We recently reported that RNAi-mediated off-target effects are important drivers of the hepatotoxicity observed for a subset of GalNAc–siRNA conjugates in rodents, and that these findings could be mitigated by seed-pairing destabilization using a single GNA nucleotide placed within the seed region of the guide strand. Here, we report further investigation of the unique and poorly understood GNA/RNA cross-pairing behavior to better inform GNA-containing siRNA design. A reexamination of published GNA homoduplex crystal structures, along with a novel structure containing a single (S)-GNA-A residue in duplex RNA, indicated that GNA nucleotides universally adopt a rotated nucleobase orientation within all duplex contexts. Such an orientation strongly affects GNA-C and GNA-G but not GNA-A or GNA-T pairing in GNA/RNA heteroduplexes. Transposition of the hydrogen-bond donor/acceptor pairs using the novel (S)-GNA-isocytidine and -isoguanosine nucleotides could rescue productive base-pairing with the complementary G or C ribonucleotides, respectively. GalNAc-siRNAs containing these GNA isonucleotides showed an improved in vitro activity, a similar improvement in off-target profile, and maintained in vivo activity and guide strand liver levels more consistent with the parent siRNAs than those modified with isomeric GNA-C or -G, thereby expanding our toolbox for the design of siRNAs with minimized off-target activity.

中文翻译：

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使用异核苷酸克服 GNA/RNA 碱基配对限制可提高 ESC+ GalNAc-siRNA 的药效学活性

我们最近报道，RNAi 介导的脱靶效应是在啮齿动物中观察到的一部分 GalNAc-siRNA 缀合物的肝毒性的重要驱动因素，并且这些发现可以通过使用放置在种子中的单个 GNA 核苷酸的种子配对失稳来减轻引导链的区域。在这里，我们报告了对独特且知之甚少的 GNA/RNA 交叉配对行为的进一步调查，以更好地为含 GNA 的 siRNA 设计提供信息。重新检查已发表的 GNA 同源双链晶体结构，以及在双链 RNA 中含有单个 (S)-GNA-A 残基的新结构，表明 GNA 核苷酸在所有双链环境中普遍采用旋转的核碱基方向。这种方向强烈影响 GNA-C 和 GNA-G，但不影响 GNA/RNA 异源双链中的 GNA-A 或 GNA-T 配对。使用新的 (S)-GNA-异胞苷和异鸟苷核苷酸对氢键供体/受体对进行转座可以分别挽救与互补 G 或 C 核糖核苷酸的生产性碱基配对。与用异构体 GNA-C 或 -G 修饰的 siRNA 相比，含有这些 GNA 异核苷酸的 GalNAc-siRNA 显示出改善的体外活性，类似的脱靶特征改善，并保持体内活性和引导链肝水平与亲本 siRNA 更一致，从而扩展了我们的工具箱，用于设计具有最小化脱靶活性的 siRNA。