Nucleobase analogs for next generation siRNAs

使用siRNA触发的RNAi途径来研究基因功能在分子中已变得普遍 生物学，并为新疗法的发展提供了希望。然而，存在一些具体问题。 这限制了这项前景光明的技术的应用。这些问题包括出现的偏离目标的影响 来自siRNAs与先天免疫系统蛋白质相互作用的能力，以及 SiRNA引导链作为miRNA发挥作用。此外，针对miRNAs的反义寡核苷酸 (抗miRs)具有显著的治疗潜力。在这次R01项目的竞争性续签中，我们 建议使用核酸化学、分子和结构生物学的工具来推进我们的 了解siRNA脱靶效应，并对siRNA和抗miRs提供新的修改，以 提高效力和选择性。 因为siRNA的引导链可以作为miRNA发挥作用，所以siRNA经常抑制 在种子区域外的引导-靶标双链中存在错配的基因靶标的表达 (指南的第2-8个核苷酸)。SiRNA研究领域的一个挑战是开发引导链 将导致更具体靶向的修改(即减少类似miRNA的脱靶效应)。这里 我们将使用X射线结晶学的组合(与路易斯安那州斯克里普斯的Ian MacRae教授合作 Jolla)、计算筛选、RNA修饰、hAgo2结合研究和RNAi实验 为hAgo2-导向络合物选择性靶向奠定基础并发现新的核苷 减少miRNA样效应的类似物。 通过结合Toll样受体(TLRs)调节先天免疫反应是另一个重要的因素 SiRNA脱靶效应将在本项目中解决。在组成链中发现的序列基序 已知某些siRNA可以激活TLRs并在人类细胞中诱导免疫反应。 修饰siRNA主干上的核糖(如2‘-OME、2’-F、LNA)可以减少TLR介导的 免疫刺激。然而，修饰的寡核苷酸通常作为TLR拮抗剂发挥作用 表明它们保持TLR结合活性。在这里，我们建议开发鸟苷类似物， 真正的“免疫沉默”，即缺乏TLR结合活性，以至于修饰的寡核苷酸既不是 受体激动剂或拮抗剂。为了实现这一目标，提出了两种不同的方法。 抑制miRNA功能也是一种有前途的治疗策略，因为增加了 特定的miRNAs与人类疾病有关。与miRNAs结合的反义寡核苷酸 Watson-Crick配对(抗miRs)是miRNA抑制剂的候选药物。由于函数分数的 MiRNA被装载到ArgAerte(AGO)蛋白中，这是对抗miR的修饰，以增强结合 TO AGO可能增强抗miR活性和选择性。结合的hAgo2的晶体结构 MiRNA-靶向复合体将被用于指导新的抗miR修饰的设计。我们会 为此，在抗miR中修饰两个不同的核苷酸位置。

Use of the siRNA-triggered RNAi pathway to study gene function has become common in molecular biology and holds promise for the development of new therapeutics. However, specific issues exist that limit the application of this promising technology. These issues include off-target effects that arise from the ability of siRNAs to interact with proteins of the innate immune system and the ability of an siRNA guide strand to function as a miRNA. In addition, antisense oligonucleotides targeting miRNAs (anti-miRs) have significant therapeutic potential. In this competitive renewal of an R01 project, we propose to use tools from nucleic acid chemistry, molecular and structural biology to advance our understanding of siRNA off-target effects and provide new modifications to siRNAs and anti-miRs to improve potency and selectivity. Because the guide strand of an siRNA can function as a miRNA, siRNAs often repress the expression of gene targets that have mismatches in the guide-target duplex outside the seed region (nucleotides 2-8 of the guide). A challenge in the field of siRNA research is to develop guide strand modifications that will lead to more specific targeting (i.e. reduced miRNA-like off target effects). Here we will use a combination of x-ray crystallography (in collaboration with Prof. Ian MacRae, Scripps, La Jolla), computational screening, RNA modification, hAgo2 binding studies and RNAi experiments to establish the basis for selective targeting by hAgo2-guide complexes and discover new nucleoside analogs that reduce miRNA-like effects. Regulation of innate immune responses by binding Toll-like receptors (TLRs) is another important siRNA off target effect to be addressed in this project. Sequence motifs found in the component strands of certain siRNAs are known to activate TLRs and induce an immune response in human cells. Modification of riboses in the backbone of siRNA (e.g. 2’-OMe, 2’-F, LNA), can reduce TLR-mediated immune stimulation. However, the modified oligonucleotides typically function as TLR antagonists indicating they maintain TLR-binding activity. Here we propose to develop guanosine analogs that are truly “immune silent” i.e. lacking TLR-binding activity such that the modified oligonucleotides are neither receptor agonists nor antagonists. Two different approaches are proposed to achieve this goal. Inhibition of miRNA function is also a promising therapeutic strategy since increased expression of specific miRNAs has been linked to human disease. Antisense oligonucleotides that bind miRNAs by Watson-Crick pairing (anti-miRs) are candidates as miRNA inhibitors. Since the functional fraction of a miRNA is loaded into an Argonaute (Ago) protein, modifications of the anti-miR that enhance binding to Ago are likely to enhance anti-miR activity and selectivity. Crystal structures of hAgo2 bound to miRNA-target complexes will be used to inform the design of new anti-miR modifications. We will modify two different nucleotide positions in the anti-miR for this purpose.