Isolation of new self-cleaving ribozymes with in vitro selection

Abstract

In vitro selection was used to isolate Mg2+-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 Mg2+ were isolated. The selected ribozymes can carry out the self-cleavage reaction in the presence of 100 mM Mg2+ but not in 10 mM Mg2+. The cleavage sites of the ribozymes are located at +3 and +4 of tRNAPhe, 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 tRNAPhe. Our results suggest that the selected ribozyme is not structural-specific for tRNA.