Superose 6 increase 5 150 gl

DDM, CHS, LMNG, GDN and A8-35 were purchased from Anatrace (Maumee, OH, USA). ESF 921 Insect Cell Culture Medium was obtained from Expression Systems (Davis, CA, USA). Size exclusion chromatography columns (Superose 6 Increase 10/300 GL and Superose 6 Increase 5/150 GL) were purchased from Cytiva (Marlborough, MA, USA). Strep-Tactin^®XT beads and BioLock were purchased from IBA Life Sciences (Göttingen, Germany).

To refold pre-miR-15a RNA, it was heated for 3 min at 95°C and snap-cooled on ice for 5 min. The complex was formed by mixing 1.5 nmol of pre-miR-15a and 0.5 nmol of catalytically inactive Dicer or Dicer^O variant in 50 μl of 50 mM Tris (pH 8.0), 100 mM NaCl, 1 mM DTT, and 2 mM MgCl₂. After 30 min incubation on ice, the mixture was applied onto Superose 6 Increase 5/150 GL (Cytiva) column attached to an ÄKTA Purifier (Cytiva). Fractions containing the complex were collected and concentrated to 0.2 mg/ml. The complex of the wild-type Dicer with pre-miR-15a and TARBP2 was prepared by direct mixing of 150 pmol of pre-miR-15a, 50 pmol of Dicer and 55 pmol of TARBP2 in 50 μl of 50 mM Tris (pH 8.0), 100 mM NaCl, 1 mM DTT, and 2 mM CaCl₂. The mixture was incubated on ice for 30 min and applied on CryoEM grid. The purity and homogeneity of the protein was assessed by SDS-PAGE, while RNA was verified by denaturing gel electrophoresis (20% polyacrylamide gel containing 8 M urea) and visualized using SYBR Gold dye (ThermoFisher Scientific).