Effect of the terminal structure of dsRNA for the initiation of RNA replication. Two kinds of RNA template (10 pmol) ((i) Temp(GGG-UCCCC) and (ii) Temp(GGG-UCCC) (A)), of which secondary structures were predicted by Mfold (B), were incubated with NV3Dpol (4 pmol), respectively (reaction volume = 40 μL), and analyzed on a non-denaturing 10% PAGE. The PAGE image was visualized by SYBRgreenII staining (C). M; 10 bp DNA step ladder (Promega). Ref; both RNA templates were electrophoresed as references. Mfold predicted many other structures, but the stem-end (3’end and 5’end hybrid) structures were almost always conserved, namely, the probability of other 3’ structures (for (i), first appearance as #7 stable structure (ΔG of #1, #2,---,and -#7 structures were -12.83, -11.08, -10.49, -10.10, -9.39, -8.52, and -7.99 kcal/mol, respectively) was less than 0.03% .