In this study, we report the synthesis of 2′-formamidonucleoside phosphoramidite derivatives and their incorporation into siRNA strands to reduce seed-based off-target effects of small interfering RNAs (siRNAs). Formamido derivatives of all four nucleosides (A, G, C and U) were synthesized in 5–11 steps from commercial compounds. Introducing these derivatives into double-stranded RNA slightly reduced its thermodynamic stability, but X-ray crystallography and CD spectrum analysis confirmed that the RNA maintained its natural A-form structure. Although the introduction of the 2′-formamidonucleoside derivative at the 2nd position in the guide strand of the siRNA led to a slight decrease in the on-target RNAi activity, the siRNAs with different sequences incorporating 2′-formamidonucleoside with four kinds of nucleobases into any position other than 2nd position in the seed region revealed a significant suppression of off-target activity while maintaining on-target RNAi activity. This indicates that 2′-formamidonucleosides represent a promising approach for mitigating off-target effects in siRNA therapeutics.

中文翻译：

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合成 2′-甲酰胺核酸糖苷亚磷酰胺用于抑制 siRNA 基于种子的脱靶效应


在这项研究中，我们报道了 2'-甲酰胺核苷亚磷酰胺衍生物的合成及其掺入 siRNA 链以减少小干扰 RNA （siRNA） 基于种子的脱靶效应。所有四种核苷（A、G、C 和 U）的甲酰胺衍生物均以 5-11 步从商业化合物合成。将这些衍生物引入双链 RNA 中略微降低了其热力学稳定性，但 X 射线晶体学和 CD 光谱分析证实 RNA 保持了其天然的 A 型结构。尽管在 siRNA 引导链的第 2 位引入 2'-甲酰胺核苷衍生物导致靶向 RNAi 活性略有降低，但将 2'-甲酰胺核酸苷与四种核碱基掺入种子区域第 2 位以外的任何位置的具有不同序列的 siRNA 显示出对脱靶活性的显着抑制，同时保持对靶向 RNAi 活性。这表明 2′-甲酰胺核苷代表了减轻 siRNA 治疗药物中脱靶效应的一种有前途的方法。