FIG. 3.

Optimization of conditions for the WNV N-7 and 2′-O cap methylations. Reactions for N-7 (•) and 2′-O methylation (○) contained substrate G*pppA-RNA and m⁷G*pppA-RNA and were incubated for 5 and 60 min, respectively. The optimal conditions were determined by individually titrating pH (A), temperature (B), MgCl₂ (C), and NaCl (D), while keeping the other three parameters constant at the optimal levels. The reaction mixtures were then treated with nuclease P1, analyzed on TLC plates, and quantified by PhosphorImager analysis. For each parameter, relative activities were presented using the optimal level as 100%. Average results from two independent experiments are shown.