Catalytic antisense RNAs produced by incorporating ribozyme cassettes into cDNA

Abstract

A simple strategy is described for the generation of catalytic hammerhead-type ribozymes (Rz) that can be used as highly specific endoribonucleases to cleave a particular target RNA. The technique requires that a cloned cDNA fragment is available which encodes at least a part of the target RNA. About 25 different restriction recognition sequences can be utilized to incorporate specifically designed DNA cassettes into the cDNA. Besides some nucleotides which are specific for a certain restriction site, the DNA cassettes contain a sequence corresponding to the catalytic domain of the hammerhead Rz and, optionally, selectable marker genes, that are removable. The resulting recombinant DNA constructs permit the in vitro and in vivo synthesis of novel 'catalytic antisense RNAs' or 'antisense Rz (Az)', which combine two features: (i) they bind like antisense RNA to their specific substrate RNA, and (ii) they cleave their target as hammerhead Rz do. The utility of the strategy to generate Rz was demonstrated experimentally by incorporating a synthetic SalI-specific DNA ribozyme (Rz) cassette into a unique SalI site of a cloned cDNA fragment of plum pox virus (PPV), which is a single-stranded positive sense plant RNA virus, belonging to the group of potyviruses. The resulting Az constructs delivered Az that were directed against the PPV (+) or (-) RNA, respectively, which cleaved their corresponding target RNAs in the expected manner. Besides the synthetic Rz cassette, a comparable SalI-specific Rz cassette, that had been prepared from a specifically designed plasmid and that contained the tet gene inserted into the sequence of the catalytic domain of the Rz, was also incorporated into the SalI site of the PPV cDNA.(ABSTRACT TRUNCATED AT 250 WORDS)