Department of Chemistry, University of California, Davis , One Shields Ave, Davis, California 95616, United States.
J Am Chem Soc. 2013 Nov 13;135(45):17069-77. doi: 10.1021/ja4079754. Epub 2013 Nov 5.
Short interfering RNAs (siRNAs) are promising drug candidates for a wide range of targets including those previously considered "undruggable". However, properties associated with the native RNA structure limit drug development, and chemical modifications are necessary. Here we describe the structure-guided discovery of functional modifications for the guide strand 5'-end using computational screening with the high-resolution structure of human Ago2, the key nuclease on the RNA interference pathway. Our results indicate the guide strand 5'-end nucleotide need not engage in Watson-Crick (W/C) H-bonding but must fit the general shape of the 5'-end binding site in MID/PIWI domains of hAgo2 for efficient knockdown. 1,2,3-Triazol-4-yl bases formed from the CuAAC reaction of azides and 1-ethynylribose, which is readily incorporated into RNA via the phosphoramidite, perform well at the guide strand 5'-end. In contrast, purine derivatives with modified Hoogsteen faces or N2 substituents are poor choices for 5'-end modifications. Finally, we identified a 1,2,3-triazol-4-yl base incapable of W/C H-bonding that performs well at guide strand position 12, where base pairing to target was expected to be important. This work expands the repertoire of functional nucleotide analogues for siRNAs.
短干扰 RNA（siRNA）是一种很有前途的候选药物，可用于多种靶点，包括以前被认为“不可成药”的靶点。然而，与天然 RNA 结构相关的特性限制了药物的开发，需要进行化学修饰。在这里，我们使用高分辨率的人 Ago2 结构进行计算筛选，描述了针对向导链 5'端的功能修饰的结构导向发现。Ago2 是 RNA 干扰途径中的关键核酸酶。我们的结果表明，向导链 5'端核苷酸不需要参与 Watson-Crick（W/C）氢键，但必须适合 hAgo2 的 MID/PIWI 结构域中 5'端结合位点的一般形状，以实现有效的敲低。叠氮化物和 1-乙炔核糖通过 CuAAC 反应形成的 1,2,3-三唑-4-基碱基很容易通过亚磷酰胺掺入 RNA，在向导链 5'端表现良好。相比之下，具有修饰的 Hoogsteen 面或 N2 取代基的嘌呤衍生物不是 5'端修饰的理想选择。最后，我们确定了一种不能进行 W/C 氢键的 1,2,3-三唑-4-基碱基，它在向导链位置 12 处表现良好，预计碱基配对到靶标在该处很重要。这项工作扩展了 siRNA 的功能性核苷酸类似物的范围。
