Designing siRNA that distinguish between genes that differ by a single nucleotide.
Designing siRNA that distinguish between genes that differ by a single nucleotide.
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DOI：
10.1371/journal.pgen.0020140
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发表时间：
2006-09-08
期刊：
影响因子：
4.5
通讯作者：
Zamore PD
中科院分区：
文献类型：
--
作者：
Schwarz DS;Ding H;Kennington L;Moore JT;Schelter J;Burchard J;Linsley PS;Aronin N;Xu Z;Zamore PD
小干扰RNA（SiRNA）是指导RNA干扰（RNAi）的向导，为减少人类细胞中单个基因的表达提供了强大的工具。理想地，显性的功能获得性人类疾病可以使用特异性沉默突变疾病等位基因的siRNA来治疗，同时使野生型等位基因的表达不受干扰。以前的报道表明，siRNA可以设计为具有单核苷酸特异性，但没有提出设计具有单核苷酸区分的siRNA的合理基础。我们系统地鉴定了区分两种疾病基因的野生型和突变等位基因的siRNA：人类Cu，Zn超氧化物歧化酶（SOD 1）基因，其通过获得毒性特性而导致遗传性肌萎缩侧索硬化症的进展，以及亨廷顿蛋白（HTT）基因，当其CAG重复区域扩展超过约35个重复时，其导致亨廷顿病。使用果蝇胚胎裂解物中的无细胞RNAi反应和报告基因测定以及培养的人类细胞中脱靶效应的微阵列分析，我们确定了siRNA内对错配最敏感的位置。我们还表明，嘌呤：嘌呤错配的siRNA具有更大的歧视权力比其他类型的碱基错配。其中G：U摆动或错配位于“种子”序列（负责靶结合的特异性siRNA引导区）中的siRNA显示出比错配位于种子3′端的siRNA更低的选择性水平; siRNA的该区域对于靶切割而不是siRNA结合至关重要。我们的数据表明，siRNA可以被设计成区分许多基因的野生型和突变型等位基因，这些基因的差异仅为一个核苷酸。RNA干扰（RNAi）技术首先在线虫中发现，现已成为研究哺乳动物基因功能的重要工具。由小干扰RNA（siRNA）（21 nt，双链RNA）指导的RNAi靶向互补mRNA以进行破坏。siRNA可以被引入培养的哺乳动物细胞中，或者甚至静脉内给予啮齿动物或灵长类动物，在那里它们抑制靶基因产物的产生。因此，siRNA指导的RNAi作为人类治疗策略具有巨大的潜力。显性遗传疾病，其中基因的突变等位基因在存在第二个正常拷贝的情况下引起疾病，可以用治疗性siRNA治疗，只要siRNA可以被设计成破坏突变的致病mRNA，同时保持正常mRNA完整。在这里，施瓦茨和他的同事描述了一种实验验证的设计这种siRNA的策略。他们的设计策略应该有助于设计靶向显性遗传疾病如肌萎缩侧索硬化症和亨廷顿病的siRNA。
Small interfering RNAs (siRNAs), the guides that direct RNA interference (RNAi), provide a powerful tool to reduce the expression of a single gene in human cells. Ideally, dominant, gain-of-function human diseases could be treated using siRNAs that specifically silence the mutant disease allele, while leaving expression of the wild-type allele unperturbed. Previous reports suggest that siRNAs can be designed with single nucleotide specificity, but no rational basis for the design of siRNAs with single nucleotide discrimination has been proposed. We systematically identified siRNAs that discriminate between the wild-type and mutant alleles of two disease genes: the human Cu, Zn superoxide dismutase (SOD1) gene, which contributes to the progression of hereditary amyotrophic lateral sclerosis through the gain of a toxic property, and the huntingtin (HTT) gene, which causes Huntington disease when its CAG-repeat region expands beyond approximately 35 repeats. Using cell-free RNAi reactions in Drosophila embryo lysate and reporter assays and microarray analysis of off-target effects in cultured human cells, we identified positions within an siRNA that are most sensitive to mismatches. We also show that purine:purine mismatches imbue an siRNA with greater discriminatory power than other types of base mismatches. siRNAs in which either a G:U wobble or a mismatch is located in the “seed” sequence, the specialized siRNA guide region responsible for target binding, displayed lower levels of selectivity than those in which the mismatch was located 3′ to the seed; this region of an siRNA is critical for target cleavage but not siRNA binding. Our data suggest that siRNAs can be designed to discriminate between the wild-type and mutant alleles of many genes that differ by just a single nucleotide. First discovered in nematodes, RNA interference (RNAi) has become an essential tool in the study of mammalian gene function. RNAi directed by small interfering RNAs (siRNAs), 21 nt, double-stranded RNAs target complementary mRNAs for destruction. siRNAs can be introduced into mammalian cells grown in culture, or even administered intravenously to rodents or primates, where they repress production of the targeted gene product. Thus, siRNA-directed RNAi has tremendous potential as a human therapeutic strategy. Dominant genetic disorders, in which a mutant allele of a gene causes disease in the presence of a second, normal copy, might be treated with therapeutic siRNAs, provided that the siRNAs could be designed to destroy the mutant, disease-causing mRNA, while leaving the normal mRNA intact. Here, Schwarz and colleagues describe an experimentally validated strategy for the design of such siRNAs. Their design strategy should facilitate the design of siRNAs targeting dominant genetic disorders such as amyotrophic lateral sclerosis and Huntington disease.
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