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

• Published in: Chembiochem : a European journal of chemical biology Vol. 21; no. 17; pp. 2527 - 2532
• Main Authors: Loibl, Natalia, Arenz, Christoph, Seitz, Oliver
• Format: Journal Article
• Language: English
• Published: Germany Wiley Subscription Services, Inc 01.09.2020; John Wiley and Sons Inc
• Subjects: Argonaute 2 protein; Argonaute Proteins - chemistry; Argonaute Proteins - metabolism; Color; Dicer; DNA probes; FIT PNA probes; Fluorescence; Fluorescent Dyes - chemical synthesis; Fluorescent Dyes - chemistry; Humans; Instrumentation; Maturation; MicroRNAs - analysis; MicroRNAs - metabolism; miR21; miRNA; miRNA maturation; pre-miR21; Probes; Ribonucleic acid; RNA; RNA-Induced Silencing Complex - chemistry; RNA-Induced Silencing Complex - metabolism; RNA-mediated interference; Unwinding; FIT PNA probes; miRNA maturation; Dicer; miR21; pre-miR21
• ISSN: 1439-4227; 1439-7633; 1439-7633
• DOI: 10.1002/cbic.202000173

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. Combined use of two orthogonally labelled FIT PNA probes allows unambiguous monitoring of miRNA‐21 maturation. One fluorogenic hybridization probe signals the presence of intact pre‐miR21, the other probe shows enhanced fluorescence upon formation of the matured miRNA passenger strand. PNA‐FIT probes also provide a read‐out for monitoring the Ago2‐mediated unwinding of the miR21 duplex in real‐time.