Substitution of the 2′-hydroxyl group at position 2.1 by an amino group interferes with Mg2+ binding and efficient cleavage by hammerhead ribozyme

Recently we have demonstrated that hammerhead ribozymes can be fully substituted with 2'-amino pyrimidines without detriment to the catalytic activity, provided that positions 2.2 and/or 2.1 are not modified. We now report on the potential molecular mechanisms by which 2'-amino groups at these positions inhibit the ribozyme cleavage activity. In the presence of Mg2+, the 2'-amino modification at positions 2.2 and/or 2.1 had no significant effect on substrate binding. Detailed analysis of the ribozyme initial cleavage rates in the presence of various Mg2+ concentrations indicated that Mg2+ binding is inhibited by the 2'-amino group at position 2.1. Furthermore, preannealed substrate molecules to the modified ribozyme are not effectively cleaved upon Mg2+ addition, indicating an alteration of the ribozyme cleavage step. Surprisingly, the cleavage activity of the modified ribozymes was substantially increased when Mg2+ ions were replaced by the thiophilic Mn2+ ions, whereas only a moderate cleavage enhancement occurred with its unmodified version. Taken together, our findings indicate that changes in the sugar at position 2.1 alter Mg2+-promoting ribozyme cleavage. (C) 1999 Academic Press.