RNA interference (RNAi) offers great promise in life science research and therapeutic development, as it easily achieves a potent target gene knockdown with high specificity. Since the conventional small interfering RNA (siRNA) structure, known as 19 bp double-stranded RNA (dsRNA) with 2-nucleotide (nt) 3' overhang, has been introduced to successfully elicit the RNAi in mammalian cells, a variety of structural variants of RNAi trigger have been developed. Our group previously reported branched, tripodal interfering RNA (tiRNA) structures as a multigene targeting RNA structure inducing RNAi. However, the immune stimulatory effect of branched tiRNA structure has not been thoroughly evaluated. In this study, we show that tiRNA with blunt ends triggers innate immune response in T98G cell and mouse macrophage cells, which is dependent upon the retinoic acid-inducible gene I (RIG-I), a well-known cytoplasmic dsRNA sensor. Interestingly, immune response triggered by tiRNA can be suppressed by the introduction of 2-nt 3' overhang structure. Our finding expands the structural diversity of RIG-I ligands and provides a guide to develop a safe multitargeting RNA structure for therapeutic application.

中文翻译：

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具有3'突出端的三脚架RNA阻止RIG-I介导的先天免疫反应。

RNA干扰（RNAi）可以轻松实现具有高度特异性的有效靶基因敲除，因此在生命科学研究和治疗开发中具有广阔的前景。自从引入具有2个核苷酸（nt）3'突出端的称为19 bp双链RNA（dsRNA）的常规小分子干扰RNA（siRNA）结构成功在哺乳动物细胞中诱导RNAi以来，各种结构变异已开发出RNAi触发因子。我们的研究小组先前报道了分支，三脚架干扰RNA（tiRNA）结构作为靶向RNA结构的诱导RNAi的多基因。但是，尚未完全评估分支的tiRNA结构的免疫刺激作用。在这项研究中，我们显示了末端钝的tiRNA会触发T98G细胞和小鼠巨噬细胞的先天免疫应答，它依赖于视黄酸诱导基因I（RIG-I），一种众所周知的细胞质dsRNA传感器。有趣的是，通过引入2-nt 3'突出结构可以抑制由tiRNA触发的免疫反应。我们的发现扩展了RIG-1配体的结构多样性，并为开发用于治疗应用的安全多靶RNA结构提供了指导。