Hesse Marlen, Davies Brian P, Arenz Christoph
Institute for Chemistry, Humboldt Universität zu Berlin, Berlin, Germany.
Methods Mol Biol. 2014;1095:95-102. doi: 10.1007/978-1-62703-703-7_7.
Assaying Dicer-mediated miRNA maturation is a valuable tool not only for validating miRNA maturation itself but also for testing Dicer activity in cell lysate and for screening small molecules inhibiting miRNA maturation in a high-throughput format. The classical assay for miRNA maturation relies on radioactive labeling of a pre-miRNA and subsequent gel electrophoresis and autoradiography. Here we present a fluorescently labeled and quenched pre-miRNA beacon that can be ligated easily out of two single labeled RNA strands. Upon Dicer cleavage of the beacon, fluorophore and quencher are separated, which results in an increase of fluorescence over time. Unlike (32)P-labeled probes, our fluorescently labeled pre-miRNA beacon is stable for at least 5 years under storage conditions. Dicer or miRNA maturation assays can be easily performed in a 384-well plate format, consuming less than 1 pmol of RNA beacon per reaction.
检测Dicer介导的miRNA成熟不仅是验证miRNA成熟本身的重要工具，也是检测细胞裂解物中Dicer活性以及高通量筛选抑制miRNA成熟的小分子的重要工具。经典的miRNA成熟检测方法依赖于对前体miRNA进行放射性标记，随后进行凝胶电泳和放射自显影。在此，我们展示了一种荧光标记且淬灭的前体miRNA信标，它可以很容易地由两条单标记的RNA链连接而成。在Dicer切割信标后，荧光团和淬灭剂分离，导致荧光随时间增加。与（32）P标记的探针不同，我们的荧光标记前体miRNA信标在储存条件下至少可稳定保存5年。Dicer或miRNA成熟检测可以很容易地在384孔板中进行，每个反应消耗的RNA信标少于1 pmol。
