteristics of RNA silencing in plants: similarities

and differences across kingdoms. Plant Mol Biol. 54:157-74.

Cottrell, T. and T. Doering. (2003) Silence of the strands: RNA interference in eukaryotic pathogens. TrendsMicrobiol. 11:37-43.

Tan, F. and J. Yin. (2004) RNAi, a new therapeutic strategy against viral infection. Cell Res. 14:460-6.

CONSIDERATIONS AND ELEMENTS OF siRNA DESIGNDesigning your experiment: A list of references describing key parameters of siRNA design.

Recommendations for experimental setup

Editorial (2003) Whither RNAi? In Nat Cell Biol, pp. 489-490.A concise editorial proposing adoption of good experimental practices for RNAi research by academia andindustry. These include the use of appropriate controls, biological replicates, confirmation of knockdown withmultiple, independent RNAi effectors, and confirmation via distinct detection methods (mRNA levels, proteinlevels, and phenotype).

Huppi, K., Martin, S. E., and Caplen N.J. (2004) Defining and Assaying RNAi in Mammalian Cells. Mol Cell17, 1-10.A review that reiterates good experimental practice with emphasis on attributes associated with expressedshRNA (i.e., confirming sequence fidelity, expression levels, regulation of expression, consideration of the mRNAtarget and its biological context turnover rates, redundancy and feedback regulation)

Aspects of siRNA design

Schwarz, D. S., Hutvagner, G., Du, T., Xu, Z., Aronin, N., and Zamore, P. D. (2003). Unexpected Asymmetry inthe assembly of the RNAi Enzyme Complex. Cell 115, 199-208.

Biochemical studies that identified a bias or asymmetry in functional siRNAs that was not present in non-functional duplexes.

Khvorova, A., Reynolds, A., and Jayasena, S. (2003). Functional siRNAs and miRNAs exhibit strand bias. Cell115, 209-216.The authors compare closely related classes of small regulatory RNAs, microRNAs, and siRNAs to identify keyattributes for efficient processing by the RISC.

Reynolds, A., Leake, D., Scaringe, S., Marshall, W. S., Boese, Q., and Khvorova, A. (2004). Rational siRNAdesign for RNA interference. Nature Biotechnology 22(3), 326 330.

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Recommended Reading insiRNA Research

This work represents the first rigorous, proof-of-concept study in a peer-reviewed journal documenting thefeasibility of rational siRNA design. The rational design detailed in the publication was extended to include manyadditional parameters now used inDharmacon'sproprietarySMARTselection

TMtechnology platform.

Kim, D-H, Behlke, M. A., Rose, S.D., Chang, M-S., Choi S., and Rossi, J.J. (2004) Synthetic dsRNA Dicersubstrates enhance RNAi potency and efficacy. Nature BiotechnologyAdvanced Online Publication January2004.

Despina Siolas, D., Lerner, C., Burchard, J., Ge, W., Linsley, P. S., Paddison, P.J. Hannon, G.J. and Cleary,M.A. (2004) Synthetic shRNAs as potent RNAi triggers. Nature BiotechnologyAdvanced Online PublicationJanuary 2004.

Saetrom, P. and O. Snove. (2004) A comparison of siRNA efficacy predictors. Biochem Biophys ResCommun. 321:247-53.

Amarzguioui, M. and H. Prydz. (2004) An algorithm for selection of functional siRNA sequences. BiochemBiophys Res Commun. 316:1050-8.

Ui-Tei, K., Y. Naito, F. Takahashi, T. Haraguchi, H. Ohki-Hamazaki, A. Juni, R. Ueda and K. Saigo. (2004)

Guidelines for the selection of highly effective siRNA sequences for mammalian and chick RNA interference.Nucleic Acids Res. 32:936-948.

Experimental considerations: countering potential off-target effects

Chi J-T, Chang H. Y., Wang N. N., Chang D. S., Dunphy N., Brown P. O. (2003) Genome-wide view of genesilencing by small interfering RNAs. PNAS100, 63436346.These authors used conventionally designed siRNAs and microarray gene profiling to assess potential off-targeteffects but observed no consistent silencing pattern for unintended targets.

Jackson A. L., Bartz S. R., Schelter J., Kobayashi S. V., Burchard J., Mao M., Li B., Cavet G., Linsley P. S.(2003) Expression profiling reveals off-target gene regulation by RNAi. Nature Biotechnology 21, 635637.Conventionally designed siRNAs demonstrated off-target, homology-directed effects by microarray geneprofiling, however, in an on-going collaboration withDharmacon, sense-strand off-target effects were diminishedwith the application ofON-TARGETmodifications to the cognate siRNAs (unpublished work).

Semizarov D., Frost L., Sarthy A., Kroeger P., Halbert D. N., Fesik S. W. Specificity of short interfering RNAdetermined through gene expression signatures. PNAS 100, 63476352.Observations of siRNA induced off-target effects were diminished by reducing concentrations. Of note, siRNAswere selected by the application of additional thermodynamic criteria to conventional design further highlightingthe utility of rational, SMARTselectiondesign.

SELECTED REFERENCES DOCUMENTING IN VIVOSTUDIESFor additional information please see our flier in vivo RNA interference publications at www.biosyn.com

Soutschek, J., et al. (2004) Therapeutic silencing of an endogenous gene by systemic administration of modifiedsiRNAs. Nature. 432:173-8.

Watanabe, H., H. Saito, P. Rychahou, T. Uchida and B. M. Evers. (2005) Aging is Associated with Decreased

Pancreatic Acinar Cell Regeneration and Phosphatidylinositol 3-kinase/Akt Activation. Gastroenterology.128:1391-404.