The antisense sequence of the HIV-1 TAR stem-loop structure covalently linked to the hairpin ribozyme enhances its catalytic activity against two artificial substrates

This work is an in vitro study of the efficiency of catalytic antisense RNA s whose catalytic domain is the wildtype sequence of the hairpin ribozyme, derived from the minus strand of the tobacco ringspot virus satellite RNA. The sequence in the target RNA recognized by the antisense molecule was the stem-loop structure of the human immunodeficiency virus-1 (HIV-1) TAR regi on. This region was able to form a complex with its antisense RNA with a bi nding rate of 2 x 10(4) M-1 s(-1). Any deletion of the antisense RNA compri sing nucleotides of the stem-loop resulted in a decrease in binding rate. S equences 3' of the stem in the sense RNA also contributed to binding. This stem-loop TAR-antisense segment, covalently linked to a hairpin ribozyme, e nhanced its catalytic activity. The highest cleavage rate was obtained when the stem-loop structure was present in both ribozyme and substrate RNAs an d they were complementary. Similarly, an extention at the 5'-end of the hai rpin ribozyme increased the cleavage rate when its complementary sequence w as present in the substrate. Inclusion of the stem-loop at the 3'-end and t he extension at the 5'-end of the hairpin ribozyme abolished the positive e ffect of both antisense units independently. These results may help in the design of hairpin ribozymes for gene silencing.