Fluorescence-based high-throughput screening of dicer cleavage activity
Language English Country United States Media print-electronic
Document type Journal Article, Research Support, Non-U.S. Gov't
PubMed
23945873
DOI
10.1177/1087057113497400
PII: 1087057113497400
Knihovny.cz E-resources
- Keywords
- Dicer, high-throughput screening, siRNA,
- MeSH
- Cell Line MeSH
- Spectrometry, Fluorescence methods MeSH
- Small Molecule Libraries MeSH
- Humans MeSH
- Drug Discovery MeSH
- Reproducibility of Results MeSH
- Ribonuclease III antagonists & inhibitors genetics metabolism MeSH
- High-Throughput Screening Assays * MeSH
- Substrate Specificity MeSH
- Animals MeSH
- Check Tag
- Humans MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Names of Substances
- Small Molecule Libraries MeSH
- Ribonuclease III MeSH
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.
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