J. Tsang et Gf. Joyce, SPECIALIZATION OF THE DNA-CLEAVING ACTIVITY OF A GROUP-I RIBOZYME THROUGH IN-VITRO EVOLUTION, Journal of Molecular Biology, 262(1), 1996, pp. 31-42
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