Selected classes of minimised hammerhead ribozyme have very high cleavage rates at low Mg2+ concentration.
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.
D000818:Animals; D004268:DNA-Binding Proteins / Q000235:genetics; D019281:Dimerization; D004331:Drosophila melanogaster; D006801:Humans; D007376:Interleukin-2 / Q000235:genetics; D051741:Kruppel-Like Transcription Factors; D008274:Magnesium / Q000378:metabolism*; D009690:Nucleic Acid Conformation; D016337:RNA, Catalytic / Q000145:classification / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism; D012097:Repressor Proteins; D014157:Transcription Factors / Q000235:genetics