Synthesis and investigation of chemically-modified oligonucleotides as gene-silencing substrates

作为基因沉默底物的化学修饰寡核苷酸的合成和研究

• 批准号: RGPIN-2014-04127
• 负责人: Desaulniers, JeanPaul
• 金额: $ 2.19万
• 依托单位: University of Ontario Institute of Technology
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2014
• 资助国家: 加拿大
• 起止时间: 2014-01-01 至 2015-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=566311
• 关键词: Synthesis; investigation; chemically; modified; oligonucleotides

This research program investigates new approaches for lowering the levels of gene expression using short interfering RNAs (siRNAs) as substrates for the natural RNA interference (RNAi) pathway. Natural siRNAs fall short in that they are a) unstable to their environment and are degraded easily; b) exhibit relatively poor cell membrane permeability; c) exhibit off-target effects which causes non-specific toxicity; d) activate the immune system inadvertently; and e) have poor biodistribution. This research program continues to offer novel innovative methods to combat these challenges by making unique chemical modifications within the siRNA to alter its stability and specificity profile. The overall long-term goals of the research program are to understand, characterize, and predict the impact that chemical modifications have on the efficacy, potency, and specificity of siRNAs. The short-term objectives of this interdisciplinary research program utilize organic chemistry, biophysical analysis, and cell biology to generate new classes of chemically-modified siRNAs. This will be achieved by studying siRNAs bearing triazole backbones and functionalized spacer groups. In our previous work, we synthesized siRNAs bearing neutrally-charged triazole and amide backbones, and these chemically-modified siRNAs were highly active as gene-silencing substrates. These studies were the first to highlight RNAi compatibility with neutrally-charged backbone modifications within the Watson-Crick region of the siRNA. This proposal focuses on expanding the scope of backbone modifications by synthesizing second-generation triazole-backbone siRNA derivatives. These include the synthesis of a) chiral triazole-backbone modifications, b) polymeric triazole modifications, and c) triazole-backbone modifications in conjunction with base-modified variants. These molecules are designed to improve the structure-activity relationship of the siRNA and this work will provide new knowledge in designing novel backbone-modified siRNAs. In another proof-of-concept study that we published, we discovered a promiscuous area within the central region of siRNAs that are highly active when up to three nucleobases were replaced with a non-cleavable carbon-based spacer linker. Given this high-level of promiscuity, this approach involves expanding the scope of chemical functionality located at the centre of the siRNA duplex in order to fine-tune the chemical and functional characteristics of the duplex RNA. Molecular functional groups such as amines, amides, alcohols will be installed in order to improve the overall charge of the molecule. Functional groups such as allyl and alkyne groups will be installed due to their compatibility and reactivity with metal-catalyzed reactions used to covalently link molecules of interest such as cholesterol, guanidium groups and other related modifications to this area. In addition, other variants that will be studied that span the central region of the siRNA include moieties such as aromatic groups, amino acids, fluorescent probes and molecules that isomerize when photo-induced. These studies will provide new knowledge in an area of siRNA development that has not been explored and will generate new chemically-modified siRNAs with novel properties. The potential of harnessing this natural pathway would have enormous impact on research and medicine for not only Canada, but also worldwide. The research outlined in this proposal will provide new and novel approaches to chemically-modify siRNAs and will provide an outstanding interdisciplinary training environment in organic chemistry, bioorganic chemistry, biophysical chemistry, and biochemistry for both graduate and undergraduate students.

本研究计划探索利用短干扰RNA （sirna）作为天然RNA干扰（RNAi）途径的底物来降低基因表达水平的新方法。天然sirna的不足之处在于它们a)对环境不稳定且容易降解；B)细胞膜通透性相对较差；C)表现出脱靶效应，引起非特异性毒性；D)无意中激活免疫系统；e)生物分布差。该研究项目继续提供新颖的创新方法，通过在siRNA中进行独特的化学修饰来改变其稳定性和特异性，以应对这些挑战。研究计划的总体长期目标是了解、表征和预测化学修饰对sirna的功效、效力和特异性的影响。这个跨学科研究项目的短期目标是利用有机化学、生物物理分析和细胞生物学来产生新型的化学修饰sirna。这将通过研究带有三唑骨架和功能化间隔基团的sirna来实现。在我们之前的工作中，我们合成了带有中性电荷的三唑和酰胺骨架的sirna，这些化学修饰的sirna作为基因沉默底物具有很高的活性。这些研究首次强调了RNAi与siRNA沃森-克里克区域中中性带电的骨干修饰的相容性。本提案的重点是通过合成第二代三唑-主链siRNA衍生物来扩大主链修饰的范围。这些包括a)手性三唑-骨架修饰的合成，b)聚合三唑修饰，以及c)三唑-骨架修饰与碱基修饰变体的合成。这些分子被设计用于改善siRNA的结构-活性关系，这项工作将为设计新的骨干修饰siRNA提供新的知识。在我们发表的另一项概念验证研究中，我们发现sirna中心区域有一个混杂区域，当多达三个核碱基被不可切割的碳基间隔连接器取代时，该区域高度活跃。考虑到这种高度的混杂性，该方法涉及扩展位于siRNA双工中心的化学功能范围，以微调双工RNA的化学和功能特征。分子官能团如胺、酰胺、醇将被安装，以提高分子的整体电荷。官能团（如烯丙基和炔基）将被安装，因为它们与金属催化反应的相容性和反应性，这些反应用于将感兴趣的分子（如胆固醇、胍基团和其他相关修饰）共价连接到该区域。此外，将研究跨越siRNA中心区域的其他变异，包括芳香基团、氨基酸、荧光探针和光诱导时异构化的分子等部分。这些研究将为尚未探索的siRNA开发领域提供新知识，并将产生具有新特性的新的化学修饰siRNA。利用这一自然途径的潜力将不仅对加拿大，而且对全世界的研究和医学产生巨大影响。本提案中概述的研究将为化学修饰sirna提供新的和新颖的方法，并将为研究生和本科生提供有机化学，生物有机化学，生物物理化学和生物化学的优秀跨学科培训环境。