Circularizing ribozymes and decoy-competitors by autocatalytic splicing in vitro and in vivo.

Important note: Information in this article was accurate in 1996. The state of the art may have changed since the publication date.

Circularizing ribozymes and decoy-competitors by autocatalytic splicing in vitro and in vivo.

SAAS Bull Biochem Biotechnol. 1996;9:77-82. Unique Identifier : AIDSLINE MED/96261112
Puttaraju M; Been MD; Department of Biochemistry, Duke University Medical Center,; Durham, NC-27710, USA.

Abstract: An Anabaena group I intron was circularly permuted at loop 5, loop 6 and loop 8, and tested for self-splicing activity. Precursor RNAs from these constructs spliced efficiently and produced circular exons in vitro. Using group I permuted-intron-exon sequences, circular forms of the HDV ribozyme, the RNA component of RNaseP from B. subtilis, the HIV TAR and a short HIV Rev-binding element were generated and tested for activity and stability. The activity of circular ribozymes is comparable to the linear counterparts with similar core sequences. Circular forms of the TAR and Rev-binding element showed specific binding to Tfr-38 and Rev(35-50) peptide, respectively. To explore the potential for using this methodology to express circular RNA in vivo, circular forms of the HDV ribozyme and RNaseP RNA were produced in E. coli. Analysis of total RNA indicated that the precursor RNA spliced efficiently and accurately to produce circular ribozymes. The activity of in vivo expressed circular ribozymes could be demonstrated indicating that they fold into active conformation. These results suggest that self-splicing group I PIE sequences could prove useful for expressing small stable circular ribozyme/decoy-competitor or antisense RNAs in cells.

Keywords: Anabaena/*GENETICS Animal Binding, Competitive Catalysis Escherichia coli/GENETICS Exons Gene Expression Introns Plasmids RNA/*METABOLISM *RNA Splicing RNA, Catalytic/*METABOLISM Support, U.S. Gov't, P.H.S. Tetrahymena/GENETICS Transfection JOURNAL ARTICLE
961030
M96A1467

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