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

本研究计划探索利用短干扰RNA(SiRNAs)作为自然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：a)它们对环境不稳定，容易降解；b)表现出相对较差的细胞膜通透性；c)表现出非靶标效应，导致非特异性毒性；d)无意中激活免疫系统；e)生物分布不良。该研究计划继续提供新颖的创新方法来应对这些挑战，方法是在siRNA中进行独特的化学修饰，以改变其稳定性和特异性。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的效力、效力和特异性的影响。这一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的siRNA。这将通过研究含有三唑骨架和官能化间隔基的siRNAs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氮唑和酰胺骨架的siRNAs，这些经过化学修饰的siRNAs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的Watson-Crick区域内中性带电的主干修饰的兼容性。这项建议的重点是通过合成第二代三氮唑主链siRNA衍生物来扩大主链修饰的范围。这些包括a)手性三唑主链修饰的合成，b)聚合物三唑主链修饰，以及c)结合碱基修饰的三唑主链修饰的合成。这些分子旨在改善siRNA的构效关系，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，我们发现在siRNAs的中心区域有一个混杂区域，当最多三个碱基被不可切割的碳基间隔区连接时，该区域非常活跃。鉴于这种高度的混杂，这种方法涉及扩大位于siRNA双链中心的化学功能的范围，以微调双链RNA的化学和功能特性。分子官能团如胺、酰胺、醇等将被安装，以提高分子的整体电荷。将安装诸如烯丙基和炔基的官能团，因为它们与金属催化的反应具有兼容性和反应性，用于共价连接感兴趣的分子，如胆固醇、金刚烷基和其他相关修饰到这一区域。此外，其他将被研究的跨越siRNA中心区域的变体包括部分，如芳香基、氨基酸、荧光探针和当光诱导时异构化的分子。这些研究将在尚未探索的siRNA开发领域提供新的知识，并将产生具有新特性的新的化学修饰的siRNA。利用这一自然途径的潜力不仅对加拿大，而且对世界各地的研究和医学都将产生巨大影响。这项建议中概述的研究将为化学修饰siRNAs提供新的和新的方法，并将为研究生和本科生提供一个出色的有机化学、生物有机化学、生物物理化学和生物化学的跨学科培训环境。

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.