Fig. 3. Quantitative analysis of thiophilic metal ion rescue of R_P and S_P phosphorothioate modifications at A67. (A) A plot of k_obs versus Mn²⁺ concentration is shown for the unmodified ribozyme (PT50; diamonds) and for ribozymes with a site-specific S_P phosphorothioate modification at A67 (A67S_P; circles). (B) Plot of k_obs versus Mn²⁺ concentration for unmodified PT50 (PT50, diamonds) and for ribozymes with a site- specific R_P phosphorothioate modification at A67 (A67R_P; squares). Rate constants for all reactions were determined in a constant background of 10 mM Mg²⁺, 3 M NaCl, 50 mM PIPES pH 5.5 at 50°C. (C) Relative reaction rates (k_rel) for the pro-S_P (A67S_P; circles) or pro-R_P (A67R_P; squares) phosphate oxygen at A67 are plotted as a function of added Mn²⁺ and fit to an equation for binding of a single metal ion (Equation 1, Materials and methods). For A67S_P, K^{Mn, app} = 5.7 ± 0.8 mM; for A67R_P, K^Mn,app >>15 mM. Error bars reflect standard deviation of at least four experiments.