Activities and relative affinities of divalent metals in unmodified and phosphorothioate-substituted hammerhead ribozymes

The roles of metals in the phosphodiester bond cleavage reaction performed by the hammerhead ribozyme are under investigation. In this study, the appa rent affinities and the abilities of several different metals to support ri bozyme activity are reported. The relative affinities of divalent cations f or the hammerhead ribozyme are determined by measuring their ability to rel ease bound Mn2+. The EPR-detected Mn2+ competition studies give an order of apparent affinity of Mn2+ similar to Co2+ similar to Zn2+ > Cd2+ much grea ter than Mg2+. This ordering generally follows the trend of maximum rates o f cleavage determined at pH 7.0, 0.1 M NaCl, and saturating metal concentra tions, of Mn2+ > Co2+ > Cd2+ > Mg2+. The maximum rate is observed for Mn2under these conditions and may be related to the high affinity, low pK(a) a nd low Delta H-hyd of this ion. Substitution of phosphorothioates 5' to eac h of the nine adenosines in the enzyme strand yields a change in the Mn2+ b inding properties of the hammerhead complex. In the phosphorothioate-substi tuted hammerhead complex, eight to nine Mn2+ bind in two types of classes: 'type 1' (n = 1+/-0.3, K-d = 1.1+/-1 mu M) and weaker 'type 2' (n = 7.7+/-0 .3, K-d = 125+/-27 mu M). The multiple phosphorothioate substitutions resul t in the loss of two to three of the higher affinity sites observed in the unmodified ribozyme. Metal competition studies with the phosphorothioate-su bstituted ribozyme indicate that the relative affinities of the metals are Cd2+ > Zn2+ > Co2+, Mg2+ with the number of Mn2+ displaced and apparent aff inity of the thiophilic Cd2+ most affected by the phosphorothioate substitu tions. (C) 2000 Elsevier Science S.A. All rights reserved.