Figure 3.

Fluorescence anisotropy based measurements of the binding of NS3h to short oligonucleotides. (A) The binding of NS3h to 5′-F-dT₆ (circles) and 5′-dT₆-F (squares) as a function of the total concentration of NS3h in solution monitored by the changes in the fluorescence anisotropy of the fluorophore that occur upon the binding of NS3h to the oligonucleotides. The increase in the fluorescence anisotropy of the fluorophore is consistent with the NS3h bound oligonucleotide having a slower rotational diffusion time than the free oligonucleotide, as expected. (B) In experiments where non-fluorophore labeled oligonucleotide is included as a competitor for NS3h binding, the fluorescence anisotropy of the fluorophore labeled oligonucleotides decreases with increasing total concentration of the competitor. As shown in Table 1 and Table 2, the estimate of the affinity of NS3h binding to the unlabeled competitor molecule is independent of whether 5′-F-dT₆ or 5′-dT₆-F is used.