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）的活细胞内部RNA的选择性核酸酶消化 - 触发的RNA干扰途径已成为分子生物学的中流stay，以研究基因功能，并具有开发新疗法的希望。但是，我们对基本RNA干扰机制的理解以及siRNA与细胞内RNA结合蛋白相互作用的能力，尤其是参与先天免疫反应的蛋白，限制了这项有希望的新技术的应用。在这里，我们建议开发新的合成方法来修饰RNA寡核苷酸，并使用所得分子来携带基因沉默，并减少不良的脱靶效应。该项目具有以下特定目的：1）我们将合成投影到主要凹槽或次要凹槽中的吊体烷基的siRNAS。化学修饰旨在改变底座的形状，同时保持其参与Watson-Crick样碱基配对的能力。这些siRNA双链体将用于定义基因沉默中核碱烷基化的影响。预计这些修饰还会阻止依赖序列的脱靶效应，并由Toll样受体和DSRBM蛋白的序列独立的脱靶效应介导。 2）我们假设改性鸟嘌呤的抗同胞构象变化将充当分子开关（当watson-crick配对时）或OFF（当Hoogsteen配对时）在双层RNA小凹槽中的空间阻滞。我们将研究两种烷基化的鸟嘌呤衍生物作为开关，共同氧化的碱病变8-氧气葡萄糖氨酸（8-oxog）和添加的烷基基团和丙烯醛烷基化的嘌呤。这些修饰将使用沃森 - 奇异的感觉链进行反义链中。反义链将设计为通过Hoogsteen配对靶向互补的mRNA，以在修饰部位靶向嘌呤。 3）我们将评估改良的siRNA在RNAi中参与序列依赖性和序列非靶向效应的能力。序列依赖性效应将通过分析含有免疫刺激序列基序的修饰的siRNA双链体来评估，以刺激小鼠和包括髓样树枝状细胞的培养的鼠免疫细胞中的免疫反应的能力。序列非依赖性效应将通过确定基础修饰对细胞双链RNA结合蛋白的结合的结果进行评估。