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）：通过短干扰RNA（siRNA）触发的RNA干扰（RNAi）途径对活细胞内的信使RNA进行选择性核酸酶消化已成为研究基因功能的分子生物学支柱，并有望开发出强大且广泛适用的新型治疗方法。然而，存在的具体问题限制了这一有前途的技术的应用。其中的关键是脱靶效应，其产生于siRNA与参与先天免疫应答的蛋白质相互作用的能力，以及与siRNA引导链不完全匹配的mRNA也被靶向的事实。在R 01项目的竞争性更新中，我们将通过应用合成核酸化学以及生物化学、分子和细胞生物学技术来提高我们对这些脱靶效应的理解，以及如何控制它们。siRNA研究领域的一个挑战是开发导向链修饰，其将导致更特异的靶向（即，降低导向-靶双链体中错配的耐受性）。在这里，我们将研究三种不同的方法来增加Ago-向导+靶链结合的特异性。首先，我们将鉴定调节对Ago 2 PAZ结构域的亲和力的对引导链3'端的修饰。从该结构域中分离3'端被认为是形成沉默能力复合物的重要步骤。我们在此提出，增强引导链3'端和PAZ结构域之间的相互作用将需要种子外的稳定配对以进行3'脱离，从而使复合物对错配的耐受性降低。第二，我们将开发新的核碱基类似物，同时识别目标中的两个相邻核苷酸。我们提出，与使用标准沃森-克里克互补碱基相比，使用这样的类似物，靶中任一位置的错配将使复合物不稳定的程度更大，因为错配的不稳定效应将通过接头翻译。最后，我们将利用向导-靶双链体在与Ago 2蛋白的复合物内形成的事实。我们在这里提出，具有附加基团的核碱基类似物可以通过附件和蛋白质之间的相互作用为错配对和/或匹配对提供额外的不稳定性。通过结合Toll样受体刺激先天免疫应答是本项目中要解决的另一个重要的siRNA脱靶效应。已知在某些siRNA的组分链中发现的序列基序激活TLR 7和8并诱导人细胞中的免疫应答。在上一个资助期间，我们发现对免疫刺激siRNA中的核碱基的修饰减少了人PBMC中细胞因子的产生。虽然现在已知许多不同的RNA序列具有免疫刺激作用，并且不同的化学修饰显示出阻断该作用，但是没有针对配体/受体相互作用的简明模型可以解释为什么某些序列比其他序列更受青睐，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siRNA中的免疫受体配体，以测试免疫受体结合的新假设，并提供新的核苷类似物，其可以阻断TLR激活或增加RNAi效应的效力。!