Nucleobase analogs for next generation siRNAs

描述(申请人提供)：通过短干扰RNA(SiRNA)触发的RNA干扰(RNAi)途径选择性地消化活细胞内的信使RNA已成为研究基因功能的分子生物学的支柱，并有望开发出功能强大且广泛适用的新疗法。然而，存在一些特定的问题，限制了这项前景光明的技术的应用。其中的关键是由于siRNAs与参与先天性免疫反应的蛋白质相互作用而产生的脱靶效应，以及与siRNA引导链不完全匹配的mRNAs也是靶向的事实。在这次R01项目的竞争性更新中，我们将增进我们对这些非目标效应的理解，以及如何通过应用合成核酸化学以及生物化学和分子和细胞生物学的技术来控制它们。SiRNA研究领域的一个挑战是开发能够导致更特异性靶向的引导链修饰(即降低对引导-靶双链中错配的耐受性)。在这里，我们将研究三种不同的方法来提高AGO-GUIDE+靶链结合的特异性。首先，我们将确定对GUID支架3‘端的修饰，以调节对Ago2PAZ结构域的亲和力。从这个结构域中分离出3‘端被认为是形成沉默能力复合体的重要一步。我们在这里提出，要加强导链3‘端与PAZ结构域之间的相互作用，需要在种子外稳定的配对才能使3’解离，从而使复合体对错配的耐受性降低。其次，我们将开发新的碱基类似物，同时识别靶标中的两个相邻核苷酸。我们认为，有了这样的类似物，靶上任何一个位置的错配都会比使用标准Watson-Crick互补碱基时更大程度地破坏复合体的不稳定，因为错配的不稳定效应将通过连接子转化。最后，我们将利用这样一个事实，即引导-靶向双链在与Ago2蛋白的复合体中形成。我们在这里提出，带有附加基团的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提供额外的失稳和/或为匹配对提供稳定。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。已知在某些siRNAs的组成链中发现的序列基序可以激活TLR7和TLR8，并在人类细胞中诱导免疫反应。在上一次资助期间，我们发现对免疫刺激siRNAs中碱基的修饰减少了人外周血单核细胞中细胞因子的产生。虽然目前已知许多不同的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，但还没有关于配体/受体相互作用的简明模型来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效果。我们将进行实验，进一步确定免疫刺激siRNAs中的免疫受体配体，测试免疫受体结合的新假说，并提供新的核苷类似物来阻断TLR的激活或增加RNAi效应的效力。好了！

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. !