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Bulletin of the Korean Chemical Society (BKCS)

ISSN 0253-2964(Print)
ISSN 1229-5949(Online)
Volume 26, Number 12
BKCSDE 26(12)
December 20, 2005 

Isolation of New Self-Cleaving Ribozymes with in vitro Selection
Bongrae Cho*, Younghoon Lee
SELEX, Self-cleavage ribozyme, tRNA, H1 RNA
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 tRNAPhe. 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 tRNALeu 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.
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