Related Experiment Video
Updated: May 8, 2026
08:16
In Vitro Cleavage Assays using Purified Recombinant Drosophila Caspases for Substrate Screening
Published on: October 6, 2022
Fluorescence-based high-throughput screening of dicer cleavage activity.
Katerina Podolska1, David Sedlak, Petr Bartunek
11Institute of Molecular Genetics of the ASCR, v.v.i., Prague 4, Czech Republic.
Journal of Biomolecular Screening
|August 16, 2013
Summary
Researchers developed a high-throughput screening assay to identify small-molecule modulators of Dicer, an enzyme crucial for microRNA and RNA interference pathways. This assay enables efficient discovery of compounds that regulate gene silencing mechanisms.
Area of Science:
- Molecular Biology
- Biochemistry
- Genetics
Background:
- Dicer enzyme is essential for microRNA (miRNA) and RNA interference (RNAi) pathways.
- miRNA and RNAi pathways utilize Dicer-processed small RNAs for sequence-specific gene silencing.
- Identifying small-molecule modulators of Dicer activity is crucial for further research and therapeutic applications.
Purpose of the Study:
- To develop and validate a fluorescence-based in vitro Dicer cleavage assay.
- To adapt the assay for high-throughput screening (HTS) for small-molecule modulators.
- To demonstrate the assay's utility by screening a library of bioactive compounds.
Main Methods:
- A fluorescence-based in vitro Dicer cleavage assay was established.
- The assay was optimized for single-turnover conditions with low substrate and enzyme concentrations (35 nM substrate, 70 nM Dicer).
- The assay was adapted for HTS in a 1536-well format using a small reaction volume (5 µL).
Main Results:
- The developed assay is suitable for HTS of Dicer activity.
- Single-turnover conditions and small reaction volumes facilitate efficient screening.
- A proof-of-principle screen of bioactive compounds demonstrated the assay's potential.
Conclusions:
- A robust and scalable fluorescence-based Dicer assay for HTS has been developed.
- This assay facilitates the discovery of novel small-molecule regulators of miRNA and RNAi pathways.
- The assay holds significant promise for advancing research in gene silencing and related therapeutic strategies.
