DICER cleavage fidelity is governed by 5′-end binding pockets
Nature • • science
Key Points:
- Researchers constructed plasmids encoding human DICER and Drosophila Dicer-1 (Dcr-1) using molecular cloning techniques, confirmed mutations by sequencing, and prepared bacmids for protein expression.
- Wild-type and mutant DICER proteins were expressed in HEK293E cells, while Dcr-1 was produced in Sf9 insect cells via baculovirus infection; proteins were purified using Ni-NTA affinity, ion exchange, and gel-filtration chromatography.
- In vitro assays were conducted to analyze DICER cleavage of synthetic pre-miRNAs, including massively parallel dicing assays with randomized pre-mir-324 variants, followed by high-throughput sequencing and bioinformatic analysis to assess cleavage efficiency and accuracy.
