esiRNAs purified with chromatography suppress homologous gene expression with high efficiency and specificity

Many preclinical studies have shown RNA interference (RNAi) as a new promising way to treat various human diseases including cancer and virus infection and there is an increasing demand for the large-scale preparation of short interfering RNAs (siRNAs) at low cost. Data are accumulating to show that endoribonuclease-prepared siRNAs (esiRNAs) are superior to chemically synthesized siRNAs in terms of expense, efficiency, and specificity. Yet all procedures available for esiRNA purification were designed to produce small amount of siRNAs for laboratory use. In this article, a new method of purification of esiRNAs based on ion exchange chromatography and size exclusion chromatography is reported. The esiRNAs prepared with this method are shown here to be of high purity and specifically suppress homologous gene expression without activating interferon response and with higher efficiency than chemically synthesized siRNAs. We can expect that the new method can be scaled up easily to provide large quantities of esiRNAs to meet the requirement of preclinical and clinical studies.     

Computational detection and suppression of sequence-specific off-target phenotypes from whole genome RNAi screens

A challenge for large-scale siRNA loss-of-function studies is the biological pleiotropy resulting from multiple modes of action of siRNA reagents. A major confounding feature of these reagents is the microRNA-like translational quelling resulting from short regions of oligonucleotide complementarity to many different messenger RNAs. We developed a computational approach, deconvolution analysis of RNAi screening data, for automated quantitation of off-target effects in RNAi screening data sets. Substantial reduction of off-target rates was experimentally validated in five distinct biological screens across different genome-wide siRNA libraries. A public-access graphical-user-interface has been constructed to facilitate application of this algorithm.     

用Rnase Ⅲ制备的小干涉RNA降解SARS冠状病毒基因的细胞内转录物

SARS冠状病毒是引起重症急性呼吸综合症的主要原因,目前尚没有特效药物或疫苗对抗这种新病毒。RNA干涉是指双链RNA可以特异地降解细胞内同源基因的Mrna。在哺乳动物细胞中,<30bp的小双链RNA能引起RNA干涉,又可以避免干扰素反应。通过体外转录得到SARS病毒3种基因RNA依赖的RNA聚合酶、刺突蛋白及核衣壳蛋白部分片段的长双链RNA,然后用Rnase Ⅲ有限切割成长度<30bp的小干涉RNA。同时把上述3种基因片段分别连接到质粒Pgl3-Control中,得到的3个质粒Pgl-R、Pgl-S和Pgl-N可以分别在细胞内转录出荧光素酶RNA依赖的RNA聚合酶、刺突蛋白、核衣壳蛋白的杂合Mrna。上述质粒分别和相应的小干涉RNA共转染HEK293F细胞,测定荧光素酶活性,结果小干涉RNA使相应质粒表达荧光素酶的活性显著下降;用逆转录定量PCR反应测量Mrna丰度,结果表明上述小干涉RNA可以特异地降解相应的病毒基因转录物。     

Off-target and a portion of target-specific siRNA mediated mRNA degradation is Ago2 ‘Slicer’ independent and can be mediated by Ago1

It is known that siRNAs are capable of reducing expression of non-target genes due to the interaction of the siRNA guide strand with a partially complementary site on the ‘off-target’ mRNA. In the current study, we show that reduction of cellular Ago2 levels has no effect on off-target reduction of endogenous genes and that off-target degradation of mRNA can occur even in an Ago2 knockout cell line. Using antisense mediated reduction of Ago proteins and chemically modified cleavage- and binding-deficient siRNAs, we demonstrate that siRNA mediated off-target reduction is Ago2 cleavage independent, but does require siRNA interaction with either Ago1 or Ago2 and the RISC-loading complex. We also show that depletion of P-body associated proteins results in a reduction of off-target siRNA-mediated degradation of mRNA. Finally, we present data suggesting that a significant portion of on-target siRNA activity is also Ago2 cleavage independent, however, this activity does not appear to be P-body associated.     

Molecular mechanisms that funnel RNA precursors into endogenous small-interfering RNA and microRNA biogenesis pathways in Drosophila

In Drosophila, three types of endogenous small RNAs—microRNAs (miRNAs), PIWI-interacting RNAs (piRNAs), and endogenous small-interfering RNAs (endo-siRNAs or esiRNAs)—function as triggers in RNA silencing. Although piRNAs are produced independently of Dicer, miRNA and esiRNA biogenesis pathways require Dicer1 and Dicer2, respectively. Recent studies have shown that among the four isoforms of Loquacious (Loqs), Loqs-PB and Loqs-PD are involved in miRNA and esiRNA processing pathways, respectively. However, how these Loqs isoforms function in their respective small RNA biogenesis pathways remains elusive. Here, we show that Loqs-PD associates specifically with Dicer2 through its C-terminal domain. The Dicer2–Loqs-PD complex contains R2D2, another known Dicer2 partner, and excises both exogenous siRNAs and esiRNAs from their corresponding precursors in vitro. However, Loqs-PD, but not R2D2, enhanced Dicer2 activity. The Dicer2–Loqs-PD complex processes esiRNA precursor hairpins with long stems, which results in the production of AGO2-associated small RNAs. Interestingly, however, small RNAs derived from terminal hairpins of esiRNA precursors are loaded onto AGO1; thus, they are classified as a new subset of miRNAs. These results suggest that the precursor RNA structure determines the biogenesis mechanism of esiRNAs and miRNAs, thereby implicating hairpin structures with long stems as intermediates in the evolution of Drosophila miRNA.     

siRNA-mediated off-target gene silencing triggered by a 7 nt complementation

A growing body of evidence suggests that siRNA could generate off-target effects through different mechanisms. However, the full impact of off-target gene regulation on phenotypic induction and accordingly on data interpretation in the context of large-scale siRNA library screen has not been reported. Here we report on off-target gene silencing effects observed in a large-scale knockdown experiment designed to identify novel regulators of the HIF-1 pathway. All of the three ‘top hits’ from our screen have been demonstrated to result from off-target gene silencing. Two of the three ‘siRNA hits’ were found to directly trigger down-regulation of hif-1α mRNA through a 7 nt motif, AGGCAGT, that is present in both the hif-1α mRNA and the siRNAs. Further analysis revealed that the generation of off-target gene silencing via this 7 nt motif depends on the characteristics of the target mRNA, including the sequence context surrounding the complementary region, the position of the complementary region in the mRNA and the copy number of the complementary region. Interestingly, the off-target siRNA against hif-1α was also shown to trigger mRNA degradation with high probability of other genes that possess multiple copies of the AGGCAGT motif in the 3′-untranslated region. Lessons learned from this study will be a valuable asset to aid in designing siRNAs with more stringent target selectivity and improving ‘hits-follow-up’ strategies for future large-scale knockdown experiments.     

Whole-Genome Thermodynamic Analysis Reduces siRNA Off-Target Effects

Small interfering RNAs (siRNAs) are important tools for knocking down targeted genes, and have been widely applied to biological and biomedical research. To design siRNAs, two important aspects must be considered: the potency in knocking down target genes and the off-target effect on any nontarget genes. Although many studies have produced useful tools to design potent siRNAs, off-target prevention has mostly been delegated to sequence-level alignment tools such as BLAST. We hypothesize that whole-genome thermodynamic analysis can identify potential off-targets with higher precision and help us avoid siRNAs that may have strong off-target effects. To validate this hypothesis, two siRNA sets were designed to target three human genes IDH1, ITPR2 and TRIM28. They were selected from the output of two popular siRNA design tools, siDirect and siDesign. Both siRNA design tools have incorporated sequence-level screening to avoid off-targets, thus their output is believed to be optimal. However, one of the sets we tested has off-target genes predicted by Picky, a whole-genome thermodynamic analysis tool. Picky can identify off-target genes that may hybridize to a siRNA within a user-specified melting temperature range. Our experiments validated that some off-target genes predicted by Picky can indeed be inhibited by siRNAs. Similar experiments were performed using commercially available siRNAs and a few off-target genes were also found to be inhibited as predicted by Picky. In summary, we demonstrate that whole-genome thermodynamic analysis can identify off-target genes that are missed in sequence-level screening. Because Picky prediction is deterministic according to thermodynamics, if a siRNA candidate has no Picky predicted off-targets, it is unlikely to cause off-target effects. Therefore, we recommend including Picky as an additional screening step in siRNA design.     

MISSION esiRNA for RNAi Screening in Mammalian Cells

RNA interference (RNAi) is a basic cellular mechanism for the control of gene expression. RNAi is induced by short double-stranded RNAs also known as small interfering RNAs (siRNAs). The short double-stranded RNAs originate from longer double stranded precursors by the activity of Dicer, a protein of the RNase III family of endonucleases. The resulting fragments are components of the RNA-induced silencing complex (RISC), directing it to the cognate target mRNA. RISC cleaves the target mRNA thereby reducing the expression of the encoded protein^1,2,3. RNAi has become a powerful and widely used experimental method for loss of gene function studies in mammalian cells utilizing small interfering RNAs.Currently two main methods are available for the production of small interfering RNAs. One method involves chemical synthesis, whereas an alternative method employs endonucleolytic cleavage of target specific long double-stranded RNAs by RNase III in vitro. Thereby, a diverse pool of siRNA-like oligonucleotides is produced which is also known as endoribonuclease-prepared siRNA or esiRNA. A comparison of efficacy of chemically derived siRNAs and esiRNAs shows that both triggers are potent in target-gene silencing. Differences can, however, be seen when comparing specificity. Many single chemically synthesized siRNAs produce prominent off-target effects, whereas the complex mixture inherent in esiRNAs leads to a more specific knockdown¹⁰.In this study, we present the design of genome-scale MISSION esiRNA libraries and its utilization for RNAi screening exemplified by a DNA-content screen for the identification of genes involved in cell cycle progression. We show how to optimize the transfection protocol and the assay for screening in high throughput. We also demonstrate how large data-sets can be evaluated statistically and present methods to validate primary hits. Finally, we give potential starting points for further functional characterizations of validated hits.Download video file.^{(167M, mp4)}     

siRNA specificity searching incorporating mismatch tolerance data

Artificially synthesized short interfering RNAs (siRNAs) are widely used in functional genomics to knock down specific target genes. One ongoing challenge is to guarantee that the siRNA does not elicit off-target effects. Initial reports suggested that siRNAs were highly sequence-specific; however, subsequent data indicates that this is not necessarily the case. It is still uncertain what level of similarity and other rules are required for an off-target effect to be observed, and scoring schemes have not been developed to look beyond simple measures such as the number of mismatches or the number of consecutive matching bases present. We created design rules for predicting the likelihood of a non-specific effect and present a web server that allows the user to check the specificity of a given siRNA in a flexible manner using a combination of methods. The server finds potential off-target matches in the corresponding RefSeq database and ranks them according to a scoring system based on experimental studies of specificity. AVAILABILITY: The server is available at http://informatics-eskitis.griffith.edu.au/SpecificityServer.     

siRNA脱靶效应研究进展

RNA干扰过程中,siRNA和mRNA特异结合能够使得靶基因沉默。但研究证实,siRNA可能与非靶基因结合而导致非靶基因沉默,这种现象称为siRNA脱靶效应。多种真核生物中的RNA干扰实验证实了脱靶效应的存在。对脱靶机制的研究发现脱靶可能与模体匹配、结构和长dsRNA等有关,很多新方法被提出来预测脱靶概率和检测脱靶基因。通过利用siRNApool、化学修饰和生物信息学方法能够尽可能地降低脱靶效应,提高RNAi实验的质量。对脱靶效应方面的研究进行了总结论述。     

Fabrication of cell-based arrays using micropatterned alkanethiol monolayers for the parallel silencing of specific genes by small interfering RNA

The emergence of small interfering RNA (siRNA) opened a new opportunity to study gene functions in a genome. However, large-scale loss-of-function analyses further require cell-based high-throughput methods that allow simultaneous silencing of the huge number of genes by siRNA. In this study, we aim at fabricating the cell-based siRNA arrays that facilitate parallel introduction of multiple siRNAs into cultured mammalian cells. The siRNA arrays were prepared using surface chemical processes including the micropatterning of a self-assembled monolayer and the layer-by-layer assembly of siRNA and cationic lipid. We examined the feasibility of the siRNA array for the sequence-specific gene silencing in an array format. Furthermore, the effects of siRNA loading and culture period after transfection were studied to optimize cell-based assays on the siRNA arrays. The results obtained in this study demonstrated that our method provides the siRNA arrays with spatial specificity in gene silencing, which will serve to obtain a quantitative data set from the cell-based screens on siRNA arrays.