Chemical Modifications of siRNA Bases to Control Off-target Effects

siRNA 碱基的化学修饰以控制脱靶效应

• 批准号: 7390349
• 负责人: PETER A. BEAL
• 金额: $ 25.75万
• 依托单位: UNIVERSITY OF CALIFORNIA AT DAVIS
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-04-01 至 2011-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7390349
• 关键词: 8-hydroxyguanosine; Acrolein; Alkylation; Base Pairing; Binding; Cells; Chemicals; Collaborations; Dendritic Cells; Development; Digestion; Gene Expression; Gene Silencing; Guanine; Guanosine; Health Sciences; Human; Immune; Immune response; Lesion; Life; Major Groove; Mediating; Messenger RNA; Microarray Analysis; Minor Groove; Modification; Molecular; Molecular Biology; Mus; Myelogenous; Oligonucleotides; Pathology; Pattern; Positioning Attribute; Protein Binding; Proteins; Purines; RNA; RNA Interference; RNA Interference Pathway; RNA Sequences; RNA-Binding Proteins; Research Personnel; Ribonucleotides; Shapes; Site; Toll-like receptors; Universities; Utah; alkyl group; base; design; gene function; human TLR7 protein; new technology; novel therapeutics; nuclease; nucleobase; phosphoramidite; professor; purine

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.

描述（由申请人提供）：通过短干扰RNA（siRNA）触发的RNA干扰途径对活细胞内的信使RNA进行选择性核酸酶消化已成为研究基因功能的分子生物学支柱，并有望开发新的治疗方法。然而，我们对基本RNA干扰机制和siRNA与细胞内RNA结合蛋白相互作用的能力的理解存在差距，特别是那些参与先天免疫反应的蛋白，限制了这种有前途的新技术的应用。在这里，我们建议开发新的合成方法来修饰RNA寡核苷酸，并使用所得分子进行基因沉默，减少不良的脱靶效应。本项目的具体目标如下：1）合成具有突出于大沟或小沟的侧链烷基的siRNA。化学修饰旨在改变碱基的形状，同时保持其参与沃森-克里克样碱基配对的能力。这些siRNA双链体将用于确定核碱基烷基化在基因沉默中的作用。还预期所述修饰阻断由Toll样受体介导的序列依赖性脱靶效应和来自dsRBM蛋白的序列非依赖性脱靶效应。2)我们假设，在修改鸟嘌呤的反syn构象变化将作为分子开关打开（沃森-克里克配对时）或关闭（Hoogsteen配对时）在双链体RNA小沟的空间阻滞。我们将研究两个烷基化的鸟嘌呤衍生物作为开关，常见的氧化碱基病变8-oxoguanosine（8-oxoG）与添加烷基，和丙烯醛烷基化嘌呤。这些修饰将使用Watson-Crick配对的正义链置于反义链中进行递送;反义链将被设计为通过Hoogsteen配对靶向互补mRNA，以在修饰位点处与嘌呤配对。3)我们将评估修饰的siRNA在RNAi中参与序列依赖性和序列非依赖性脱靶效应的能力。将通过分析含有免疫刺激序列基序的修饰的siRNA双链体在小鼠和培养的鼠免疫细胞（包括骨髓树突细胞）中刺激免疫应答的能力来评估序列依赖性效应。将通过确定碱基修饰对细胞双链体RNA结合蛋白结合的影响来评价序列非依赖性效应。