Chemical Modifications of siRNA Bases to Control Off-target Effects

描述(申请人提供)：通过短干扰RNA(SiRNA)触发的RNA干扰途径选择性核酸酶消化活细胞内的信使RNA已成为分子生物学研究基因功能的支柱，并有望开发新的治疗方法。然而，由于我们对基本的RNA干扰机制以及siRNAs与细胞内RNA结合蛋白的相互作用能力的了解存在差距，特别是那些参与先天性免疫反应的蛋白，限制了这项有前景的新技术的应用。在这里，我们建议开发新的合成方法来修饰RNA寡核苷酸，并使用所得到的分子来进行基因沉默，以减少不良的脱靶效应。本项目有以下具体目标：1)我们将合成含有侧链烷基的siRNAs，这些基团既可以伸入主槽，也可以投射到副槽。化学修饰旨在改变碱基的形状，同时保持其参与沃森-克里克类碱基配对的能力。这些siRNA双链将被用来确定碱基烷基化在基因沉默中的作用。这些修饰还有望阻止Toll样受体介导的序列依赖的脱靶效应，以及dsRBM蛋白介导的序列无关的脱靶效应。2)我们假设修饰鸟嘌呤中的反同步构象变化将作为分子开关，开启(当Watson-Crick配对时)或关闭(当Hoogsteen配对时)双链RNA小槽中的空间位阻。我们将研究两个烷基化的鸟嘌呤类化合物作为开关，常见的氧化碱基损伤8-氧鸟苷(8-oxoG)添加了烷基，以及丙烯醛烷基化的嘌呤。这些修饰将使用Watson-Crick配对的正义链放置在反义链中进行传递；反义链将被设计为通过Hoogsteen与修饰部位的嘌呤配对来靶向互补的mRNA。3)我们将评估修饰的siRNAs在RNAi中参与序列依赖和序列非依赖的脱靶效应的能力。将通过分析含有免疫刺激序列基序的修改的siRNA双链来评估它们在小鼠和培养的小鼠免疫细胞(包括髓系树突状细胞)中刺激免疫反应的能力，以评估序列依赖的效应。将通过确定碱基修饰对细胞双链RNA结合蛋白结合的影响来评估序列无关的效应。

DESCRIPTION (provided by applicant): Selective nuclease digestion of messenger RNAs inside living cells via the short interfering RNA (siRNA)- triggered RNA interference pathway has become a mainstay in molecular biology to study gene function and holds promise for the development of new therapeutics. However, gaps in our understanding of the basic RNA interference mechanism and the ability of siRNAs to interact with intracellular RNA-binding proteins, particularly those involved in the innate immune response, limit the application of this promising new technology. Here we propose to develop new synthetic approaches to modified RNA oligonucleotides and to use the resulting molecules to carryout gene silencing with reduced undesirable off-target effects. This project has the following specific aims: 1) We will synthesize siRNAs bearing pendant alkyl groups projecting into either the major groove or the minor groove. The chemical modifications are designed to change the shape of the base while maintaining its ability to engage in Watson-Crick-like base pairing. These siRNA duplexes will be used to define the effects of nucleobase alkylation in gene silencing. The modifications are also expected to block sequence-dependent off-target effects mediated by Toll-like receptors and sequence- independent off-target effects from dsRBM proteins. 2) We hypothesize that anti-syn conformational changes in modified guanines will act as molecular switches to turn on (when Watson-Crick paired) or off (when Hoogsteen paired) steric blockades in the duplex RNA minor groove. We will investigate two alkylated guanine derivatives as switches, the common oxidized base lesion 8-oxoguanosine (8-oxoG) with added alkyl groups, and the acrolein alkylated purines. These modifications will be placed in the antisense strand using a Watson-Crick-paired sense strand for delivery; the antisense strand will be designed to target a complementary mRNA via Hoogsteen pairing to a purine at the site of modification. 3) We will evaluate the ability of modified siRNAs to engage in sequence-dependent and sequence-independent off-target effects in RNAi. Sequence-dependent effects will be assessed by analyzing modified siRNA duplexes containing immunostimulatory sequence motifs for their ability to stimulate immune responses in mice and in cultured murine immune cells including myeloid dendritic cells. Sequence-independent effects will be evaluated by determining the consequence of base modifications on the binding of cellular duplex RNA-binding proteins.