X-ray structure and activities of an essential Mononegavirales L-protein domain

Paesen, Guido C.; Collet, Axelle; Sallamand, Corinne; Debart, Françoise; Vasseur, Jean-Jacques; Canard, Bruno; Decroly, Etienne; Grimes, Jonathan M.

Thin-layer chromatography analysis of cap structures. G*pppGGGACAAGU (in which the asterisk indicates the [ 32 P]-labelled phosphate) was synthesized by incubating pppGGGACAAGU (10 mM) with vaccinia virus capping enzyme (New England Biolabs) in the presence of 1.65 mCi [a-32 P]-GTP (Perkin Elmer). The capped RNA was purified by precipitation in 3 M sodium acetate supplemented with 1 mg ml À 1 of glycogen (Thermo Scientific), and submitted to methylation by CR-VI þ (as above), after which it was precipitated again (stopping the reactions), and digested with 1 U of Nuclease P1 (US Biologicals) in 30 mM sodium acetate (pH 5.3), 5 mM ZnCl 2 and 50 mM NaCl (4 h, 37°C). The products were spotted onto polyethylenimine cellulose thin-layer chromatography plates (Macherey Nagel), and resolved in two steps, first using 0.65 M LiCl, then 0.45 M (NH 2 ) 2 SO 4 as mobile phase. The radiolabelled caps released by nuclease P1 were visualized using a Fluorescent Image Analyzer FLA3000 (Fuji) phosphor-imager.