RNA interference (RNAi) has developed into one of. New technologies from sirna world
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1 MINERVA BIOTEC 2008;20:3-11 New technologies from sirna world F. MARTINEZ DI MONTEMUROS, P. PARISE RNA interference (RNAi) has developed into one of the most exciting and important methods for analyzing gene function and metabolic pathways in the past 15 years. The ability to assess gene function via sirnamediated methods represents an exciting and valuable tool that has already begun to accelerate critical investigations across abroad range of biomedical and biological research. Recently, widespread, non-specific effects that complicate the interpretation of data generated from sirna-mediated knockdown studies have been observed. Off-target effects occur when a sirna is processed by the RISC and down-regulates unintended targets. As these changes in gene expression can lead to measurable phenotypes (i.e. false positives) it is of great importance to understand the mechanism behind off-targeting so that strategies can be developed to minimize their effects. The research on off-target effects found that a combination of bioinformatics, chemical modifications, and sirna pooling significantly reduce unintended targeting, leading to the development of new technologies. Characterized by the same pathway of RNAi, micrornas (mirnas) have rapidly emerged as an important class of short endogenous RNAs that act as post-transcriptional regulators of gene expression by base-pairing with their target mrnas. Several technologies for assessing expression profiling and function of micrornas are available to the research community and presented in this review. Key words: RNA interference - MicroRNA - Biotechnology. RNA interference (RNAi) has developed into one of the most exciting and important methods for analyzing gene function and metabolic pathways in the past 15 years. During the 1990 s, this phenomenon Address reprint requests to: F. Martinez di Montemuros, Celbio S.p.A., via Figino 20/22, Pero, Milan, Italy. fmartinez@celbio.it Biotechnology Division-Molecular Biology Celbio S.p.A., Pero, Milan, Italy of gene-specific silencing was described in a variety of model organisms before its recognition as a widespread, perhaps universal, mechanism. In plants, the RNA mediated process was termed co-suppression and post-transcriptional or homologous gene silencing (PTGS or HGS). 1 In fungi, it was defined as quelling, 2 and in nematodes, it became known as RNAi. 3 Further studies implicate the RNAi mechanism as an evolutionarily conserved ancient cellular line of defense directed against invading viral genomes or as a method to clear a cell of aberrant transcription products. Further analyses of the RNAi pathway revealed that it is triggered by double stranded RNA (dsrna) molecules sharing sequence-specific homology to particular target mrnas. 4-8 Target mrnas include transcription products of an invading viral genome, transfected transgenes, parasitic transposons or repetitive elements within the host genome itself. Subsequent biochemical studies in Drosophila cell-free lysates revealed that the mediators of RNA-dependent gene silencing are nucleotide small interfering RNA duplexes (sirnas). These sirnas are derived from processing of the trigger dsrna by an enzyme known as Dicer in a RNase III-like fashion. The mechanism involves the recruitment of sirna duplex products into a multi-protein sirna complex, known as the RNA-Induced Silencing Complex (RISC). The RISC is then thought to be guided to the target mrna of inter- Vol No 1. MINERVA BIOTECNOLOGICA 3
2 MARTINEZ DI MONTEMUROS NEW TECHNOLOGIES FROM SIRNA WORLD shrna-mir shrna Pri-miRNA DROSHA Pre-miRNA Nucleus EXPORTIN 5 Pre-miRNA DICER Cytoplasm sirna mrna expression level (%) Cell viability (%) mirna mrna RISC RISC Endogenous protein expression Figure 1. The RNAi pathway. Entry points of sirna, shrna and shrnamir are showed. 0 MAP2K1-1 MAP2K1-2 MAP2K1-3 MAP2K1-4 MAP2K2-1 Figure 2. Off-target effects are sequence dependent and target independent. A side-by-side comparison of toxicity and gene knockdown of eight sirnas targeting two separate genes (MAP2K1 and MAP2K2). While all four duplexes in each cluster provide equivalent levels of gene silencing (>80%), only a single sirna in each group is toxic. MAP2K2-2 MAP2K2-3 MAP2K2-4 Lipidi Untreated 0 est where the sirna duplex interacts in a sequencespecific manner to mediate catalytic cleavage (Figure 1). Preliminary attempts to use RNAi in mammalian systems employed long dsrnas as triggers, but led to the induction of a non-specific Type I interferon response rather than sequence-specific silencing. This interferon response resulted in widespread changes in protein expression, masking any sequence-specific effects and eventually leading to apoptosis. This global phenomenon prevented initial efforts to employ RNAi as a viable means to study individual gene function. In subsequent landmark studies using chemically synthesized sirnas, investigators showed that sirna could effectively bypass the mammalian interferon response and sequence-specifically silence gene expression Clearly, the ability to assess gene function via sirnamediated methods represents an exciting and valuable tool that has already begun to accelerate critical investigations across abroad range of biomedical and biological research. Functionality and off-target effects of sirnas As discussed, RNAi is a post-transcriptional gene regulatory mechanism that can be mediated by endogenously encoded small RNA molecules (micrornas), or synthetic duplexes (sirnas). In both cases, these molecules partner with the RISC to target messenger RNAs for degradation and/or translation attenuation. Since the discovery that sirnas can enter the RNAi pathway and mediate gene knockdown, researchers have invested considerable effort in optimizing the technology for functional genomics studies. 12, 13 As is the case with most new technologies, the transition of RNAi into a research tool has had its obstacles. Initial problems associated with inconsistent sirna functionality were addressed by detailed studies that identified functional attributes associated with duplex performance More recently, widespread, non-specific effects that complicate the interpretation of data generated from sirna-mediated knockdown studies have been observed. Off-target effects occur when an sirna is processed by the RISC and downregulates unintended targets As these changes in gene expression can lead to measurable phenotypes (i.e. false positives) it is of great importance to understand the mechanism behind off-targeting so that strategies can be developed to minimize their effects (Figure 2). Non-specific effects resulting from the introduction of sirna appear to have three separate origins: 1) lipid-mediated response; 4 MINERVA BIOTECNOLOGICA March 2008
3 NEW TECHNOLOGIES FROM SIRNA WORLD MARTINEZ DI MONTEMUROS 2) interferon response; 3) RISC-dependent off-target effects. As demonstrated by Fedorov et al., 22 cationic lipids typically used to deliver sirna can induce broad changes in gene expression profiles. Separately, multiple labs have observed that particular sequence motifs and dsrna lengths can induce the interferon response pathway in a cell-type specific manner. While these unanticipated effects can be eliminated by adopting stringent sirna design filters and optimizing lipid concentrations and compositions, a more challenging contributor to non-specific sirna-mediated gene modulation, off-target effects, presents the research community with a surprisingly complex problem Dharmacon, having conducted considerable research on off-target effects, found that a combination of bioinformatics, chemical modifications, and sirna pooling significantly reduce unintended targeting. 31 These three innovations are included in the new ON-TARGETplus SMARTpool sirna reagents. Off-target effects were first described by Jackson et al. in Using genome-wide microarray profiling as a method of detection, the authors identified modest, 1.5- to 3-fold changes in the expression of dozens of genes following transfection of individual sirna. The levels of complementarity between the sense or antisense strand of the sirna and the off-targeted genes varied considerably and the overall off-target expression profile was unique for each sirna, suggesting a sequence specific component to the phenomena. Initially, these modest changes in off-target gene expression led many to dismiss the event as inconsequential. Unfortunately, this optimism was recently dispelled by reports that off-target effects could induce measurable phenotypes. In a broad RNAibased phenotypic screen designed to identify kinase regulators of the Hypoxia-inducible factor 1 (HIF-1), Lin et al. discovered that the phenotypic effects inducedby several of their top candidates were the consequenceof off-targeting of HIF Similarly, Fedorov et al. demonstrated that a significant fraction of sirnas targeting the housekeeping gene diazepam binding inhibitor (DBI) produced cell viability phenotypes thathad characteristic features of off-target effects, i.e. target independence, sequence dependence, and RISC-reliance. 22 These findings demonstrated a clear shortcoming of the technology. TARGET OFF - TARGETS sirna1 sirna2 sirna3 sirna4 SMARTpool 75% 0% % Knockdown Figure 3. Pooling of four sirna duplexes greatly reduces off-target effects of individual sirnas, while maintaining high levels of target silencing. Heat maps from microarray expression studies of HeLa cells transfected four Cyclophilin B (NM_000942) SMARTselection designed sirnas and the corresponding SMARTpool sirna reagent at 24 hours. White represents no change in gene expression, while greyblack represents greater than or equal to 75% reduction in mrna level. False positives generated during phenotypic screens can lead to false leads and the unnecessary use of resources to explore non-productive research tracts. From a therapeutics standpoint, off-target phenotypes represent a drawback that can severely limit applications of sirnas as future therapeutic agents. For these reasons, understanding the mechanism underlying off-target effects and identifying measures that can enhance sirna specificity is critical for developing RNAi into a reliable research and therapeutic tool. The first strategy originated from observations that off-target effects were concentration-dependent. As the concentration of the sirna is reduced, off-target effects faded. Unfortunately, attempts to employ this approach with all but the most potent sirnas on a broad scale have failed due to the simultaneous depletion of target gene knockdown along with off-target effects. This impasse is resolved by adopting the very simple strategy known as pooling. Dharmacon SMARTpool sirna reagents pool four highly functional SMARTselection designed sirnas targeting the same gene. Studies show that strong on target gene knockdown can be achieved with minimal offtarget effects if a pool consisting of highly functional multiple sirna is substituted for individual duplexes. This finding is in contrast to speculation that mixtures Vol No 1. MINERVA BIOTECNOLOGICA 5
4 MARTINEZ DI MONTEMUROS NEW TECHNOLOGIES FROM SIRNA WORLD Accell sirna (stock solution) Accell delivery mix (1 μm sirna 1 2 Accell sirna delivery media nucleotides that are essential for off-targeting were identified. Modification of these positions eliminates the majority of antisense strand off-target effects while preserving on-target knockdown. The combination of both sense and antisense strand modifications has recently been validated. The last approach toward eliminating off-target effects is associated with sirna design. In the studies by both Birmingham et al. 30 and Lin et al., 28 the investigators observed that off-targeted genes often contained matches between the seed region of the sirna (positions 2-7) and sequences inthe 3 UTR of the offtargeted gene. Furthermore, Birmingham found evidence that the likelihood of a gene being off-targeted is elevated by the presence of multiple 3 UTR seed matches. micrornas also utilize the seed region to mediate gene knockdown, thus these findings intimate a strong mechanistic parallel between sirna off-targeting and microrna-mediated gene regulation. The role of the 3 UTR seed match can be taken into consideration during sirna design, allowing users to minimize off-target effects. Delivery of sirnas Figure 4. Accell sirna protocol. Two simple steps with low optimization and improved stability. of sirnas can compound off-target effects. As shown in Figure 3, while individual duplexes delivered at 100 nm can induce varying numbers of off-targeted genes, transfection of the corresponding SMARTpool sirna (100 nm total concentration) induces only a fraction of the total off-target profile. Importantly, the reduction in off target gene modulation is achieved without jeopardizing target specific knockdown. A second approach to minimizing off-target effects involves recent discoveries in the field of sirna chemical modification. 31, 32 In the process of performing a structure function analysis of chemically modified sirna, Dharmacon scientists identified a unique combination of modifications that eliminate as much as 80% of off-target effects. The modification pattern includes both sense and antisense strand enhancements. On the sense strand, modifications have been added to impede strand entry into RISC, thus eliminating off-target effects by this strand. To address offtargets generated by the antisense strand, key Over the years, gene silencing by RNAi has relied heavily on the use of cationic lipid-based delivery reagents for transfecting cells with small interfering RNAs (sirnas). In many cases, lipid-based transfection is sufficient and provides efficient levels of gene knockdown. Yet, in a significant fraction of cases, cells are either refractory to lipid mediated delivery of sirnas oraversely sensitive to the presence of lipids. During the last years new transfection reagents based on synthetic non-lipidic polymers have been developed to reduce the amount of sirna to get functional effect into the cell over 100 times. In particular, INTERFERin TM (Polyplus Transfection, Strasbourg, France) is a powerful sirna transfection reagent that ensures efficient gene silencing and reproducible transfection to mammalian cells. For most adherent cell lines and primary cells, 1 nm sirna is sufficient to obtain over 90% gene silencing, as observed for HeLa, MCF7 and NIH-3T3. In any case, by using transfection reagents it still remains impossibile to avoid cell toxicity, especially in primary and hard-to-transfect cells. The Accell sirna represents a recently developed technology that promotes delivery of sirna indepen- 6 MINERVA BIOTECNOLOGICA March 2008
5 NEW TECHNOLOGIES FROM SIRNA WORLD MARTINEZ DI MONTEMUROS dent of viral vectors or lipid-based transfection reagents (also referred to as passive delivery ) to virtually any cell type. Through the incorporation of a unique combination of chemical and bioinformatic enhancements that increase functionality, stability, and lipid-independent delivery, Accell sirnas can be used in a wide range of human, mouse, and rat cell lines that have been traditionally identified as difficult to transfect (Figure 4). This novel delivery technology permits gene silencing with minimal off-target events (as assessed by genome-wide profiling), has limited repercussions on cell viability, and triggers a negligible innate immune response as assessed by quantitation of inflammatory response proteins. Thus, unlike electroporation, which is historically the first alternative delivery method for cells found to be averse to lipid based transfection reagents, Accell sirna can be employed without significant effects on cell physiology. Over the course of investigations, it is often necessary to knockdown gene expression for extended periods of time to accurately assess the contribution of a particular protein to a given biological event. Long-term reduction of gene expression can be problematic, particularly in cases where there are large intracellular reservoirs of the target or the half-life of the proteinis drawn out. In some instances, researchers have attempted multiple, consecutive lipid-mediated transfections of sirnas into cultured cells. Unfortunately, this approach is frequently cytotoxic and can induce brief (24-72 h) changes in the state of the innate immune response. To overcome these barriers, DNA-mediated RNAi, commonly exploiting viral delivery platforms, has been adopted. This approach can be successful, but is costly and requires significant development time. For a solution that requires neither lipid transfection nor viral delivery, Accell sirna may be applied to provide long-term knockdown of gene expression. Microporation is a novel and proprietary electroporation technology developed by DigitalBio Technology (NanoEnTek Inc., Seoul, Korea). Microporation is a unique technology using a pipette tip as an electroporation space instead of plate type electrode and cuvette style chamber. In doing so, the uniform electric field is generated accompanying minimal heat generation, metal ion dissolution, ph variation and oxide formation, which are harmful events to the cell during conventional electroporation process. Great transfection efficiency and cell survival rate is only a few benefits from the Microporation technology. Microporation technology eliminated the entire 1 RNA mrna DNA Gene targeting duplex Protein expression 5 3 Reverse transcription 2 Preintegration complex bothering sample handling processes by integrating a pipet and an electric chamber as one. Viral systems Among viral systems, SMARTvector shrna lentiviral platform merges innovations that greatly improve the functionality and specificity of DNA-mediated RNAi and reduce the toxicity associated with low titer lentiviral preparations. With the advancements embodied in the SMARTvector technology, functional silencing sequences can be delivered into a broad range of cell types for long-term knockdown. This includes cells that are resistant to standard transfection such as stem cells, primary cells and neuronal cells (Figure 5). These vectors include: unique microrna expression scaffold for efficient processing of the shrna; ground breaking SMARTvector shrna design algorithm. Maximizes selection of functional silencing sequences. Seed-based bioinformatic filters and strand bias increase specificity; ready-to-use lentiviral particles allow ease of application. Viral vector system with broad tropism enables delivery into many cell types including difficult-to-transfect cells; expression of turbogfp permits visualization of transduction efficiency. Generation of stable cell lines facilitated by puromycin selection marker; 4 Dicer Nucleus shrna expression Integration Figure 5. SMARTvector delivery system. Pre-packaged SMARTvector are ready to transduce high-titer (>10 8 TU) lentiviral particles. Vol No 1. MINERVA BIOTECNOLOGICA 7
6 MARTINEZ DI MONTEMUROS NEW TECHNOLOGIES FROM SIRNA WORLD CMV Ψ cppt Zeo tgfp IRES Puror shrnamir WRE 5 LTR pgipz mir5' 22 mir3' sinltr Ampr puc Ori sv40 Ori Figure 6. pgipz lentiviral shrnamir vector. The vector contains CMV promoter, lentiviral backbone, turbogfp and Puromycin cassettes for selection. TRE UBC Ψ cppt Zeo trfp shrnamir rtta3 IRES Puro WRE 5 LTR mir30' 22 mir30' ptrpz sinltr Amp puc sv40 Ori Figure 7. TRIPZ inducile lentiviral shrnamir vector. Combines the advantages of shrnamir design, Tet-On inducibility and powerful lentiviral delivery to produce an RNAi trigger that allows tightly regulatable RNAi in most cell types. TurboRFP marks inducible shrnamir expression. lentigen proprietary manufacturing process yields high-titer ( 10 8 TU per ml) purified and concentrated lentiviral particle stocks. The Expression Arrest microrna-adapted shrna (shrnamir) libraries were developed in collaboration with Greg Hannon (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, USA) and Steve Elledge (Harvard University, Boston, MA, USA). shrnamir triggers for mammalian RNAi are based on current knowledge of the endogenous microrna biogenesis pathway. shrnamir constructs (Figures 6, 7) are designed to mimic a natural microrna primary transcript, enabling specific processing by the endogenous RNAi pathway and producing more effective knockdown. 33, 34 Genome wide shrnamir libraries incorporate several features aimed at increasing the efficiency and specificity of gene knockdown providing solutions for diverse RNAi applications. 35, 36 GeneNet pooled sirna Libraries enable to perform high-throughput gene knockdown studies on a genome-wide basis. You can simultaneously identify multiple genes that alter a specific cellular phenotype in a single experiment. 37 By merging effector expression libraries with sirna technology and the efficiency of lentiviral transduction, GeneNet sirna Libraries enable to conveniently introduce and see the effect of many thousands of sirna molecules on cell funtions at once. After selecting transduced cells 8 MINERVA BIOTECNOLOGICA March 2008
7 NEW TECHNOLOGIES FROM SIRNA WORLD MARTINEZ DI MONTEMUROS that express a desired phenotype (such as resistance to apoptosis or irradiation, or a growth response to a chemical additive, etc.), you can identify the sets of genes that control the response Controls Controls are a critical part of a gene silencing experiment. They enable accurate interpretation of knockdown data and provide confidence in the specificity of the response. Several companies have developed and validated positive and negative controls either for sirna and for shrnamir libraries. Negative controls Changes in the mrna or protein levels in cells treated with negative or non-silencing controls reflect nonspecific responses in cells and can be used as a baseline against which specific knockdown can be measured. Positive controls They are useful to demonstrate that your experimental system is functional and your sirna is successfully activating the RNAi pathway. Transfection efficiency Knowledge of transfection efficiency in gene knockdown experiments is also critical in understanding the results. Factors affecting transfection efficiency include purity of sirna or plasmid DNA, health of transfected cells, inconsistencies in number of cells plated, insufficient mixing of transfection complexes, different cell types or change in the cell passage number. Determining the transfection efficiency for every RNAi experiment is thus essential to enable valid comparisons across sets of data. Recommended controls for RNAi studies are based on suggested controls detailed in an editorial published in Nature Cell Biology. 12 Detection of sirna and mirna The field of non-coding RNAs has gained increasing attention in recent years, particularly due to the discovery of sirnas and micro RNAs. As discussed, these RNAs are short (typically nucleotides) single stranded moieties that regulate the expression of target genes by interacting with complementary sites within the target mrnas and either repressing translation or eliciting target mrna degradation. The current, standard methods for detecting and quantifying novel mirna and sirna molecules involve Northern blotting with hybridization. Detecting and quantitating known mirnas can be done using pre-designed reverse priming and reverse transcription followed by primer sets built for the specific mirna for Realtime PCR analysis. These sets require many steps and can take several hours to complete and trouble-shoot. The QuantiMir TM RT kit (System Biosciences, Mountain View, CA, USA) provides all the reagents necessary to anchor-tail and convert small, non-coding RNAs into cdna starting from total RNA samples. Once performed the reactions on RNA samples, the cdnas are ready to use for either end-point PCR experiments or to perform Real-time qpcr analysis. The method consists of designing the forward primer which corresponds to the mirna or sirna of interest and provide Real-time PCR SYBR green master mix or typical PCR mastermix with Taq DNA polymerase for end-point PCR expression profiling. Recently, previously unknown germline specific classes of mirna-like molecules were identified in mouse testes and mouse oocytes, illustrating the need for continued and indepth mirna discovery efforts across a wide range of tissues. These facts taken together demonstrate an ever-increasing need for simple, robust, and sensitive methods that enable discovery and quantitation of micrornas and their precursors. The versatility of this method allows it to be used in detecting levels of sirna and shrna, particularly useful in inducible experiments. Conclusions RNAi remains a promising new approach for gene silencing both in the context of post-genomic research on target validation and for potential therapeutic applications. As a tool for target validation, synthetic and DNA-based RNAi strategies have been applied in both academia and industry, with significant recent advances in understanding selection of sirna sequences, optimization for potency and specificity, mechanisms for activation of innate immuni- Vol No 1. MINERVA BIOTECNOLOGICA 9
8 MARTINEZ DI MONTEMUROS NEW TECHNOLOGIES FROM SIRNA WORLD ty, and methods for whole-genome sirna screenings. In this light, Whitehurst et al., 41 have recently combined a high-throughput cell-based one-well/onegene screening platform with a genome-wide synthetic library of chemically synthesized small interfering RNAs for systematic interrogation of the molecular underpinnings of cancer cell chemoresponsiveness. Using a stringent objective statistical algorithm to reduce false discovery rates below 5%, they isolated a panel of 87 genes that represent major focal points of the autonomous response of cancer cells to the abrogation of microtubule dynamics identifying mechanistic relationships between cancerassociated aberrant gene expression programmes and the basic cellular machinery required for robust mitotic progression. As a therapeutic strategy, synthetic and DNA-based RNAi strategies have advanced to pre-clinical animal models and early clinical testing. These approaches have explored multiple strategies to address the delivery hurdle, and include both local and systemic administration opportunities. 42, 43 References 1. Napoli C, Lemieux C, Jorgensen R. Introduction of a chimeric chalcone synthase gene into petunia results in reversible co-suppression of homologous genes in trans. Plant Cell 1990;2: Romano N, Macino G. Quelling: transient inactivation of gene expression in Neurospora crassa by transformation with homologous sequences. Mol Microbiol 1992;6: Fire A, Xu S, Montgomery MK, Kostas SA, Driver SE, Mello CC. Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature 1998;391: Elbashir SM, Harborth J, Lendeckel W, Yalcin A, Weber K, Tuschl T. Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature 2001;411: Zamore PD. RNA interference: listening to the sound of silence. Nat Struct Biol 2001;8: Tuschl T. RNA Interference and Small Interfering RNAs. Chembiochem Europ J Chem Biol 2001;2: Hannon GJ. RNA interference. Nature 2002;418: Agrawal N, Dasaradhi PVN, Mohmmed A, Malhotra P, Bhatnagar RK, Mukherjee SK. RNA Interference: Biology, Mechanism, and Applications. Microbiol Mol Biol Rev 2003;67: Caplen NJ, Parrish S, Imani F, Fire A, Morgan RA. Specific inhibition of gene expression by small double-stranded RNAs in invertebrate and vertebrate systems. Proc Natl Acad Sci U S A 2001;98: Bantounas I, Phylactou LA, Uney JB. RNA interference and the use of small interfering RNA to study gene function in mammalian systems. J Mol Endocrinol 2004;33: McCaffrey AP, Meuse L, Pham TT, Conklin DS, Hannon GJ, Kay MA. Gene expression: RNA interference in adult mice. Nature 2002;418: Anonymous. Whither RNAi? Nat Cell Biol 2003;5: Huppi K, Martin SE, Caplen NJ. Defining and assaying RNAi in mammalian cells. Mol Cell 2004;17: Schwarz DS, Hutvagner G, Du T, Xu Z, Aronin N, Zamore PD. Unexpected Asymmetry in the assembly of the RNAi Enzyme Complex. Cell 2003;115: Khvorova A, Reynolds A, Jayasena S. Functional sirnas and mirnas exhibit strand bias. Cell 2003;115: Reynolds A, Leake D, Scaringe S, Marshall WS, Boese Q, Khvorova A. Rational sirna design for RNA interference. Nat Biotech 2004;22: Amarzguioui M, Prydz H. An algorithm for selection of functional sirna sequences. Biochem Biophys Res Commun 2004;316: Ui-Tei K, Naito Y, Takahashi F, Haraguchi T, Ohki-Hamazaki H, Juni A et al. Guidelines for the selection of highly effective sirna sequences for mammalian and chick RNA interference. Nucleic Acids Res 2004;32: Chi J-T, Chang HY, Wang NN, Chang DS, Dunphy N, Brown PO. Genome-wide view of gene silencing by small interfering RNAs. Proc Natl Acad Sci U S A 2003;100: Jackson AL, Bartz SR, Schelter J, Kobayashi SV, Burchard J, Mao M et al. Expression profiling reveals off-target gene regulation by RNAi. Nat Biotech 2003;21: Jackson AL, Linsley PS. Noise amidst the silence: off-target effects of sirnas? Trends Genet 2004;20: Fedorov Y, Anderson EM, Birmingham A, Reynolds A, Karpilow J, Robinson K et al. Off-target effects by sirna can induce toxic phenotype. RNA 2006;12: Saxena S, Jónsson ZO, Dutta A. Small RNAs with imperfect match to endogenous mrna repress translation: implications for off-target activity of small inhibitory RNA in mammalian cells. J Biol Chem 2003;278: Persengiev SP, Zhu X, Green MR. Nonspecific, concentrationdependent stimulation and repression of mammalian gene expression by small interfering RNAs (sirnas). RNA 2004;10: Semizarov D, Frost L, Sarthy A, Kroeger P, Halbert DN, Fesik SW. Specificity of short interfering RNA determined through gene expression signatures. Proc Natl Acad Sci U S A 2003;100: Scacheri PC, Rozenblatt-Rosen O, Caplen NJ, Wolfsberg TG, Umayam L, Lee JC et al. Short interfering RNAs can induce unexpected and divergent changes in the levels of untargeted proteins in mammalian cells. Proc Natl Acad Sci U S A 2004;101: Qiu S, Adema CM, Lane T. A computational study of off-target effects of RNA interference. Nucleic Acids Res 2005;33: Lin X, Ruan X, Anderson MG, McDowell JA, Kroeger PE, Fesik SW et al. sirna-mediated off-target gene silencing triggered by a 7 nt complementation. Nucleic Acids Res 2005;33: Naito Y, Yamada T, Matsumiya T, Ui-Tei K, Saigo K, Morishita S. dscheck: highly sensitive off-target search software for doublestranded RNA-mediated RNA interference. Nucleic Acids Res 2005;33:W Birmingham A, Anderson EM, Reynolds A, Ilsley-Tyree D, Leake D, Fedorov Y et al. 3 UTR seed matches, but not overall identity, are associated with RNAi off-targets. Nat Meth 2006;3: Jackson AL, Burchard J, Leake D, Reynolds A, Schelter J, Guo J et al. Position-specific chemical modification increases specificity of sirna-mediated gene silencing. RNA 2006;12: Anderson EM, Birmingham A, Baskerville S, Reynolds A, Maksimova E, Leake D et al. Experimental validation of the importance of seed complement frequency to sirna specificity. RNA 2008;14: Paddison PJ, Cleary M, Silva JM, Chang K, Sheth N, Sachidanandam R et al. Cloning of short hairpin RNAs for gene knockdown in mammalian cells. Nat Meth 2004;1: Silva JM, Li MZ, Chang K, Ge W, Golding MC, Rickles RJ et al. Second-generation shrna libraries covering the mouse and human genomes. Nat Genet 2005;37: Stegmeier F, Hu G, Rickles RJ, Hannon GJ, Elledge SJ. A lentiviral microrna-based system for single-copy polymerase II-regulated RNA interference in mammalian cells. Proc Natl Acad Sci U S A 2005;102: MINERVA BIOTECNOLOGICA March 2008
9 NEW TECHNOLOGIES FROM SIRNA WORLD MARTINEZ DI MONTEMUROS 36. Silva JM, Marran K, Parker JS, Silva J, Golding M, Schlabach MR et al. Profiling Essential Genes in Human Mammary Cells by Multiplex RNAi Screening. Science 2008;319: Gazin C, Wajapeyee N, Gobeil S, Virbasius CM, Green MR. An elaborate pathway required for Ras-mediated epigenetic silencing. Nature 2007;449: Schlabach MR, Luo J, Solimini NL, Hu G, Xu Q, Li MZ et al. Cancer Proliferation Gene Discovery Through Functional Genomics. Science 2008;319: Dickins RA, Hemann MT, Zilfou JT, Simpson DR, Ibarra I, Hannon GJ et al. Probing tumor phenotypes using stable and regulated synthetic microrna precursore. Nat Genet 2005;37: Westbrook TF, Martin ES, Schlabach MR, Leng Y, Liang AC, Feng B et al. A genetic screen for candidate tumor suppressors identifies REST. Cell 2005;121: Whitehurst AW, Bodemann BO, Cardenas J, Ferguson D, Girard L, Peyton M et al. Synthetic lethal screen identification of chemosensitizer loci in cancer cells. Nature 2007;446: Zimmermann TS, Lee AC, Akinc A, Bramlage B, Bumcrot D, Fedoruk MN et al. RNAi-mediated gene silencing in non-human primates. Nature 2006;441: Akinc A, Zumbuehl A, Goldberg M, Leshchiner ES, Busini V, Hossain N et al. A combinatorial library of lipid-like materials for delivery of RNAi therapeutics. Nat Biotechnol 2008;26: Vol No 1. MINERVA BIOTECNOLOGICA 11
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