Nucleic Acids Research, Vol 27, Issue 11 2400-2407, Copyright © 1999 by Oxford University Press
J Conaty, P Hendry and T Lockett
In vitro selection was used to enrich for highly efficient RNA
phosphodiesterases within a size-constrained (18 nt) ribonucleotide domain.
The starting population (g0) was directed in trans against an RNA
oligonucleotide substrate immobilised to an avidin-magnetic phase. Four
rounds of selection were conducted using 20 mM Mg2+to fractionate the
population on the basis of divalent metal ion-dependent phosphodiesterase
activity. The resulting generation 4 (g4) RNA was then directed through a
further two rounds of selection using low concentrations of Mg2+.
Generation 6 (g6) was composed of sets of active, trans cleaving minimised
ribozymes, containing recognised hammerhead motifs in the conserved
nucleotides, but with highly variable linker domains (loop II-L.1-L.4).
Cleavage rate constants in the g6 population ranged from 0.004 to 1.3
min-1at 1 mM Mg2+(pH 8.0, 37 degrees C). Selection was further used to
define conserved positions between G(10.1) and C(11.1) required for high
cleavage activity at low Mg2+concentration. At 10 mM MgCl2the kinetic
phenotype of these molecules was comparable to a hammerhead ribozyme with 4
bp in helix II. At low Mg2+concentration, the disparity in cleavage rate
constants increases in favour of the minimised ribozymes. Favourable
kinetic traits appeared to be a general property for specific selected
linker sequences, as the high rates of catalysis were transferable to a
different substrate system.
ARTICLES
Selected classes of minimised hammerhead ribozyme have very high cleavage rates at low Mg2+ concentration
CSIRO Division of Molecular Science, PO Box 184, North Ryde, NSW 1670, Australia and School of Biochemistry and Molecular Genetics, University of New South Wales, Kensington, NSW 2052, Australia.
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