Berkhout Ben, Herrera-Carrillo Elena
Laboratory of Experimental Virology, Department of Medical Microbiology, Amsterdam UMC, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands.
Methods Mol Biol. 2021;2167:225-252. doi: 10.1007/978-1-0716-0716-9_13.
Since the first application of RNA interference (RNAi) in mammalian cells, the expression of short hairpin RNA (shRNA) molecules for targeted gene silencing has become a benchmark technology. Plasmid and viral vector systems can be used to express shRNA precursor transcripts that are processed by the cellular RNAi pathway to trigger sequence-specific gene knockdown. Intensive RNAi investigations documented that only a small percentage of computationally predicted target sequences can be used for efficient gene silencing, in part because not all shRNA designs are active. Many factors influence the shRNA activity and guidelines for optimal shRNA design have been proposed. We recently described an alternatively processed shRNA molecule termed AgoshRNA with a ~18 base pairs (bp) stem and a 3-5 nucleotides (nt) loop. This molecule is alternatively processed by the Argonaute (Ago) protein into a single guide RNA strand that efficiently induces the RNAi mechanism. The design rules proposed for regular shRNAs do not apply to AgoshRNA molecules and therefore new rules had to be defined. We optimized the AgoshRNA design and managed to create a set of active AgoshRNAs targeted against the human immunodeficiency virus (HIV). In an attempt to enhance the silencing activity of the AgoshRNA molecules, we included the hepatitis delta virus (HDV) ribozyme at the 3' terminus, which generates a uniform 3' end instead of a 3' U-tail of variable length. We evaluated the impact of this 3'-end modification on AgoshRNA processing and its gene silencing activity and we demonstrate that this novel AgoshRNA-HDV design exhibits enhanced antiviral activity.
自RNA干扰（RNAi）首次应用于哺乳动物细胞以来，用于靶向基因沉默的短发夹RNA（shRNA）分子的表达已成为一项基准技术。质粒和病毒载体系统可用于表达shRNA前体转录本，这些转录本通过细胞RNAi途径进行加工，以触发序列特异性基因敲低。大量的RNAi研究表明，只有一小部分通过计算预测的靶序列可用于高效的基因沉默，部分原因是并非所有的shRNA设计都具有活性。许多因素会影响shRNA的活性，并且已经提出了优化shRNA设计的指导原则。我们最近描述了一种经过另类加工的shRNA分子，称为AgoshRNA，其具有约18个碱基对（bp）的茎和3 - 5个核苷酸（nt）的环。该分子由Argonaute（Ago）蛋白另类加工成单链引导RNA，从而有效地诱导RNAi机制。针对常规shRNA提出的设计规则不适用于AgoshRNA分子，因此必须定义新的规则。我们优化了AgoshRNA的设计，并成功创建了一组针对人类免疫缺陷病毒（HIV）的活性AgoshRNA。为了增强AgoshRNA分子的沉默活性，我们在3'末端加入了丁型肝炎病毒（HDV）核酶，它会产生一个统一的3'末端，而不是可变长度的3' U尾。我们评估了这种3'末端修饰对AgoshRNA加工及其基因沉默活性的影响，并且证明这种新型的AgoshRNA - HDV设计具有增强的抗病毒活性。
