Department of Chemistry, Graduate School of Science, Nagoya University, Furo-cho, Chikusa-ku, Nagoya, Aichi 464-8602, Japan.
Department of Computational Biology and Medical Sciences, Graduate School of Frontier Sciences, The University of Tokyo, Chiba 277-8561, Japan.
Nucleic Acids Res. 2024 Oct 14;52(18):10754-10774. doi: 10.1093/nar/gkae741.
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.
在这项研究中，我们报告了 2'-甲酰胺核苷亚磷酰胺衍生物的合成及其在 siRNA 链中的掺入，以降低小干扰 RNA（siRNA）的基于种子的脱靶效应。所有四种核苷（A、G、C 和 U）的甲酰胺衍生物均由商业化合物经 5-11 步合成得到。将这些衍生物引入双链 RNA 中会略微降低其热力学稳定性，但 X 射线晶体学和 CD 光谱分析证实 RNA 保持其天然的 A 型结构。尽管在 siRNA 向导链的第 2 位引入 2'-甲酰胺核苷衍生物会导致针对靶 RNAi 活性略有下降，但在种子区域的第 2 位以外的任何位置引入带有四种碱基的 2'-甲酰胺核苷的不同序列的 siRNA 会显著抑制脱靶活性，同时保持针对靶 RNAi 活性。这表明 2'-甲酰胺核苷代表了一种有前途的方法，可以减轻 siRNA 治疗中的脱靶效应。
