Pbe 5g

All the wild-type and mutant proteins of PUF-8^172–535 and PUF-8^172–522 were expressed in Escherichia coli BL21-GOLD (DE3) cells (Novagen). Cells were grown in Luria-Bertani (LB) medium at 37°C to an OD₆₀₀ of 0.7–1.0 and induced with 0.4 mM isopropyl β-d-thiogalactopyranoside (IPTG) for 22 h at 16°C. The cells were pelleted and resuspended in lysis buffer (25 mM Tris–HCl, pH 8.5, 500 mM NaCl, 20 mM imidazole, and 5% (v/v) glycerol) and lysed by sonication. The supernatant were purified by His tag purification resin (Roche), followed by size exclusion chromatography on a HiLoad Superdex 200 16/60 column (GE Healthcare) in buffer A (25 mM Tris–HCl, pH 8.5, 250 mM NaCl).
Native RNA: PBE-5A (5′-UGUAAAUA-3′), PBE-5U (5′-UGUAUAUA-3′), PBE-5C (5′-UGUACAUA-3′), PBE-5G (5′-UGUAGAUA-3′), PBE-1G (5′-GGUAAAUA-3′), PBE-2U (5′-UUUAUAUA-3′), RNA_ctrl (5′-AUAAAUGU-3′), and the 5′-Fam-tagged RNAs (PBE-5A, PBE-5U, PBE-5C, PBE-5G, PBE-1G and Ctrl) were purchased from Takara Bio Inc. (Dalian, China). RNA oligomers were dissolved in diethyl pyrocarbonate (DEPC)-treated water to a concentration of 2 mM as the stock solution.