Fluorescence-Based High-Throughput Screening of Dicer Cleavage Activity

Abstract

Production of small RNAs by ribonuclease III Dicer is a key step in microRNA and RNA interference pathways, which employ Dicer-produced small RNAs as sequence-specific silencing guides. Further studies and manipulations of microRNA and RNA interference pathways would benefit from identification of small-molecule modulators. Here, we report a study of a fluorescence-based in vitro Dicer cleavage assay, which was adapted for high-throughput screening. The kinetic assay can be performed under single-turnover conditions (35 nM substrate and 70 nM Dicer) in a small volume (5 µL), which makes it suitable for high-throughput screening in a 1536-well format. As a proof of principle, a small library of bioactive compounds was analyzed, demonstrating potential of the assay.

Biochemical Comparison of dsRNA Degrading Nucleases in Four Different Insects

“…The continuous fluorescence intensity measurement method developed for the Dicer cleavage assay was first reported by Podolska et al (2014) . The principle is illustrated in Figure 1 .…”

“…In this study, the continuous fluorescence intensity measurement method developed by Podolska et al (2014) for Dicer cleavage assays was adopted and modified for monitoring dsRNA degrading activity. Owing to different combinations of fluorophores and quenchers with varying fluorescent intensity ( Podolska et al, 2014 ), selection experiments were performed, which found 5-FAM fluorescent donor- and 3-BHQ1 quencher-labeled 24 bp dsRNA to be the most sensitive substrate. With this substrate, the fluorescence method was validated using traditional gel electrophoresis and a well-accepted micro-quantitative PCR method.…”

“…This method has the disadvantage of being impractical when large quantities of samples must be analyzed. However, the continuous fluorescence intensity measurement method developed for Dicer cleavage assays is applicable for such analyses ( Podolska et al, 2014 ). Here, we modified the method by selecting a suitable fluorophore and quencher pair and developed a fluorescence method for quantification of dsRNA degrading activity ( Figure 1 ).…”

Development and Implementation of an HTS-Compatible Assay for the Discovery of Selective Small-Molecule Ligands for Pre-microRNAs

“…This finding was also observed by researchers who carried out a fluorescence-based assay of Dicer cleavage of a long, double-stranded RNA substrate. 32 Thus, tetracyclines may be a better scaffold for the future rational design of potentially selective pre-miRNA binders. Of the remaining small-molecule hits ( Fig.…”

Cell-Based Reporter System for High-Throughput Screening of MicroRNA Pathway Inhibitors and Its Limitations

“…Of the nine previously reported compounds that showed inhibitory activity in HTS but were not picked up for dose-response analysis, two were anthracyclines (doxorubicin and daunorubicin), which were reported to interfere with pre-miRNA processing by Dicer (Maiti et al, 2012 ). While our earlier HTS for Dicer inhibitors (Podolska et al, 2014 ) did not identify these compounds (no overlap was found between Bioactives affecting Dicer in vitro and luciferase reporters in vivo ), two other anthracyclines showed inhibitory effects in HTS. One of them (idarubicin) yielded at least three-fold luciferase stimulation in four miRNA-sensitive reporter assays, while it showed a weak stimulation (maximum 2.1-fold) of only one of the three non-targeted reporters (Figure 7B ), which makes anthracyclines interesting candidates for further studies.…”