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Volume 271, Number 42, Issue of October 18, 1996 pp. 26081-26087
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.

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Circular Ribozymes Generated in Escherichia coli Using Group I Self-splicing Permuted Intron-Exon Sequences

(Received for publication, March 19, 1996, and in revised form, July 24, 1996)

From the Department of Biochemistry, Duke University Medical Center, Durham, North Carolina 27710

A circularly permuted self-splicing group I intron from Anabaena was used to generate covalently closed circular trans-acting ribozymes in Escherichia coli. The RNA component of Bacillus subtilis RNaseP and an artificial trans-acting hepatitis delta virus ribozyme were expressed as the exon portion of the permuted intron. RNA isolated from these cells contained circular forms of the ribozymes, indicating that circles were generated from precursors expressed in these cells. Total RNA isolated from cells producing the circular RNA contained ribozyme activity. In contrast, a linear form of the delta virus ribozyme expressed as part of an unprocessed transcript yielded no detectable activity. These data extend previous in vitro and in vivo studies on splicing-mediated RNA circularization by demonstrating the intracellular production of circular ribozymes. These results have implications for the development of systems expressing therapeutic forms of small RNAs such as ribozymes and decoy-type competitors. Circular RNAs generated by splicing are devoid of flanking sequences that could potentially interfere with function. Also, because circular RNAs are not primary substrates for exonucleases, they may have increased in vivo half-lives relative to linear molecules with similar sequences.

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