Nucleobase analog for the next generation siRNa

使用siRNA触发的RNAi途径来研究基因功能在分子生物学中已经变得普遍。 并有望开发新的治疗方法。然而，存在一些具体问题， 限制了这项有前途的技术的应用。这些问题包括出现的脱靶效应 从siRNA与先天免疫系统的蛋白质相互作用的能力和免疫系统的蛋白质相互作用的能力， siRNA引导链作为miRNA起作用。此外，靶向miRNAs的反义寡核苷酸 （抗miR）具有显著的治疗潜力。在R 01项目的竞争性更新中，我们 我建议使用核酸化学、分子和结构生物学的工具来推进我们的研究。 了解siRNA脱靶效应，并提供siRNA和抗miR的新修饰， 提高效力和选择性。 因为siRNA的引导链可以作为miRNA起作用，所以siRNA通常抑制miRNA的表达。 在种子区域外的向导-靶标双链体中具有错配的基因靶标的表达 （指导序列的核苷酸2-8）。siRNA研究领域的一个挑战是开发指导链， 这些修饰将导致更特异性的靶向（即减少的miRNA样脱靶效应）。这里 我们将结合使用X射线晶体学（与路易斯安那州斯克里普斯的伊恩·麦克雷教授合作 Jolla）、计算筛选、RNA修饰、hAgo 2结合研究和RNAi实验，以 为hAgo 2导向复合物的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（TLR）调节先天免疫应答是另一个重要的免疫调节机制。 siRNA脱靶效应将在本项目中得到解决。在组成链中发现的序列基序 已知某些siRNA可激活TLR并诱导人类细胞中的免疫应答。 siRNA骨架中核糖的修饰（例如2 '-OMe、2'-F、LNA）可以减少TLR介导的细胞凋亡。 免疫刺激然而，修饰的寡核苷酸通常用作TLR拮抗剂， 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，使得修饰的寡核苷酸既不 受体激动剂或拮抗剂。提出了两种不同的方法来实现这一目标。 miRNA功能的抑制也是一种有前景的治疗策略，因为miRNA的表达增加。 特定的miRNAs与人类疾病有关。反义寡核苷酸结合miRNA， Watson-Crick配对（anti-miR）是作为miRNA抑制剂的候选物。由于函数分数 将miRNA加载到Argonaute（Ago）蛋白中，抗miR的修饰增强结合 到Ago的基因可能增强抗miR活性和选择性。hAgo 2的晶体结构与 miRNA-靶复合物将用于设计新的抗miR修饰。我们将 为此目的修饰抗miR中的两个不同核苷酸位置。

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.