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.     

Identification of sequence features that predict competition potency of siRNAs

Small interfering RNAs (siRNAs) specifically knock-down target mRNAs via RNA interference (RNAi) mechanism. During this process, introduction of excess amount of exogenous siRNAs could lead to the saturation of cellular RNAi machinery. One consequence of RNAi machinery saturation is the competition between two simultaneously introduced siRNAs, during which one siRNA loses gene silencing activity. Although competition phenomena have been well characterized, the molecular and sequence features of siRNAs that specify the competition potency remain poorly understood. Here, for the first time, we performed a large-scale siRNA competition potency analysis by measuring the competition potency of 56 different siRNAs and ranking them based on their competition potency. We have also established an algorithm to predict the competition potency of siRNAs based upon the conserved sequence features of strong and weak competitor siRNAs. The present study supports our hypothesis that the competition potency of siRNAs is specified by the 5′-half antisense sequence and provides a useful guideline to design siRNAs with minimal RNAi machinery saturation.     

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。     

Competition potency of siRNA is specified by the 5'-half sequence of the guide strand

Small-interfering RNAs (siRNAs) execute specific cellular gene silencing by exploiting the endogenous RNA interference (RNAi) pathway. Therefore, excess amounts of siRNAs can saturate cellular RNAi machineries. Indeed, some siRNAs saturate the RNA-induced silencing complex (RISC) and competitively inhibit silencing by other siRNAs. However, the molecular feature of siRNAs that specifies competition potency has been undetermined. While previous reports suggested a correlation between the competition potency and silencing efficiency of siRNAs, we found that the silencing efficiency was insufficient to explain the competition potency. Instead, we show that the nucleotide sequence of the 5′-half of the guide strand determines the competition potency of an siRNA. Our finding provides important information for understanding the mechanistic basis of competition in combinatorial RNAi treatment.     

RNAi‐mediated endogene silencing in strawberry fruit: detection of primary and secondary siRNAs by deep sequencing

RNA interference (RNAi) has been exploited as a reverse genetic tool for functional genomics in the nonmodel species strawberry (Fragaria × ananassa) since 2006. Here, we analysed for the first time different but overlapping nucleotide sections (>200 nt) of two endogenous genes, FaCHS (chalcone synthase) and FaOMT (O‐methyltransferase), as inducer sequences and a transitive vector system to compare their gene silencing efficiencies. In total, ten vectors were assembled each containing the nucleotide sequence of one fragment in sense and corresponding antisense orientation separated by an intron (inverted hairpin construct, ihp). All sequence fragments along the full lengths of both target genes resulted in a significant down‐regulation of the respective gene expression and related metabolite levels. Quantitative PCR data and successful application of a transitive vector system coinciding with a phenotypic change suggested propagation of the silencing signal. The spreading of the signal in strawberry fruit in the 3′ direction was shown for the first time by the detection of secondary small interfering RNAs (siRNAs) outside of the primary targets by deep sequencing. Down‐regulation of endogenes by the transitive method was less effective than silencing by ihp constructs probably because the numbers of primary siRNAs exceeded the quantity of secondary siRNAs by three orders of magnitude. Besides, we observed consistent hotspots of primary and secondary siRNA formation along the target sequence which fall within a distance of less than 200 nt. Thus, ihp vectors seem to be superior over the transitive vector system for functional genomics in strawberry fruit.     

Dual-target gene silencing by using long,synthetic siRNA duplexes without triggering antiviral responses

Chemically synthesized small interfering RNAs (siRNAs) can specifically knock-down expression of target genes via RNA interference (RNAi) pathway. To date, the length of synthetic siRNA duplex has been strictly maintained less than 30 bp, because an early study suggested that double-stranded RNAs (dsRNAs) longer than 30 bp could not trigger specific gene silencing due to the induction of nonspecific antiviral interferon responses. Contrary to the current belief, here we show that synthetic dsRNA as long as 38 bp can result in specific target gene silencing without nonspecific antiviral responses. Using this longer duplex structure, we have generated dsRNAs, which can simultaneously knock-down expression of two target genes (termed as dual-target siRNAs or dsiRNAs). Our results thus demonstrate the structural flexibility of gene silencing siRNAs, and provide a starting point to construct multifunctional RNA structures. The dsiRNAs could be utilized to develop a novel therapeutic gene silencing strategy against diseases with multiple gene alternations such as viral infection and cancer.     

Selective silencing by RNAi of a dominant allele that causes amyotrophic lateral sclerosis

RNA interference (RNAi) can achieve sequence-selective inactivation of gene expression in a wide variety of eukaryotes by introducing double-stranded RNA corresponding to the target gene. Here we explore the potential of RNAi as a therapy for amyotrophic lateral sclerosis (ALS) caused by mutations in the Cu, Zn superoxide dismutase (SOD1) gene. Although the mutant SOD1 is toxic, the wild-type SOD1 performs important functions. Therefore, the ideal therapeutic strategy should be to selectively inhibit the mutant, but not the wild-type SOD1 expression. Because most SOD1 mutations are single nucleotide changes, to selectively silence the mutant requires single-nucleotide specificity. By coupling rational design of small interfering RNAs (siRNAs) with their validation in RNAi reactions in vitro and in vivo, we have identified siRNA sequences with this specificity. A similarly designed sequence, when expressed as small hairpin RNA (shRNA) under the control of an RNA polymerase III (pol III) promoter, retains the single-nucleotide specificity. Thus, RNAi is a promising therapy for ALS and other disorders caused by dominant, gain-of-function gene mutations.     

RNA Interference and Development

RNA interference consists in specific mRNA degradation in response to introduction of a double-stranded RNA, homologous in nucleotide sequence. RNA interference was found in eukaryotes and is used in genomics as a powerful method to determine the functions of genes with known nucleotide sequences. RNA interference is considered as a tool of protection against viruses and harmful consequences of mobile elements' transposals. The involvement of the components of RNA interference is considered in spermatogenesis of Drosophila melanogaster and regulation of the expression of genes in Caenorhabditis elegans responsible for temporal patterns of development. The role of RNA interference in stem cell formation and functioning is also considered.     

In vitro analysis of RNA interference in Drosophila melanogaster

Double-stranded RNA (dsRNA) triggers the destruction of mRNA sharing sequence with the dsRNA, a phenomenon termed RNA interference (RNAi). The dsRNA is converted by endonucleolytic cleavage into 21- to 23-nt small interfering RNAs (siRNAs), which direct a multiprotein complex, the RNA-induced silencing complex to cleave RNA complementary to the siRNA. RNAi can be recapitulated in vitro in lysates of syncytial blastoderm Drosophila embryos. These lysates reproduce all of the known steps in the RNAi pathway in flies and mammals. Here we explain how to prepare and use Drosophila embryo lysates to dissect the mechanism of RNAi.     

siRNA抑制丙型肝炎病毒IRES介导的基因表达

以HCVIRES为靶位 ,应用T7RNA多聚酶体外转录合成了 5条小干扰RNA(siRNA) .脂质体转染法将其导入HCVIRES介导萤光素酶表达的转基因细胞 (HepG2 .970 6 )中 ,通过测定萤光素酶的量 ,评价T7siRNA对HCV介导基因表达的抑制作用 .结果表明 ,所合成的 5条T7siRNAs ,均能特异性地抑制萤光素酶基因的表达 ,抑制率分别为 94 31%、80 0 1%、78 0 1%、80 33%、85 6 4% ,其中以靶向HCVIRES第二茎环结构的T7siRNA1抑制率最高 ,且对HCV基因的抑制作用有剂量依赖性 ,随T7siRNA1量的增加 ,抑制率逐渐增强 .siRNA抑制HCV基因的作用具有良好的特异性 ,改变其中 1个核苷酸即无显著抑制作用 .