SPECIALIZATION OF THE DNA-CLEAVING ACTIVITY OF A GROUP-I RIBOZYME THROUGH IN-VITRO EVOLUTION

In an earlier study, an in vitro evolution procedure was applied to a large population of variants of the Tetrahymena group I ribozyme to ob tain individuals with a 10(5)-fold improved ability to cleave a target single-stranded DNA substrate under simulate physiological conditions . The evolved ribozymes also showed a twofold improvement, compared to the wild-type, in their ability to cleave a single-stranded RNA subst rate. Here, we report continuation of the in vitro evolution process u sing a new selection strategy to achieve both enhanced DNA and diminis hed RNA-cleavage activity. Our strategy combines a positive selection for DNA cleavage with a negative selection against RNA binding. After 36 ''generations'' of in vitro evolution, the evolved population showe d an similar to 100-fold increase in the ratio of DNA to RNA-cleavage activity. Site-directed mutagenesis experiments confirmed the selectiv e advantage of two covarying mutations within the catalytic core of th e ribozyme that are largely responsible for this modified behavior. Th e population of ribozymes has now undergone a total of 63 successive g enerations of evolution, resulting in an average of 28 mutations relat ive to the wild-type that are responsible for the altered phenotype. ( C) 1996 Academic Press Limited