Department of Chemistry, Humbolt-Universität zu Berlin, Brook-Taylor-Strase 2, 12489, Berlin, Germany.
Chembiochem. 2020 Sep 1;21(17):2527-2532. doi: 10.1002/cbic.202000173. Epub 2020 May 26.
The inhibition of micro RNA (miRNA) maturation by Dicer and loading matured miRNAs into the RNA-induced silencing complex (RISC) is envisioned as a modality for treatment of cancer. Existing methods for evaluating maturation either focus on the conversion of modified precursors or detect mature miRNA. Whereas the former is not applicable to native pre-miRNA, the latter approach underestimates maturation when both nonmatured and matured miRNA molecules are subject to cleavage. We present a set of two orthogonally labelled FIT PNA probes that distinguish between cleaved pre-miRNA and the mature miRNA duplex. The probes allow Dicer-mediated miR21 maturation to be monitored and Ago2-mediated unwinding of the miR21 duplex to be assayed. A two-channel fluorescence readout enables measurement in real-time without the need for specialized instrumentation or further enzyme mediated amplification.
通过 Dicer 抑制微 RNA (miRNA) 的成熟并将成熟的 miRNA 装载到 RNA 诱导的沉默复合物 (RISC) 中，被认为是治疗癌症的一种方式。现有的评估成熟度的方法要么专注于修饰前体的转化，要么检测成熟的 miRNA。虽然前者不适用于天然的 pre-miRNA，但当非成熟和成熟的 miRNA 分子都受到切割时，后者的方法会低估成熟度。我们提出了一组正交标记的 FIT PNA 探针，它们可以区分切割的 pre-miRNA 和成熟的 miRNA 双链体。这些探针允许监测 Dicer 介导的 miR21 成熟，并测定 miR21 双链体的 Ago2 介导的解旋。双通道荧光读数可实现实时测量，无需专用仪器或进一步的酶介导扩增。
