Fluorescence-based high-throughput screening of dicer cleavage activity.
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.
1Institute of Molecular Genetics of the ASCR, v.v.i., Prague 4, Czech Republic.
Katerina
K
Podolska
David
D
Sedlak
Petr
P
Bartunek
Petr
P
Svoboda
D000818:Animals; D002460:Cell Line; D055808:Drug Discovery; D057166:High-Throughput Screening Assays; D006801:Humans; D015203:Reproducibility of Results; D043244:Ribonuclease III / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism*; D054852:Small Molecule Libraries; D013050:Spectrometry, Fluorescence / Q000379:methods*; D013379:Substrate Specificity
D054852:Small Molecule Libraries; D043244:Ribonuclease III
Dicer; high-throughput screening; siRNA