Selected classes of minimised hammerhead ribozyme have very high cleavage rates at low Mg2+ concentration

Abstract

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-1 at 1 mM Mg2+ (pH 8.0, 37°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 MgCl2 the 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 he a general property for specific selected linker sequences, as the high rates of catalysis were transferable to a different substrate system.