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Random mutagenesis followed by an in vitro selection procedure was shown to be capable of identifying important bases of the hairpin ribozyme for cleavage of an RNA target sequence. The selection scheme enriched the RNA population for those molecules capable of efficient site-specific self-cleavage in the absence of ligation. Cleavable mutants were selected for all positions in loop 4 except for position A38, supporting the notion that A38 is an important base in the hairpin ribozyme. This has been confirmed by direct mutagenesis, validating the utility of this procedure. Thus, the method developed and reported here has utility for the selection of efficient hairpin ribozymes capable of highly efficient cleavage of a substrate RNA without a requirement for ribozyme-catalyzed ligation, conditions desired for many applications of catalytic RNA such as gene therapy.

Screening for Important Base Identities in the Hairpin Ribozyme by In Vitro Selection for Cleavage