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Abstract. -- Antisense oligonucleotides (ASOs) are used as therapeutic agents in medicine and antisense genes are used in agriculture to modulate the gene expression in transgenic crops. One mechanism of antisense action is by interfering with ribosome function and blocking translation. In this study, the inhibition of translation caused by a series of ASOs was tested, using RNA 3 of the multipartite brome mosaic virus as a model system. RNA 3 hybridized to a series of ASOs was used as a template for in vitro translation using rabbit reticulocyte lysates. Using 25mers, it was found that ASOs complementary to the AUG site, the cap region, and the first 5' stem loop provided strong inhibition. In order to more exactly map these inhibitory sites, a series of 15-20mers were tested. These were less inhibitory than the 25mers, but the AUG site was again one of the most susceptible sites. Only certain ASOs targeted to the 5' stem loop were effective, one of which "clamped" two stem flanking regions together, perhaps stabilizing the stem loop. This study demonstrated the efficient inhibition of in vitro translation of this plant virus by relatively short antisense sequences, and suggested avenues of investigation for the control of plant viruses in transgenic plants.
For each gene in a DNA double helix, only one strand codes for useful information, while the other strand is the complement to the first strand. The sequence of the mRNA coded for by a gene is termed the "sense" version, while the complementary version is termed "antisense." It was realized fairly early that a short synthesized segment of antisense DNA could be used to disrupt RNA function. This was first demonstrated with retroviruses in cell culture (Zamecnik & Stephenson 1978). These antisense oligonucleotides (ASOs) and antisense genes (expressing longer antisense mRNAs) have since been tested as novel therapeutic tool against cancers, viral infections and genetic disorders in human cells (Agrawal 1996; Temsamani & Guinot 1997), as well as against plant mRNAs (Smith et al. 1988) and plant viruses (Nelson et al. 1993) for agricultural purposes.
Antisense has been shown to operate by either of two mechanisms to control mammalian or bacterial gene expression. First, antisense binding can sterically block the ribosome and/or translation factors from binding to the 5' end of mRNA and initiating translation. Some naturally occurring control systems are based on this mechanism, such as the control the expression of the transposase of IS10 in bacteria (Ma & Simons 1990) or the N-myc gene in human cells (Krystal et al. 1990). Synthesized ASOs designed to bind to the initiation codon and block translation have been effective against coxsackievirus B3 (Yang et al. 1997) and the E2 gene of papilloma viruses (Cowsert et al. 1993). A second mechanism involves the destruction of double stranded RNAs in the cell by ribonuclease H (RNase H). The binding of mRNA by ASOs induces the cleavage of the mRNA at the binding site. A natural system exemplifying this is bacteriophage lambda and the cII gene (Krinke & Wulff 1987). This RNase mechanism has served as the focus of much work to design therapeutic ASOs (e.g., Duroux et al. 1995).
In plant systems, artificial control of gene expression has been attributed to a variety of mechanisms. Partial or full viral genes in sense orientation have been used quite successfully to control viral …