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

For in vitro Dicer cleavage assays, the RNA and Dicer solutions were prepared separately at twice their final desired concentration in 30 μL of the cleavage reaction buffer (50 mM HEPES pH 7.4, 50 mM NaCl, 5 mM MgCl 2 and 0.05% NP-40). The RNA solutions, containing 80 pM of 32 P-labeled pre-let-7a-1 and cold RNA at concentrations ranging from 0.1× to 10× the estimated K M , were heated at 95°C for 2 min and snap-cooled at 4°C for a minimum of 5 min to refold the RNA. The concentration of the Dicer solution was adjusted for each pre-let-7a-1 substrate concentration to allow measurement of the initial velocity of the reaction (5-10% substrate cleavage in 30 min) under multiple turnover conditions (substrate/Dicer molar ratio ≥ 200). Both RNA and protein solutions were pre-heated for 5 min at 37°C, and cleavage reactions were initiated by adding 30 μL of the protein solution to 30 μL of the RNA solution. At specific time points, a 5-μL aliquot of the cleavage reaction was taken, immediately mixed with 5 μL of stop buffer (95% formamide and 50 mM EDTA). For each reaction, 6 time points were taken at less than 10% reaction completion and over at least 2 full turnovers of the enzyme pool to ascertain the possible use of the steady-state assumption. Samples were analyzed by denaturating gel electrophoresis [20% acrylamide:bis-acrylamide (19:1) /7 M urea gel]. The amounts of substrate (S) and product (P) were quantified from radioactive bands as for the binding assay, and the fraction of cleaved product [F = P/ (S + P)] was plotted against time. The resulting time courses were fitted by linear regression and the slope was taken as the initial velocity