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。     

小干扰RNA的研究进展

RNA干扰(RNA interference,RNAi)是近年发展起来的一种新技术。RNAi是指通过外源性或内源性的双链RNA在体内诱导靶基因mRNA产生特异性降解,进而引起不同水平的基因沉默,其效应分子主要是小干扰RNA(siRNA)。siRNA是生物界普遍存在的一种抵御外来基因和病毒感染的基因调控方式,也是一种重要的研究工具。大量的研究工作致力于设计合理的siRNA片段用于基因功能研究,并将其作为一种治疗方法用于肿瘤、病毒性疾病等基因治疗以及药物靶向研究。因此本文对siRNA的作用机制、设计原则及其在临床应用中的缺点和解决方法进行综述。     

基于BP神经网络的siRNA活性预测

RNA干扰(RNAinterference,RNAi)是由双链RNA(dsRNA)引起的基因沉默现象,它通过降解具有同源序列的mRNA来起作用,特殊设计的siRNA能使靶基因发生特异性沉默,起到确定基因功能或沉默致病基因从而治疗疾病的目的。在RNAi技术的应用中,通常采用的是长度为19bp,正、反义链3'端各有2个不配对碱基的双链RNA(siRNA)。但针对靶基因不同位点设计的siRNA作用效果差别很大。影响siRNA效果的因素是多方面的,这些因素的作用又是非线性的。本文在研究影响siRNA作用效果的各种因素的基础上,对已经公开发表的实验数据进行特征提取,作为BP神经网络的训练数据,并将训练好的BP神经网络用于siRNA活性预测。     

基因枪在RNA干扰中的应用研究进展

RNA干扰（RNA interference,RNAi）是指双链RNA（double-strand RNA,dsRNA）特异性降解同源mRNA,从而引发基因转录后水平沉默的现象,是一种高效、高特异性抑制基因表达的途径。自1998年Fire等发现RNA干扰现象以来,其特异性降解目的基因的优势吸引了众多研究者的目光。本文在简要综述RNAi技术在基因功能研究、抗病毒治疗,肿瘤基因治疗等领域的应用后,重点归纳了基因枪技术在RNAi研究即siRNA导入细胞中的应用,并简单分析其优势与意义。     

小干扰RNA的研究进展

RNA干扰（RNA interference,RNAi）是近年发展起来的一种新技术。RNAi是指通过外源性或内源性的双链RNA在体内诱导靶基因mR_NA产生特异性降解,进而引起不同水平的基因沉默。其效应分子主要是小干扰RNA（siRNA）。siRNA是生物界普遍存在的一种抵御外来基因和病毒感染的基因调控方式,也是一种重要的研究工具。大量的研究工作致力于设计合理的siRNA片段用于基因功能研究,并将其作为一种治疗方法用于肿瘤、病毒性疾病等基因治疗以及药物靶向研究。因此本文对siRNA的作用机制、设计原则及其在临床应用中的缺点和解决方法进行综述。     

干扰RNA的非靶免疫副作用及其siRNA序列设计与化学修饰

siRNA介导的RNA干扰技术已经成为基因功能研究和开展疾病治疗的有用工具.近年发现,siRNA在哺乳动物体内可激活天然免疫系统,诱导干扰素等炎症因子的分泌,并且可非特异性抑制某些非靶基因的表达,有可能极大限制RNA干扰技术的应用.进行高效特异性siRNA的设计和修饰,以保持或者增强siRNA的特异性靶基因沉默作用,又消除siRNA对机体的非靶免疫副作用,成为使siRNA安全有效应用于临床治疗的关键.     

特异小干扰RNA敲除PLK1基因的表达

为研究特异小干扰RNA（siRNA）作用于大肠癌细胞株SW480中PLK1 (Polo-like kinase 1)基因表达的mRNA对该细胞分裂生长的影响,设计了对应于PLK1基因表达mRNA不同位点的10种特异siRNA,经化学合成后,用脂质体转染SW480细胞,实时定量PCR检测PLK1基因的表达,观察不同的siRNA作用强度,并计数细胞了解相应细胞的生长情况,western-blot观察PLK1表达蛋白的变化和流式细胞计数分析细胞周期改变。发现10种siRNA均可敲除PLK1基因表达的20 %以上,其中P1、P4和P9 3组敲除mRNA达80 ％以上,这3种siRNA及其混合物对PLK1基因mRNA的作用具有相应浓度效应,在25 nmol/L时达到最佳作用效果,而且相同浓度的混合物作用效果更好（超过95%）,PLK1表达蛋白质明显降低,细胞周期在G2期受到阻碍。72 h后的各种siRNA浓度下细胞生长变化与PLK1基因的mRNA水平变化相一致。结果表明化学合成的特异siRNA对SW480细胞中PLK1基因表达具有消除作用,混合物作用更强,在细胞水平上抑制了SW480细胞的分裂生长。     

鸡传染性支气管炎病毒的RNA干扰

为探讨短的双链RNA(siRNA)对鸡传染性支气管炎病毒(IBV)增殖的干扰作用,利用软件设计siRNA1280个,75%位于Pol基因内。通过同源比较和保守性分析,筛选到针对Pol、M、N基因的12个siRNA(每个基因3～4个)作为后选目的片段,分别在Vero细胞、9日龄SPF鸡胚上进行基因干扰试验。结果,来自Pol、N靶序列的2个siRNA在Vero细胞上及鸡胚上均对IBV增殖产生明显的干扰作用,并与siRNA剂量有一定相关性,依赖于与mRNA互补的负链siRNA存在。本研究首次证实IBV增殖过程中存在siRNA干扰现象,为利用RNA干扰(RNAi)技术控制IBV提供了新手段。     

RNA干扰分子的制作

小干扰RNA是一种能够在各种生物体和细胞(包括蠕虫、果蝇、植物、哺乳动物)中减弱基因表达的有效工具。在哺乳动物中转染的siRNA能够抑制特殊基因的表达,这已经证明是探索基因功能、基因敲除、抗病毒研究、基因治疗的有效方法。简单、有效、特异性地抑制基因的表达具有巨大的科学、商业和医学治疗价值。如何设计和制作siRNA是影响RNA干扰效率的一个很重要的方面。本文就siRNA的设计和制作等方面作扼要的介绍。     

Use of bicistronic vectors in combination with flow cytometry to screen for effective small interfering RNA target sequences

The efficacy and specificity of small interfering RNAs (siRNAs) are largely dependent on the siRNA sequence. Since only empirical strategies are currently available for predicting these parameters, simple and accurate methods for evaluating siRNAs are needed. To simplify such experiments, target genes are often tagged with reporters for easier readout. Here, we used a bicistronic vector expressing a target gene and green fluorescent protein (GFP) to create a system in which the effect of an siRNA sequence was reflected in the GFP expression level. Cells were transduced with the bicistronic vector, expression vectors for siRNA and red fluorescent protein (RFP). Flow cytometric analysis of the transduced cells revealed that siRNAs for the target gene silenced GFP from the bicistronic vector, but did not silence GFP transcribed without the target gene sequence. In addition, the mean fluorescence intensities of GFP on RFP-expressing cells correlated well with the target gene mRNA and protein levels. These results suggest that this flow cytometry-based method enables us to quantitatively evaluate the efficacy and specificity of siRNAs. Because of its simplicity and effectiveness, this method will facilitate the screening of effective siRNA target sequences, even in high-throughput applications.     

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.     

Efficient and targeted delivery of siRNA in vivo

RNA interference (RNAi) has been regarded as a revolutionary tool for manipulating target biological processes as well as an emerging and promising therapeutic strategy. In contrast to the tangible and obvious effectiveness of RNAi in vitro, silencing target gene expression in vivo using small interfering RNA (siRNA) has been a very challenging task due to multiscale barriers, including rapid excretion, low stability in blood serum, nonspecific accumulation in tissues, poor cellular uptake and inefficient intracellular release. This minireview introduces major challenges in achieving efficient siRNA delivery in vivo and discusses recent advances in overcoming them using chemically modified siRNA, viral siRNA vectors and nonviral siRNA carriers. Enhanced specificity and efficiency of RNAi in vivo via selective accumulations in desired tissues, specific binding to target cells and facilitated intracellular trafficking are also commonly attempted utilizing targeting moieties, cell-penetrating peptides, fusogenic peptides and stimuli-responsive polymers. Overall, the crucial roles of the interdisciplinary approaches to optimizing RNAi in vivo, by efficiently and specifically delivering siRNA to target tissues and cells, are highlighted.     

Combination of active and inactive siRNA targeting the mitotic kinesin Eg5 impairs silencing efficiency in several cancer cell lines

Gene silencing by RNA interference (RNAi) has proven to be a powerful tool for investigating gene function in mammalian cells. Combination of several short interfering RNA (siRNA) targeting the same gene is commonly used to improve RNA interference. However, in contrary to the well-described mechanism of RNAi, efficiency of single siRNA compared to pool remains poorly documented. We addressed this issue using several active and inactive siRNA targeting Eg5, a kinesin-related motor involved in mitotic spindle assembly. These siRNA, used alone or in combination, were tested for their silencing efficiency in several cancer cell lines. Here we show that presence of inactive Eg5 siRNA in a pool dramatically decreases knockdown efficacy in a cell line- and dose-dependent manner. Lack of inhibition by unrelated siRNA suggests that a competition may occur during siRNA incorporation into RNA-induced silencing complexes (RISCs) along with the target mRNA. Altogether, our results, which need to be confirmed with additional inactive siRNA, indicate that combination of siRNA may not increase but instead decrease silencing efficiency.     

不同长度茎部shRNA表达载体构建与鉴定

本研究针对同一目的基因设计构建不同茎部长度的shRNA表达载体,并对其在细胞及胚胎水平的干扰效应做一比较。以绿色荧光蛋白基因为沉默效应的靶基因,设计茎部长度分别为21bp、27bp、29bp的干扰片段,退火后连入带有H6启动子的真核表达载体psiSTRIKE中(分别命名为EGFP-21siRNA、EGFP-27siRNA和EGFP-29siRNA),将构建成功的载体以脂质体法转染小鼠胚胎成纤维细胞,利用荧光定量PCR对其荧光表达进行精确定量。不同茎部长度的shRNA载体均使绿色荧光蛋白基因表达降低,茎部为29bp时比21bp、27bp表现出更明显的沉默效应。细胞水平沉默效应的初步验证,为筛选适合小鼠个体水平的最佳发夹结构奠定了基础。     

Analysis of siRNA specificity on targets with double-nucleotide mismatches

Although RNA interference as a tool for gene knockdown is a great promise for future applications, the specificity of small interfering RNA (siRNA)-mediated gene silencing needs to be thoroughly investigated. Most research regarding siRNA specificity has involved analysis of affected off-target genes instead of exploring the specificity of the siRNA itself. In this study we have developed an efficient method for generating a siRNA target library by combining a siRNA target validation vector with a nucleotide oligomix. We have used this library to perform an analysis of the silencing effects of a functional siRNA towards its target site with double-nucleotide mismatches. The results indicated that not only the positions of the mismatched base pair have an impact on silencing efficiency but also the identity of the mismatched nucleotide. Our data strengthen earlier observations of widespread siRNA off-target effects and shows that ~35% of the double-mutated target sites still causes knockdown efficiency of >50%. We also provide evidence that there may be substantial differences in knockdown efficiency depending on whether the mutations are positioned within the siRNA itself or in the corresponding target site.