Nucleobase analogs for next generation siRNAs

描述（由申请人提供）：通过短干扰RNA （siRNA）触发的RNA干扰（RNAi）途径在活细胞内选择性核酸酶消化信使RNA已成为分子生物学研究基因功能的支柱，并有望开发出一种强大且广泛适用的新型治疗方法。然而，存在一些具体问题，限制了这项有前途的技术的应用。其中的关键是脱靶效应，这种脱靶效应是由siRNA与先天免疫反应中涉及的蛋白质相互作用的能力引起的，而且与siRNA引导链不完全匹配的mrna也是靶向的。在这个R01项目的竞争性更新中，我们将推进我们对这些脱靶效应的理解，以及如何通过应用合成核酸化学与生物化学、分子和细胞生物学的技术相结合来控制它们。siRNA研究领域的一个挑战是开发引导链修饰，从而导致更特异性的靶向（即降低引导-靶标双工的错配容忍度）。在这里，我们将研究三种不同的方法来增加Ago-guide +靶链结合的特异性。首先，我们将确定调节Ago2 PAZ结构域亲和力的引导基3'端修饰。从该结构域分离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，为了增强引导链3‘端与PAZ结构域之间的相互作用，需要在种子外稳定配对以实现3’分离，从而降低复合物对错配的容错性。其次，我们将开发新的核碱基类似物，同时识别靶标中的两个相邻核苷酸。我们提出，与使用标准的沃森-克里克互补碱基相比，使用这类类似物，靶中任何位置的失配都会使复合物失稳，因为失配的失稳效应将通过连接子传递。最后，我们将利用在Ago2蛋白的复合体内形成导靶双工的事实。我们在此提出，带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不匹配对提供额外的不稳定性和/或稳定化对。通过结合toll样受体刺激先天免疫反应是本项目要解决的另一个重要的siRNA脱靶效应。在某些sirna的组成链中发现的序列基序可以激活TLRs 7和8，并在人类细胞中诱导免疫反应。在上一个资助期内，我们发现免疫刺激sirna的核碱基修饰减少了人类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被证明可以阻断这种作用，但没有一个简明的配体/受体相互作用模型可以解释为什么某些序列比其他序列更受欢迎，或者可以解释化学修饰的作用。我们将开展实验来进一步确定免疫刺激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，以阻断TLR激活或增加RNAi效应的效力。！

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. !