Common seed analysis to identify off-target effects in siRNA screens

Genome-scale small interfering RNA (siRNA) screens have become an increasingly popular approach to new target identification and pathway elucidation. However, the large data sets generated from siRNA screens have demonstrated high false-positive rates and the requirement for extensive experimental triage to distinguish true hits. A number of groups have independently reported the presence of siRNAs with identical seed sequences among their top screening hits. Based on these observations, we have developed a comprehensive technique for detecting and visualizing seed-based off-target effects in siRNA screening data. This is accomplished by analyzing the behavior of siRNAs that share identical seed sequences, which we refer to as common seed analysis (CSA). By applying these techniques to primary screening data of the Wnt pathway, we identify 158 distinct seed sequences that have a statistically significant effect on the assay. The promiscuous seed sequences identified in this manner can then be discounted in the analysis of follow-up experiments using single siRNAs. The ability to detect off-target effects when sufficient numbers of siRNAs share a common seed has significant implications for the design of siRNA screening experiments, data analysis, hit selection, and library design.     

High-throughput RNAi screening in vitro: from cell lines to primary cells

Small interfering RNAs (siRNAs) are being used to induce sequence-specific gene silencing in cultured cells to study mammalian gene function. Libraries of siRNAs targeting entire human gene classes can be used to identify genes with specific cellular functions. Here we describe high-throughput siRNA delivery methods to facilitate siRNA library screening experiments with both immortalized and primary cells. We adapted chemical reverse transfection for immortalized adherent cell lines in a 96-well format. The method is fast, robust, and exceptionally effective for many cell types. For primary cells and immortalized cells that are recalcitrant to lipofection-based methods, we developed electropermeabilization (electroporation) conditions that facilitate siRNA delivery to a broad range of cell types, including primary human T-cells, hMSC, NHA, NDHF-Neo, HUVEC, DI TNC1, RPTEC, PC12, and K562 cells. To enable high-throughput electropermeabilization of primary cells, we developed a novel 96-well electroporation device that provides highly efficient and reproducible delivery of siRNAs. The combination of high-throughput chemical reverse transfection and electroporation makes it possible to deliver libraries of siRNAs to virtually any cell type, enabling gene function analysis and discovery on a genome scale.     

Effective small interfering RNAs and phosphorothioate antisense DNAs have different preferences for target sites in the luciferase mRNAs

Antisense DNA target sites can be selected by the accessibility of the mRNA target. It remains unknown whether a mRNA site that is accessible to an antisense DNA is also a good candidate target site for a siRNA. Here, we reported a parallel analysis of 12 pairs of antisense DNAs and siRNA duplexes for their potency to inhibit reporter luciferase activity in mammalian cells, both of the antisense DNA and siRNA agents in a pair being directed to same site in the mRNA. Five siRNAs and two antisense DNAs turned out to be effective, but the sites targeted by those effective siRNAs and antisense DNAs did not overlap. Our results indicated that effective antisense DNAs and siRNAs have different preferences for target sites in the mRNA.     

Multiplexing siRNAs to compress RNAi-based screen size in human cells

Here we describe a novel strategy using multiplexes of synthetic small interfering RNAs (siRNAs) corresponding to multiple gene targets in order to compress RNA interference (RNAi) screen size. Before investigating the practical use of this strategy, we first characterized the gene-specific RNAi induced by a large subset (258 siRNAs, 129 genes) of the entire siRNA library used in this study (~800 siRNAs, ~400 genes). We next demonstrated that multiplexed siRNAs could silence at least six genes to the same degree as when the genes were targeted individually. The entire library was then used in a screen in which randomly multiplexed siRNAs were assayed for their affect on cell viability. Using this strategy, several gene targets that influenced the viability of a breast cancer cell line were identified. This study suggests that the screening of randomly multiplexed siRNAs may provide an important avenue towards the identification of candidate gene targets for downstream functional analyses and may also be useful for the rapid identification of positive controls for use in novel assay systems. This approach is likely to be especially applicable where assay costs or platform limitations are prohibitive.     

Identification of the suppressive factors for human immunodeficiency virus type-1 replication using the siRNA mini-library directed against host cellular genes

We performed the screening to find the novel host factors affecting human immunodeficiency virus type-1 (HIV-1) replication using the siRNA mini-library consisted with 257 siRNAs directed against cellular genes. J111 cells, a human acute monocytic leukemia cell line, were transfected with individual siRNA, followed by either infected or transfected with the HIV-1 molecular clone with luciferase reporter gene in 96-well plate format. The results showed that six siRNAs significantly enhanced the HIV-1 replication in J111 cells, indicating that the target cellular genes of those siRNAs may negatively regulate HIV-1 replication in normal cell culture condition. We also discuss the possible mechanisms by which those cellular proteins regulate viral replication.     

RNA interference by mixtures of siRNAs prepared using custom oligonucleotide arrays

RNA interference (RNAi) is a process in which double-strand RNA (dsRNA) directs the specific degradation of a corresponding target mRNA. The mediators of this process are small dsRNAs, of ~21 bp in length, called small interfering RNAs (siRNAs). siRNAs, which can be prepared in vitro in a number of ways and then transfected into cells, can direct the degradation of corresponding mRNAs inside these cells. Hence, siRNAs represent a powerful tool for studying gene functions, as well as having the potential of being highly specific pharmaceutical agents. Some limitations in using this technology exist because the preparation of siRNA in vitro and screening for siRNAs efficient in RNAi can be expensive and time-consuming processes. Here, we demonstrate that custom oligonucleotide arrays can be efficiently used for the preparation of defined mixtures of siRNAs for the silencing of exogenous and endogenous genes. The method is fast, inexpensive, does not require siRNA optimization and has a number of advantages over methods utilizing enzymatic preparation of siRNAs by digestion of longer dsRNAs, as well as methods based on chemical synthesis of individual siRNAs or their DNA templates.     

Identification of druggable targets for radiation mitigation using a small interfering RNA screening assay

Currently, there is a serious absence of pharmaceutically attractive small molecules that mitigate the lethal effects of an accidental or intentional public exposure to toxic doses of ionizing radiation. Moreover, cellular systems that emulate the radiobiologically relevant cell populations and that are suitable for high-throughput screening have not been established. Therefore, we examined two human pluripotent embryonal carcinoma cell lines for use in an unbiased phenotypic small interfering RNA (siRNA) assay to identify proteins with the potential of being drug targets for the protection of human cell populations against clinically relevant ionizing radiation doses that cause acute radiation syndrome. Of the two human cell lines tested, NCCIT cells had optimal growth characteristics in a 384 well format, exhibited radiation sensitivity (D(0) = 1.3 ± 0.1 Gy and ? = 2.0 ± 0.6) comparable to the radiosensitivity of stem cell populations associated with human death within 30 days after total-body irradiation. Moreover, they internalized siRNA after 4 Gy irradiation enabling siRNA library screening. Therefore, we used the human NCCIT cell line for the radiation mitigation study with a siRNA library that silenced 5,520 genes known or hypothesized to be potential therapeutic targets. Exploiting computational methodologies, we identified 113 siRNAs with potential radiomitigative properties, which were further refined to 29 siRNAs with phosphoinositide-3-kinase regulatory subunit 1 (p85α) being among the highest confidence candidate gene products. Colony formation assays revealed radiation mitigation when the phosphoinositide-3-kinase inhibitor LY294002 was given after irradiation of 32D cl 3 cells (D(0) = 1.3 ± 0.1 Gy and ? = 2.3 ± 0.3 for the vehicle control treated cells compared to D(0) = 1.2 ± 0.1 Gy and ? = 6.0 ± 0.8 for the LY294002 treated cells, P = 0.0004). LY294002 and two other PI3K inhibitors, PI 828 and GSK 1059615, also mitigated radiation-induced apoptosis in NCCIT cells. Treatment of mice with a single intraperitoneal LY294002 dose of 30 mg/kg at 10 min, 4, or 24 h after LD(50/30) whole-body dose of irradiation (9.25 Gy) enhanced survival. This study documents that an unbiased siRNA assay can identify new genes, signaling pathways, and chemotypes as radiation mitigators and implicate the PI3K pathway in the human radiation response.     

An efficient and fully automated high-throughput transfection method for genome-scale siRNA screens

RNA interference (RNAi), combined with the availability of genome sequences, provides an unprecedented opportunity for the massive and parallel investigations of gene function. Small interfering RNA (siRNA) represents a popular and quick approach of RNAi for in vitro loss-of-function genetic screens. Efficient transfection of siRNA is critical for unambiguous interpretation of screen results and thus overall success of any siRNA screen. A high-throughput, lipid-based transfection method for siRNA was developed that can process eighty 384-well microplates in triplicate (for a total of 30,720 unique transfections) in 8 h. Transfection throughput was limited only by the speed of robotics, whereas the cost of screening was reduced. As a proof of principle, a genome-scale screen with a library of 22,108 siRNAs was performed to identify the genes sensitizing cells to mitomycin C at concentrations of 0, 20, and 60 nM. Transfection efficiency, performances of control siRNAs, and other quality metrics were monitored and demonstrated that the new, optimized transfection protocol produced high-quality results throughout the screen.     

Local RNA target structure influences siRNA efficacy: a systematic global analysis

The efficiency with which small interfering RNAs (siRNAs) down-regulate specific gene expression in living cells is variable and a number of sequence-governed, biochemical parameters of the siRNA duplex have been proposed for the design of an efficient siRNA. Some of these parameters have been clearly identified to influence the assembly of the RNA-induced silencing complex (RISC), or to favour the sequence preferences of the RISC endonuclease. For other parameters, it is difficult to ascertain whether the influence is a determinant of the siRNA per se, or a determinant of the target RNA, especially its local structural characteristics. In order to gain an insight into the effects of local target structure on the biological activity of siRNA, we have used large sets of siRNAs directed against local targets of the mRNAs of ICAM-1 and survivin. Target structures were classified as accessible or inaccessible using an original, iterative computational approach and by experimental RNase H mapping. The effectiveness of siRNA was characterized by measuring the IC50 values in cell culture and the maximal extent of target suppression. Mean IC50 values were tenfold lower for accessible local target sites, with respect to inaccessible ones. Mean maximal target suppression was improved. These data illustrate that local target structure does, indeed, influence the activity of siRNA. We suggest that local target screening can significantly improve the hit rate in the design of biologically active siRNAs.     

Generation of shrnas from randomized oligonucleotides

Suppression of gene expression by small interfering RNA (siRNA) has proved to be a gene-specific and cost effective alternative to other gene suppression technologies. Short hairpin RNAs (shRNAs) generated from the vector-based expression are believed to be processed into functional siRNAs in vivo, leading to gene silencing. Since an shRNA library carries a large pool of potential siRNAs, such a library makes it possible to knock down gene expression at the genome wide scale. Although much of research has been focused on generating shRNA libraries from either individually made gene specific sequences or cDNA libraries, there is no report on constructing randomized shRNA libraries, which could provide a good alternative to these existing libraries. We have developed a method of constructing shRNAs from randomized oligonucleotides. Through this method, one can generate a partially or fully randomized shRNA library for various functional analyses. We validated this procedure by constructing a p53-specific shRNA. Western blot revealed that the p53-shRNA successfully suppressed expression of the endogenous p53 in MCF-7 cells. We then made a partially randomized shRNA library. Sequencing of 15 randomly picked cloned confirmed the randomness of the library. Therefore, the library can be used for various functional assays, such as target validation when a suitable screening or selection method is available.     

A new method with flexible and balanced control of false negatives and false positives for hit selection in RNA interference high-throughput screening assays

The z-score method and its variants for testing mean difference are commonly used for hit selection in high-throughput screening (HTS) assays. Strictly standardized mean difference (SSMD) offers a way to measure and classify the short interfering RNA (siRNA) effects. In this article, based on SSMD, the authors propose a new testing method for hit selection in RNA interference (RNAi) HTS assays. This SSMD-based method allows the differentiation between siRNAs with large and small effects on the assay output and maintains flexible and balanced control of both the false-negative rate, in which the siRNAs with strong effects are not selected as hits, and the restricted false-positive rate, in which the siRNAs with weak or no effects are selected as hits. This method directly addresses the size of siRNA effects represented by the strength of difference between an siRNA and a negative reference, whereas the classic z-score method and t-test of testing no mean difference address whether the mean of an siRNA is exactly the same as the mean of a negative reference. This method can readily control the false-negative rate, whereas it is nontrivial for the classic z-score method and t-test to control the false-negative rate. Therefore, theoretically, the SSMD-based method offers better control of the sizes of siRNA effects and the associated false-positive and false-negative rates than the commonly used z-score method and t-test for hit selection in HTS assays. The SSMD-based method should generally be applicable to any assay in which the end point is a difference in signal compared to a reference sample, including those for RNAi, receptor, enzyme, and cellular function.     

Identification of new genes involved in human adipogenesis and fat storage

Since the worldwide increase in obesity represents a growing challenge for health care systems, new approaches are needed to effectively treat obesity and its associated diseases. One prerequisite for advances in this field is the identification of genes involved in adipogenesis and/or lipid storage. To provide a systematic analysis of genes that regulate adipose tissue biology and to establish a target-oriented compound screening, we performed a high throughput siRNA screen with primary (pre)adipocytes, using a druggable siRNA library targeting 7,784 human genes. The primary screen showed that 459 genes affected adipogenesis and/or lipid accumulation after knock-down. Out of these hits, 333 could be validated in a secondary screen using independent siRNAs and 110 genes were further regulated on the gene expression level during adipogenesis. Assuming that these genes are involved in neutral lipid storage and/or adipocyte differentiation, we performed InCell-Western analysis for the most striking hits to distinguish between the two phenotypes. Beside well known regulators of adipogenesis and neutral lipid storage (i.e. PPARγ, RXR, Perilipin A) the screening revealed a large number of genes which have not been previously described in the context of fatty tissue biology such as axonemal dyneins. Five out of ten axonemal dyneins were identified in our screen and quantitative RT-PCR-analysis revealed that these genes are expressed in preadipocytes and/or maturing adipocytes. Finally, to show that the genes identified in our screen are per se druggable we performed a proof of principle experiment using an antagonist for HTR2B. The results showed a very similar phenotype compared to knock-down experiments proofing the "druggability". Thus, we identified new adipogenesis-associated genes and those involved in neutral lipid storage. Moreover, by using a druggable siRNA library the screen data provides a very attractive starting point to identify anti-obesity compounds targeting the adipose tissue.     

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.     

Improved and automated prediction of effective siRNA

Short interfering RNAs are used in functional genomics studies to knockdown a single gene in a reversible manner. The results of siRNA experiments are highly dependent on the choice of siRNA sequence. In order to evaluate siRNA design rules, we collected a database of 398 siRNAs of known efficacy from 92 genes. We used this database to evaluate previously proposed rules from smaller datasets, and to find a new set of rules that are optimal for the entire database. We also trained a regression tree with full cross-validation. It was however difficult to obtain the same precision as methods previously tested on small datasets from one or two genes. We show that those methods are overfitting as they work poorly on independent validation datasets from multiple genes. Our new design rules can predict siRNAs with efficacy >/= 50% in 91% of cases, and with efficacy >/=90% in 52% of cases, which is more than a twofold improvement over random selection. Software for designing siRNAs is available online via a web server at or as a standalone version for high-throughput applications.     

VEGF-specific siRNAs modified with 2′-deoxy effectively suppress VEGF expression and inhibit growth of nasopharyngeal carcinoma xenograft in a mouse model

Vascular endothelial growth factor (VEGF) is up-regulated in the vast majority of human tumors. The up-regulation of VEGF not only plays important roles in tumor angiogenesis, but also provides a target for tumor treatment with small interfering RNA (siRNA) that targets VEGF; however, it is unclear whether a quite high up-regulation of VEGF will affect the efficiency of RNA interference strategies targeting VEGF. A high level expression of VEGF was found in CNE cells from a nasopharyngeal car-cinoma cell line. In this study, we investigate whether VEGF-specific siRNAs can effectively suppress VEGF expression in CNE cells, and study the methods for the use of VEGF-specific siRNAs as potential therapeutic agents. CNE cells with high VEGF expression induced by hypoxia were transfected with VEGF-specific siRNAs. The expression of VEGF was effectively suppressed by VEGF-specific siRNAs, measured by ELISA, Western blot analysis and RT-PCR. Furthermore, experiments in nude mice bear-ing nasopharyngeal carcinoma xenograft were initiated 5 d after injection of CNE cells. VEGF-specific siRNAs were modified with 2′-deoxy, then injected into the tumors, and a liposome-mediated siRNA transfection system and ultrasound exposure were used to help delivery of the siRNAs. Tumor growth was reduced significantly after 3 weeks’ treatment. These studies suggest that VEGF-specific siRNAs still can effectively suppress VEGF expression even in tumor cell lines with a relatively high level of VEGF expression, such as CNE, and VEGF-specific siRNAs modified with 2′-deoxy can be used as po-tential agents for tumor therapy.     

高效siRNA设计的研究进展

RNA干扰(RNA interference, RNAi) 是生物界普遍存在的一种抵御外来基因和病毒感染的保守进化机制, 其本质是siRNA与靶向mRNA特异结合、并由RISC介导其降解, 从而阻止mRNA的翻译, 导致基因沉默。因此, RNAi可以作为基因功能研究、基因治疗等的新工具。但是, 随机设计的siRNA之间沉默效应差别很大。如何针对靶基因设计特异、高效的siRNA就成了一个关键的问题。文章对siRNA设计原则的研究进展进行了总结论述。