Fluorescence spectroscopic analysis for the association of CCHFV N protein with the vRNA panhandle, 5’ and 3’ NCR sequences.

<p>(A). As described in the Materials and Methods, a fixed concentration of N protein in RNA binding buffer was excited at 295 nm, and fluorescence spectrum from 300–500 nm was recorded. The fluorescence signal from binding buffer without N protein was subtracted from all the spectra shown in this panel. Increasing concentrations of the panhandle, shown by arrows, were added to the N protein and the fluorescence spectra (shown by different colors) were recorded. The fluorescence intensity value at 334 nm from each spectra was plotted verses input concentration of the panhandle to generate the binding profile for the calculation of K_d (shown at the bottom), as discussed in materials and methods. All fluorescence experiments were carried out at room temperature. (B and C). The fluorescence experiment shown in these two panels was carried out similar to the panel A, except the vRNA panhandle was replaced by 5’ NCR and the 3’ NCR in panels B and C, respectively. The increasing input concentrations of the 5’ and 3’ NCR are shown by the arrows. The corresponding binding profiles are shown at the bottom. The insets in the binding profiles show the double reciprocal plot for the calculation of ΔF_max that was used in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0184935#pone.0184935.e004" target="_blank">Eq 4</a> for the calculation of K_d values, as mentioned in materials and methods.