Mutisya Daniel, Selvam Chelliah, Lunstad Benjamin D, Pallan Pradeep S, Haas Amanda, Leake Devin, Egli Martin, Rozners Eriks
Department of Chemistry, Binghamton University, The State University of New York, Binghamton, NY 13902, USA.
Global Research and Development in Molecular Biology, Thermo Fisher Scientific Bioscience Division, Lafayette, CO 80026, USA.
Nucleic Acids Res. 2014 Jun;42(10):6542-51. doi: 10.1093/nar/gku235. Epub 2014 May 9.
RNA interference (RNAi) has become an important tool in functional genomics and has an intriguing therapeutic potential. However, the current design of short interfering RNAs (siRNAs) is not optimal for in vivo applications. Non-ionic phosphate backbone modifications may have the potential to improve the properties of siRNAs, but are little explored in RNAi technologies. Using X-ray crystallography and RNAi activity assays, the present study demonstrates that 3'-CH2-CO-NH-5' amides are excellent replacements for phosphodiester internucleoside linkages in RNA. The crystal structure shows that amide-modified RNA forms a typical A-form duplex. The amide carbonyl group points into the major groove and assumes an orientation that is similar to the P-OP2 bond in the phosphate linkage. Amide linkages are well hydrated by tandem waters linking the carbonyl group and adjacent phosphate oxygens. Amides are tolerated at internal positions of both the guide and passenger strand of siRNAs and may increase the silencing activity when placed near the 5'-end of the passenger strand. As a result, an siRNA containing eight amide linkages is more active than the unmodified control. The results suggest that RNAi may tolerate even more extensive amide modification, which may be useful for optimization of siRNAs for in vivo applications.
RNA干扰（RNAi）已成为功能基因组学中的一项重要工具，具有引人关注的治疗潜力。然而，目前的小干扰RNA（siRNA）设计对于体内应用而言并非最优。非离子型磷酸骨架修饰可能具有改善siRNA性质的潜力，但在RNAi技术中却鲜有探索。通过X射线晶体学和RNAi活性测定，本研究表明3'-CH2-CO-NH-5'酰胺是RNA中磷酸二酯核苷间连接的优异替代物。晶体结构显示，酰胺修饰的RNA形成典型的A-型双链体。酰胺羰基指向大沟，并呈现出与磷酸连接中P-OP2键相似的取向。酰胺键通过连接羰基和相邻磷酸氧的串联水得到良好的水合作用。酰胺在siRNA的引导链和过客链的内部位置均可耐受，并且当置于过客链5'-端附近时可能会增强沉默活性。因此，含有八个酰胺键的siRNA比未修饰的对照更具活性。结果表明，RNAi可能耐受甚至更广泛的酰胺修饰，这对于优化用于体内应用的siRNA可能是有用的。
