Mapping of antisense inhibition sites in the leader region of brome mosaicvirus RNA 3

Antisense oligonucleotides (ASOs) are used as therapeutic agents in medicin e and antisense genes are used in agriculture to modulate the gene expressi on in transgenic crops. One mechanism of antisense action is by interfering with ribosome function and blocking translation. In this study, the inhibi tion of translation caused by a series of ASOs was tested, using RNA 3 of t he 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 rabbi t 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 t he 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 ste m 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 in vestigation for the control of plant viruses in transgenic plants.