Isolation of New Self-Cleaving Ribozymes with in vitro Selection

摘要

In vitro selection was used to isolate Mg~(2+)-dependent self-cleaving ribozymes with cis-cleavage activity from a pre-tRNA library having 40-mer random sequences attached to 5'-end of E.coli tRNA~(Phe).After 8 rounds of SELEX (Systematic Evolution of Ligands by Exponential Enrichment),RNA molecules which can self-cleave at the high concentration of Mg~(2+) were isolated.The selected ribozymes can carry out the self-cleavage reaction in the presence of 100 mM Mg~(2+)but not in 10 mM Mg~(2+).The cleavage sites of the ribozymes are located at +3 and +4 of tRNA~(Phe),compared with +1 position of 5'-end cleavage site of pre-tRNA by RNase P.New RNA constructs deprived of its D stem-loop,anticodon stem-loop,variable loop and T stem-loop,respectively showed the cleavage specificity identical to a ribozyme having the intact tRNA structure.Also,the new ribozyme fused with both a ribozyme and tRNA~(Leu) showed the cleavage activities at the various sites within its sequences,different from two sites of position +3 and +4 observed in the ribozyme with tRNA~(Phe).Our results suggest that the selected ribozyme is not structural-specific for tRNA.