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Science 18 December 1992:
Vol. 258. no. 5090, pp. 1910 - 1915
DOI: 10.1126/science.1470913
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Articles
Science, Vol 258, Issue 5090, 1910-1915
Copyright © 1992 by American Association for the Advancement of Science
Selection of a ribozyme that functions as a superior template in a self-copying reaction
R Green
and
JW Szostak
Department of Molecular Biology, Massachusetts General Hospital, Boston 02114.
The sunY ribozyme is derived from a self-splicing RNA group I intron. This ribozyme was chosen as a starting point for the design of a self-replicating RNA because of its small size. As a means of facilitating the self-replication process, the size of this ribozyme was decreased by the deletion of nonconserved structural domains; however, when such deletions were made, there were severe losses of enzymatic activity. In vitro genetic selection was used to identify mutations that reactivate a virtually inactive sunY deletion mutant. A selected mutant with five substitution mutations scattered throughout the primary sequence showed greater catalytic activity than the original ribozyme under the selection conditions. The sunY ribozyme and its small selected variant can both catalyze template-directed oligonucleotide assembly. The small size and reduced secondary structure of the selected variant results in an enhancement, relative to that of the original ribozyme, of its rate of self-copying. This engineered ribozyme is able to function effectively both as a catalyst and as a template in self-copying reactions.
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- Isolation of new ribozymes from a large pool of random sequences [see comment].
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