Active siRNA Research Articles

(650/Paper 312): Small interfering RNAs-(siRNAs) that target expression of the NMDAR1 subunit gene in the spinal cord dorsal horn prevent inflammatory allodynia

NR1 is an essential subunit of the NMDA receptor which, at the spinal level, is involved in injury-induced pain hypersensitivity and morphine tolerance. An in vitro luciferase assay was used to identify candidate and control (inactive) siRNA sequences that are expressed by a recombinant adeno-associated virus (rAAV) plasmid. rAAV vectors targeting the NR1 subunit were prepared that express active or control (mismatch) siRNA sequences and injected into the mouse spinal cord dorsal horn (SCDH). Three weeks after vector administration, GFP labeling of the ipsilateral SCDH confirmed the spatial localization of the viral transduction.

Tolerance of RNA Interference Toward Modifications of the 5′ Antisense Phosphate of Small Interfering RNA

Bringing RNA interference (RNAi) under the control of light will allow the spacing, timing, and degree of gene expression to be controlled. We have previously shown that RNAi by small interfering (si) RNA can be modulated through randomly incorporated photolabile groups. Our and others interest is to find key locations on siRNA that can completely block RNAi until irradiation releases completely active siRNA. Some literature suggests that the 5' phosphate of the antisense strand of siRNA cannot be modified without completely blocking RNAi. We have examined this site as a potential switch for light control of RNAi and present evidence that siRNA modified at the 5' antisense phosphate can still cause RNAi, although not at the level effected by fully native siRNA. This contrasts with results from the literature, which suggest that modification of the 5' antisense phosphate will completely abrogate RNAi in siRNA. We have used mass spectrometry to identify and quantitate possible impurities that may be responsible for residual RNAi and show that they are present at 1% or less. Our results suggest that there is an inherent tolerance of the RNAi machinery toward modification of the 5' antisense phosphate.

Suppression of mouse rhodopsin expression in vivo by AAV mediated siRNA delivery

Purpose: The purpose of this study is to demonstrate that the expression of rhodopsin can be down regulated in vivo by AAV-delivered siRNA. This is the first step in an RNA replacement strategy for the allele-independent treatment of Autosomal Dominant Retinitis Pigmentosa (ADRP). Methods: HEK 293 cells were co-transfected with a plasmid carrying mouse RHO cDNA driven by the CMV promoter and a chemically synthesized siRNA duplex of 21 nucleotides. Reduction of RHO mRNA was confirmed by RT-PCR. One active siRNA and a control siRNA were embedded in a small hairpin RNA (shRNA) and cloned in Adeno-associated virus (AAV) vector under regulation of the H1 promoter and containing a GFP reporter. AAV5 expressing either active siRNA or an irrelevant siRNA were subretinaly injected into the right eyes of wild-type or RHO+/− heterozygote mice at post-natal day 16. At 1 and 2 months post-injection, animals were analyzed by electroretinography (ERG). Animals were then sacrificed, and retinas were examined by Western blot, RT-PCR, histology and immunohistochemistry. Results: All of the siRNAs tested in HEK 293 cells caused degradation of RHO mRNA, although the efficiency varied from 25% to 80%. In vivo siRNA delivery to the retina led to more than 40% reduction of scotopic a- and b-wave amplitudes in RHO+/− heterozygotes. Although the reduction of RHO mRNA was estimated at 30% compared to control animals, Western blots revealed 60% decrease in rhodopsin content. Histological analysis showed significant reduction in the thickness of the ONL, ranging between 53% and 86%. Conclusions: AAV-siRNA delivery into the subretinal space resulted in the reduction of retinal function caused by diminished RHO mRNA and protein content. This level of reduction may permit the replacement of endogenous mRNA with siRNA-resistant mRNA encoding wild-type RHO.

Inhibition of HCV subgenomic replicons by siRNAs derived from plasmids with opposing U6 and H1 promoters

Hepatitis C virus (HCV) is a main cause of chronic liver disease, which may lead to the development of liver cirrhosis and hepatocellular carcinoma. Therapeutic options are still limited in a significant proportion of patients. Small interfering RNAs (siRNAs) are an efficient tool to inhibit gene expression by RNA interference. As HCV RNA replicates in the cytoplasm of liver cells without integration into the genome, RNA-directed antiviral strategies are likely to successfully block its replication cycle. In this study, a panel of siRNAs was used to target various important regions of the HCV genome [5' untranslated region (UTR), NS3, NS4A, NS4B, NS5B, 3' UTR]. Convergent opposing human H1 and U6 polymerase III promoters were used to generate siRNAs. Target genes in sense and antisense orientation were attached to a luciferase reporter system to test the inhibitory efficiency of both siRNA strands. Our data revealed effective RNA interference against the HCV(+)-strand, the HCV(-)-strand or both strands simultaneously up to 65%. Subsequently, active siRNAs were tested in HCV subgenomic replicon cells and suppression of HCV RNA and NS5B protein levels up to 75% was confirmed. Interestingly, siRNAs that were effective against the sense as well as the antisense strand revealed the greatest inhibitory effects on HCV subgenomic replicons. Additionally, combinations of siRNAs induced a greater inhibition of HCV subgenomic replication of up to 90% proving the potential of this combined antiviral approach.

More complete gene silencing by fewer siRNAs: transparent optimized design and biophysical signature

Highly accurate knockdown functional analyses based on RNA interference (RNAi) require the possible most complete hydrolysis of the targeted mRNA while avoiding the degradation of untargeted genes (off-target effects). This in turn requires significant improvements to target selection for two reasons. First, the average silencing activity of randomly selected siRNAs is as low as 62%. Second, applying more than five different siRNAs may lead to saturation of the RNA-induced silencing complex (RISC) and to the degradation of untargeted genes. Therefore, selecting a small number of highly active siRNAs is critical for maximizing knockdown and minimizing off-target effects. To satisfy these needs, a publicly available and transparent machine learning tool is presented that ranks all possible siRNAs for each targeted gene. Support vector machines (SVMs) with polynomial kernels and constrained optimization models select and utilize the most predictive effective combinations from 572 sequence, thermodynamic, accessibility and self-hairpin features over 2200 published siRNAs. This tool reaches an accuracy of 92.3% in cross-validation experiments. We fully present the underlying biophysical signature that involves free energy, accessibility and dinucleotide characteristics. We show that while complete silencing is possible at certain structured target sites, accessibility information improves the prediction of the 90% active siRNA target sites. Fast siRNA activity predictions can be performed on our web server at .

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.

Knockdown of Neutrophil p29 Peroxiredoxin (Prx) in Transgenic K562 Cells Expressing Phox Proteins Results in Diminished Oxidase Activity.

Introduction: Production of reactive oxygen species through a respiratory burst is critical to the microbicidal activity of the neutrophil. The respiratory burst is initiated by assembly of the components of the NADPH oxidase enzyme system (p47phox, p67phox, p40phox, gp91phox, p22phox and Rac) and expression of the activity of the system to produce superoxide anion (O2 −). We recently identified a neutrophil protein with an approximate MW of 29 kDa which binds to the p67phox, is classified as a peroxiredoxin (Prx) and translocates to the plasma membrane during stimulation of the neutrophil. Additional studies demonstrate that this protein (p29 Prx) increases production of O2 − in a cell-free system of oxidase activity in a specific, stoichiometric manner and that the cysteine residues of p29 Prx, at amino acid positions 47 and 91, are required for this activity. The current studies demonstrate the role of p29 Prx in oxidase activity using the technique of small interfering RNA (siRNA) to degrade specific mRNA and decrease the expression of the protein.Methods: siRNA probes for p29 Prx were constructed based on standard constraints for unique 19 nucleotide binding sites along with other sequence selection criteria. Six probes were constructed based on the cDNA sequence of p29 Prx; one resulted in significant knockdown of p29 Prx. Inactive siRNA fluorescently labeled was obtained commercially. K562 cells, stably transfected with the p67phox, p47phox, gp91phox and low affinity fMLP receptor, were cultured under standard conditions and expressed p29 Prx message by RT-PCR and protein by Western blot. Transgenic K562 cells were transfected with siRNA or GFP labeled control siRNA with Nucleofector technology. Cell counts and viability were determined by standard techniques. For Western blots, proteins from cell lysates were separated on 10% SDS-PAGE and blotted onto nitrocellulose, and specific proteins were detected with polyclonal antibodies to p29 Prx, actin or p67phox, p47phox, gp91phox, and p22phox by chemiluminescent technique.Results: After transfection with the active siRNA for p29 Prx, inactive or labeled siRNA, the K562 cells were harvested at 24, 48, and 72 hours. A knockdown by one of the 6 siRNAs resulted in decreased levels of p29 Prx by Western blot. Optimum knockdown was achieved by transfection of 6 μg siRNA and the decrease in p29 Prx observed at 24 hours but was optimum after 48 hours. Under these conditions, a decrease in p29 Prx by 50–60% detected by Western blot was achieved with no differences in levels of actin or any of the phox proteins. The viability of control cells and siRNA transfected cells was not different. Cells transfected with siRNA for p29 Prx which demonstrated a knockdown of this protein exhibited decreased respiratory burst (O2 −) in response to fMLP (measured by chemiluminescence) or PMA (cytochrome c reduction) compared to control porated or transfected cells.Conclusion: These results correlate with in vitro studies of recombinant p29 Prx in the SDS cell-free system of oxidase activation. Decreased levels of p29 Prx result in decreased oxidase activity on transgenic K562 cells. p29 Prx may be important for the expression of the oxidase enzyme system through its antioxidant or signaling activity.

Selecting effective siRNAs based on guide RNA structure.

In RNA interference, guide RNAs direct RNA-induced silencing complexes to mRNA targets, mediating cleavage and ultimately leading to gene silencing. We have observed that unstructured guide strands, which either completely lack complementary bases or in which internal base pairing is thermodynamically unlikely, confer strongest silencing, whereas structures with base-paired ends are inactive. Thus, the structure of the guide strand represents a major determinant of small interfering RNA activity. Here we describe a detailed computational protocol for identification of unstructured guide strands for a given mRNA target sequence. Sequentially, all guide sequences with target complementarity are simulated, their corresponding structures are folded and unstructured guide strands are selected and rated according to thermodynamic parameters. Although this procedure is new and remains to be validated by the community, it allows reliable identification of highly active siRNAs that can be used for functional target validation or drug development.

Silencing of HIV-1 genes using siRNA-expressing genetic constructs

Today antiviral chemotherapy is the main methodto control HIV infection. Common medications influ-ence primarily the key enzymes of HIV-1, such asreverse transcriptase, integrase, and protease. How-ever, in spite of a large quantity of existing anti-HIVdrugs, antiviral therapy is often ineffective. The mainreason for ineffectiveness of treatment is adaptivemutagenesis of the virus, causing the appearance ofnew HIV-1 variants resistant to antiviral drugs. Thetoxicity and high cost of such drugs also present aserious problem.It is known that RNA interference is a powerfulnatural mechanism involved in the regulation of geneexpression and protection of cells against foreignRNA; it is also called ancient immunity operating atthe RNA level [1]. Recently, it has been found thatRNA interference is regulated during the developmentof an organism [2]. Viruses have the capacity to regu-late RNA interference. In the course of evolution, theyacquired the ability to turn off RNA interference in thehost cell to ensure their own reproduction [3]. Severalyears ago, we proposed artificial RNA interference forboth the investigation of its mechanisms and the use ofsilencing of HIV-1 genes in gene therapy. Now severalresearch groups are working in this field [4–6]. In thispaper, we report the results of testing three geneticconstructs for suppression of HIV-1 production inhuman cells.We induced artificial RNA interference using shorthairpin genetic constructs. Such constructs containshort palindromic sequences separated by a shortloop. The palindromic regions of RNA moleculesform hairpins. Owing to the existing cell mechanismof RNA monitoring, such double-stranded RNAs(dsRNAs) are detected and processed by Dicer(RNase III) to yield double-stranded small interferingRNA (siRNA). At the second stage of RNA interfer-ence, only the antisense siRNA strand remains withinthe RNA-induced silencing complex (RISC). A nucle-otide sequence of the siRNA within RISC is used tosearch for complementary regions in cell RNAs. In thecase of an HIV-1-specific siRNA, RISC recognizes anavailable single-stranded region in viral transcriptsand cuts it.The efficiency of siRNA is determined by a num-ber of factors. In particular, it depends on the siRNAnucleotide composition, which should meet the fol-lowing empiric requirements [7, 8]: a GC content of30–52%, at least three A/U in positions 15–19 of thesense strand, lack of internal repeats, A in positions 19and 3 of the sense strand, U in position 10 of the sensestrand, lack of G/C in position 19 of the sense strand,lack of G in position 13 of the sense strand, etc. Theavailability of target RNA is the second factor deter-mining the efficiency of siRNA. If the mRNA regioncomplementary to siRNA is unavailable (for example,it is bound to a protein or included in a duplex), thenit cannot be degraded.We began our study when these requirements wereunknown, so we proceeded from the assumptions thatthe best targets in HIV-1 transcripts are the most con-served regions within the functionally importantdomains and that biologically active siRNAs can beisolated in direct tests for suppression of virus produc-tion.We searched for HIV-1 conserved regions with theuse of the BLAST program and multiple sequencealignment. Within the AF316544 sequence of HIV-1subtype A, we found the most conserved regions cor-responding to the protease, reverse transcriptase, andTat domains. These regions were chosen as targets forsiRNA-encoding constructs. The resulting siRNAs

Kinetic and Efficacy Analysis of RNA Interference in Stably and Transiently Expressing Cell Lines

RNA interference, particularly through the use of small interfering RNA (siRNA), has become an important laboratory tool for both fundamental and applied investigations. However, it is currently unknown whether siRNA-mediated knockdown of transiently expressed proteins is an acceptable quantitative surrogate for stably expressed proteins. Further, the best means by which to transfect cells with functionally active siRNA are poorly defined, and determination of the best reagent and transfection conditions for a particular cell line is a burdensome prerequisite for RNA interference studies. We therefore established the optimal transfection conditions for six commercial siRNA delivery reagents in three cell lines (HR5-CL11, HeLa, and NIH/3T3) transiently or stably expressing the firefly luciferase gene. The delivery efficiency, knockdown kinetics, and cytotoxicity of the reagents were evaluated. siPORT Amine, X-tremeGENE, and TransIT-siQUEST achieved the best knockdown and consistency of performance among the three cell lines. Delivery efficiency varied and was cell line dependent in some cases. The knockdown kinetics were reagent-dependent, and knockdown was generally more rapid in the stably transfected cells. Cytotoxicity of the reagents was variable. GeneSilencer was the least cytotoxic reagent for all three cell lines, and TransIT-siQUEST was the most cytotoxic to the HeLa and HR5-CL11 cell lines. These comparative results provide an initial basis for reagent selection and experimental design for RNA interference studies in HeLa, NIH/3T3, and their respective derivative cell lines.

Efficient prediction of siRNAs with siRNArules 1.0: An open-source JAVA approach to siRNA algorithms

RNAi interference and siRNA have become useful tools for investigation of gene function. However, the discovery that not all siRNA are equally efficient made necessary screens or design algorithms to obtain high activity siRNA candidates. Several algorithms have been published, but they remain inefficient, obscure, or commercially restricted. This article describes an open-source JAVA program that is surprisingly efficient at predicting active siRNAs (Pearson correlation coefficient r = 0.52, n = 526 siRNAs). Furthermore, this version 1.0 sets the stage for further improvement of the free code by the open-source community (http://sourceforge.net/).

Biophysical and Structural Characterization of Polyethylenimine-Mediated siRNA Delivery in Vitro

The goals of this study were as follows: 1) to evaluate the efficacy of different polyethylenimine (PEI) structures for siRNA delivery in a model system, and 2) to determine the biophysical and structural characteristics of PEI that relate to siRNA delivery. Biophysical characterization (effective diameter and zeta potential), cytotoxicities, relative binding affinities and in vitro transfection efficiencies were determined using nano-complexes formed from PEI's of 800, 25,000, (both branched) and 22,000 (linear) molecular weights at varying N:P ratios and siRNA concentrations. The HR5-CL11 cell line stably expressing luciferase was used as a model system in vitro. Successful siRNA delivery was observed within a very narrow window of conditions, and only with the 25,000 branched PEI at an N:P ratio of 6:1 and 8:1 and with 200 nM siRNA. While the zeta potential and size of PEI:siRNA complexes correlated to transfection efficacy in some cases, complex stability may also affect transfection efficacy. The ability of PEI to transfer functionally active siRNA to cells in culture is surprisingly dependent on its biophysical and structural characteristics when compared to its relative success and ease of use for DNA delivery.

Ex vivo and In vivo Delivery of Anti-Tissue Factor Short Interfering RNA Inhibits Mouse Pulmonary Metastasis of B16 Melanoma Cells

The coagulation trigger tissue factor has been implicated in tumor growth, angiogenesis, and metastasis. In this study, we explore the effects of ex vivo and in vivo delivery of short interfering RNA (siRNA) targeting tissue factor on B16 melanoma colonization of the lung in a murine model for metastasis. The purposes of this work are to establish a noncytotoxic in vivo model for investigation of tissue factor function and provide preclinical assessment of the therapeutic potential of tissue factor siRNA for prevention of metastasis. C57BL/6 mice were evaluated for pulmonary metastases following tail vein injection of B16 cells transfected with either active or inactive siRNA. Mice receiving cells transfected with active siRNA had significantly lower numbers of pulmonary tumors compared with mice injected with control cells (transfected with inactive siRNA). The average time point at which the mice started to exhibit tumor-associated stress was also increased significantly from 22 days for the control group to 27 days for the experimental group (P = 0.01). In a therapeutically more relevant model, where the siRNA was delivered i.p. and the cells (untransfected) by tail vein injection, an inhibitory effect on metastasis was observed when the siRNA treatment was initiated either before or at the time of cell injection. The results suggest that tissue factor has a crucial function in promoting lung tumor metastasis of blood-borne tumor cells in the early stages of the tumor take process and further suggest that treatment with tissue factor siRNA may become a viable clinical strategy for prevention of tumor metastasis.

In situ fluorescence analysis demonstrates active siRNA exclusion from the nucleus by Exportin 5

Two types of short double-stranded RNA molecules, namely microRNAs (miRNAs) and short interfering RNAs (siRNAs), have emerged recently as important regulators of gene expression. Although these molecules show similar sizes and structural features, the mechanisms of action underlying their respective target silencing activities appear to differ: siRNAs act primarily through mRNA degradation, whereas most miRNAs appear to act primarily through translational inhibition. Our understanding of how these overlapping pathways are differentially regulated within the cell remains incomplete. In the present work, quantitative fluorescence microscopy was used to study how siRNAs are processed within human cells. We found that siRNAs are excluded from non-nucleolar areas of the nucleus in an Exportin-5 dependent process that specifically recognizes key structural features shared by these and other small RNAs such as miRNAs. We further established that the Exportin-5-based exclusion of siRNAs from the nucleus can, when Exp5 itself is inhibited, become a rate-limiting step for siRNA-induced silencing activity. Exportin 5 therefore represents a key point of intersection between the siRNA and miRNA pathways, and, as such, is of fundamental importance for the design and interpretation of RNA interference experimentation.

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.

179. In Vivo Delivery of DNA or siRNA Formulated into Nanoparticles with Linear PEI

Top of pageAbstract One of the most promising non-viral vectors is the cationic polymer polyethylenimine (PEI), a well-known cell culture transfection reagent. In particular, the latest generation of linear PEI is more efficient than the first generation of branched polymers. Various delivery routes of small size defined PEI polyplexes formulated in a 5% glucose solution can be used (intravenous, intracerebral, intraperitoneal, intracardiac, instillation |[hellip]|). Intravenous injection (IV) is the route of choice in many gene therapy applications. Among the different routes of IV injection tested (caudal, jugular and retro-orbital), the retro-orbital route appears to be the simplest and the most efficient one to deliver the gene of interest in the organs studied (i.e. lung, liver, spleen, heart and kidney). The purpose of our work was to compare different parameters of DNA injection complexed with L-PEI injected by retro-orbital route. Different amount of pCMVLuc plasmid was injected complexed with in vivo-jetPEI|[trade]| in 22|[ndash]|24 grams OF1 female mice. Among the DNA quantity injected (10 |[mu]|g to 70 |[mu]|g) at N/P ratio of 8, we have shown that the quantity of 40 or 50 |[mu]|g gives the most efficient plasmid expression. For this amount, the best result was obtained by injecting 400 or 600 |[mu]|l instead of 200 |[mu]|l. As the efficiency and the cytotoxicity depend on the molecular weight of the polymer, we compared different polymer length and showed that the commercial in vivo-jetPEI|[trade]| by being noncytotoxic is the most efficient in gene delivery. The pharmacokinetic and organ distribution profile was also determined with 40 |[mu]|g of plasmid between 6 and 72 hours after the injection of the plasmid. Taken together, our data showed that the IV retro-orbital injection is the route of choice to inject plasmid DNA complexed with linear polyethylenimine. Moreover, we have also explored the promising potential of in vivo-jetPEI|[trade]| to deliver active siRNAs injected by retro-orbital route and shown that in vivo-jetPEI|[trade]| can deliver siRNAs silencing a co-transfected target gene expressed in lungs after intravenous injection in mice.

Phosphorothioate‐stimulated uptake of short interfering RNA by human cells

The cellular delivery of short interfering RNA (siRNA) is a main hurdle in therapeutic drug development. Here, we describe that phosphorothioate (PTO)-derived oligonucleotides stimulate the physical cellular uptake of siRNA in trans in human cells. This is reflected by an apparent dose-dependent siRNA-mediated suppression of lamin A/C in primary human umbilical vein endothelial cells. The PTO-stimulated cellular uptake in trans is concentration dependent, length dependent, related to the phosphorothioate chemistry but not sequence specific. We provide experimental evidence to support a caveolin-mediated uptake mechanism. In sum, this work strongly suggests the exploration of PTOs as facilitators in the delivery of biologically active siRNA to mammalian cells.

Small interfering RNAs containing full 2′-O-methylribonucleotide-modified sense strands display Argonaute2/eIF2C2-dependent activity

RNA interference (RNAi) is a process by which short interfering RNAs (siRNAs) direct the degradation of complementary single-strand RNAs. In this study, we investigated the effects of full-strand phosphorothioate (PS) backbone and 2'-O-methyl (2'-OMe) sugar modifications on RNAi-mediated silencing. In contrast to previous reports, we have identified active siRNA duplexes containing full 2'-OMe-modified sense strands that display comparable activity to the unmodified analog of similar sequence. The structure of these modified siRNAs is the predominant determinant of their activity, with sequence and backbone composition being secondary. We further show, by using biotin-tagged siRNAs and affinity-tagged hAgo2/eIF2C2, that activity of siRNA duplexes containing full 2'-OMe substitutions in the sense strand is mediated by the RNA-induced silencing complex (RISC) and that strand-specific loading (or binding) to hAgo2 may be modulated through selective incorporation of these modifications.

Tolerated wobble mutations in siRNAs decrease specificity, but can enhance activity in vivo

RNA interference (RNAi) has become an invaluable tool for functional genomics. A critical use of this tool depends on an understanding of the factors that determine the specificity and activity of the active agent, small interfering RNA (siRNA). Several studies have concluded that tolerance of mutations can be considerable and hence lead to off-target effects. In this study, we have investigated in vivo the toleration of wobble (G:U) mutations in high activity siRNAs against Flap Endonuclease 1 (Fen1) and Aquaporin-4 (Aqp4). Mutations in the central part of the antisense strand caused a pronounced decrease in activity, while mutations in the 5′ and 3′ends were tolerated very well. Furthermore, based on analysis of nine different mutated siRNAs with widely differing intrinsic activities, we conclude that siRNA activity can be significantly enhanced by wobble mutations (relative to mRNA), in the 5′ terminal of the antisense strand. These findings should facilitate design of active siRNAs where the target mRNA offers limited choice of siRNA positions.

A systematic analysis of the silencing effects of an active siRNA at all single-nucleotide mismatched target sites

A systematic analysis of the silencing effects of an active siRNA at all single-nucleotide mismatched target sites