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

该研究项目研究了使用短干扰RNA（siRNA）作为天然RNA干扰（RNAi）途径底物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）对其环境不稳定并且容易降解; B）表现出相对差的细胞膜渗透性; c）表现出导致非特异性毒性的脱靶效应; d）无意中激活免疫系统;以及e）具有差的生物分布。这项研究计划继续提供新的创新方法，通过在siRNA内进行独特的化学修饰来改变其稳定性和特异性来应对这些挑战。该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。这个跨学科研究计划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的化学修饰siRNA。 这将通过研究带有三唑骨架和官能化间隔基团的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高度活性。 这些研究首次突出了RNAi与siRNA的Watson-Crick区域内的中性电荷骨架修饰的相容性。 该提案的重点是通过合成第二代三唑骨架siRNA衍生物来扩大骨架修饰的范围。 这些包括合成a）手性三唑骨架修饰，B）聚合三唑修饰，和c）三唑骨架修饰结合碱基修饰变体。 这些分子被设计用于改善siRNA的结构-活性关系，并且这项工作将为设计新型骨架修饰的siRNA提供新的知识。** 在我们发表的另一项概念验证研究中，我们发现了siRNA中心区域内的一个混杂区域，当多达三个核碱基被不可切割的碳基间隔连接子取代时，该区域高度活跃。鉴于这种高水平的混杂性，该方法涉及扩大位于siRNA双链体中心的化学功能性的范围，以便微调双链体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总电荷。将安装官能团如烯丙基和炔基，这是由于它们与用于共价连接感兴趣的分子如胆固醇、胍基和其他相关修饰的金属催化反应的相容性和反应性。 此外，将被研究的跨越siRNA中心区域的其他变体包括诸如芳香族基团、氨基酸、荧光探针和在光诱导时异构化的分子的部分。 这些研究将在siRNA开发领域提供新的知识，该领域尚未探索，并将产生具有新特性的新化学修饰的siRNA。利用这一自然途径的潜力将对加拿大乃至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提供化学修饰siRNA的新方法，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方面的优秀跨学科培训环境。

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.