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We have previously reported the development of a group I intron-derived ribozyme that can bind an exogenous RNA substrate and excise from that substrate an internal segment in vitro, which allows for sequence-specific modification of RNA molecules. In this report, the activity of this  trans  excision-splicing ribozyme in a cellular environment, specifically  Escherichia coli , was investigated. The ribozyme was re-engineered to target for excision a single-base insertion in the transcript of a green fluorescent protein, and fluorescence was exploited as a reporter for  trans  excision-splicing. We show that the ribozyme is able to catalyze the  trans  excision-splicing reaction in vivo and can repair the mutant transcripts. On average, 12% correction is observed as measured by fluorescence and at least 0.6% correction as confirmed through sequence analysis. This represents the first report of a biomolecule (in this case a ribozyme) that can selectively excise a targeted nucleotide from within an mRNA transcript in vivo. This new class of biochemical tools makes possible a wide variety of new experimental strategies, perhaps including a new approach to molecular-based therapeutics.

In vivo excision of a single targeted nucleotide from an mRNA by a trans excision-splicing ribozyme