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 apparent affinities and the abilities of several different metals to support ribozyme activity are reported. The relative affinities of divalent cations for the hammerhead ribozyme are determined by measuring their ability to release bound Mn2+. The EPR-detected Mn2+ competition studies give an order of apparent affinity of Mn2+ approximately Co2+ approximately Zn2+ > Cd2+ >> Mg2. This ordering generally follows the trend of maximum rates of cleavage determined at pH 7.0, 0.1 M NaCl, and saturating metal concentrations, of Mn2+ > Co2+ > Cd2+ > Mg2+. The maximum rate is observed for Mn2+ under these conditions and may be related to the high affinity, low pKa and low deltaHhyd of this ion. Substitution of phosphorothioates 5' to each of the nine adenosines in the enzyme strand yields a change in the Mn2+ binding properties of the hammerhead complex. In the phosphorothioate-substituted hammerhead complex, eight to nine Mn2+ bind in two types of classes: 'type 1' (n = 1+/-0.3, Kd = 1.1+/-1 microM) and weaker 'type 2' (n = 7.7+/-0.3, Kd = 125+/-27 microM). The multiple phosphorothioate substitutions result in the loss of two to three of the higher affinity sites observed in the unmodified ribozyme. Metal competition studies with the phosphorothioate-substituted ribozyme indicate that the relative affinities of the metals are Cd2+ > Zn2+ > Co2+, Mg2+ with the number of Mn2+ displaced and apparent affinity of the thiophilic Cd2+ most affected by the phosphorothioate substitutions.
Hunsicker
L M
LM
Department of Chemistry, Texas A&M University, College Station 77842-3012, USA.
DeRose
V J
VJ
D002104:Cadmium / Q000737:chemistry; D002413:Cations, Divalent / Q000737:chemistry / Q000378:metabolism*; D003035:Cobalt / Q000737:chemistry; D004247:DNA / Q000378:metabolism; D004578:Electron Spin Resonance Spectroscopy; D007700:Kinetics; D008274:Magnesium; D008345:Manganese / Q000737:chemistry / Q000378:metabolism*; D015394:Molecular Structure; D009690:Nucleic Acid Conformation; D016337:RNA, Catalytic / Q000737:chemistry / Q000378:metabolism*; D013873:Thionucleotides / Q000737:chemistry*; D015032:Zinc / Q000737:chemistry
D002413:Cations, Divalent; D016337:RNA, Catalytic; D013873:Thionucleotides; D002104:Cadmium; D003035:Cobalt; D008345:Manganese; D004247:DNA; D008274:Magnesium; D015032:Zinc