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 （sirna）特性的兴趣。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。目前的建议侧重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)可以很容易地通过固相合成引入RNA中，(2)是RNA磷酸主链的极好模拟物，(3)将增加酶的稳定性和sirna的细胞摄取，而不影响其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。通过采用和优化固相肽、PNA和RNA合成方法，开发合成方法，在RNA的任何期望位置引入连续的酰胺键。2. 与范德比尔特大学的Martin Egli教授合作，利用紫外光谱和X射线晶体学技术证实酰胺链RNA可以模拟天然RNA的结构。3. 与Dr. Devin Leake （Dharmacon）合作，在每条链的3'端合成具有多个酰胺键的sirna，并测试其生物学特性和RNAi活性。酰胺类化合物可能为体内RNAi应用提供了几个优势：(1)由于缺乏天然磷酸盐，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，生物分布和药代动力学得到改善。尽管有这些潜在的有益特性，酰胺和任何其他非离子键都没有在RNAi中测试过。如果被RNAi蛋白接受，酰胺可以显著改善sirna的性质，并可用于设计一类新的化学修饰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.