RNA interference in mammalian cells using siRNAs synthesized with T7 RNA polymerase

Methods that allow the specific silencing of a desired gene are invaluable tools for research. One of these is based on RNA interference (RNAi), a process by which double-stranded RNA (dsRNA) specifically suppresses the expression of a target mRNA. Recently, it has been reported that RNAi also works in mammalian cells if small interfering RNAs (siRNAs) are used to avoid activation of the interferon system by long dsRNA. Thus, RNAi could become a major tool for reverse genetics in mammalian systems. However, the high cost and the limited availability of the short synthetic RNAs and the lack of certainty that a designed siRNA will work present major drawbacks of the siRNA technology. Here we present an alternative method to obtain cheap and large amounts of siRNAs using T7 RNA polymerase. With multiple transfection procedures, including calcium phosphate co-precipitation, we demonstrate silencing of both exogenous and endogenous genes.     

Guidelines for the selection of highly effective siRNA sequences for mammalian and chick RNA interference

In the present study, the relationship between short interfering RNA (siRNA) sequence and RNA interference (RNAi) effect was extensively analyzed using 62 targets of four exogenous and two endogenous genes and three mammalian and Drosophila cells. We present the rules that may govern siRNA sequence preference and in accordance with which highly effective siRNAs essential for systematic mammalian functional genomics can be readily designed. These rules indicate that siRNAs which simultaneously satisfy all four of the following sequence conditions are capable of inducing highly effective gene silencing in mammalian cells: (i) A/U at the 5′ end of the antisense strand; (ii) G/C at the 5′ end of the sense strand; (iii) at least five A/U residues in the 5′ terminal one-third of the antisense strand; and (iv) the absence of any GC stretch of more than 9 nt in length. siRNAs opposite in features with respect to the first three conditions give rise to little or no gene silencing in mammalian cells. Essentially the same rules for siRNA sequence preference were found applicable to DNA-based RNAi in mammalian cells and in ovo RNAi using chick embryos. In contrast to mammalian and chick cells, little siRNA sequence preference could be detected in Drosophila in vivo RNAi.     

针对leader序列的siRNA能够有效抑制SARS冠状病毒多个mRNA的表达

小干扰RNAs(siRNAs)能够有效降解具有互补序列的RNA.在SARS-CoV的基因组RNA和所有亚基因组RNA的5′端均有一段共同的leader序列,而且该leader序列在不同的病毒分离物中高度保守,因此leader序列可作为一个用于抑制SARS-CoV复制的有效靶点.研究表明,针对leader序列化学合成的siRNA和DNA载体表达的shRNA都可以有效抑制SARS-CoV mRNA的表达.Leader序列特异的siRNA或shRNA不仅可以有效抑制leader与报告基因EGFP融合基因的表达,而且还可以有效抑制leader与刺突蛋白(spikeprotein)、膜蛋白(membrane protein)和核衣壳蛋白(nucleocapsid protein)基因的融合转录产物的表达.结果表明,针对leader序列的RNA干扰可以发展成为一种抗SARS-CoV治疗的有效策略.     

Potent RNAi by short RNA triggers

RNA interference (RNAi) is a gene-silencing mechanism by which a ribonucleoprotein complex, the RNA-induced silencing complex (RISC) and a double-stranded (ds) short-interfering RNA (siRNA), targets a complementary mRNA for site-specific cleavage and subsequent degradation. While longer dsRNA are endogenously processed into 21- to 24-nucleotide (nt) siRNAs or miRNAs to induce gene silencing, RNAi studies in human cells typically use synthetic 19- to 20-nt siRNA duplexes with 2-nt overhangs at the 3′-end of both strands. Here, we report that systematic synthesis and analysis of siRNAs with deletions at the passenger and/or guide strand revealed a short RNAi trigger, 16-nt siRNA, which induces potent RNAi in human cells. Our results indicate that the minimal requirement for dsRNA to trigger RNAi is an ~42 Å A-form helix with ~1.5 helical turns. The 16-nt siRNA more effectively knocked down mRNA and protein levels than 19-nt siRNA when targeting the endogenous CDK9 gene, suggesting that 16-nt siRNA is a more potent RNAi trigger. In vitro kinetic analysis of RNA-induced silencing complex (RISC) programmed in HeLa cells indicates that 16-nt siRNA has a higher RISC-loading capacity than 19-nt siRNA. These results suggest that RISC assembly and activation during RNAi does not necessarily require a 19-nt duplex siRNA and that 16-nt duplexes can be designed as more potent triggers to induce RNAi.     

A simple strategy for generation of gene knockdown constructs with convergent H1 and U6 promoters

RNA interference (RNAi) is a powerful tool for functional genetic studies in model organisms and mammalian cells. To facilitate rapid construction of gene knockdown constructs and RNAi libraries for known genes of mammalian cells, a new and simple strategy to produce small interfering RNA (siRNA) expression vectors with two opposing polymerase III promoters was developed. The design involved a one-step PCR amplification and single cloning procedure to construct a dual promoter siRNA expression vector. The forward primer is identical for all PCR reactions, only a single reverse primer that contains the siRNA targeting sequence has to be synthesized in the construction of each individual vector. This single primer design is cost-effective and it reduces the risk of sequence errors during synthesis of long oligos. Sense and antisense strands of siRNA duplexes were transcribed from the same template and this eliminated the need to synthesize long hairpin-forming oligonucleotides. Our study demonstrated that this vector design could mediate potent inhibition of expression of both exogenous and endogenous genes in mammalian cells.     

Enzymatically Produced Pools of Canonical and Dicer-Substrate siRNA Molecules Display Comparable Gene Silencing and Antiviral Activities against Herpes Simplex Virus

RNA interference (RNAi)-based sequence-specific gene silencing is applied to identify gene function and also possesses great potential for inhibiting virus replication both in animals and plants. Small interfering RNA (siRNA) molecules are the inducers of gene silencing in the RNAi pathway but may also display immunostimulatory activities and promote apoptosis. Canonical siRNAs are 21 nucleotides (nt) in length and are loaded to the RNA Induced Silencing Complex when introduced into the cells, while longer siRNA molecules are first processed by endogenous Dicer and thus termed Dicer-substrate siRNA (DsiRNA). We have applied RNA polymerases from bacteriophages T7 and phi6 to make high-quality double-stranded RNA molecules that are specific for the UL29 gene of herpes simplex virus (HSV). The 653 nt long double-stranded RNA molecules were converted to siRNA and DsiRNA pools using Dicer enzymes originating from human or Giardia intestinalis, producing siRNAs of approximately 21 and 27 nt in length, respectively. Chemically synthesised 21 and 27 nt single-site siRNA targeting the UL29 were used as references. The impact of these siRNAs on cell viability, inflammatory responses, gene silencing, and anti-HSV activity were assayed in cells derived from human nervous system and skin. Both pools and the canonical single-site siRNAs displayed substantial antiviral activity resulting in four orders of magnitude reduction in virus titer. Notably, the pool of DsiRNAs caused lower immunostimulation than the pool of canonical siRNAs, whereas the immunostimulation effect was in relation to the length with the single-site siRNAs. Our results also propose differences in the processivity of the two Dicers.     

Functional and intracellular localization properties of U6 promoter-expressed siRNAs, shRNAs, and chimeric VA1 shRNAs in mammalian cells

RNA polymerase III (Pol III) expression systems for short hairpin RNAs (U6 shRNAs or chimeric VA1 shRNAs) or individually expressed sense/antisense small interfering RNA (siRNA) strands have been used to trigger RNA interference (RNAi) in mammalian cells. Here we show that individually expressed siRNA expression constructs produce 21-nucleotide siRNAs that strongly accumulate as duplex siRNAs in the nucleus of human cells, exerting sequence-specific silencing activity similar to cytoplasmic siRNAs derived from U6 or VA1-expressed hairpin precursors. In contrast, 29-mer siRNAs separately expressed as sense/antisense strands fail to elicit RNAi activity, despite accumulation of these RNAs in the nucleus. Our findings delineate different intracellular accumulation patterns for the three expression strategies and suggest the possibility of a nuclear RNAi pathway that requires 21-mer duplexes.     

Isoform-specific phosphorylation of metabotropic glutamate receptor 5 by protein kinase C (PKC) blocks Ca2+ oscillation and oscillatory translocation of Ca2+-dependent PKC

Prolonged activation of metabotropic glutamate receptor 5a (mGluR5a) causes synchronized oscillations in intracellular calcium, inositol 1,4,5-trisphosphate production, and protein kinase C (PKC) activation. Additionally, mGluR5 stimulation elicited cyclical translocations of myristoylated alanine-rich protein kinase C substrate, which were opposite to that of gammaPKC (i.e. from plasma membrane to cytosol) and dependent on PKC activity, indicating that myristoylated alanine-rich protein kinase C substrate is repetitively phosphorylated by oscillating gammaPKC on the plasma membrane. Mutation of mGluR5 Thr(840) to aspartate abolished the oscillation of gammaPKC, but the mutation to alanine (T840A) did not. Cotransfection of gammaPKC with betaIIPKC, another Ca2+-dependent PKC, resulted in synchronous oscillatory translocation of both classical PKCs. In contrast, cotransfection of deltaPKC, a Ca2+-independent PKC, abolished the oscillations of both gammaPKC and inositol 1,4,5-trisphosphate. Regulation of the oscillations was dependent on deltaPKC kinase activity but not on gammaPKC. Furthermore, the T840A-mGluR5-mediated oscillations were not blocked by the deltaPKC overexpression. These results revealed that activation of mGluR5 causes translocation of both gammaPKC and deltaPKC to the plasma membrane. deltaPKC, but not gammaPKC, phosphorylates mGluR5 Thr(840), leading to the blockade of both Ca2+ oscillations and gammaPKC cycling. This subtype-specific targeting proposes the molecular basis of the multiple functions of PKC.     

SiRNAs conjugated with aromatic compounds induce RISC-mediated antisense strand selection and strong gene-silencing activity

Short interference RNA (siRNA) is a powerful tool for suppressing gene expression in mammalian cells. In this study, we focused on the development of siRNAs conjugated with aromatic compounds in order to improve the potency of RNAi and thus to overcome several problems with siRNAs, such as cellular delivery and nuclease stability. The siRNAs conjugated with phenyl, hydroxyphenyl, naphthyl, and pyrenyl derivatives showed strong resistance to nuclease degradation, and were thermodynamically stable compared with unmodified siRNA. A high level of membrane permeability in HeLa cells was also observed. Moreover, these siRNAs exhibited enhanced RNAi efficacy, which exceeded that of locked nucleic acid (LNA)-modified siRNAs, against exogenous Renilla luciferase in HeLa cells. In particular, abundant cytoplasmic localization and strong gene-silencing efficacy were found in the siRNAs conjugated with phenyl and hydroxyphenyl derivatives. The novel siRNAs conjugated with aromatic compounds are promising candidates for a new generation of modified siRNAs that can solve many of the problems associated with RNAi technology.     

Sequence,chemical, and structural variation of small interfering RNAs and short hairpin RNAs and the effect on mammalian gene silencing

Small interfering RNAs (siRNAs) induce sequence-specific gene silencing in mammalian cells and guide mRNA degradation in the process of RNA interference (RNAi). By targeting endogenous lamin A/C mRNA in human HeLa or mouse SW3T3 cells, we investigated the positional variation of siRNA-mediated gene silencing. We find cell-type-dependent global effects and cell-type-independent positional effects. HeLa cells were about 2-fold more responsive to siRNAs than SW3T3 cells but displayed a very similar pattern of positional variation of lamin A/C silencing. In HeLa cells, 26 of 44 tested standard 21-nucleotide (nt) siRNA duplexes reduced the protein expression by at least 90%, and only 2 duplexes reduced the lamin A/C proteins to <50%. Fluorescent chromophores did not perturb gene silencing when conjugated to the 5'-end or 3'-end of the sense siRNA strand and the 5'-end of the antisense siRNA strand, but conjugation to the 3'-end of the antisense siRNA abolished gene silencing. RNase-protecting phosphorothioate and 2'-fluoropyrimidine RNA backbone modifications of siRNAs did not significantly affect silencing efficiency, although cytotoxic effects were observed when every second phosphate of an siRNA duplex was replaced by phosphorothioate. Synthetic RNA hairpin loops were subsequently evaluated for lamin A/C silencing as a function of stem length and loop composition. As long as the 5'-end of the guide strand coincided with the 5'-end of the hairpin RNA, 19-29 base pair (bp) hairpins effectively silenced lamin A/C, but when the hairpin started with the 5'-end of the sense strand, only 21-29 bp hairpins were highly active.     

siRNA function in RNAi: a chemical modification analysis

Various chemical modifications were created in short-interfering RNAs (siRNAs) to determine the biochemical properties required for RNA interference (RNAi). Remarkably, modifications at the 2'-position of pentose sugars in siRNAs showed the 2'-OHs were not required for RNAi, indicating that RNAi machinery does not require the 2'-OH for recognition of siRNAs and catalytic ribonuclease activity of RNA-induced silencing complexes (RISCs) does not involve the 2'-OH of guide antisense RNA. In addition, 2' modifications predicted to stabilize siRNA increased the persistence of RNAi as compared with wild-type siRNAs. RNAi was also induced with chemical modifications that stabilized interactions between A-U base pairs, demonstrating that these types of modifications may enhance mRNA targeting efficiency in allele-specific RNAi. Modifications altering the structure of the A-form major groove of antisense siRNA-mRNA duplexes abolished RNAi, suggesting that the major groove of these duplexes was required for recognition by activated RISC*. Comparative analysis of the stability and RNAi activities of chemically modified single-stranded antisense RNA and duplex siRNA suggested that some catalytic mechanism(s) other than siRNA stability were linked to RNAi efficiency. Modified or mismatched ribonucleotides incorporated at internal positions in the 5' or 3' half of the siRNA duplex, as defined by the antisense strand, indicated that the integrity of the 5' and not the 3' half of the siRNA structure was important for RNAi, highlighting the asymmetric nature of siRNA recognition for initiation of unwinding. Collectively, this study defines the mechanisms of RNAi in human cells and provides new rules for designing effective and stable siRNAs for RNAi-mediated gene-silencing applications.     

Small interfering RNAs suppress the expression of endogenous and GFP-fused epidermal growth factor receptor (erbB1) and induce apoptosis in erbB1-overexpressing cells

Deregulated and excessive expression of epidermal growth factor receptor (EGFR or erbB1), a transmembrane receptor tyrosine kinase specific for the epidermal growth factor (EGF), is a feature and/or cause of a wide range of human cancers, and thus inhibition of its expression is potentially therapeutic. In RNA interference (RNAi), duplexes of 21-nucleotide RNAs (small interfering RNA, siRNA) corresponding to mRNA sequences of particular genes are used to efficiently inhibit the expression of the target proteins in mammalian cells. Here we show that by using RNAi the expression of endogenous erbB1 can be specifically and extensively (90%) suppressed in A431 human epidermoid carcinoma cells. As a consequence, EGF-induced tyrosine phosphorylation was inhibited and cell proliferation was reduced due to induction of apoptosis. We established an inverse correlation between the level of expressed erbB1 and EGF sensitivity on a cell-by-cell basis using flow cytometry. A431 cells expressing endogenous erbB1 were transfected with erbB1 fused C-terminally to enhanced green fluorescent protein (EGFP). Selective inhibition of the expression of the fusion protein was achieved with an siRNA specific for the EGFP mRNA, whereas the erbB1-specific siRNAs inhibited the expression of both molecules. siRNA-mediated inhibition of erbB1 and other erbB tyrosine kinases may constitute a useful therapeutic approach in the treatment of human cancer.     

siRNAs generated by recombinant human Dicer induce specific and significant but target site-independent gene silencing in human cells

RNA interference has emerged as a powerful tool for the silencing of gene expression in animals and plants. It was reported recently that 21 nt synthetic small interfering RNAs (siRNAs) specifically suppressed the expression of endogenous genes in several lines of mammalian cells. However, the efficacy of siRNAs is dependent on the presence of a specific target site within the target mRNA and it remains very difficult to predict the best or most effective target site. In this study, we demonstrate that siRNAs that have been generated in vitro by recombinant human Dicer (re-hDicer) significantly suppress not only the exogenous expression of a puromycin-resistance gene but also the endogenous expression of H-ras, c-jun and c-fos. In our system, selection of a target site is not necessary in the design of siRNAs. However, it is important to avoid homologous sequences within a target mRNA in a given protein family. Our diced siRNA system should be a powerful tool for the inactivation of genes in mammalian cells.     

Selective gene silencing of rat ATP-binding cassette G2 transporter in an in vitro blood-brain barrier model by short interfering RNA

The aim of the present study was to specifically silence the rat ATP-binding cassette transporter G2 (rABCG2) gene in brain capillary endothelial cells by transfection of short interfering RNA (siRNA). Four different siRNAs designed to target rABCG2 were each transfected into HEK293 cells with myc-tagged rABCG2 cDNA. Quantitative real-time PCR and western blot analyses revealed that three of the siRNAs were able to reduce exogenous rABCG2 mRNA and protein levels in HEK293 cells. Moreover, rABCG2-mediated mitoxantrone efflux transport was suppressed by the introduction of these three siRNAs into HEK293 cells. In contrast, the other siRNA and non-specific control siRNA did not significantly affect the mRNA expression, the protein level or the transport activity. Endogenous rABCG2 mRNA and protein expression in a conditionally immortalized rat brain capillary endothelial cell line (TR-BBB13) was suppressed by the most potent siRNA among the four siRNAs tested. Furthermore, this siRNA did not affect the mRNA levels of other ABC transporters, such as ABCB1, ABCC1 and ABCG1, and the protein level of ABCB1 in TR-BBB13 cells, suggesting that it can selectively silence rABCG2 at the blood-brain barrier. This should be a useful and novel strategy for clarifying the contribution of rABCG2 to brain-to-blood transport of substrate drugs and endogenous compounds across the blood-brain barrier.