Analysis of DENV2 NS5 binding to 5′ SLA or 3′SL RNA. (A) Schematic illustration of DENV2 genome showing the position, sequence, and size of 5′ terminal stem–loop A (5′ SLA) and 3′ terminal stem–loop (3′SL) within the 5′ and 3′ untranslated regions (UTRs). The purity of the in vitro synthesized and purified 5′ SLA and 3′SL RNA samples were analyzed by agarose gel electrophoresis. Secondary structures of 5′SLA and 3′SL were predicted by mfold web server (Zuker 2003) with the thermodynamic free energy ΔG of the folding process at 37°C indicated. (B,C) RNA electrophoretic mobility shift assay (REMSA) for 5′ SLA (B) or 3′ SL (C) with WT NS5 or NS5 R888A mutant, analyzed on 1.2% agarose gel with lane number indicated at the bottom. Lane 1 in each of the four gels represented 5′ SLA (B) or 3′ SL (C) RNA alone in binding buffer. Lanes 2–7 represented binding reactions of NS5 with RNA at molar ratios of 0:1, 0.5:1, 1:1, 2:1, 4:1, 8:1, and 16:1 as indicated on top of each lane. (D) Binding affinity of 5′ SLA or 3′ SL with WT NS5 or NS5 mutants Y838F, R888A, D146A, GAA, D146A + GAA reflected by apparent dissociation constant (Kd) was calculated from band intensities using ImageJ from two to four independent repeat experiments represented as average ± SD. The statistical significance of difference between the two groups was evaluated by Student's t-test and a P-value <0.05 was considered significant (≤0.0001, ≤0.001, ≤0.01, ≤0.05 were represented by [****], [***], [**], [*], respectively).
