High-yield production of human Dicer by transfection of human HEK293-EBNA1 cells grown in suspension

Bouvette, Jonathan; Korkut, Dursun Nizam; Fouillen, Aurélien; Amellah, Soumiya; Nanci, Antonio; Durocher, Yves; Omichinski, James G.; Legault, Pascale

Dicer is known to bind pre-miRNA substrates and then cleave at two specific phosphodiester bonds to yield miRNA-5p/3p species. To evaluate the RNA binding and cleavage activity of the purified recombinant Dicer, we performed binding and kinetic studies using the pre-let-7a-1 substrate, one of the most commonly used pre-miRNA substrate for Dicer characterization. Binding studies were conducted via electrophoretic mobility shift assay (EMSA) using 32 P-labeled pre-let-7a-1 and increasing concentration of Dicer, either wild-type (WT) Dicer supplemented with 0.5 mM EDTA to prevent cleavage or with the catalytically-inactive D1320/1709A variant (Fig. 4) . When fitting the binding data to the Hill equation, dissociation constants (K d ) of 5 ± 1 nM (WT Dicer) and 9 ± 1 nM (D1320A/D1709A Dicer) were obtained with Hill coefficient of 1.3 ± 0.2 and 1.4 ± 0.3, respectively.