A design principle for a single-stranded RNA genome that replicates with less double-strand formation

Usui, Kimihito; Ichihashi, Norikazu; Yomo, Tetsuya

RNA replications of the RNA species shown in Figure 2 were performed in Q␤ replicase buffer (125 mM Tris-HCl, pH 7.8, 5 mM MgCl 2 ), 0.005% BSA, 1.25 mM NTPs, [ 32 P]-UTP with each RNA (100 nM) and Q␤ replicase (100 nM) for 20 min at 37 • C. The mixture was applied to 8% polyacrylamide gels for electrophoresis, and the ssRNAs and dsRNAs were quantified by autoradiography according to a previous study (5) . RNA replication of the RNAs containing an ␣-domain gene was performed in an amino aciddepleted translation system (28) containing [ 32 P]-UTP with each RNA (100 nM) and Q␤ replicase (100 nM) for 25 min at 37 • C. The Q␤ replicase was purified according to a previous study (29) . To obtain the time course data ( Figure 7C ), the reaction was encapsulated in a water-in-oil emulsion to repress the amplification of nonspecific RNA according to a previous study (4) . The quantification of ssRNA and dsRNA was performed by autoradiography after agarose gel electrophoresis according to a previous study (28) .