Herrera-Carrillo E, Harwig A, Berkhout B
Laboratory of Experimental Virology, Department of Medical Microbiology, Center for Infection and Immunity Amsterdam (CINIMA), Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands.
Gene Ther. 2017 Aug;24(8):453-461. doi: 10.1038/gt.2017.44. Epub 2017 May 29.
RNA interference (RNAi) is a sequence-specific gene silencing mechanism that is triggered by the expression of a short hairpin RNA (shRNA). shRNA molecules enter the RNAi pathway at the Dicer processing step. Recent studies indicated that the cellular microRNA miR-451 is not recognized by Dicer, but that it is processed instead by the Argonaute 2 (Ago2) protein. Subsequently, Dicer-independent shRNAs were described that rely on Ago2 for processing, as well as the subsequent silencing step. We called these AgoshRNA molecules because they depend on Ago2 both for maturation and activation. Processing of an AgoshRNA yields only a single active RNA strand, thus reducing the chance of adverse off-target effects induced by the passenger strand of regular shRNAs. In this study, we converted several anti-HIV-1 shRNAs into AgoshRNAs. Seven of the 21 designed AgoshRNAs were potent anti-HIV molecules, although their RNAi activity is generally somewhat reduced compared with the matching shRNAs. The AgoshRNA candidates revealed no cellular toxicity. This may relate to the absence of passenger strand expression, which was verified for these AgoshRNA candidates. Furthermore, we demonstrate that a toxic shRNA can be converted into a non-toxic AgoshRNA.
RNA干扰（RNAi）是一种序列特异性基因沉默机制，由短发夹RNA（shRNA）的表达触发。shRNA分子在Dicer加工步骤进入RNAi途径。最近的研究表明，细胞微小RNA miR-451不被Dicer识别，而是由Argonaute 2（Ago2）蛋白进行加工。随后，描述了不依赖Dicer的shRNA，其加工以及随后的沉默步骤依赖于Ago2。我们将这些分子称为AgoshRNA，因为它们的成熟和激活都依赖于Ago2。AgoshRNA的加工仅产生一条活性RNA链，从而降低了由常规shRNA的过客链诱导的不良脱靶效应的可能性。在本研究中，我们将几种抗HIV-1 shRNA转化为AgoshRNA。21种设计的AgoshRNA中有7种是有效的抗HIV分子，尽管与匹配的shRNA相比，它们的RNAi活性通常有所降低。AgoshRNA候选物未显示细胞毒性。这可能与过客链表达的缺失有关，这已在这些AgoshRNA候选物中得到证实。此外，我们证明了一种有毒的shRNA可以转化为无毒的AgoshRNA。
