RNA Interference Efficiency Research Articles

Transcripts for combined synthetic microRNA and gene delivery

MicroRNAs (miRNAs) are a class of short noncoding RNAs which are endogenously expressed in many organisms and regulate gene expression by binding to messenger RNA (mRNA). MicroRNAs are either produced from their independent transcription units in intergenic regions or lie in intragenic regions. Intragenic miRNAs and their host mRNAs are produced from the same transcript by the microprocessor and the spliceosome protein complex respectively. The details and exact timing of the processing events have implications for downstream RNA interference (RNAi) efficiency and mRNA stability. Here we engineer and study in mammalian cells a range of synthetic intragenic miRNAs co-expressed with their host genes. Furthermore, we study transcripts which carry the target of the miRNA, thereby emulating a common regulation mechanism. We perform fluorescence microscopy and flow cytometry to characterize the engineered transcripts and investigate the properties of the underlying biological processes. Our results shed additional light on miRNA and pre-mRNA processing but importantly provide insight into engineering transcripts customized for combined delivery and use in synthetic gene circuits.

MicroRNAs that interfere with RNAi

A recent study by Massirer et al. in the nematode C. elegans has shown that a family of microRNAs (miRNAs), miR-35-41, regulates the efficiency of RNA interference (RNAi), revealing a new connection between these small RNA pathways. In this commentary, we discuss the potential mechanisms for cross regulation in the miRNA and RNAi pathways and the implications for gene expression. While miRNAs are genetically encoded, the small interfering RNAs (siRNAs) that function in RNAi can originate from processing of exogenous dsRNA (exo-RNAi) or from the production of siRNAs from endogenous transcripts (endo-RNAi). These small RNA pathways involve Dicer and Argonaute proteins and typically use antisense base pairing to target mRNAs for downregulated expression. The discovery that loss of miR-35–41 results in enhanced exo-RNAi sensitivity and reduced endo-RNAi effectiveness suggests that these miRNAs normally help balance the RNAi pathways. The effect of mir-35–41 on RNAi is largely through lin-35, the C. elegans homolog of the tumor suppressor Retinoblastoma (Rb) gene. lin-35/Rb previously has been shown to regulate RNAi sensitivity through unclear mechanisms and the new finding that accumulation of LIN-35/Rb protein is dependent on miR-35–41 adds another layer of complexity to this process. The utilization of miRNAs to control the responsiveness of RNAi exemplifies the cross-regulation embedded in small RNA-directed pathways.

A mesoporous silica nanoparticle – PEI – Fusogenic peptide system for siRNA delivery in cancer therapy

RNA interference (RNAi) is widely regarded as a promising technology for disease treatment, yet one major obstacle for its clinical application is the lack of efficient siRNA delivery vehicles. In this study, we described a magnetic mesoporous silica nanoparticles (M-MSNs)-based, polyelectrolyte (polyethylenimine, PEI) and fusogenic peptide (KALA)-functionalized siRNA delivery system (denoted as M-MSN_siRNA@PEI-KALA), which was highly effective for initiating target gene silencing both in vitro and in vivo. The construction of this delivery system began with the encapsulation of siRNA within the mesopores of M-MSNs, followed by the coating of PEI on the external surface of siRNA-loaded M-MSNs and the chemical conjugation of KALA peptides. The as-prepared delivery vehicles, with notable siRNA protective effect and negligible cytotoxicity, could be easily internalized into cells, readily escape from the endolysosomes and release the loaded siRNA into the cytoplasm. As a result, the knockdown of enhanced green fluorescent protein (EGFP) and vascular endothelial growth factor (VEGF) in tumor cells were observed, both with excellent RNAi efficiencies. In the following in vivo experiments, the intratumoral injection of M-MSN_VEGF siRNA@PEI-KALA significantly inhibited the tumor growth, possibly by the suppression of neovascularization in tumors. To sum up, we have established a highly effective MSNs-based delivery system, which has great potential to serve as therapeutic siRNA formulation for cancer treatment.

Coexpression of Escherichia coli RNase III in silkworm cells improves the efficiency of RNA interference induced by long hairpin dsRNAs

Long hairpin dsRNA transcribed from chromosomal DNA can induce RNA interference in Bombyx mori cells, although its gene silencing efficiency is lower than that of exogenously introduced double-stranded RNAs (dsRNAs). To solve this problem, we monitored the nuclear cytoplasmic translocation of the transcribed hairpin dsRNA and analyzed the processing efficiency into mature small interfering RNA (siRNA). Northern blot analysis revealed that the transcribed hairpin dsRNAs were spliced and transported into the cytoplasm, but were not effectively diced into siRNAs. Interestingly, RNAi with hairpin dsRNAs from genome-integrated IR transgene was stimulated by the coexpression of Escherichia coli RNase III, although this exogenous enzyme seemed to bring about nonspecific cleavage of cellular mRNA.

Molecular characterization and gene functional analysis of Dicer‐2 gene from Nilaparvata lugens (Hemiptera: Geometroidea)

Nilaparvata lugens (Stål) (Hemiptera: Geometroidea), a serious rice pest in many countries of Asia, causes a great loss in rice production every year. RNA interference (RNAi) is a powerful technology for gene function study in insects and a potential tool for pest control. As a core component of RNAi pathway, Dicer-2 (Dcr-2) protein determines the production of small interfering RNA (siRNA) and is crucial for the efficiency of RNAi. In this study, the full-length complementary DNA (cDNA) of N. lugens Dcr-2 (NlDcr-2) was first cloned and analyzed, and then the RNAi experiment was conducted to explore the function of NlDcr-2 gene. The complete Dcr-2 cDNA of N. lugens was 4 971 bp in length with an open reading frame (ORF) of 1,656 amino acids. Phylogenetic and protein domain analysis showed that the predicted NlDcr-2 protein was similar to Tribolium castaneum. In the RNAi experiment, the messenger RNA level of NlDcr-2 was significantly reduced by NlDcr-2 double-stranded RNA (dsRNA) (dsDcr-2). Fifty-five per cent decrease of NlDcr-2 was found after 4 days of unremitting feeding. No significant effect was observed on the development of N. lugens after dsRNA ingestion.

Comparative Genomics Reveals Two Novel RNAi Factors in Trypanosoma brucei and Provides Insight into the Core Machinery

The introduction ten years ago of RNA interference (RNAi) as a tool for molecular exploration in Trypanosoma brucei has led to a surge in our understanding of the pathogenesis and biology of this human parasite. In particular, a genome-wide RNAi screen has recently been combined with next-generation Illumina sequencing to expose catalogues of genes associated with loss of fitness in distinct developmental stages. At present, this technology is restricted to RNAi-positive protozoan parasites, which excludes T. cruzi, Leishmania major, and Plasmodium falciparum. Therefore, elucidating the mechanism of RNAi and identifying the essential components of the pathway is fundamental for improving RNAi efficiency in T. brucei and for transferring the RNAi tool to RNAi-deficient pathogens. Here we used comparative genomics of RNAi-positive and -negative trypanosomatid protozoans to identify the repertoire of factors in T. brucei. In addition to the previously characterized Argonaute 1 (AGO1) protein and the cytoplasmic and nuclear Dicers, TbDCL1 and TbDCL2, respectively, we identified the RNA Interference Factors 4 and 5 (TbRIF4 and TbRIF5). TbRIF4 is a 3′-5′ exonuclease of the DnaQ superfamily and plays a critical role in the conversion of duplex siRNAs to the single-stranded form, thus generating a TbAGO1-siRNA complex required for target-specific cleavage. TbRIF5 is essential for cytoplasmic RNAi and appears to act as a TbDCL1 cofactor. The availability of the core RNAi machinery in T. brucei provides a platform to gain mechanistic insights in this ancient eukaryote and to identify the minimal set of components required to reconstitute RNAi in RNAi-deficient parasites.

Molecular cloning of BmTUDOR-SN and analysis of its role in the RNAi pathway in the silkworm, Bombyx mori (Lepidoptera: Bombycidae)

Tudor-sn, a conserved nuclease, was first isolated from RNA-induced silencing complex (RISC) and was subsequently implicated in the RNA interference (RNAi) pathway in humans, flies and nematodes. However, in the silkworm, Bombyx mori L, the RNAi mechanism and the components of RISC were quite unclear. Here, we cloned the full-length cDNA of TUDOR-SN (BmTUDOR-SN) from the silkworm. Phylogenetic analysis revealed that BmTudor-sn had a high homology with Tudor-sn proteins in other insects. Fluorescent microscopic observation indicated that the subcellular localization of enhanced green fluorescent protein fused BmTudor-sn was mainly in the cytoplasm of silkworm BmN4 cells. Knockdown of BmTUDOR-SN did not, however, affect the RNAi efficiency in BmN4 cells.

C. elegans rrf-1 Mutations Maintain RNAi Efficiency in the Soma in Addition to the Germline

Gene inactivation through RNA interference (RNAi) has proven to be a valuable tool for studying gene function in C. elegans. When combined with tissue-specific gene inactivation methods, RNAi has the potential to shed light on the function of a gene in distinct tissues. In this study we characterized C. elegans rrf-1 mutants to determine their ability to process RNAi in various tissues. These mutants have been widely used in RNAi studies to assess the function of genes specifically in the C. elegans germline. Upon closer analysis, we found that two rrf-1 mutants carrying different loss-of-function alleles were capable of processing RNAi targeting several somatically expressed genes. Specifically, we observed that the intestine was able to process RNAi triggers efficiently, whereas cells in the hypodermis showed partial susceptibility to RNAi in rrf-1 mutants. Other somatic tissues in rrf-1 mutants, such as the muscles and the somatic gonad, appeared resistant to RNAi. In addition to these observations, we found that the rrf-1(pk1417) mutation induced the expression of several transgenic arrays, including the FOXO transcription factor DAF-16. Unexpectedly, rrf-1(pk1417) mutants showed increased endogenous expression of the DAF-16 target gene sod-3; however, the lifespan and thermo-tolerance of rrf-1(pk1417) mutants were similar to those of wild-type animals. In sum, these data show that rrf-1 mutants display several phenotypes not previously appreciated, including broader tissue-specific RNAi-processing capabilities, and our results underscore the need for careful characterization of tissue-specific RNAi tools.

Metformin sensitizes endometrial cancer cells to chemotherapy by repressing glyoxalase I expression

Metformin plays an important role in the inhibition of cancer cell growth and prolongs remission durations. It reverses progestin-resistance in endometrial cancer cells by downregulating glyoxalase I (GloI) expression. This study aimed to investigate the effect of metformin on endometrial cancer cell chemotherapeutic sensitivity and explore the underlying molecular mechanisms. MTT assay was performed to determine the rate of cell death after cisplatin and paclitaxel with or without metformin. Western blot was carried out to analyze GloI expression. SiRNA-targeting of GloI was used to knockdown GloI expression before further treatment with chemotherapeutic agents to examine the effect of GloI downregulation on chemotherapy-induced cell killing. In addition, plasmid transfection was used to overexpress GloI and determine whether high GloI levels blocked metformin-enhanced cell sensitivity to chemotherapy. PCR was used to analyze the efficiency of RNA interference and plasmid transfection. The addition of metformin enhanced the sensitivity of endometrial cells to cisplatin and paclitaxel, which was associated with reduced levels of GloI expression. Moreover, low-dose chemotherapeutic drugs alone could not significantly reduce GloI expression, whereas the addition of metformin potently downregulated GloI protein levels. Cisplatin and paclitaxel markedly inhibited the proliferative ability of GloI-depleted endometrial cancer cells. However, the overexpression of GloI abolished the effect of metformin-enhanced cell sensitivity to chemotherapeutic drugs. Metformin enhances the rate of cell-killing induced by chemotherapeutic agents by repressing GloI expression.

Nuclear localization signal-enhanced RNA interference of EZH2 and Oct4 in the eradication of head and neck squamous Cell carcinoma-derived cancer stem cells

Metastasis is the major cause of high mortality in head and neck squamous cell carcinoma (HNSCC), in which HNSCC-derived cancer stem cells (CSCs) may be involved. Several reports have coupled non-viral gene delivery with RNA interference (RNAi) to target specific genes in cancer cells. However, the delivery efficiency of RNAi is limited and remained to be improved. Moreover, the therapeutic effect of non-viral gene delivery approaches on HNSCC-derived CSCs is still uncertain. In this study, we found that EZH2 and Oct4 are upregulated in HNSCC-derived ALDH1+/CD44+ CSC-like cells. Polyurethane-short branch PEI (PU-PEI)-based administration of double-stranded DNA (dsDNA) encoding small interfering RNA (siRNA) against EZH2 and Oct4 (siEZH2/siOct4) led to partial anti-cancer capacity and mild suppression of CSC-like properties. By pre-conjugation of nuclear localization signal (NLS) to siRNA-expressing dsDNA, the anti-cancer efficacy was enhanced due to elevated nuclear delivery. Notably, the NLS-preconjugated siEZH2/siOct4 constructs remarkably repressed epithelial-mesenchymal transdifferentiation (EMT) and radioresistance in ALDH1+/CD44+ CSC-like cells, in which Wnt5A and CyclinD1 may be involved respectively. We furthermore demonstrated that this improved method was capable of reducing tumor growth and metastasis in vivo. Our findings may provide a feasible non-viral gene delivery method to eradicate HNSCC-derived CSCs and improve HNSCC therapy.

Dye selection for live cell imaging of intact siRNA

Investigations into the fate of small interfering RNA (siRNA) after transfection may unravel new ways to improve RNA interference (RNAi) efficiency. Because intracellular degradation of RNA may prevent reliable observation of fluorescence-labeled siRNA, new tools for fluorescence microscopy are warranted to cover the considerable duration of the RNAi effect. Here, the characterization and application of new fluorescence resonance energy transfer (FRET) dye pairs for sensing the integrity of duplex siRNA is reported, which allows an assessment of the degradation status of an siRNA cell population by live cell imaging. A panel of high-yield fluorescent dyes has been investigated for their suitability as FRET pairs for the investigation of RNA inside the cell. Nine dyes in 13 FRET pairs were evaluated based on the performance in assays of photostability, cross-excitation, bleed-through, as well as on quantified changes of fluorescence as a consequence of, e.g., RNA strand hybridization and pH variation. The Atto488/Atto590 FRET pair has been applied to live cell imaging, and has revealed first aspects of unusual trafficking of intact siRNA. A time-lapse study showed highly dynamic movement of siRNA in large perinuclear structures. These and the resulting optimized FRET labeled siRNA are expected to have significant impact on future observations of labeled RNAs in living cells.

Roles of target site location and sequence complementarity in trans‐acting siRNA formation in Arabidopsis

In plants, many mRNAs and non-coding RNAs are cleaved by RNA-induced silencing complexes. After cleavage, only a limited number of RNAs are processed into trans-acting siRNAs (tasiRNAs). One reason is that 22 nt small RNAs, but not the more common 21 nt small RNAs, can efficiently trigger tasiRNA formation. The characteristics of the target transcripts may also affect tasiRNA production. Here we report the effects of target site location and sequence complementarity on tasiRNA formation. A synthetic sequence that included a miR173 target site and two siRNAs targeting an endogenous mRNA encoding PHYTOENE DESATURASE3 was introduced into a protein-coding (GFP) gene in the coding region or 3' UTR. tasiRNAs were generated in the transgenic seedlings, and the PDS3 mRNA level was reduced, leading to a photobleaching phenotype. It was found that tasiRNAs were most efficiently produced when the miR173 target site was placed immediately after the stop codon. Introducing premature stop codons caused a dramatic reduction of tasiRNAs and over-accumulation of 3' cleavage products, suggesting positive effects of translation on processing the 3' cleavage products into tasiRNAs. By systematically mutating the miR173 target site, we found that perfect complementarity between the 3' end of miR173 and the 5' end of the target sequence was crucial. Mismatches at that position abolished tasiRNA formation, but mismatches at the 5' end of miR173 had less effect. These data suggest important roles for translation and specific sequence complementarity in tasiRNA formation, providing new insights into tasiRNA biogenesis as well as a strategy for improving the efficiency of RNA interference (RNAi) using tasiRNAs.

SiRNA-Aptamer Chimeras on Nanoparticles: Preserving Targeting Functionality for Effective Gene Silencing

SiRNA-aptamer chimeras are emerging as a highly promising approach for cell-type specific delivery of siRNA due to the outstanding targeting capability of aptamers and the compatibility of chimeras with native ribonuclease (Dicer) processing. For efficient RNA interference (RNAi), however, additional challenges must be addressed, in particular how to get siRNA out of the endosome after cell entry and how to preserve aptamer targeting specificity when chimeras are combined with delivery carriers. Here, we report a rationally designed nanoparticle vector that simultaneously displays large surface area for high siRNA payload, exposed aptamer for specific targeting, proton sponge effect for endosome escape, and fluorescence for imaging and quantification. A key concept of this work is to graft chimeras onto nanoparticle surface via a two-step process: first immobilizing siRNA onto nanoparticle via noncovalent interactions to facilitate intracellular unpackaging and reduce nanoparticle surface charge (avoiding nonspecific electrostatic interactions between aptamers and nanoparticles) and then coupling siRNA and aptamer with retained conformation and high accessibility. Compared with conventional one-step adsorption of siRNA-aptamer chimeras onto nanoparticles with random orientations and conformations, which does not elicit much improved RNAi effect than nontargeted nanoparticle-siRNA complexes (∼6-8% improvement of the total cell population), under the same RNA concentration our approach shows selective gene silencing and enables 34% more silenced cells of the total cell population over nontargeted nanoparticle-siRNA complexes. This remarkable difference in RNAi efficiency using nanoparticle-chimera complexes is directly related to cell uptake discrepancy resulting from aptamer conformation on the nanoparticle surface (intact vs random).

Mitochondria Associate with P-bodies and Modulate MicroRNA-mediated RNA Interference

P-bodies are cytoplasmic granules that are linked to mRNA decay, mRNA storage, and RNA interference (RNAi). They are known to interact with stress granules in stressed cells, and with late endosomes. Here, we report that P-bodies also interact with mitochondria, as previously described for P-body-related granules in germ cells. The interaction is dynamic, as a large majority of P-bodies contacts mitochondria at least once within a 3-min interval, and for about 18 s. This association requires an intact microtubule network. The depletion of P-bodies does not seem to affect mitochondria, nor the mitochondrial activity to be required for their contacts with P-bodies. However, inactivation of mitochondria leads to a strong decrease of miRNA-mediated RNAi efficiency, and to a lesser extent of siRNA-mediated RNAi. The defect occurs during the assembly of active RISC and is associated with a specific delocalization of endogeneous Ago2 from P-bodies. Our study reveals the possible involvement of RNAi defect in pathologies involving mitochondrial deficiencies.

Search for Limiting Factors in the RNAi Pathway in Silkmoth Tissues and the Bm5 Cell Line: The RNA-Binding Proteins R2D2 and Translin

RNA interference (RNAi), an RNA-dependent gene silencing process that is initiated by double-stranded RNA (dsRNA) molecules, has been applied with variable success in lepidopteran insects, in contrast to the high efficiency achieved in the coleopteran Tribolium castaneum. To gain insight into the factors that determine the efficiency of RNAi, a survey was carried out to check the expression of factors that constitute the machinery of the small interfering RNA (siRNA) and microRNA (miRNA) pathways in different tissues and stages of the silkmoth, Bombyx mori. It was found that the dsRNA-binding protein R2D2, an essential component in the siRNA pathway in Drosophila, was expressed at minimal levels in silkmoth tissues. The silkmoth-derived Bm5 cell line was also deficient in expression of mRNA encoding full-length BmTranslin, an RNA-binding factor that has been shown to stimulate the efficiency of RNAi. However, despite the lack of expression of the RNA-binding proteins, silencing of a luciferase reporter gene was observed by co-transfection of luc dsRNA using a lipophilic reagent. In contrast, gene silencing was not detected when the cells were soaked in culture medium supplemented with dsRNA. The introduction of an expression construct for Tribolium R2D2 (TcR2D2) did not influence the potency of luc dsRNA to silence the luciferase reporter. Immunostaining experiments further showed that both TcR2D2 and BmTranslin accumulated at defined locations within the cytoplasm of transfected cells. Our results offer a first evaluation of the expression of the RNAi machinery in silkmoth tissues and Bm5 cells and provide evidence for a functional RNAi response to intracellular dsRNA in the absence of R2D2 and Translin. The failure of TcR2D2 to stimulate the intracellular RNAi pathway in Bombyx cells is discussed.

A SIRT1-LSD1 Corepressor Complex Regulates Notch Target Gene Expression and Development

Epigenetic regulation of gene expression by histone-modifying corepressor complexes is central to normal animal development. The NAD(+)-dependent deacetylase and gene repressor SIRT1 removes histone H4K16 acetylation marks and facilitates heterochromatin formation. However, the mechanistic contribution of SIRT1 to epigenetic regulation at euchromatic loci and whether it acts in concert with other chromatin-modifying activities to control developmental gene expression programs remain unclear. We describe here a SIRT1 corepressor complex containing the histone H3K4 demethylase LSD1/KDM1A and several other LSD1-associated proteins. SIRT1 and LSD1 interact directly and play conserved and concerted roles in H4K16 deacetylation and H3K4 demethylation to repress genes regulated by the Notch signaling pathway. Mutations in Drosophila SIRT1 and LSD1 orthologs result in similar developmental phenotypes and genetically interact with the Notch pathway in Drosophila. These findings offer new insights into conserved mechanisms of epigenetic gene repression and regulation of development by SIRT1 in metazoans.

Dual mode polyspermine with tunable degradability for plasmid DNA and siRNA delivery

Stimuli-responsive degradability is an indispensable design component for polymeric gene carriers. In order to obtain enhanced, non-cytotoxic, and molecularly tunable nonviral gene delivery, spermine, a bioavailable small cationic molecule, was polymerized with diacrylate cross-linkers with or without acid-degradable ketal linkages for controlled dual mode-degradability (i.e., differential degradations in the endosome and the cytosol). The effects of ketal to ester ratios in the polymeric backbone on degradation rate, condensation of both plasmid DNA and siRNA, cellular uptake, intracellular disassembly, and consequent DNA transfection and RNA interference efficiency in vitro and in vivo were investigated. Limited nucleic acid complexation and cellular uptake but efficient intracellular release of nucleic acids were obtained with poly(spermine ketal ester) (PSKE), the most acid-degradable polyspermine. In contrast, poly(spermine ester) (PSE), which is not acid-degradable, demonstrated efficient nucleic acid complexation and cellular uptake but inefficient intracellular release of nucleic acids. The highest in vitro DNA transfection was obtained by the random co-polymer of PSKE and PSE at an equal ratio (PSKE-PSE), attributed to its balanced DNA complexation and acid-responsive release efficiency, while efficient siRNA unpackaging by PSKE resulted in the highest gene silencing efficiency. Preliminary in vivo studies demonstrated that the highest DNA transfection was obtained by using PSE, while both PSKE and PSE silenced GFP expression at the similar level. In conclusion, dual mode-degradable polyspermine is a non-cytotoxic nonviral gene carrier, and its acid-degradability can be molecularly tuned for differentially controlled transfection and gene silencing in vitro and in vivo.

Bioreducible poly (β-amino esters)/shRNA complex nanoparticles for efficient RNA delivery

RNA interference (RNAi) mediating gene silencing is a promising approach in the area of gene therapy, but it still is a major challenge to find new non-viral vectors with high transfection efficiency and low toxicity until today. In this work, three novel bioreducible poly (β-amine esters) (PAEs) with different amino monomers in the main chain were designed and synthesized by Michael addition polymerization. All PAEs could condense shRNA into complex nanoparticles with particle size (60–200 nm) and positive surface charges (>+10 mV). The PAEs/shRNA complex nanoparticles (PAENs) were stable under the extracellular physiological condition, while it would degrade in the reductive environment due to the cleavage of the disulfide bonds in the PAEs main chain. PAENs could achieve efficient cellular uptake and EGFP silencing in HEK-293 cells and U-87 MG cells with low cytotoxicity. The high accumulation of PAENs in tumor and high silencing efficiency of intra-tumor EGFP expression occurred when PAENs were intravenously injected into BALB/c mice bearing U-87 MG-GFP tumor. The relationship between the polymer structure and RNAi efficiency and cytotoxicity showed that the density of nitrogen atoms in PAEs backbone and the existence of disulfide bonds demonstrated the remarkable influence on in vitro and in vivo gene silencing efficiency and cytotoxicity. These experimental results suggested that the PAENs could be a promising non-viral vector for efficient RNA delivery.

Cell stress is related to re-localization of Argonaute 2 and to decreased RNA interference in human cells

Various kinds of stress on human cells induce the formation of endogenous stress granules (SGs). Human Argonaute 2 (hAgo2), the catalytic core component of the RNA-induced silencing complex (RISC), can be recruited to SGs as well as P-bodies (PBs) indicating that the dynamic intracellular distribution of hAgo2 in SGs, in PBs or at other sub-cellular sites could be related to the efficiency of the RNA interference (RNAi) machinery. Here, we studied the influence of heat shock, sodium arsenite (NaAsO2), cycloheximide (CHX) and LipofectamineTM 2000-mediated transfection of phosphorothioate (PS)-modified oligonucleotides (ON) on the intracellular localization of hAgo2 and the efficiency of RNAi.Fluorescence microscopy and sedimentation analysis of cell fractions indicate stress-induced accumulation of hAgo2 in SGs and the loss of distinctly composed complexes containing hAgo2 or their sub-cellular context. Transfection of cells with PS-ON induces cell stress that is phenotypically similar to the established inducers heat shock and NaAsO2. The intracellular re-distribution of hAgo2 is related to its increased metabolic stability and to decreased RNAi directed by microRNA or by short interfering RNA. Here, we propose a functional model of the relationship between cell stress, translocation of hAgo2 to SGs providing a depot function, and loss of RNAi activity.

Efficient and targeted delivery of siRNA in vivo

RNA interference (RNAi) has been regarded as a revolutionary tool for manipulating target biological processes as well as an emerging and promising therapeutic strategy. In contrast to the tangible and obvious effectiveness of RNAi in vitro, silencing target gene expression in vivo using small interfering RNA (siRNA) has been a very challenging task due to multiscale barriers, including rapid excretion, low stability in blood serum, nonspecific accumulation in tissues, poor cellular uptake and inefficient intracellular release. This minireview introduces major challenges in achieving efficient siRNA delivery in vivo and discusses recent advances in overcoming them using chemically modified siRNA, viral siRNA vectors and nonviral siRNA carriers. Enhanced specificity and efficiency of RNAi in vivo via selective accumulations in desired tissues, specific binding to target cells and facilitated intracellular trafficking are also commonly attempted utilizing targeting moieties, cell-penetrating peptides, fusogenic peptides and stimuli-responsive polymers. Overall, the crucial roles of the interdisciplinary approaches to optimizing RNAi in vivo, by efficiently and specifically delivering siRNA to target tissues and cells, are highlighted.