Evolution in vitro of an RNA enzyme with altered metal dependence

Abstract

THE Tetrahymena group I ribozyme catalyses a sequence-specific phosphodiester cleavage reaction on an external RNA oligonucleotide substrate in the presence of a divalent metal cation cofactor¹. This reaction proceeds readily with either Mg²⁺ or Mn²⁺, but no detectable reaction has been reported when other divalent cations are used as the sole cofactor^2–5. Cations such as Ca²⁺, Sr²⁺ and Ba²⁺ can stabilize the correct folded conformation of the ribozyme, thereby partially alleviating the Mg²⁺ or Mn²⁺ requirement^2–5. But catalysis by the ribozyme involves coordination of either Mg²⁺ or Mn²⁺ at the active site, resulting in an overall requirement for one of these two cations⁵. Here we use an in vitro evolution process^6,7 to obtain variants of the Tetrahymena ribozyme that are capable of cleaving an RNA substrate in reaction mixtures containing Ca²⁺ as the divalent cation. These findings extend the range of different chemical environments available to RNA enzymes and illustrate the power of in vitro evolution in generating macromolecular catalysts with desired properties.