Continuous fluorescence-based method for assessing Dicer cleavage efficiency reveals 3' overhang nucleotide preference

The cleavage of double-stranded RNA (dsRNA) molecules by Dicer is a critical step in silencing genes by the RNA induced silencing complex (RISC). The development of Dicer substrates as nucleic acid–based therapeutics brings about a need to rapidly evaluate chemically modified RNA molecules for their ability to be processed by Dicer. This study outlines a quantitative fluorescence quencher-based assay for studying the ability of Dicer substrates to be processed by Dicer. By using a dsRNA probe labeled with Cy5–Iowa Black RQ, a panel of unlabeled test substrates can be rapidly assessed in heterologous competition assays without the need for electrophoresis or radiolabeling. This assay was piloted by evaluation of 196 unlabeled 27-mer Dicer substrates with various overhang structures in a purified Dicer enzyme assay system. Results indicate that Dicer has no preference for the sequence of RNA in the main double-stranded region of the substrate. However, a preference for Dicer substrate RNAs (D-siRNAs) contai...