Monitoring Dicer‐Mediated miRNA‐21 Maturation and Ago2 Loading by a Dual‐Colour FIT PNA Probe Set

Abstract

Inhibition of micro RNA (miRNA) maturation by Dicer and loading of matured miRNAs into the RNA‐induced silencing complex (RISC) is envisioned as a modality for treatment of cancer. The existing methods for evaluation of maturation either focus on the conversion of modified precursors or detect the mature miRNA. While the former is not applicable to native pre‐miRNA the latter approach underestimates maturation when both non‐matured and matured miRNA molecules are subject to cleavage. We present a set of two orthogonally labeled FIT PNA probes that distinguishes between cleaved pre‐miRNA and the mature miRNA duplex. The probes allow monitoring Dicer‐mediated miR21 maturation and assaying Ago2‐mediated unwinding of the miR21 duplex. A two‐channel fluorescence readout enables measurements in real‐time without the need for specialized instrumentation or further enzyme mediated amplification.