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

描述（由申请人提供）：RNA干扰（RNAi）的发现重新激发了对化学修饰的兴趣，以优化小干扰RNA（siRNA）的特性。我们研究的长期目标是利用化学方法探索RNA的结构和功能，并开发用于RNAi实际应用的修饰。本提案集中于核苷间酰胺作为磷酸二酯键的非离子模拟物，并将测试以下假设：酰胺（1）可以使用固相合成容易地引入RNA中，（2）是RNA的磷酸骨架的优异模拟物，以及（3）将增加siRNA的酶稳定性和细胞摄取而不损害其RNAi活性。我们还设想酰胺可以改善siRNA的生物分布和药代动力学。我们提出了一个跨学科（有机化学，结构生物化学和RNA生物学）的研究，具体目标是：1。开发合成方法，通过采用和优化固相肽，PNA和RNA合成方法，在RNA中的任何所需位置引入连续的酰胺键。2.与Martin Egli教授（范德比尔特大学）合作，使用紫外光谱和X射线晶体学技术确认酰胺连接的RNA可以模拟天然RNA的结构。3.与迪文泄漏博士（Dharmacon）合作，合成每条链3 '端具有几个酰胺键的siRNA，并测试其生物学特性和RNAi活性。酰胺可以为体内RNAi应用提供几个优点：（1）由于不存在天然磷酸盐而具有高核酸酶抗性;（2）由于负电荷的减少而增强的细胞摄取;（3）由于增加的疏水性而改善的生物分布和药代动力学。尽管有这些潜在的有益特性，但在RNAi中既没有测试酰胺也没有测试任何其他非离子键。如果被RNAi蛋白接受，酰胺可以显著改善siRNA的性质，并且可以用于设计一类新的化学修饰的siRNA。合成化学、结构研究和RNA生物学的结合将为化学修饰（酰胺）如何影响RNA的构象、水合作用和热稳定性提供独特的见解。这些知识对于合理设计核酸类似物和开发用于癌症、病毒感染、遗传病症和神经变性疾病等长期存在的问题的基因选择性治疗剂是重要的。

DESCRIPTION (provided by applicant): Discovery of RNA interference (RNAi) has reinvigorated interest in chemical modifications to optimize properties of small interfering RNAs (siRNAs). The long-term goal of our research is to explore RNA's structure and function using chemical approaches and to develop modifications for practical application in RNAi. The present proposal focuses on internucleoside amides as non-ionic mimics of the phosphodiester linkages and will test the hypotheses that amides (1) can be readily introduced in RNA using solid-phase synthesis, (2) are excellent mimics of the phosphate backbone of RNA, and (3) will increase enzymatic stability and cellular uptake of siRNAs without compromising their RNAi activity. We also envision that amides may improve biodistribution and pharmacokinetics of siRNAs. We propose an interdisciplinary (organic chemistry, structural biochemistry and RNA biology) study with the specific aims to: 1. Develop synthetic methods to introduce consecutive amide linkages at any desired location in RNA by adopting and optimizing solid-phase peptide, PNA, and RNA synthesis methods. 2. Confirm that amide-linked RNA can mimic the structure of natural RNA using UV spectroscopic and X- ray crystallographic techniques in collaboration with Prof. Martin Egli (Vanderbilt University). 3. Synthesize siRNAs having several amide linkages at the 3'-end of each strand and test their biological properties and RNAi activity in collaboration with Dr. Devin Leake (Dharmacon). Amides may offer several advantages for in vivo RNAi applications: (1) high nuclease resistance due to the absence of the natural phosphate; (2) enhanced cellular uptake due to the reduction of the negative charge; (3) improved biodistribution and pharmacokinetics due to the increased hydrophobicity. Despite these potentially beneficial properties, neither amides nor any other non-ionic linkages have been tested in RNAi. If accepted by RNAi proteins, amides may significantly improve properties of siRNAs and may be used to design a novel class of chemically modified siRNAs. Combination of synthetic chemistry, structural studies and RNA biology will provide unique insights into how chemical modifications (amides) influence conformation, hydration, and thermal stability of RNA. Such knowledge is important for rational design of nucleic acid analogues and for developing gene selective therapeutic agents for such long standing problems as cancer, viral infections, genetic disorders, and neurodegenerative diseases.