Jani Saumya, Jackson Alexis, Davies-Sala Carol, Chiem Kevin, Soler-Bistué Alfonso, Zorreguieta Angeles, Tolmasky Marcelo E
Center for Applied Biotechnology Studies, College of Natural Sciences and Mathematics, California State University Fullerton, Fullerton, CA, USA.
Fundación Instituto Leloir, IIBBA-CONICET, and FCEyN, University of Buenos Aires, Aires, Argentina.
Methods Mol Biol. 2018;1737:89-98. doi: 10.1007/978-1-4939-7634-8_6.
RNase P is a ribozyme consisting of a catalytic RNA molecule and, depending on the organism, one or more cofactor proteins. It was initially identified as the enzyme that mediates cleavage of precursor tRNAs at the 5'-end termini to generate the mature tRNAs. An important characteristic of RNase P is that its specificity depends on the structure rather than the sequence of the RNA substrate. Any RNA species that interacts with an antisense molecule (called external guide sequence, EGS) and forms the appropriate structure can be cleaved by RNase P. This property is the basis for EGS technology, an antisense methodology for inhibiting gene expression by eliciting RNase P-mediated cleavage of a target mRNA molecule. EGS technology is being developed to design therapies against a large variety of diseases. An essential milestone in developing EGSs as therapies is the assessment of the efficiency of antisense molecules to induce cleavage of the target mRNA and evaluate their effect in vivo. Here, we describe simple protocols to test the ability of EGSs to induce cleavage of a target mRNA in vitro and to induce a phenotypic change in growing cells.
核糖核酸酶P是一种核酶，由一个催化RNA分子以及根据生物体不同而有的一种或多种辅助因子蛋白组成。它最初被鉴定为在5'-末端介导前体tRNA切割以产生成熟tRNA的酶。核糖核酸酶P的一个重要特征是其特异性取决于RNA底物的结构而非序列。任何与反义分子（称为外部引导序列，EGS）相互作用并形成适当结构的RNA种类都可以被核糖核酸酶P切割。这一特性是EGS技术的基础，EGS技术是一种通过引发核糖核酸酶P介导的靶标mRNA分子切割来抑制基因表达的反义方法。EGS技术正在被开发用于设计针对多种疾病的疗法。将EGS开发为疗法的一个关键里程碑是评估反义分子诱导靶标mRNA切割的效率并评估它们在体内的作用。在此，我们描述了简单的方案来测试EGS在体外诱导靶标mRNA切割以及在生长细胞中诱导表型变化的能力。
