Zhou Chuanzheng, Liu Yi, Andaloussi Mounir, Badgujar Naresh, Plashkevych Oleksandr, Chattopadhyaya Jyoti
Department of Bioorganic Chemistry, Box 581, Biomedical Center, Uppsala University, SE-751 23 Uppsala, Sweden.
J Org Chem. 2009 Jan 2;74(1):118-34. doi: 10.1021/jo8016742.
In the antisense (AS) and RNA interference (RNAi) technologies, the native single-stranded 2'-deoxyoligonucleotides (for AS) or double-stranded RNA (for RNAi) are chemically modified to bind to the target RNA in order to give improved downregulation of gene expression through inhibition of RNA translation. It is shown here how the fine adjustment of the electrostatic interaction by alteration of the substituents as well as their stereochemical environment around the internucleotidic phosphodiester moiety near the edge of the minor grove of the antisense oligonucleotides (AON)-RNA heteroduplex can lead to the modulation of the antisense properties. This was demonstrated through the synthesis of various modified carbocyclic-locked nucleic acids (LNAs) and -ethylene-bridged nucleic acids (ENAs) with hydroxyl and/or methyl substituents attached at the carbocyclic part and their integration into AONs by solid-phase DNA synthesis. The target affinity toward the complementary RNA and DNA, nuclease resistance, and RNase H elicitation by these modified AONs showed that both the nature of the modification (-OH versus -CH(3)) and their respective stereochemical orientations vis-a-vis vicinal phosphate play a very important role in modulating the AON properties. Whereas the affinity to the target RNA and the enzymatic stability of AONs were not favored by the hydrophobic and sterically bulky modifications in the center of the minor groove, their positioning at the edge of the minor groove near the phosphate linkage resulted in significantly improved nuclease resistance without loss of target affinity. On the other hand, hydrophilic modification, such as a hydroxyl group, close to the phosphate linkage made the internucleotidic phosphodiester especially nucleolytically unstable, and hence was not recommended. The substitutions on the carbocyclic moiety of the carba-LNA and -ENA did not affect significantly the choice of the cleavage sites of RNase H mediated RNA cleavage in the AON/RNA hybrid duplex, but the cleavage rate depended on the modification site in the AON sequence. If the original preferred cleavage site by RNase H was included in the 4-5nt stretch from the 3'-end of the modification site in the AON, decreased cleavage rate was observed. Upon screening of 52 modified AONs, containing 13 differently modified derivatives at C6' and C7' (or C8') of the carba-LNAs and -ENAs, two excellent modifications in the carba-LNA series were identified, which synergistically gave outstanding antisense properties such as the target RNA affinity, nuclease resistance, and RNase H activity and were deemed to be ideal candidates as potential antisense or siRNA therapeutic agents.
在反义（AS）技术和RNA干扰（RNAi）技术中，天然的单链2'-脱氧寡核苷酸（用于AS）或双链RNA（用于RNAi）会进行化学修饰，以与靶RNA结合，从而通过抑制RNA翻译来增强基因表达的下调作用。本文展示了如何通过改变反义寡核苷酸（AON）-RNA异源双链体小沟边缘附近核苷酸间磷酸二酯部分周围的取代基及其立体化学环境，对静电相互作用进行精细调节，进而实现对反义特性的调控。这是通过合成各种在碳环部分带有羟基和/或甲基取代基的修饰碳环锁定核酸（LNA）和乙烯桥连核酸（ENA），并通过固相DNA合成将它们整合到AON中来证明的。这些修饰的AON对互补RNA和DNA的靶标亲和力、核酸酶抗性以及RNase H诱导作用表明，修饰的性质（-OH对-CH(3)）及其相对于相邻磷酸的各自立体化学取向在调节AON特性方面起着非常重要的作用。虽然小沟中心的疏水和空间位阻较大的修饰不利于AON对靶RNA的亲和力和酶稳定性，但它们位于靠近磷酸连接的小沟边缘时，能显著提高核酸酶抗性且不损失靶标亲和力。另一方面，靠近磷酸连接的亲水性修饰，如羟基，会使核苷酸间磷酸二酯特别容易被核酸酶降解，因此不推荐使用。碳环-LNA和-ENA碳环部分的取代对AON/RNA杂交双链体中RNase H介导的RNA切割的切割位点选择没有显著影响，但切割速率取决于AON序列中的修饰位点。如果RNase H原来优先的切割位点包含在AON修饰位点3'端的4 - 5个核苷酸范围内，则会观察到切割速率降低。在筛选了52个修饰的AON后，这些AON在碳环-LNA和-ENA的C6'和C7'（或C8'）处含有13种不同修饰的衍生物，在碳环-LNA系列中鉴定出了两种优异的修饰，它们协同赋予了出色的反义特性，如靶RNA亲和力、核酸酶抗性和RNase H活性，被认为是潜在反义或siRNA治疗剂的理想候选物。
