Lee Hye-Sook, Seok Heeyoung, Lee Dong Ha, Ham Juyoung, Lee Wooje, Youm Emilia Moonkyung, Yoo Jin Seon, Lee Yong-Seung, Jang Eun-Sook, Chi Sung Wook
Division of Life Sciences, College of Life Sciences and Biotechnology, Korea University, Seoul 136-701, Korea.
Department of Health Sciences and Technology, Samsung Advanced Institute for Health Sciences and Technology, Sungkyunkwan University, Seoul 135-710, Korea.
Nat Commun. 2015 Dec 18;6:10154. doi: 10.1038/ncomms10154.
Gene silencing via RNA interference inadvertently represses hundreds of off-target transcripts. Because small interfering RNAs (siRNAs) can function as microRNAs, avoiding miRNA-like off-target repression is a major challenge. Functional miRNA-target interactions are known to pre-require transitional nucleation, base pairs from position 2 to the pivot (position 6). Here, by substituting nucleotide in pivot with abasic spacers, which prevent base pairing and alleviate steric hindrance, we eliminate miRNA-like off-target repression while preserving on-target activity at ∼ 80-100%. Specifically, miR-124 containing dSpacer pivot substitution (6pi) loses seed-mediated transcriptome-wide target interactions, repression activity and biological function, whereas other conventional modifications are ineffective. Application of 6pi allows PCSK9 siRNA to efficiently lower plasma cholesterol concentration in vivo, and abolish potentially deleterious off-target phenotypes. The smallest spacer, C3, also shows the same improvement in target specificity. Abasic pivot substitution serves as a general means to harness the specificity of siRNA experiments and therapeutic applications.
通过RNA干扰实现的基因沉默会意外地抑制数百种脱靶转录本。由于小干扰RNA（siRNA）可以发挥微小RNA（miRNA）的功能，避免类似miRNA的脱靶抑制是一项重大挑战。已知功能性miRNA-靶标相互作用预先需要过渡成核，即从第2位到枢轴位置（第6位）的碱基对。在这里，我们通过用无碱基间隔物取代枢轴位置的核苷酸，这种间隔物可防止碱基配对并减轻空间位阻，从而消除了类似miRNA的脱靶抑制，同时保持了约80%-100%的靶向活性。具体而言，含有dSpacer枢轴取代（6pi）的miR-124失去了种子介导的全转录组范围的靶标相互作用、抑制活性和生物学功能，而其他常规修饰则无效。应用6pi可使PCSK9 siRNA在体内有效降低血浆胆固醇浓度，并消除潜在有害的脱靶表型。最小的间隔物C3在靶标特异性方面也表现出相同的改善。无碱基枢轴取代是一种利用siRNA实验和治疗应用特异性的通用方法。
