Unsmooth SHAPE probing data for the Mg2+ and 2′-dG titrations. (A) Representative capillary electrophoresis traces are shown for 2′-dG titrations at 20°C without Mg2+. The overlaid traces depict the data without 2′-dG (green) and with 20 µM 2′-dG (red). The nucleotides from the different structural regions analyzed here are indicated. The SHAPE reactivities as a function of nucleotide position are evaluated by subtracting the integrated area for individual nucleotides corresponding to the blank sample (RNA treated with neat DMSO) from SHAPE data (1-methyl-7-nitroisatoic anhydride [1M7] treated). To normalize the areas, the first 10% of the data corresponding to the highest reactivity values are considered outliers and are temporarily excluded from the analysis. From the remaining data (areas), 10% of highly reactive nucleotides are averaged to calculate a normalization factor. The entire profile, including the previously excluded outliers, is then normalized by that factor. The SHAPE reactivities for (B) 20 µM 2′-dG (red) and (C) without 2′-dG (green) are shown. (D) Representative capillary electrophoresis traces are shown for 2′-dG titrations at 20°C in the presence of 5 mM Mg2+. The overlaid traces depict the data without 2′-dG (green) and with 20 µM 2′-dG (red). The nucleotides from the different structural regions analyzed here are indicated. SHAPE reactivities as a function of nucleotide position are shown for (E) 20 µM 2′-dG (red) and (F) without 2′-dG (green) in the presence of 5 mM Mg2+.
