Effects on RNAi of the tight structure, sequence and position of the targeted region

RNA interference (RNAi) is a gene-silencing phenomenon that involves the double-stranded RNA-mediated cleavage of mRNA, and small interfering RNAs (siRNAs) can cause RNAi in mammalian cells. There have been many attempts to clarify the mechanism of RNAi, but information about the relationship between the sequence and structure, in particular, a tight structure, of the target RNA and the activities of siRNAs are limited. In the present study, we examined this relationship by introducing the TAR element, which adopts a very stable secondary structure, at different positions within target RNAs. Our results suggested that the activities of siRNAs were affected by the tight stem–loop structure of TAR. In contrast, the position of the target within the mRNA, the binding of the Tat protein to the TAR, and the location of the target within a translated or a noncoding region had only marginal effects on RNAi. When the target sequence was placed in two different orientations, only one orientation had a significant effect on the activities of siRNA, demonstrating that the presence of certain nucleotides at some specific positions was favorable for RNAi. Systematic analysis of 47 different sites within 47 plasmids under identical conditions indicated that it is the target sequence itself, rather than its location, that is the major determinant of siRNA activity.     

A systematic analysis of the silencing effects of an active siRNA at all single-nucleotide mismatched target sites

The specificity of small interfering RNA (siRNA)-mediated gene silencing is a critical consideration for the application of RNA interference (RNAi). While the discovery of potential off-target effects by siRNAs is of concern, no systematic analysis has been conducted to explore the specificity of RNAi. Here, we present a study where a functionally validated siRNA (siCD46) was examined for silencing specificity on all possible 57 permutated target sites, each carrying a single-nucleotide mutation that would generate a mismatch when paired with siRNA antisense strand. We found that it was not only the position of the mismatched base pair, but also the identity of the nucleotides forming the mismatch that influenced silencing. Surprisingly, mismatches formed between adenine (A) and cytosine (C), in addition to the G:U wobble base pair, were well tolerated and target sites containing such mismatches were silenced almost as efficiently as its fully matched counterpart by siCD46. Northern blots showed that the silencing of fusion genes harboring the mutated target sites involved target mRNA degradation. This study provides direct evidence that the target recognition of siRNA is far more degenerative than previously considered. This finding is instrumental in the understanding of RNAi specificity and may aid the computational prediction of RNA secondary structure.     

Local RNA target structure influences siRNA efficacy: systematic analysis of intentionally designed binding regions

Contradictory reports in the literature have emphasised either the sequence of small interfering RNAs (siRNA) or the structure of their target molecules to be the major determinant of the efficiency of RNA interference (RNAi) approaches. In the present study, we analyse systematically the contributions of these parameters to siRNA activity by using deliberately designed mRNA constructs. The siRNA target sites were included in well-defined structural elements rendering them either highly accessible or completely involved in stable base-pairing. Furthermore, complementary sequence elements and various hairpins with different stem lengths and designs were used as target sites. Only one of the strands of the siRNA duplex was found to be capable of silencing via its respective target site, indicating that thermodynamic characteristics intrinsic to the siRNA strands are a basic determinant of siRNA activity. A significant obstruction of gene silencing by the same siRNA, however, was observed to be caused by structural features of the substrate RNA. Bioinformatic analysis of the mRNA structures suggests a direct correlation between the extent of gene-knockdown and the local free energy in the target region. Our findings indicate that, although a favourable siRNA sequence is a necessary prerequisite for efficient RNAi, complex target structures may limit the applicability even of carefully chosen siRNAs.     

Uncoupling of RNAi from active translation in mammalian cells

Small inhibitory RNAs (siRNAs) are produced from longer RNA duplexes by the RNAse III family member Dicer. The siRNAs function as sequence-specific guides for RNA cleavage or translational inhibition. The precise mechanism by which siRNAs direct the RNA-induced silencing complex (RISC) to find the complementary target mRNA remains a mystery. Some biochemical evidence connects RNAi with translation making attractive the hypothesis that RISC is coupled with the translational apparatus for scanning mRNAs. Such coupling would facilitate rapid alignment of the siRNA antisense with the complementary target sequence. To test this hypothesis we took advantage of a well-characterized translational switch afforded by the ferritin IRE-IRP to analyze RNAi mediated cleavage of a target mRNA in the presence and absence of translation. Our results demonstrate that neither active translation nor unidirectional scanning is required for siRNA mediated target degradation. Our findings demonstrate that nontranslated mRNAs are highly susceptible to RNAi, and blocking scanning from both the 5' and 3' ends of an mRNA does not impede RNAi. Interestingly, RNAi is about threefold more active in the absence of translation.     

A comparison of siRNA efficacy predictors

Short interfering RNA (siRNA) efficacy prediction algorithms aim to increase the probability of selecting target sites that are applicable for gene silencing by RNA interference. Many algorithms have been published recently, and they base their predictions on such different features as duplex stability, sequence characteristics, mRNA secondary structure, and target site uniqueness. We compare the performance of the algorithms on a collection of publicly available siRNAs. First, we show that our regularized genetic programming algorithm GPboost appears to have a higher and more stable performance than other algorithms on the collected datasets. Second, several algorithms gave close to random classification on unseen data, and only GPboost and three other algorithms have a reasonably high and stable performance on all parts of the dataset. Third, the results indicate that the siRNAs' sequence is sufficient input to siRNA efficacy algorithms, and that other features that have been suggested to be important may be indirectly captured by the sequence.     

Similar behaviour of single-strand and double-strand siRNAs suggests they act through a common RNAi pathway

RNA interference (RNAi), mediated by either long double-stranded RNA (dsRNA) or short interfering RNA (siRNA), has become a routine tool for transient knockdown of gene expression in a wide range of organisms. The antisense strand of the siRNA duplex (antisense siRNA) was recently shown to have substantial mRNA depleting activity of its own. Here, targeting human Tissue Factor mRNA in HaCaT cells, we perform a systematic comparison of the activity of antisense siRNA and double-strand siRNA, and find almost identical target position effects, appearance of mRNA cleavage fragments and tolerance for mutational and chemical backbone modifications. These observations, together with the demonstration that excess inactive double-strand siRNA blocks antisense siRNA activity, i.e. shows sequence-independent competition, indicate that the two types of effector molecules share the same RNAi pathway. Interest ingly, both FITC-tagged and 3′-deoxy antisense siRNA display severely limited activity, despite having practically wild-type activity in a siRNA duplex. Finally, we find that maximum depletion of target mRNA expression occurs significantly faster with antisense siRNA than with double-strand siRNA, suggesting that the former enters the RNAi pathway at a later stage than double-strand siRNA, thereby requiring less time to exert its activity.     

Designing highly active siRNAs for therapeutic applications

The discovery of RNA interference (RNAi) generated considerable interest in developing short interfering RNAs (siRNAs) for understanding basic biology and as the active agents in a new variety of therapeutics. Early studies showed that selecting an active siRNA was not as straightforward as simply picking a sequence on the target mRNA and synthesizing the siRNA complementary to that sequence. As interest in applying RNAi has increased, the methods for identifying active siRNA sequences have evolved from focusing on the simplicity of synthesis and purification, to identifying preferred target sequences and secondary structures, to predicting the thermodynamic stability of the siRNA. As more specific details of the RNAi mechanism have been defined, these have been incorporated into more complex siRNA selection algorithms, increasing the reliability of selecting active siRNAs against a single target. Ultimately, design of the best siRNA therapeutics will require design of the siRNA itself, in addition to design of the vehicle and other components necessary for it to function in vivo. In this minireview, we summarize the evolution of siRNA selection techniques with a particular focus on one issue of current importance to the field, how best to identify those siRNA sequences likely to have high activity. Approaches to designing active siRNAs through chemical and structural modifications will also be highlighted. As the understanding of how to control the activity and specificity of siRNAs improves, the potential utility of siRNAs as human therapeutics will concomitantly grow.     

Accurate transcriptome-wide prediction of microRNA targets and small interfering RNA off-targets with MIRZA-G

Small interfering RNA (siRNA)-mediated knock-down is a widely used experimental approach to characterizing gene function. Although siRNAs are designed to guide the cleavage of perfectly complementary mRNA targets, acting similarly to microRNAs (miRNAs), siRNAs down-regulate the expression of hundreds of genes to which they have only partial complementarity. Prediction of these siRNA ‘off-targets’ remains difficult, due to the incomplete understanding of siRNA/miRNA–target interactions. Combining a biophysical model of miRNA–target interaction with structure and sequence features of putative target sites we developed a suite of algorithms, MIRZA-G, for the prediction of miRNA targets and siRNA off-targets on a genome-wide scale. The MIRZA-G variant that uses evolutionary conservation performs better than currently available methods in predicting canonical miRNA target sites and in addition, it predicts non-canonical miRNA target sites with similarly high accuracy. Furthermore, MIRZA-G variants predict siRNA off-target sites with an accuracy unmatched by currently available programs. Thus, MIRZA-G may prove instrumental in the analysis of data resulting from large-scale siRNA screens.     

R2D2 leads the silencing trigger to mRNA's death star

During RNA interference (RNAi), Dicer generates short interfering RNAs (siRNAs), which then guide target mRNA cleavage by the RISC complex. Now, Liu et al. identify R2D2, a Dicer-associated protein that is important for siRNA incorporation into RISC, thus linking the initiation and execution phases of RNAi.     

应用RNAi技术沉默大转录本基因LRP1B

低密度脂蛋白受体相关蛋白1B(Lipoprotein receptor-related protein 1 B, LRP1B)是脂蛋白受体家族庞大受体亚群的成员之一, LRP1B基因编码一个长达16.5 kb的大转录本。由于LRP1B的转录产物过大限制了其功能研究。为研究LRP1B基因与肿瘤转移的相关性, 文章应用RNAi技术特异地封闭了LRP1B基因的表达。在设计siRNA过程中, 根据高效siRNA序列特征和靶序列处的mRNA二级结构特点, 对LRP1B基因多达1 100个的siRNA候选靶序列进行双重评价, 最终在基因转录本的不同位置选取了6个siRNA靶位点。针对这些靶位点, 文章构建了6个表达shRNA的pSilencer4.1重组体, 稳定转染HEK 293细胞并采用半定量RT-PCR检测各表达载体的沉默效果。结果表明, 所构建的6个shRNA-pSilencer4.1重组体中有5个对LRP1B基因形成了有效沉默(＞50%), 并得到了多个完全封闭LRP1B基因表达的HEK 293细胞单克隆。     

Analysis of acyclic nucleoside modifications in siRNAs finds sensitivity at position 1 that is restored by 5′-terminal phosphorylation both in vitro and in vivo

Small interfering RNAs (siRNAs) are short, double-stranded RNAs that use the endogenous RNAi pathway to mediate gene silencing. Phosphorylation facilitates loading of a siRNA into the Ago2 complex and subsequent cleavage of the target mRNA. In this study, 2′, 3′ seco nucleoside modifications, which contain an acylic ribose ring and are commonly called unlocked nucleic acids (UNAs), were evaluated at all positions along the guide strand of a siRNA targeting apolipoprotein B (ApoB). UNA modifications at positions 1, 2 and 3 were detrimental to siRNA activity. UNAs at positions 1 and 2 prevented phosphorylation by Clp1 kinase, abrogated binding to Ago2, and impaired Ago2-mediated cleavage of the mRNA target. The addition of a 5′-terminal phosphate to siRNA containing a position 1 UNA restored ApoB mRNA silencing, Ago2 binding, and Ago2 mediated cleavage activity. Position 1 UNA modified siRNA containing a 5′-terminal phosphate exhibited a partial restoration of siRNA silencing activity in vivo. These data reveal the complexity of interpreting the effects of chemical modification on siRNA activity, and exemplify the importance of using multiple biochemical, cell-based and in vivo assays to rationally design chemically modified siRNA destined for therapeutic use.     

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。     

Rational siRNA design for RNA interference

Short-interfering RNAs suppress gene expression through a highly regulated enzyme-mediated process called RNA interference (RNAi). RNAi involves multiple RNA-protein interactions characterized by four major steps: assembly of siRNA with the RNA-induced silencing complex (RISC), activation of the RISC, target recognition and target cleavage. These interactions may bias strand selection during siRNA-RISC assembly and activation, and contribute to the overall efficiency of RNAi. To identify siRNA-specific features likely to contribute to efficient processing at each step, we performed a systematic analysis of 180 siRNAs targeting the mRNA of two genes. Eight characteristics associated with siRNA functionality were identified: low G/C content, a bias towards low internal stability at the sense strand 3'-terminus, lack of inverted repeats, and sense strand base preferences (positions 3, 10, 13 and 19). Further analyses revealed that application of an algorithm incorporating all eight criteria significantly improves potent siRNA selection. This highlights the utility of rational design for selecting potent siRNAs and facilitating functional gene knockdown studies.     

抗HPV11 E2核酶的计算机设计

 借助计算机软件分析 ,设计出能特异性切割HPV11型 6 4 4ntE2mRNA的核酶 (ribozyme) .遵循Symon′s锤头状核酶结构和GUX剪切位点原则 ,靶序列存在 32个这样的剪切位点 .通过计算机软件分析出核酶的最佳剪切位点 ,并对底物及核酶的二级结构进行预测及进行相应基因生物学功能和基因同源性分析 ,筛选出 2个锤头结构核酶 .针对这两位点设计的核酶分别命名为RZ2 777和RZ32 81.计算机分析显示 ,两核酶与底物切点两翼碱基形成锤头状结构 ,切点所在基因序列具有相对松弛的二级结构 ,位于该基因重要生物功能区内 ,是核酶的理想攻击区域 .通过基因库检索 ,在已知人类基因排除了与上述两核酶切点两翼碱基有基因同源性序列的可能性 .将两核酶用于体外剪切实验取得了良好的实验结果 ,认为借助计算机分析可帮助尽快从多个剪切位点选择出最适核酶     

The impact of target site accessibility on the design of effective siRNAs

Small-interfering RNAs (siRNAs) assemble into RISC, the RNA-induced silencing complex, which cleaves complementary mRNAs. Despite their fluctuating efficacy, siRNAs are widely used to assess gene function. Although this limitation could be ascribed, in part, to variations in the assembly and activation of RISC, downstream events in the RNA interference (RNAi) pathway, such as target site accessibility, have so far not been investigated extensively. In this study we present a comprehensive analysis of target RNA structure effects on RNAi by computing the accessibility of the target site for interaction with the siRNA. Based on our observations, we developed a novel siRNA design tool, RNAxs, by combining known siRNA functionality criteria with target site accessibility. We calibrated our method on two data sets comprising 573 siRNAs for 38 genes, and tested it on an independent set of 360 siRNAs targeting four additional genes. Overall, RNAxs proves to be a robust siRNA selection tool that substantially improves the prediction of highly efficient siRNAs.