siRNA In Mammalian Cells Research Articles

Light-regulated RNA interference induced by p-hydroxyphenacyl-modified siRNA in mammalian cells

siRNA is an important tool for modulating gene expression in current biomedical research. It would be highly desirable for siRNA to respond to an external stimulus. In this paper, we report a convenient, photolabile caging agent to regulate siRNA functions. 2-bromo-4’-hydroxyacetophenone (BHAP) can readily modify phosphorothioate backbones and inhibit siRNAs. Mild UV irradiation will cleave the modifying moiety to generate natural nucleic acid backbones, thus activating siRNA functions. Such modification is conveniently conducted in an aqueous solution with high efficiency and is cost-effective and scalable. This approach provides a convenient tool for the controlled regulation of gene expression by deploying minimal usage of complex organic synthesis for site-specific installation of the caging group to siRNA unlike previous reported works that required a series of intricate organic synthesis and cumbersome purification techniques to achieve similar aims. This study will open new doors for optochemical regulation of a variety of genes by pHP caging group in mammalian cell culture.

Inhibition of viral suppressor of RNAi proteins by designer peptides protects from enteroviral infection in vivo

RNA interference (RNAi) is the major antiviral mechanism in plants and invertebrates, but the absence of detectable viral (v)siRNAs in mammalian cells upon viral infection has questioned the functional relevance of this pathway in mammalian immunity. We designed a series of peptides specifically targeting enterovirus A71 (EV-A71)-encoded protein 3A, a viral suppressor of RNAi (VSR). These peptides abrogated the VSR function of EV-A71 in infected cells and resulted in the accumulation of vsiRNAs and reduced viral replication. These vsiRNAs were functional, as evidenced by RISC-loading and silencing of target RNAs. The effects of VSR-targeting peptides (VTPs) on infection with EV-A71 as well as another enterovirus, Coxsackievirus-A16, were ablated upon deletion of Dicer1 or AGO2, core components of the RNAi pathway. Invivo, VTP treatmentprotected mice against lethal EV-A71 challenge, with detectable vsiRNAs. Our findings provide evidencefor the functional relevance of RNAi in mammalian immunity and present a therapeutic strategy for infectious disease.

Renal peroxiredoxin 6 interacts with anion exchanger 1 and plays a novel role in pH homeostasis.

Peroxiredoxin 6 (PRDX6) is one of six members of the PRDX family, which have peroxidase and antioxidant activity. PRDX6 is unique, containing only one conserved cysteine residue (C47) rather than the two found in other PRDXs. A yeast two-hybrid screen found PRDX6 to be a potential binding partner of the C-terminal tail of anion exchanger 1 (AE1), a Cl−/HCO3− exchanger basolaterally expressed in renal α-intercalated cells. PRDX6 immunostaining in human kidney was both cytoplasmic and peripheral and co-localized with AE1. Analysis of native protein showed it was largely monomeric, whereas expressed tagged protein was more dimeric. Two methionine oxidation sites were identified. In vitro and ex vivo pulldowns and immunoprecipitation assays confirmed interaction with AE1, but mutation of the conserved cysteine resulted in loss of interaction. Prdx6 knockout mice had a baseline acidosis with a major respiratory component and greater AE1 expression than wild type animals. After an oral acid challenge, PRDX6 expression increased in wild type mice, with preservation of AE1. However, AE1 expression was significantly decreased in knockout animals. Kidneys from acidified mice showed widespread proximal tubular vacuolation in wild type but not knockout animals. Knockdown of PRDX6 by siRNA in mammalian cells reduced both total and cell membrane AE1 levels. Thus, PRDX6-AE1 interaction contributes to maintenance of AE1 during cellular stress such as during metabolic acidosis.

A simplified and versatile system for the simultaneous expression of multiple siRNAs in mammalian cells using Gibson DNA Assembly.

RNA interference (RNAi) denotes sequence-specific mRNA degradation induced by short interfering double-stranded RNA (siRNA) and has become a revolutionary tool for functional annotation of mammalian genes, as well as for development of novel therapeutics. The practical applications of RNAi are usually achieved by expressing short hairpin RNAs (shRNAs) or siRNAs in cells. However, a major technical challenge is to simultaneously express multiple siRNAs to silence one or more genes. We previously developed pSOS system, in which siRNA duplexes are made from oligo templates driven by opposing U6 and H1 promoters. While effective, it is not equipped to express multiple siRNAs in a single vector. Gibson DNA Assembly (GDA) is an in vitro recombination system that has the capacity to assemble multiple overlapping DNA molecules in a single isothermal step. Here, we developed a GDA-based pSOK assembly system for constructing single vectors that express multiple siRNA sites. The assembly fragments were generated by PCR amplifications from the U6-H1 template vector pB2B. GDA assembly specificity was conferred by the overlapping unique siRNA sequences of insert fragments. To prove the technical feasibility, we constructed pSOK vectors that contain four siRNA sites and three siRNA sites targeting human and mouse β-catenin, respectively. The assembly reactions were efficient, and candidate clones were readily identified by PCR screening. Multiple β-catenin siRNAs effectively silenced endogenous β-catenin expression, inhibited Wnt3A-induced β-catenin/Tcf4 reporter activity and expression of Wnt/β-catenin downstream genes. Silencing β-catenin in mesenchymal stem cells inhibited Wnt3A-induced early osteogenic differentiation and significantly diminished synergistic osteogenic activity between BMP9 and Wnt3A in vitro and in vivo. These findings demonstrate that the GDA-based pSOK system has been proven simplistic, effective and versatile for simultaneous expression of multiple siRNAs. Thus, the reported pSOK system should be a valuable tool for gene function studies and development of novel therapeutics.

Synthesis and characterization of N-ethyl-N’-(3-dimethylaminopropyl)-guanidinyl-polyethylenimine polymers and investigation of their capability to deliver DNA and siRNA in mammalian cells

Recent advancements in polymeric gene delivery have raised the potential of gene therapy as treatment for various acquired and inherited diseases. Here, we report on the synthesis and characterization of N-ethyl-N’-(3-dimethylaminopropyl)-guanidinyl-polyethylenimine (sGP) polymers and investigation of their capability to carry DNA and siRNA in vitro. Zinc triflate-mediated activation of primary amines of branched polyethylenimine (bPEI) followed by reaction with varying amounts of N-ethyl-N’-(3-dimethylaminopropyl) carbodiimide (EDAC) resulted in the generation of a small series of trisubstituted guanidinyl-modified polyethylenimine polymers. Determination of primary amines on modified polymers by TNBS assay revealed 62–84% of the attempted conjugation of EDAC onto bPEI. These modified polymers were shown to condense plasmid DNA and retard its mobility on 0.8% agarose gel. Further, these polymers were evaluated for their capability to carry pDNA into the cells by performing transfection assay on various mammalian cells. All the modified polymer/pDNA complexes exhibited significantly higher levels of gene expression with one of the complexes, sGP3/pDNA complex, displayed ~1.45 to 3.0 orders of magnitude higher transfection efficiency than that observed in the native bPEI and the commercial transfection reagent, Lipofectamine™. The efficacy of sGP3 polymer was further assessed by siRNA delivery, which resulted in ~81% suppression of the target gene. In conclusion, these studies demonstrate the potential of these substituted guanidinyl-modified PEIs as efficient gene delivery vectors.

Lipoplexes versus nanoparticles: pDNA/siRNA delivery

Small interfering RNA (siRNA) has been widely used as potential therapeutic for treatment of various genetic disorders. However, rapid degradation, poor cellular uptake and limited stability in blood limit the effectiveness of the systemic delivery of siRNA. Therefore, an efficient delivery system is required to enhance its transfection and duration of therapeutics. In the present study, plasmid DNA (pEGFPN3) expressing green fluorescent protein (GFP) was used as a reporter gene. Chitosan nanoparticles/polyplexes and cationic liposomes/lipoplexes were developed and compared for their transfectivity and therapeutic activity in mammalian cell line (HEK 293). The nanoparticulates were first characterized by assessing the surface charge (zeta potential), size (dynamic light scattering) and morphology (transmission electron microscope) followed by evaluation for their DNA retardation ability, transfection efficiency and cytotoxicity on HEK 293 cell line. The chitosan nanoparticles/plasmid DNA (pDNA) complex and liposomes/pDNA complex were co-transfected with GFP-specific siRNA into HEK 293 cells and it was found that both are efficient delivery vehicles for siRNA transfection, resulting in ∼57% and ∼70% suppression of the targeted gene (GFP), respectively, as compared with the mock control (cells transfected with nanocarrier/pDNA complexes alone). This strong inhibition of GFP expression indicated that cationic liposomes are better than chitosan nanoparticles and can be used as an effective carrier of siRNA in mammalian cells.

Amphiphilic polyethylenimine polymers mediate efficient delivery of DNA and siRNA in mammalian cells

Recently, non-viral gene delivery vectors have shown promising results in the treatment of inherited as well as infectious diseases. Among various cationic polymers, branched polyethylenimine (bPEI, 25 kDa) has been the most widely used vector and is known as a gold standard in gene delivery. However, in vivo applications of PEI have been hampered by its charge-associated toxicity. Here, in order to address the toxicity concern as well as to enhance the transfection efficacy of bPEI, it has been modified by tethering a hydrophobic and positively charged 6-(N,N,N',N'-tetramethylguanidinium chloride)-hexanoic acid (TH) linker using water soluble coupling reagent, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDAC). A small series of 6-(N,N,N',N'-tetramethylguanidinium chloride)-hexanoyl-polyethylenimine (THP) polymers was synthesized by varying the amounts of TH-linker . These modified polymers were subsequently characterized by spectroscopic techniques and evaluated for their buffering capacity, transfection efficiency and cytotoxicity on HeLa, HEK293 and CHO cells and the results were compared with those obtained with the standard transfection reagent, Lipofectamine™. From these studies, it was observed that one of the formulations, the THP2-DNA complex, exhibited significantly higher transfection efficiency (∼1.5-3.6 folds) and cell viability, outcompeting native bPEI and Lipofectamine™ in the absence and presence of serum. Further, to establish the superiority of THP2, it was examined for its ability to bind and deliver plasmid DNAs (pDNAs) of different molecular weight, provide protection against nucleases present in the cellular milieu and also deliver siRNAs (GFP- and GAPDH-specific). Taken together, the results show the potential of THP polymers as promising carriers of nucleic acids for in vivo applications.

English

RNA-interference is the mechanism of sequence-specific, post-transcriptional gene silencing, initiated by small interfering RNA (siRNA), homologous to the gene being suppressed. Several techniques are utilized to transfer siRNA into cultured cells or animal models, while every method has advantages and disadvantages. In this study, a siRNA expression recombinant adeno-associated virus (AAV) was established by inserting H1 promoter into transfer plasmid of AAV Helper-Free system. To perform functional tests on siRNA, which was expressed by the viral vector, recombinant AAVs, coding for siRNA against exogenous gene, EGFP, and endogenous gene, p53, were established and added into HEK293 cells, respectively. The results proved the expression of EGFP and p53 in cells were definitely suppressed at 72 h post-infection, which suggested that the H1 promoter, inserted into the recombinant AAV, could express siRNA in mammalian cells and this siRNA delivery system could be used for long-term gene silencing.   Key words: Adeno-associated virus, RNA interference, p53, EGFP.

Regulation of TIP60 by ATF2 Modulates ATM Activation

TIP60 (HTATIP) is a histone acetyltransferase (HAT) whose function is critical in regulating ataxia-telangiectasia mutated (ATM) activation, gene expression, and chromatin acetylation in DNA repair. Here we show that under non-stressed conditions, activating transcription factor-2 (ATF2) in cooperation with Cul3 ubiquitin ligase promotes degradation of TIP60, thereby attenuating its HAT activity. Inhibiting either ATF2 or Cul3 expression by small interfering RNA stabilizes the TIP60 protein. ATF2 association with TIP60 on chromatin is decreased following exposure to ionizing radiation (IR), resulting in enhanced TIP60 stability and activity. We also identified a panel of melanoma and prostate cancer cell lines whose ATF2 expression is inversely correlated with TIP60 levels and ATM activation after IR. Inhibition of ATF2 expression in these lines restored TIP60 protein levels and both basal and IR-induced levels of ATM activity. Our study provides novel insight into regulation of ATM activation by ATF2-dependent control of TIP60 stability and activity.

Function and anatomy of plant siRNA pools derived from hairpin transgenes

BackgroundRNA interference results in specific gene silencing by small-interfering RNAs (siRNAs). Synthetic siRNAs provide a powerful tool for manipulating gene expression but high cost suggests that novel siRNA production methods are desirable. Strong evolutionary conservation of siRNA structure suggested that siRNAs will retain cross-species function and that transgenic plants expressing heterologous siRNAs might serve as useful siRNA bioreactors. Here we report a detailed evaluation of the above proposition and present evidence regarding structural features of siRNAs extracted from plants.ResultsTesting the gene silencing capacity of plant-derived siRNAs in mammalian cells proved to be very challenging and required partial siRNA purification and design of a highly sensitive assay. Using the above assay we found that plant-derived siRNAs are ineffective for gene silencing in mammalian cells. Plant-derived siRNAs are almost exclusively double-stranded and most likely comprise a mixture of bona fide siRNAs and aberrant partially complementary duplexes. We also provide indirect evidence that plant-derived siRNAs may contain a hitherto undetected physiological modification, distinct from 3' terminal 2-O-methylation.ConclusionsiRNAs produced from plant hairpin transgenes and extracted from plants are ineffective for gene silencing in mammalian cells. Thus our findings establish that a previous claim that transgenic plants offer a cost-effective, scalable and sustainable source of siRNAs is unwarranted. Our results also indicate that the presence of aberrant siRNA duplexes and possibly a plant-specific siRNA modification, compromises the gene silencing capacity of plant-derived siRNAs in mammalian cells.

Approaches for chemically synthesized siRNA and vector-mediated RNAi

Successful applications of RNAi in mammalian cells depend upon effective knockdown of targeted transcripts and efficient intracellular delivery of either preformed si/shRNAs or vector expressed si/shRNAs. We have previously demonstrated that 27 base pair double stranded RNAs which are substrates for Dicer can be up to 100 times more potent than 21mer siRNAs. In this mini-review we elaborate upon the rationale and design strategies for creating Dicer substrate RNAs that provide enhanced knockdown of targeted RNAs and minimize the utilization of the sense strand as RNAi effectors. Expression of shRNAs or siRNAs in mammalian cells can be achieved via transcription from either Pol II or Pol III promoters. There are certain constrictions in designing such vectors, and these are described here. Additionally, we review strategies for inducible shRNA expression and the various viral vectors that can be used to transduce shRNA genes into a variety of cells and tissues. The overall goal of this mini-review is to provide an overview of available approaches for optimizing RNAi mediated down regulation of gene expression in mammalian cells via RNA interference. Although the primary focus is the use of RNAi mediated cleavage of targeted transcripts, it is highly probable that some of the approaches described herein will be applicable to RNAi mediated inhibition of translation and transcriptional gene silencing.

ADAR1 RNA Deaminase Limits Short Interfering RNA Efficacy in Mammalian Cells

Double-stranded RNA induces the homology-dependent degradation of cognate mRNA in the cytoplasm via RNA interference (RNAi) but also is a target for adenosine-to-inosine (A-to-I) RNA editing by adenosine deaminases acting on RNA (ADARs). An interaction between the RNAi and the RNA editing pathways in Caenorhabditis elegans has been suggested recently, but the precise mode of interaction remains to be established. In addition, it is unclear whether this interaction is possible in mammalian cells with their somewhat different RNAi pathways. Here we show that ADAR1 and ADAR2, but not ADAR3, avidly bind short interfering RNA (siRNA) without RNA editing. In particular, the cytoplasmic full-length isoform of ADAR1 has the highest affinity among known ADARs, with a subnanomolar dissociation constant. Gene silencing by siRNA is significantly more effective in mouse fibroblasts homozygous for an ADAR1 null mutation than in wild-type cells. In addition, suppression of RNAi effects are detected in fibroblast cells overexpressing functional ADAR1 but not when overexpressing mutant ADAR1 lacking double-stranded RNA-binding domains. These results identify ADAR1 as a cellular factor that limits the efficacy of siRNA in mammalian cells.

Usage of Putative Chicken U6 Promoters for Vector-Based RNA Interference

Gene silencing with short interfering RNA (siRNA) expression vectors is a powerful method for the analysis of gene functions. For the expression of siRNA in mammalian cells, mammalian U6 small nuclear RNA (snRNA) promoters are widely used. However, the mammalian U6 promoter might not function well in other species. In this study, we cloned four putative chicken U6 promoters by PCR and analyzed their functions. First, we screened the chicken genomic database using the human U6 snRNA gene and identified four candidate sequences. The sequences contained some control elements in their promoter regions, but as we could not rule out that they were pseudogenes, we amplified these sequences and used them as promoters for short hairpin RNA (shRNA) expression. Using the firefly luciferase (Luc) gene as a target, transient expression assays were performed with chicken ovary-derived cells. All four putative chicken U6 promoters exhibited suppressive activity toward Luc, and so could act as a promoter for expression of the snRNA gene in the chicken genome. The promoter activity was not as strong as that of a commercially available siRNA expression vector. This probably reflects artificial sequences between the promoters and synthetic DNA encoding shRNA.

The Activity of siRNA in Mammalian Cells is Related to the Kinetics of siRNA-target Recognition In Vitro: Mechanistic Implications

The specificity of siRNA-mediated suppression of gene expression involves base-base recognition between siRNA and its single-stranded RNA target. We investigated the kinetics of this process in vitro by using full-length ICAM-1 target RNA and biologically active and inactive ICAM-1-directed siRNA, respectively. To mimic the situation in living cells we used the well-characterised facilitator of RNA-RNA annealing and strand exchange cetyltrimethylammonium bromide, which increases strongly the kinetics of RNA-RNA interactions at conditions that do not affect RNA structure nor the presumed structure-function relationship. For the biologically active siRNA si2B, we find faster binding, i.e. recognition of the target and a slower backward reaction when compared with the biologically inactive siRNA si1. This is reflected by an approximately 400-fold more favorable equilibrium constant of si2B. Kinetic evidence favors an associative mechanism of recognition of the target strand by the double-stranded siRNA. The minimal model for siRNA-target recognition described here is consistent with the high biological activity of si2B only if one assumes a step subsequent to target recognition, which might be degradation of the target RNA when complexed with the antisense strand of siRNA or when considering rapid destruction of the released sense strand of siRNA.

A new vector, based on the PolII promoter for the U1 snRNA gene, for the expression of siRNAs in mammalian cells

Several vectors for the induction of RNA interference in mammalian cells have been described, based mainly on polIII-dependent promoters. They transcribe short hairpin RNAs (shRNA) that, after being processed into short interfering RNAs (siRNAs), mediate the degradation of the target mRNA. Here, we describe the construction of a new siRNA-expressing vector (psiUx) based on the strong and ubiquitous polII-dependent promoter of the human U1 small nuclear RNA (snRNA) gene. In psiUx, the only constraint for the shRNA sequence is a purine at position +1, since specific 3′-end formation is achieved by a box element located downstream of the transcribed region. Several constructs were designed against the lamin A/C target. Depending on the structure of the shRNA transcribed, a preferential or exclusive accumulation of the antisense strand is obtained, thus avoiding possible nonspecific targeting by the sense strand. In all cases tested, very effective siRNAs were produced, thus providing a proof-of-principle that a snRNA-type polII promoter can be used for the expression of siRNAs. We show that psiUx ensures high levels of expression and efficient knock down of the target gene also in stable cell lines.

A new vector, based on the PolII promoter for the U1 snRNA gene, for the expression of siRNAs in mammalian cells

A new vector, based on the PolII promoter for the U1 snRNA gene, for the expression of siRNAs in mammalian cells

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 19–25 bp, 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.

Targeted gene silencing by small interfering RNA-based knock-down technology.

RNA interference (RNAi) has emerged as a powerful tool for the silencing of gene expression in animals and plants. RNAi is mediated by approximately 21-nt small interfering RNAs (siRNAs), which are originally produced from larger double stranded RNAs (dsRNAs) in vivo through the action of Dicer. Recently, many groups have reported systems designed to express siRNAs in mammalian cells through transfection of either oligonucleotides or plasmids encoding siRNAs. Although the use of siRNAs to silence genes in vertebrate cells was only reported three years ago, the emerging literature indicates that most vertebrate genes can be studied with this technology. This review summarizes some approaches to generate siRNAs, the delivery and application of siRNAs to target cells and the utility of siRNAs as analytical and potential therapeutic tools.

Expression of siRNA from a single transcript that includes multiple ribozymes in mammalian cells.

RNA interference (RNAi) has been developed recently as a powerful tool for silencing of mRNAs in various organisms. In mammalian cells, the introduction of small interfering RNAs (siRNAs) can inhibit gene expression in a sequence-specific manner without induction of the nonspecific degradation that is activated by long double-stranded RNA (dsRNA) (>30 nt). Here, we report a method for generating siRNAs in mammalian cells using a self-cleaving ribozyme-expressing vector. Four ribozymes within transcripts that were expressed under the control of a cytomegalovirus (CMV) or tRNAVal promoter excised, in cis, specific sense and antisense sequences from primary transcripts and generated siRNAs in HeLa cells. The siRNAs generated by the ribozymes were able to decrease the expression of a firefly gene for luciferase. These results suggest that polymerase II (pol II) systems, particularly in view of the availability of many potential tissue-specific promoters, and pol III systems, in which siRNAs are generated by a trimming-ribozyme (TRz) system as described here, should be useful in efforts to suppress the expression of specific genes.

Structural variations and stabilising modifications of synthetic siRNAs in mammalian cells.

Double-stranded short interfering RNAs (siRNA) induce post-transcriptional silencing in a variety of biological systems. In the present study we have investigated the structural requirements of chemically synthesised siRNAs to mediate efficient gene silencing in mammalian cells. In contrast to studies with Drosophila extracts, we found that synthetic, double-stranded siRNAs without specific nucleotide overhangs are highly efficient in gene silencing. Blocking of the 5'-hydroxyl terminus of the antisense strand leads to a dramatic loss of RNA interference activity, whereas blocking of the 3' terminus or blocking of the termini of the sense strand had no negative effect. We further demonstrate that synthetic siRNA molecules with internal 2'-O-methyl modification, but not molecules with terminal modifications, are protected against serum-derived nucleases. Finally, we analysed different sets of siRNA molecules with various 2'-O-methyl modifications for stability and activity. We demonstrate that 2'-O-methyl modifications at specific positions in the molecule improve stability of siRNAs in serum and are tolerated without significant loss of RNA interference activity. These second generation siRNAs will be better suited for potential therapeutic application of synthetic siRNAs in vivo.