Nucleobase analogs for next generation siRNAs

下一代 siRNA 的核碱基类似物

• 批准号: 8634117
• 负责人: PETER A. BEAL
• 金额: $ 30.54万
• 依托单位: UNIVERSITY OF CALIFORNIA AT DAVIS
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-04-01 至 2017-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8634117
• 关键词: Address; Affinity; Binding; Biochemistry; Cells; Chemicals; Chemistry; Complex; Coupled; Development; Digestion; Funding; Gene Silencing; Guide RNA; Human; Immune response; Immunologic Receptors; Lead; Life; Ligands; Location; Messenger RNA; MicroRNAs; Modeling; Modification; Molecular Biology; Molecular and Cellular Biology; Nucleic Acids; Nucleotides; Pattern; Production; Proteins; RNA; RNA Interference; RNA Interference Pathway; RNA Sequences; Receptor Activation; Research; Seeds; Site; Specificity; Techniques; Technology; Testing; Therapeutic; Toll-like receptors; Translating; analog; appendage; base; cytokine; gene function; next generation; novel; novel therapeutics; nuclease; nucleobase analog; nucleoside analog; public health relevance; receptor; receptor binding; research study; structural biology; synthetic nucleic acid; tool

DESCRIPTION (provided by applicant): Selective nuclease digestion of messenger RNAs inside living cells via the short interfering RNA (siRNA)-triggered RNA interference (RNAi) pathway has become a mainstay in molecular biology to study gene function and holds promise for the development of a powerful and broadly applicable new class of therapeutics. However, specific issues exist that limit the application of this promising technology. Key among these are off-target effects that arise from the ability of siRNAs to interact with proteins involved in the innate immune response and the fact that mRNAs with imperfect matches to the siRNA guide strand are also targeted. In this competitive renewal of an R01 project, we will advance our understanding of these off-target effects and how to control them through the application of synthetic nucleic acid chemistry coupled with techniques from biochemistry and molecular and cellular biology. A challenge in the field of siRNA research is to develop guide strand modifications that will lead to more specific targeting (i.e. reduced tolerance of mismatches in the guide-target duplex). Here we will investigate three different approaches to increase the specificity of Ago-guide + target strand binding. First, we will identify modifications to the guid stand 3' end that modulate affinity for the Ago2 PAZ domain. Disengaging the 3' end from this domain is proposed to be an important step in forming the silencing-competent complex. We propose here that enhancing the interaction between the guide strand 3' end and the PAZ domain will require stable pairing outside the seed for 3' disengagement, rendering the complex less tolerant of mismatches. Second, we will develop new nucleobase analogs that simultaneously recognize two adjacent nucleotides in the target. We propose that with such analogs, a mismatch at either location in the target would destabilize the complex to a greater degree than if standard Watson-Crick complementary bases were used because the destabilizing effect of the mismatch will be translated through the linker. Finally, we will take advantage of the fact that the guide-target duplex forms within a complex with the Ago2 protein. We propose here that nucleobase analogs with appended groups could provide additional destabilization to the mismatched pair and/or stabilization to the matched pair via interactions between the appendage and the protein. Stimulation of the innate immune response by binding Toll-like receptors 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 7 and 8 and induce an immune response in human cells. During the last funding period, we showed that modifications to the nucleobases in immunostimulatory siRNAs reduce cytokine production in human PBMCs. While many different RNA sequences are now known to be immunostimulatory and different chemical modifications shown to block the effect, no concise model has been presented for the ligand/receptor interaction that can explain why certain sequences are favored over others or that can explain the effects of chemical modifications. We will carry out experiments to define further the immune receptor ligand in immunostimulatory siRNAs, to test novel hypotheses for immune receptor binding and to provide new nucleoside analogs that either block TLR activation or increase potency of the RNAi effect. !

描述（由申请人提供）：通过短的干扰RNA（siRNA）触发的RNA干扰（RNAI）途径的活细胞内部RNA的选择性核酸酶消化已成为分子生物学的主要主流，以研究基因功能，并持有强大且广泛适用的新型治疗类别的有望。但是，存在限制这种有前途技术的应用的具体问题。其中的关键是脱靶效应，这是由siRNA与与先天免疫反应有关的蛋白质相互作用的能力以及具有与siRNA指南不完全匹配的mRNA也是针对性的。在R01项目的这种竞争性更新中，我们将通过应用合成核酸化学以及生物化学以及分子和细胞生物学的技术来提高对这些脱靶效应的理解，以及如何通过应用合成核酸化学来控制它们。 siRNA研究领域的一个挑战是开发指导链修饰，这将导致更具体的靶向（即，在指南目标双工中降低了不匹配的耐受性）。在这里，我们将研究三种不同的方法，以提高AGO guide +目标链结合的特异性。首先，我们将确定对GUID Stand 3'端的修改，以调节对AGO2 PAZ域的亲和力。提议将3'端与该领域脱离3'的末端是形成竞争激动的复合物的重要步骤。我们在这里提出，增强导向链3'端与PAZ域之间的相互作用将需要在种子外面进行稳定的配对以进行3'脱离接触，从而使复合物对不匹配的耐受性较低。其次，我们将开发新的核碱类似物，这些类似物同时识别目标中的两个相邻的核苷酸。我们建议，使用这种类似物，与使用标准的Watson-Crick互补碱基相比，目标的任一位位置的不匹配将使复合物不稳定，因为使用不匹配的不稳定效果将通过接头翻译。最后，我们将利用一个事实，即在与AGO2蛋白的复合物中形成指南目标双链体。我们在这里建议，具有附加组的核酶类似物可以通过附属物与蛋白质之间的相互作用为不匹配的一对和/或稳定对匹配的对提供额外的不稳定。通过结合Toll样受体来刺激先天免疫反应是该项目中要解决的另一个重要的siRNA关闭目标效应。已知在某些siRNA的组分链中发现的序列基序可激活TLRS 7和8，并诱导人类细胞中的免疫反应。在最后一个资金期间，我们表明对免疫刺激性siRNA中核碱基的修饰减少了人PBMC中的细胞因子产生。尽管现在已知许多不同的RNA序列是免疫刺激性的，并且显示出不同的化学修饰可以阻止效果，但没有针对配体/受体相互作用提出简洁的模型，这些模型可以解释为什么某些序列比其他序列相对于其他序列有利，或者可以解释化学修饰的作用。我们将进行实验，以进一步定义免疫刺激性siRNA中的免疫受体配体，以检验新的假设以进行免疫受体结合，并提供新的核苷类似物，以阻止TLR激活或增加RNAI效应的能力。呢