Figure 3.

NA chaperone activity of ORF1p variants. (A) Schematic of the FRET assay to measure the annealing and exchange phases of NA chaperone activity using the assay conditions described in the ‘Materials and Methods’ section. (B) Reactions (40 μl, 0.5 μM ORF1p, 5 nM 21-nt mismatched duplex) were incubated at room temperature, irradiated every 0.7 s at 535 nm and fluorescence was measured at 590 and 680 nm (solid circles). At the indicated time (vertical arrow) 50 nM perfect complement was added. The relative concentrations of duplex and ORF1p during the annealing phase of these reactions correspond to those that produce maximal protection of the mismatched duplex (caging, Figure 2A, gray rectangle). The annealing and exchange phases data are fit to single and double exponential functions in time (solid lines and Supplementary Table S2) respectively, using the minimization of χ² method.