Enhancement of Allele Discrimination by Introduction of Nucleotide Mismatches into siRNA in Allele-Specific Gene Silencing by RNAi
Enhancement of Allele Discrimination by Introduction of Nucleotide Mismatches into siRNA in Allele-Specific Gene Silencing by RNAi
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DOI：
10.1371/journal.pone.0002248
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发表时间：
2008-05-21
期刊：
影响因子：
3.7
通讯作者：
Hohjoh, Hirohiko
中科院分区：
文献类型：
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作者：
Ohnishi, Yusuke;Tamura, Yoshiko;Hohjoh, Hirohiko
通过 RNA 干扰 (RNAi) 进行的等位基因特异性基因沉默在治疗上非常有用，可特异性抑制疾病相关等位基因的表达，而不抑制相应野生型等位基因的表达。为了实现这种等位基因特异性 RNAi (ASP-RNAi)，赋予 ASP-RNAi 的小干扰 RNA (siRNA) 双链体的设计和评估至关重要；然而，这也很困难。在之前的研究中，我们开发了一种测定系统，用于评估具有编码 Photinus 和 Renilla 荧光素酶基因的突变型和野生型报告基因等位基因的 ASP-RNAi。根据使用该系统的实验，我们意识到增强突变体和相应野生型等位基因之间的等位基因区分是必要且重要的。在这里，我们描述了通过将碱基替换引入 siRNA 序列（其中原始变异存在于中心位置），针对携带单核苷酸变异的突变等位基因的 ASP-RNAi 的改进。使用该评估系统检查了针对人类朊病毒蛋白（PRNP）基因突变等位基因的人为错配的 siRNA 或短发夹 RNA（shRNA），这些突变等位基因似乎与朊病毒疾病的易感性相关。数据表明，在 siRNA 和 shRNA 中引入一碱基错配能够增强突变型和野生型等位基因之间的区分。有趣的是，引入的错配使 ASP-RNAi 显着改善，似乎主要存在于引导 siRNA 元件中，对应于 microRNA 的“种子区域”。由于 microRNA 的“种子区域”在与靶 RNA 关联中的重要作用，可以想象，破坏相应种子区域中的碱基配对相互作用，以及向导 siRNA 元件的中心位置（涉及靶 RNA 的切割）可能会影响等位基因的辨别。此外，我们还表明，有义链 siRNA 元件 3' 端的核苷酸错配可能会增加反义链（指导）siRNA 组装成 RNA 诱导的沉默复合物 (RISC)，在惰性 siRNA 双链体的情况下，可能会增强 ASP-RNAi。因此，这里提供的数据表明，通过碱基替换对 siRNA 双链体功能部分进行结构修饰可以极大地影响等位基因区分和基因沉默，从而有助于增强 ASP-RNAi。
Allele-specific gene silencing by RNA interference (RNAi) is therapeutically useful for specifically inhibiting the expression of disease-associated alleles without suppressing the expression of corresponding wild-type alleles. To realize such allele-specific RNAi (ASP-RNAi), the design and assessment of small interfering RNA (siRNA) duplexes conferring ASP-RNAi is vital; however, it is also difficult. In a previous study, we developed an assay system to assess ASP-RNAi with mutant and wildtype reporter alleles encoding the Photinus and Renilla luciferase genes. In line with experiments using the system, we realized that it is necessary and important to enhance allele discrimination between mutant and corresponding wild-type alleles. Here, we describe the improvement of ASP-RNAi against mutant alleles carrying single nucleotide variations by introducing base substitutions into siRNA sequences, where original variations are present in the central position. Artificially mismatched siRNAs or short-hairpin RNAs (shRNAs) against mutant alleles of the human Prion Protein (PRNP) gene, which appear to be associated with susceptibility to prion diseases, were examined using this assessment system. The data indicates that introduction of a one-base mismatch into the siRNAs and shRNAs was able to enhance discrimination between the mutant and wild-type alleles. Interestingly, the introduced mismatches that conferred marked improvement in ASP-RNAi, appeared to be largely present in the guide siRNA elements, corresponding to the 'seed region' of microRNAs. Due to the essential role of the 'seed region' of microRNAs in their association with target RNAs, it is conceivable that disruption of the base-pairing interactions in the corresponding seed region, as well as the central position (involved in cleavage of target RNAs), of guide siRNA elements could influence allele discrimination. In addition, we also suggest that nucleotide mismatches at the 3'-ends of sense-strand siRNA elements, which possibly increase the assembly of antisensestrand (guide) siRNAs into RNA-induced silencing complexes (RISCs), may enhance ASP-RNAi in the case of inert siRNA duplexes. Therefore, the data presented here suggest that structural modification of functional portions of an siRNA duplex by base substitution could greatly influence allele discrimination and gene silencing, thereby contributing to enhancement of ASP-RNAi.