Amide-Modified RNA: Synthesis, Structure and Potential for RNA Interference

描述（申请人提供）：RNA干扰（RNAi）已成为分子生物学和功能基因组学中最强大和最广泛使用的研究工具之一。还有一个有趣的潜力，RNAi可能成为一种新的治疗方法。尽管取得了显著的进展，但RNAi远不是一个完美的工具，需要在体内研究和治疗应用方面进行重大改进。特别是，必须改善短干扰RNA（siRNA）的效力、递送和细胞摄取、生物分布和酶稳定性。我们研究的长期目标是开发优化siRNA性质的化学修饰，同时在更广泛的背景下研究RNA的结构和功能。目前的更新建立在我们的发现，在第一阶段的赠款，即酰胺是一个很好的结构替代磷酸在RNA。更新的知识框架建立在广泛的假设，酰胺也可以模拟磷酸-氨基酸相互作用的RNA-蛋白质复合物，并可能显着改善siRNA的性能。该提议的中心创新方面是用中性和相对疏水的酰胺键取代带负电荷和极性的磷酸酯。这种对RNA骨架的戏剧性修饰在RNAi领域几乎没有先例，如果成功，将引发我们对siRNA中什么可以容忍和什么不能容忍的思考的范式转变。siRNA电荷的减少将允许设计新的siRNA-细胞穿透肽缀合物，这对于未修饰的RNA是不可能的。初步结果强烈支持我们的假设，即用酰胺取代磷酸盐在siRNA中具有良好的耐受性。因此，该提案将挑战目前的范式，即带负电荷的磷酸盐是蛋白质识别RNA所必需的。具体目标将1）研究酰胺修饰的siRNA的RNAi活性; 2）研究Argonaute蛋白的Piwi结构域对酰胺修饰的RNA的识别;以及3）使用新型siRNA-细胞穿透肽缀合物改善细胞摄取。这些目标将测试与修饰的siRNA的分子识别和更广泛的RNAi机制相关的特定假设。这些目标将通过一项全面的多学科研究来实现，该研究涉及合成和生物物理化学（PI），结构生物化学（Egli，晶体学和Kennedy，NMR），RNA生物学（Thermo Fisher）以及细胞生物化学和荧光显微镜（Grewer和McGee）的合作努力。成功实现拟议目标的主要影响将是改进化学修饰的siRNA，用于体内应用，作为研究工具，并可能作为药物开发的先导化合物。最终目标是解决递送和细胞摄取问题，这将极大地扩展在动物中使用RNAi来研究疾病生理学的能力。拟议的研究还将推进RNA-蛋白质相互作用的基础知识，并为RNAi机制提供独特的见解。

DESCRIPTION (provided by applicant): RNA interference (RNAi) has become one of the most powerful and widely used research tools in molecular biology and functional genomics. There is also an intriguing potential that RNAi may become a new therapeutic approach. Despite the remarkable progress, RNAi is far from being a perfect tool and needs significant improvement for in vivo research and therapeutic applications. In particular, the potency, delivery and cellular uptake, biodistribution, and enzymatic stability of short interfering RNAs (siRNAs) must be improved. The long-term goal of our research is to develop chemical modifications that optimize the properties of siRNAs while studying structure and function of RNA in a broader context. The present renewal builds on our discoveries, made during the first period of this grant, that amide is an excellent structural replacement for phosphate in RNA. The intellectual framework of the renewal builds around broad hypotheses that amides may also mimic the phosphate-amino acid interactions in RNA-protein complexes and may significantly improve the properties of siRNAs. The central innovative aspect of this proposal is in replacing the negatively charged and polar phosphate with the neutral and relatively hydrophobic amide linkage. Such a dramatic modification of RNA's backbone has little precedent in the RNAi field and, if successful, will initiate a paradigm shift in our thinking of what can and what cannot be tolerated in siRNAs. Reduction of siRNA's charge will allow design of novel siRNA-cell penetrating peptide conjugates, which is not possible with unmodified RNA. The preliminary results strongly support our hypotheses that replacement of phosphates with amides will be well tolerated in siRNAs. Thus, the proposal will challenge the current paradigm that negatively charged phosphates are required for recognition of RNA by proteins. The specific aims will 1) study the RNAi activity of amide-modified siRNAs; 2) study the recognition of amide-modified RNA by the Piwi domain of Argonaute protein; and 3) improve the cellular uptake using novel siRNA-cell penetrating peptide conjugates. The aims will test specific hypotheses relevant to molecular recognition of modified siRNAs and broader RNAi mechanism. The aims will be achieved through a comprehensive and multidisciplinary study involving collaborative efforts in synthetic and biophysical chemistry (PI), structural biochemistry (Egli, crystallography and Kennedy, NMR), RNA biology (Thermo Fisher), and cell biochemistry and fluorescence microscopy (Grewer and McGee). The main impact of successfully reaching the proposed aims will be improved chemically modified siRNAs for in vivo applications as research tools and, potentially, as lead compounds for drug development. The ultimate goal is to solve the delivery and cellular uptake problems, which would dramatically expand the ability to use RNAi in animals to study physiology of disease. The proposed studies will also advance fundamental knowledge on RNA-protein interactions and provide unique insights into RNAi mechanism.