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Efficient hammerhead ribozymes targeted to the polycistronic Sendai virus P/C mRNA. Structure-function relationships
1Department of Immunology/Microbiology, Rush Medical College, Chicago, Illinois 60612, USA.
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 evidenced by the 8-fold higher resistance to ribonuclease T2 digestion of Rz13st compared with Rz13B. Our results suggest that when designing effective intracellular ribozymes, parameters that enhance formation of productive ribozyme:substrate duplexes and that increase RNA stability should be optimized.
Insights
Hammerhead ribozymes targeting Sendai virus mRNA showed varying efficacy based on design. Optimal intracellular ribozyme design requires balancing hybridization arm length and nonhybridizing extraneous sequences for stability and function.
Area of Science:
- Molecular Biology
- Virology
- Biochemistry
Background:
- Sendai virus P/C mRNA encodes essential P and C proteins via overlapping reading frames.
- Understanding protein function in virus replication necessitates control over mRNA expression.
Purpose of the Study:
- To investigate the functional impact of hammerhead ribozyme design on Sendai virus P/C mRNA cleavage.
- To compare ribozyme efficacy in both cell-free and intracellular environments.
Main Methods:
- Synthesized and expressed identical hammerhead ribozymes in vitro and in transfected cells.
- Assessed ribozyme efficiency by varying hybridization arm length (HAL) and nonhybridizing extraneous sequences (NES).
- Evaluated ribozyme stability against ribonuclease T2 digestion.
Main Results:
- Ribozymes with longer HAL (13-mer) were more efficient in cell-free assays, while shorter HAL (9-mer) were ineffective intracellularly.
- Minimizing NES enhanced in vitro efficiency but reduced intracellular activity.
- The most effective intracellular ribozyme (Rz13st) featured NES that formed stem-loop structures, increasing stability and achieving ~95% inhibition of P gene expression.
Conclusions:
- Intracellular ribozyme design must optimize parameters for productive ribozyme:substrate duplex formation.
- Enhancing RNA stability through structural elements like stem-loops is crucial for effective intracellular ribozyme function.