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Titolo:
EVOLUTIONARY OPTIMIZATION OF THE CATALYTIC PROPERTIES OF A DNA-CLEAVING RIBOZYME
Autore:
TSANG J; JOYCE GF;
Indirizzi:
SCRIPPS CLIN & RES INST,DEPT CHEM,10666 N TORREY PINES RD LA JOLLA CA92037 SCRIPPS CLIN & RES INST,DEPT CHEM LA JOLLA CA 92037 SCRIPPS CLIN & RES INST,DEPT MOLEC BIOL LA JOLLA CA 92037
Titolo Testata:
Biochemistry
fascicolo: 19, volume: 33, anno: 1994,
pagine: 5966 - 5973
SICI:
0006-2960(1994)33:19<5966:EOOTCP>2.0.ZU;2-Z
Fonte:
ISI
Lingua:
ENG
Soggetto:
TETRAHYMENA RIBOZYME; RNA-POLYMERASE; ENZYMATIC AMPLIFICATION; SUBSTRATE BINDING; SITE; CLEAVAGE; SELECTION; INVITRO; MECHANISM; INTRONS;
Tipo documento:
Article
Natura:
Periodico
Settore Disciplinare:
Science Citation Index Expanded
Citazioni:
33
Recensione:
Indirizzi per estratti:
Citazione:
J. Tsang e G.F. Joyce, "EVOLUTIONARY OPTIMIZATION OF THE CATALYTIC PROPERTIES OF A DNA-CLEAVING RIBOZYME", Biochemistry, 33(19), 1994, pp. 5966-5973

Abstract

In a previous study [Beaudry, A. A., and Joyce, G. F. (1992) Science 257, 635-641], an in vitro evolution procedure was used to obtain variants of the Tetrahymena ribozyme with 100-fold improved ability to cleave a target single-stranded DNA under physiologic conditions. Here wereport continuation of the in vitro evolution process to achieve 10(5)-fold overall improvement in DNA-cleavage activity. In addition, we demonstrate that, by appropriate manipulation of the selection constraints, one can optimize specific catalytic properties of the evolved ribozymes. We first reduced the concentration of the DNA substrate 50-fold to favor ribozymes with improved substrate binding affinity. We thenreduced the reaction time 12-fold to favor ribozymes with improved catalytic rate. In both cases, the evolving population responded as expected, first improving substrate binding 25-fold, and then improving catalytic rate about 50-fold. The population of ribozymes has undergone 27 successive generations of in vitro evolution, resulting in, on average, 17 mutations relative to the wild type that are responsible for the improved DNA-cleavage activity.

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Documento generato il 21/09/20 alle ore 03:27:45