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At least 109 records · Page 6Linked to original sources
PubMed2004

Functional comparison of single- and double-stranded siRNAs in mammalian cells.

The concept of small interfering RNA (siRNA) has been extended to include not only short double-stranded RNA of 19-25bp, but also single-stranded antisense RNA of the same length, since such single-stranded antisense siRNAs were recently found to be able to inhibit gene expression as well. We made comprehensive comparison of double- and single-stranded siRNA functions in RNA interference (RNAi), targeting multiple sites and different mRNAs, measuring RNAi effects at different time-points and in different cell lines, and examining response curves. Duplex siRNAs were found to be more potent than single-stranded antisense siRNAs. This was verified by the observation that single-stranded antisense siRNAs, which were inefficient in some cases when used alone, could be rescued from inefficiency by sequentially transfecting with the sense siRNAs. This result suggests that the structural character of siRNA molecules might be a more important determinant of siRNA efficiency than the cellular persistence of them.

Animals↗
PubMed2004

Validating siRNA using a reporter made from synthetic DNA oligonucleotides.

Only a small fraction of all siRNAs are effective in silencing their target genes, and siRNA efficacy can only be determined experimentally. Previously described reporter-based siRNA validation methods all rely on the availability of physical cDNA clones, and this limits the high throughput applicability of the method. In the current report, we used short synthetic DNA fragment containing a siRNA targeting site, instead of cDNA, to fuse with a reporter gene. When targeting such transcripts with different siRNAs, we found that such constructs can faithfully report the efficacy of the corresponding siRNAs in a sequence specific manner, even when the inserted DNA fragment is essentially only long enough to cover the targeting site. The efficacy of both vector-based siRNA and synthetic siRNA can be evaluated using this system. Since only readily available short synthetic DNA fragments are needed for forming the evaluation vector, this method provides an appealing way of validating siRNAs in high throughput.

Animals↗
PubMed2006

No induction of anti-viral responses in human cell lines HeLa and MCF-7 when transfecting with siRNA or siLNA.

Gene silencing by RNAi and siRNAs has become a well-used tool for researchers. Because of its relatively small size, siRNA was originally thought to avoid activation of anti-viral responses. Recent reports demonstrating so-called "off-target effects" are therefore alarming. One issue raised is that siRNA induces interferon-regulated genes at the transcriptional level. We characterize the anti-viral responses of synthetic siRNA and in vitro-transcribed siRNA by measuring the mRNA levels of IFN-beta and OAS2 in HeLa cells. Transfections with both traditional and LNA-modified synthetic siRNA cause no anti-viral responses, whereas transfection with either long dsRNA or in vitro-transcribed siRNA leads to greater than 1000-fold induction of these genes. The lack of response was also demonstrated at the level of phosphorylated eIF2alpha, and measuring of IFN-beta by ELISA in cell culture media from the human cell line MCF-7. Altogether, transfection with synthetic siRNA does not induce anti-viral responses in these two cell lines. Our results reinforce the role of siRNA as an effective tool for reverse genetics and strengthen siLNA as a tool for future therapeutic applications.

2',5'-Oligoadenylate Synthetase↗
PubMed2006

Selecting effective siRNA sequences based on the self-organizing map and statistical techniques.

Short interfering RNA (siRNA) has been widely used for studying gene functions in mammalian cells but varies markedly in its gene-silencing efficacy. Although many design rules/guidelines for effective siRNAs based on various criteria have been reported recently, there are only a few consistencies among them. This makes it difficult to select effective siRNA sequences in mammalian genes. Here, we propose a new method for selecting effective siRNA target sequences on the basis of the self-organizing map (SOM) technique and statistical significance analyses for a large number of effective siRNAs. In the proposed method, the score is defined as a gene degradation measure. The effectiveness for the proposed method was confirmed by evaluating effective and ineffective siRNAs for recently reported genes (12 genes, 172 siRNA sequences) and comparing with other reported scoring methods. The size (value) of this score is closely correlated with the degree of gene degradation, and the score can easily be used for selecting high-potential siRNA candidates. The evaluation results indicate that the proposed method would be useful for many other genes. It will therefore be useful for selecting siRNA sequences in mammalian genes.

Animals↗
PubMed2006

Gene silencing through RNA interference (RNAi) in vivo: strategies based on the direct application of siRNAs.

RNA interference (RNAi) offers great potential not only for in vitro target validation, but also as a novel therapeutic strategy based on the highly specific and efficient silencing of a target gene, e.g. in tumor therapy. Since it relies on small interfering RNAs (siRNAs), which are the mediators of RNAi-induced specific mRNA degradation, a major issue is the delivery of therapeutically active siRNAs into the target tissue/target cells in vivo. For safety reasons, strategies based on (viral) vector delivery may be of only limited clinical use. The more desirable approach is to directly apply catalytically active siRNAs. This review highlights the recent knowledge on the guidelines for the selection of siRNAs which show high activity in the absence of non-specific siRNA effects. It then focuses on approaches to directly use siRNA molecules in vivo and gives a comprehensive overview of in vivo studies based on the direct application of siRNAs to induce RNAi. One promising approach is the in vivo siRNA delivery through complexation of chemically unmodified siRNAs with polyethylenimine (PEI). The anti-tumoral effects of PEI/siRNA-based targeting of tumor-relevant genes in vivo are described.

Gene Silencing↗
PubMed2006

A low molecular weight fraction of polyethylenimine (PEI) displays increased transfection efficiency of DNA and siRNA in fresh or lyophilized complexes.

RNA interference (RNAi) represents a powerful method for specific gene silencing. It is mediated through small double-stranded RNA molecules (small interfering RNAs, siRNAs) which sequence-specifically trigger the cleavage and subsequent degradation of their target mRNA. One critical factor that determines the success of RNAi is the ability to deliver intact siRNAs into target cells. Polyethylenimines (PEIs) are synthetic polymers with a high cationic charge density which function as transfection reagents based on their ability to compact DNA or RNA into complexes. This paper describes the application of lyophilized PEI/siRNA complexes based on a novel polyethylenimine. By fractionation of a commercially available 25-kDa PEI using gel permeation chromatography, a low molecular weight polyethylenimine (PEI F25-LMW) with superior transfection efficacy and low toxicity in various cell lines is obtained. Complexes formed in 5% glucose, but not in 150 mM NaCl, can be lyophilized and reconstituted without loss of transfection efficacy. Furthermore, PEI F25-LMW is able to complex and fully protect siRNAs against nucleolytic degradation, and delivers siRNAs into cells where they display bioactivity. Upon lyophilization and reconstitution of PEI F25-LMW-based siRNA complexes, siRNAs are still able to efficiently induce RNAi. To further demonstrate their applicability, lyophilized PEI/siRNA complexes are employed for targeting of the growth factor VEGF. Treatment of PC-3 prostate carcinoma cells with fresh or with lyophilized complexes results in decreased cell proliferation in different assays due to the siRNA-mediated downregulation of VEGF. In conclusion, siRNAs can be applied in lyophilized formulations, and lyophilized PEI F25-LMW-based siRNA complexes represent a powerful, inexpensive, non-toxic and simple ready-to-use platform for the specific and efficient targeting of genes in vitro.

Cell Line, Tumor↗
PubMed2005

Local RNA target structure influences siRNA efficacy: systematic analysis of intentionally designed binding regions.

Contradictory reports in the literature have emphasised either the sequence of small interfering RNAs (siRNA) or the structure of their target molecules to be the major determinant of the efficiency of RNA interference (RNAi) approaches. In the present study, we analyse systematically the contributions of these parameters to siRNA activity by using deliberately designed mRNA constructs. The siRNA target sites were included in well-defined structural elements rendering them either highly accessible or completely involved in stable base-pairing. Furthermore, complementary sequence elements and various hairpins with different stem lengths and designs were used as target sites. Only one of the strands of the siRNA duplex was found to be capable of silencing via its respective target site, indicating that thermodynamic characteristics intrinsic to the siRNA strands are a basic determinant of siRNA activity. A significant obstruction of gene silencing by the same siRNA, however, was observed to be caused by structural features of the substrate RNA. Bioinformatic analysis of the mRNA structures suggests a direct correlation between the extent of gene-knockdown and the local free energy in the target region. Our findings indicate that, although a favourable siRNA sequence is a necessary prerequisite for efficient RNAi, complex target structures may limit the applicability even of carefully chosen siRNAs.

Animals↗
PubMed2005

Local RNA target structure influences siRNA efficacy: a systematic global analysis.

The efficiency with which small interfering RNAs (siRNAs) down-regulate specific gene expression in living cells is variable and a number of sequence-governed, biochemical parameters of the siRNA duplex have been proposed for the design of an efficient siRNA. Some of these parameters have been clearly identified to influence the assembly of the RNA-induced silencing complex (RISC), or to favour the sequence preferences of the RISC endonuclease. For other parameters, it is difficult to ascertain whether the influence is a determinant of the siRNA per se, or a determinant of the target RNA, especially its local structural characteristics. In order to gain an insight into the effects of local target structure on the biological activity of siRNA, we have used large sets of siRNAs directed against local targets of the mRNAs of ICAM-1 and survivin. Target structures were classified as accessible or inaccessible using an original, iterative computational approach and by experimental RNase H mapping. The effectiveness of siRNA was characterized by measuring the IC50 values in cell culture and the maximal extent of target suppression. Mean IC50 values were tenfold lower for accessible local target sites, with respect to inaccessible ones. Mean maximal target suppression was improved. These data illustrate that local target structure does, indeed, influence the activity of siRNA. We suggest that local target screening can significantly improve the hit rate in the design of biologically active siRNAs.

Cell Line↗
PubMed2006

Gene expression analysis in blood cells in response to unmodified and 2'-modified siRNAs reveals TLR-dependent and independent effects.

Ribonucleic nucleic acid recognition by Toll-like receptors (TLRs) induces innate immune responses. However, no comprehensive analysis of gene expression in human blood cells in response to unmodified and 2'-modified immunostimulatory RNAs has been reported. Using oligonucleotide microarrays, we show that around 400 genes were significantly (P<0.001) altered in peripheral blood mononuclear cells (PBMC) in response to either single-stranded (ss) or double-stranded (ds) small interfering RNAs (siRNAs). Most of the upregulated genes encode proteins involved in innate and adaptive immune responses, including proinflammatory cytokines, interferons, chemokines and chemokine receptors. Genes encoding proteins involved in lymphocyte activation (e.g. CD80, CD40, and CD69) and in regulation of the immune responses (e.g. SOCS proteins) were upregulated. Also, genes encoding for antiviral proteins (Mx1, Mx2, TRIM proteins), and interferon regulatory factors (e.g. IRF7) were upregulated. Around 90% of the genes (140 out of 160) affected by R-848, a specific ligand for TLR7 and TLR8, were also affected by ss siRNAs or ds siRNAs, indicating that the signaling pathways activated by R-848 are also activated by immunostimulatory siRNAs. In addition to immunoactivation via TLRs, ss siRNAs and ds siRNAs induced TLR-independent gene alterations. Surprisingly, replacement of only uridine bases with either 2'-fluoro or 2'-O-methyl modified counterparts abrogated all the observed bystander effects. Collectively, these microarray data offer for the first time an insight into human PMBC response to immunostimulatory RNAs such as ss siRNAs and ds siRNAs. The data should help to define strategies to either enhance or avoid the non-specific effects of siRNAs in order to develop safe therapeutics.

Gene Expression Profiling↗
PubMed2003

Fas siRNA reduces apoptotic cell death of allogeneic-transplanted hepatocytes in mouse spleen.

BACKGROUND: Synthetic siRNAs 21 to 23 nucleotides in length silence gene expression posttranscriptionally, and RNA interference targeting Fas protects mice from fulminant hepatitis. Fas-mediated apoptosis has also been implied in the mechanism of hepatocyte apoptosis upon allogenic hepatocyte transplantation (HTx), and blockade of Fas and Fas ligand interaction successfully promotes the repopulation of allogenic liver cells in recipient spleens. In the present study, we further investigated the protective effects of Fas silencing on allogeneic hepatocytes transplanted into mouse spleens. MATERIALS AND METHODS: Hepatocytes were isolated from BALB/c mice and mock transfected or transfected with Fas siRNA or GFP siRNA (n = 8/group). The expression of Fas was examined by RT-PCR and flow cytometric analysis. Forty-eight hours later, the cells were transplanted into spleens of allogenic B6 mice. Spleens were harvested on day 21 after transplantation. Apoptosis was assessed by TUNEL assay, survival of hepatocytes by alanine transaminase (ALT) assay. RESULTS: Fas siRNA transfection reduced Fas expression on hepatocytes at both mRNA and protein levels (P <.05). Upon transplanting into recipient spleens, hepatocytes transfected with Fas siRNA demonstrated a lower percentage of apoptosis detected by TUNEL (6 +/- 3% in Fas siRNA group vs 12 +/- 5% in GFP siRNA group and 10 +/- 3% in mock transfected group; P <.05), and increased survival as determined by ALT assay (38.2 +/- 10.6 IU/g in Fas siRNA group vs 21.3 +/- 8.4 IU/g in GFP siRNA group and 18.5 +/- 5.9 IU/g in mock-transfected animals). CONCLUSIONS: Fas silencing by RNA interference reduces apoptosis and increases survival of allogenic transplanted hepatocytes, and thus holds promise to inhibit acute rejection after hepatocyte transplantation.

Animals↗
PubMed2004

Lipidic carriers of siRNA: differences in the formulation, cellular uptake, and delivery with plasmid DNA.

RNA interference (RNAi) has become a popular tool for downregulating specific gene expression in many species, including mammalian cells [Novina, C. D., and Sharp, P. A. (2004) The RNAi revolution, Nature 430, 161-164]. Synthetic double-stranded RNA sequences (siRNA) of 21-23 nucleotides have been shown in particular to have the potential to silence specifically gene function in cultured mammalian cells. As a result, there has been a significant surge of interest in the application of siRNA in functional genomics programs as a means of deciphering specific gene function. However, for siRNA functional genomics studies to be valuable and effective, specific silencing of any given target gene is essential, devoid of nonspecific knockdown and toxic side effects. For this reason, we became interested in investigating cationic liposome/lipid-mediated siRNA delivery (siFection) as a meaningful and potentially potent way to facilitate effective functional genomics studies. Accordingly, a number of cationic liposome/lipid-based systems were selected, and their formulation with siRNA was studied, with particular emphasis on formulation parameters most beneficial for siRNA use in functional genomics studies. Cationic liposome/lipid-based systems were selected from a number of commercially available products, including lipofectAMINE2000 and a range of CDAN/DOPE systems formulated from different molar ratios of the cationic cholesterol-based polyamine lipid N(1)-cholesteryloxycarbonyl-3,7-diazanonane-1,9-diamine (CDAN) and the neutral helper lipid dioleoyl-L-alpha-phosphatidylethanolamine (DOPE). Parameters that were been investigated included the lipid:nucleic acid ratio of mixing, the extent of cationic liposome/lipid-nucleic acid complex (lipoplex) formation plus medium used, the lipoplex particle size, the mode of delivery, and dose-response effects. Results suggest that concentrations during siRNA lipoplex (LsiR) formation are crucial for maximum knockdown, but the efficacy of gene silencing is not influenced by the size of LsiR particles. Most significantly, results show that most commercially available cationic liposome/lipid-based systems investigated here mediate a significant nonspecific downregulation of the total cellular protein content at optimal doses for maximal specific gene silencing and knockdown. Furthermore, one pivotal aspect of using siRNA for functional genomics studies is the need for at least minimal cellular toxicity. Results demonstrate that CDAN and DOPE with and without siRNA confer low toxicity to mammalian cells, whereas lipofectAMINE2000 is clearly toxic both as a reagent and after formulation into LsiR particles. Interestingly, LsiR particles formulated from CDAN and DOPE (45:55, m/m; siFECTamine) seem to exhibit a slower cellular uptake than LsiR particles formulated from lipofectAMINE2000. Intracellularly, LsiR particles formulated from CDAN and DOPE systems also appear to behave differently, amassing in distinct but diffuse small nonlysosomal compartments for at least 5 h after siFection. By contrast, LsiR particles formulated from lipofectAMINE2000 accumulate in fewer larger intracellular vesicles.

Apoptosis↗
PubMed2006

Suppression of ocular neovascularization with siRNA targeting VEGF receptor 1.

In this study, we used small interfering RNA (siRNA) directed against vascular endothelial growth factor receptor 1 (vegfr1) mRNA to investigate the role of VEGFR1 in ocular neovascularization (NV). After evaluating many siRNAs, Sirna-027 was identified; it cleaved vegfr1 mRNA at the predicted site and reduced its levels in cultured endothelial cells and in mouse models of retinal and choroidal neovascularization (CNV). Compared to injection of an inverted control sequence, quantitative reverse transcriptase-PCR demonstrated statistically significant reductions of 57 and 40% in vegfr1 mRNA after intravitreous or periocular injection of Sirna-027, respectively. Staining showed uptake of 5-bromodeoxyuridine-labeled Sirna-027 in retinal cells that lasted between 3 and 5 days after intravitreous injection and was still present 5 days after periocular injection. In a CNV model, intravitreous or periocular injections of Sirna-027 resulted in significant reductions in the area of NV ranging from 45 to 66%. In mice with ischemic retinopathy, intravitreous injection of 1.0 mug of Sirna-027 reduced retinal NV by 32% compared to fellow eyes treated with 1.0 mug of inverted control siRNA. These data suggest that VEGFR1 plays an important role in the development of retinal and CNV and that targeting vegfr1 mRNA with siRNA has therapeutic potential.

Animals↗
PubMed2003

Variations of the 3' protruding ends in synthetic short interfering RNA (siRNA) tested by microinjection in Drosophila embryos.

Short interfering RNAs (siRNAs) are the processing product originating from long double-stranded RNAs (dsRNAs) that are cleaved by the RNase III-like ribonuclease Dicer. As siRNAs mediate cleavage of specific single-stranded target RNAs, they are essential intermediates of RNA interference (RNAi). When applied in synthetic form, siRNAs likewise can induce the silencing process in the absence of long dsRNAs. Here, we tested variations of a conventional synthetic siRNA that had been used successfully to silence the Drosophila notch gene. The variants had two 3 ' -terminal deoxynucleotides in their protruding single-stranded ends. In one case, the deoxynulceotides would match to the notch mRNA, whereas the other variant had nonmatching deoxy-T residues, representing a widely used siRNA design. siRNAs with different combinations of sense and antisense strands were injected into Drosophila embryos at two different concentrations. We found that the all-ribonucleotide siRNA gave the best inhibition of notch expression. The combination of two modified strands with 3 ' -terminal deoxynucleotides was effective, but if combined with a sense or antisense ribostrand, the efficacy dropped. The siRNAs with nonmatching 3 ' -terminal TT residues showed a reduced silencing potential, which became evident at low concentration. An siRNA with a nonmatching 3 ' -terminal ribonucleotide in the antisense strand retained most of its silencing potential in accordance with the hypothesis that primer extension for generation of ssRNA from single-stranded mRNA does not operate in Drosophila.

3' Untranslated Regions↗
PubMed2006

Inhibition of human carcinoma and neuroblastoma cell proliferation by anti-c-myc siRNA.

Suppression of c-myc proto-oncogene expression by small interfering RNA (siRNA) in human epidermoid carcinoma KB-3-1 and neuroblastoma SK-N-MC cell lines was investigated. The siRNA duplex targeted to the exon 3 of c-myc mRNA (siRNA-I) was prepared by in vitro transcription using T7 RNA polymerase and short double-stranded DNA (dsDNA) templates. siRNA-I was shown to efficiently decrease c-myc mRNA expression in both tumor cell lines and to arrest their proliferation. Incubation of KB-3-1 cells with 150 nM siRNA-I results in a 92% decrease in the c-myc mRNA level and an 83% decrease in the protein level. In SK-N-MC cells, 150 nM siRNA-I causes a 60% decrease in the c-myc mRNA level and a 55% decrease in the protein level. The reduction of the c-myc mRNA level correlates with the inhibition of cell proliferation; 150 nM siRNA-I causes a 2.5-fold reduction in the SK-N-MC proliferation rate and a 15-fold decrease in the proliferation rate and complete arrest of cell division in KB-3-1 cells. siRNA-I has little effect on proliferation of the IMR-32 cells that overexpress the N-myc but not the c-myc gene, demonstrating that siRNA-I antiproliferation activity is mediated by specific block of c-myc expression.

Carcinoma↗
PubMed2006

Combination of active and inactive siRNA targeting the mitotic kinesin Eg5 impairs silencing efficiency in several cancer cell lines.

Gene silencing by RNA interference (RNAi) has proven to be a powerful tool for investigating gene function in mammalian cells. Combination of several short interfering RNA (siRNA) targeting the same gene is commonly used to improve RNA interference. However, in contrary to the well-described mechanism of RNAi, efficiency of single siRNA compared to pool remains poorly documented. We addressed this issue using several active and inactive siRNA targeting Eg5, a kinesin-related motor involved in mitotic spindle assembly. These siRNA, used alone or in combination, were tested for their silencing efficiency in several cancer cell lines. Here we show that presence of inactive Eg5 siRNA in a pool dramatically decreases knockdown efficacy in a cell line- and dose-dependent manner. Lack of inhibition by unrelated siRNA suggests that a competition may occur during siRNA incorporation into RNA-induced silencing complexes (RISCs) along with the target mRNA. Altogether, our results, which need to be confirmed with additional inactive siRNA, indicate that combination of siRNA may not increase but instead decrease silencing efficiency.

Base Sequence↗
PubMed2005

RNA interference by mixtures of siRNAs prepared using custom oligonucleotide arrays.

RNA interference (RNAi) is a process in which double-strand RNA (dsRNA) directs the specific degradation of a corresponding target mRNA. The mediators of this process are small dsRNAs, of approximately 21 bp in length, called small interfering RNAs (siRNAs). siRNAs, which can be prepared in vitro in a number of ways and then transfected into cells, can direct the degradation of corresponding mRNAs inside these cells. Hence, siRNAs represent a powerful tool for studying gene functions, as well as having the potential of being highly specific pharmaceutical agents. Some limitations in using this technology exist because the preparation of siRNA in vitro and screening for siRNAs efficient in RNAi can be expensive and time-consuming processes. Here, we demonstrate that custom oligonucleotide arrays can be efficiently used for the preparation of defined mixtures of siRNAs for the silencing of exogenous and endogenous genes. The method is fast, inexpensive, does not require siRNA optimization and has a number of advantages over methods utilizing enzymatic preparation of siRNAs by digestion of longer dsRNAs, as well as methods based on chemical synthesis of individual siRNAs or their DNA templates.

Green Fluorescent Proteins↗
PubMed2006

Synthesis and properties of modified siRNA having amide-linked oligoribonucleosides at their 3' overhang regions.

siRNAs against luciferase gene were modified with amide-linked oligoribonucleosides (amide-linked RNA) at their 3'-overhang regions. The modified siRNAs were more stable than the unmodified siRNA against degradation by nuclease S1, which indicates that the modified overhang regions increase the nuclease resistance of the siRNAs. Tm values of the modified siRNAs increased compared with that of the unmodified siRNA. This result suggests that the modified overhang regions stabilize the duplex structure of siRNAs. The modified siRNAs reduced the target gene expression in a similar manner as the unmodified siRNA in living cells. This result indicates that the overhang modifications are tolerated for the inhibition of target gene expression.

Amides↗
PubMed2005

A pathway for the biogenesis of trans-acting siRNAs in Arabidopsis.

The Arabidopsis genes, TAS2 and TAS1a, produce structurally similar noncoding transcripts that are transformed into short (21-nucleotide [nt]) and long (24-nt) siRNAs by RNA silencing pathways. Some of these short siRNAs direct the cleavage of protein-coding transcripts, and thus function as trans-acting siRNAs (ta-siRNAs). Using genetic analysis, we defined the pathway by which ta-siRNAs and other short siRNAs are generated from these loci. This process is initiated by the miR173-directed cleavage of a primary poly(A) transcript. The 3' fragment is then transformed into short siRNAs by the sequential activity of SGS3, RDR6, and DCL4: SGS3 stabilizes the fragment, RDR6 produces a complementary strand, and DCL4 cleaves the resulting double-stranded molecule into short siRNAs, starting at the end with the miR173 cleavage site and proceeding in 21-nt increments from this point. The 5' cleavage fragment is also processed by this pathway, but less efficiently. The DCL3-dependent pathway that generates long siRNAs does not require miRNA-directed cleavage and plays a minor role in the silencing of these loci. Our results define the core components of a post-transcriptional gene silencing pathway in Arabidopsis and reveal some of the features that direct transcripts to this pathway.

Arabidopsis↗