Electron paramagnetic resonance spectroscopic measurement of Mn2+ binding affinities to the hammerhead ribozyme and correlation with cleavage activity

Efficient phosphodiester bond cleavage activity by the hammerhead ribozyme requires divalent cations. Toward understanding this metal ion requirement, the Mn2+-binding properties of hammerhead model ribozymes have been invest igated under dilute solution conditions, using electron paramagnetic resona nce spectroscopy (EPR) to detect free Mn2+ in the presence of added ribozym e. Numbers and affinities of bound Mn2+ were obtained at pH 7.8 (5 mc triet hanolamine) in the presence of 0, 0.1, and 1.0 M NaCl for an RNA-DNA model consisting of a 13-nucleotide DNA "substrate" hybridized to a 34-nucleotide RNA "enzyme" [Pley, H. W., Flaherty, K. M., and McKay, D. B. (1994) Nature 372, 68-74]. In 0.1 M NaCl, two classes of Mn2+ sites are found with n(1) = 3.7 +/- 0.4, K-d(1) = 4 +/- 1 mu M (type 1) and n(2) = 5.2 +/- 0.4, k(d(2 )) = 460 +/- 130 mu M (type 2). The high-affinity type I sites are confirme d for an active RNA-RNA hybrid (34-nucleotide RNA enzyme: 13-nucleotide RNA substrate) by EPR measurements at low Mn2+ concentrations. Decreasing NaCl concentration results in an increased number of bound Mn2+ per hammerhead. By contrast, a binding titration in 1 M NaCl indicates that a single Mn2site with apparent K-d similar to 10 mu M is populated in low concentration s of Mn2+, and apparent cooperative effects at higher Mn2+ concentrations r esult in population of a similar total number of Mn2+ sites (n(t) = 8-10) a s found in 0.1 M NaCl. Mn2+-dependent activity profiles are similar for the active RNA-RNA hybrid in 0.1 and 1 M NaCl. Correlation with binding affini ties determined by EPR indicates that hammerhead activity in 0.1 M NaCl is only observed after all four of the high-affinity Mn2+ sites are occupied, rises with population of the type 2 sites, and is independent of Mn2+ conce ntrations corresponding to >8-9 Mn2+ bound per hammerhead. Equivalent measu rements in 1 M NaCl demonstrate a rise in activity with the cooperative tra nsition observed in the Mn2+ binding curve. These measurements indicate tha t, over this NaCl concentration range, hammerhead ribozyme activity is infl uenced by population of a specific set of divalent cation sites.