Goguen Ryan P, Gatignol Anne, Scarborough Robert J
Lady Davis Institute for Medical Research, Montréal, QC, Canada.
Department of Microbiology and Immunology, McGill University, Montréal, QC, Canada.
Methods Mol Biol. 2021;2167:253-267. doi: 10.1007/978-1-0716-0716-9_14.
RNA aptamers can be used to target proteins or nucleic acids for therapeutic purposes and are candidates for RNA-mediated gene therapy. Like other small therapeutic RNAs, they can be expressed in cells from DNA templates that include a cellular promoter upstream of the RNA coding sequence. Secondary structures flanking aptamers can be used to enhance the activity or stability of these molecules. Notably, flanking self-cleaving ribozymes to remove extraneous nucleotides included during transcription as well as flanking hairpins to improve RNA stability have been used to increase the effect of therapeutic aptamers. Here we describe the cloning procedure of aptamers containing different flanking secondary structures and methods to compare their expression levels by a northern blot protocol optimized for the detection of small RNA molecules.
RNA适配体可用于靶向蛋白质或核酸以实现治疗目的，是RNA介导的基因治疗的候选物。与其他小型治疗性RNA一样，它们可以在细胞中从DNA模板表达，该模板在RNA编码序列上游包含一个细胞启动子。适配体侧翼的二级结构可用于增强这些分子的活性或稳定性。值得注意的是，侧翼的自我切割核酶用于去除转录过程中包含的多余核苷酸，以及侧翼发夹结构用于提高RNA稳定性，已被用于增强治疗性适配体的效果。在这里，我们描述了含有不同侧翼二级结构的适配体的克隆程序，以及通过针对小RNA分子检测优化的Northern印迹法比较其表达水平的方法。
