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

描述(由申请人提供)：RNA干扰(RNAi)的发现重新激发了人们对化学修饰以优化小干扰RNA(SiRNAs)特性的兴趣。我们研究的长期目标是用化学方法探索RNA的结构和功能，并为RNAi的实际应用开发修饰。本提案将重点放在核苷酰胺作为磷酸二酯键的非离子模拟物上，并将检验以下假设：(1)通过固相合成可以很容易地将酰胺引入RNA中，(2)是RNA磷酸骨架的优秀模拟物，(3)在不影响其RNAi活性的情况下提高酶的稳定性和细胞对siRNAs的摄取。我们还设想，酰胺可以改善siRNAs的生物分布和药代动力学。我们提出了一项跨学科(有机化学、结构生物化学和RNA生物学)的研究，其具体目标是：1.通过采用和优化固相肽、PNA和RNA合成方法，开发在RNA中任意位置引入连续酰胺键的合成方法。2.与Vanderbilt大学的Martin Egli教授合作，利用紫外光谱和X射线结晶学技术证实了酰胺连接的RNA可以模拟天然RNA的结构。3.合成在每条链的3‘端有几个酰胺键的siRNAs，并与Devin Leake(Dharamacon)博士合作测试它们的生物学特性和RNAi活性。酰胺类药物可用于体内RNAi应用：(1)由于没有天然磷酸盐而具有较高的核酸酶抗性；(2)由于负电荷的减少而增强细胞摄取；(3)由于疏水性增加而改善生物分布和药代动力学。尽管有这些潜在的有益性质，但酰胺或任何其他非离子连接都没有在RNAi中进行测试。如果被RNAi蛋白质接受，酰胺可以显著改善siRNAs的性质，并可用于设计一类新型的化学修饰的siRNAs。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、水化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以及开发针对癌症、病毒感染、遗传疾病和神经退行性疾病等长期存在的问题的基因选择性治疗药物具有重要意义。

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.