FIGURE 4.
The importance of nucleotide positions 15 and 39 for 2AP binding. (A) Normalized 2AP fluorescence intensities as a function of RNA concentration for G39A (squares), G39C (circles), and A15C (triangles). The inset shows the influence of an adenine at position 39 in the context of the VV1 aptamer. The best fit is not reported for A15C, as the very weak fluorescence quenching observed precludes a reliable estimation of the fitting parameters (e.g., R value). (B) Schematic representing the different 2AP binding affinity reductions observed for positions 15, 39, and 65. Note that the 2AP binding affinities of the A15C and U65C variants could not be determined due to their very low affinity. (C) Effect of A15 substitutions on loop–loop formation. Nondenaturing gel electrophoresis of the natural (wt), P2 comp, and variants mutated at position 15 in the presence of 1 mM magnesium ions. The control loop variant P2 comp exhibits a retarded electrophoretic migration rate. (D) Effect of G39 substitutions on the loop–loop formation. Nondenaturing gel electrophoresis of the natural (wt), P2 comp, and variants mutated at position 39 in the presence of 1 mM magnesium ions. The control loop variant P2 comp exhibits a retarded electrophoretic migration rate.