Efficient hammerhead ribozymes targeted to the polycistronic Sendai virus P/C mRNA. Structure-function relationships.

The Journal of Biological Chemistry
D K Gavin, K C Gupta

Abstract

The Sendai virus polycistronic P/C mRNA encodes the P and C proteins from alternate overlapping reading frames. To determine the functions of these proteins in virus replication, hammerhead ribozymes were targeted to cleave the 5'-untranslated region of the P/C mRNA. Both cell-free and intracellular assays were employed to determine ribozyme efficacy. To appropriately compare activities between cell-free and intracellular assays, identical ribozymes were synthesized in vitro as well as expressed in cells. Ribozyme parameters, namely hybridization arm length (HAL) and nonhybridizing extraneous sequences (NES), were found to have rate-determining properties. In cell-free reactions, ribozymes with 13-mer HAL were up to 10-fold more efficient than those with 9-mer HAL. Ribozymes with 9-mer HAL were relatively ineffective in transfected cells. Minimizing the number of NES increased ribozyme efficiency in vitro. However, ribozymes with minimal NES were essentially inert intracellularly. The NES at the termini of the most effective intracellular ribozyme, Rz13st ( approximately 95% inhibition of the p gene expression), were predicted to fold into stem-loop structures. These structures most likely increase ribozyme stability as evidenc...Continue Reading

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Apr 7, 1999·Antisense & Nucleic Acid Drug Development·C HammannM Tabler
Dec 7, 2007·Biotechnology & Genetic Engineering Reviews·Maung Nyan Win, Christina D Smolke
Jan 25, 2002·Critical Reviews in Oral Biology and Medicine : an Official Publication of the American Association of Oral Biologists·S P Lyngstadaas

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