A cis and trans adenine-dependent hairpin ribozyme against Tpl-2 target.
Résumé
Ribozymes are catalytic RNAs that possess the property of cutting an RNA target via site-specific cleavage after sequence-specific recognition. Ribozymes can moreover cleave multiple substrate molecules. An increasing number of studies show that ribozymes are particularly well adapted tools against cancer, silencing or down-regulating gene expression at the RNA level. We have constructed an adenine-dependent hairpin ribozyme that cleaves the sequence at nucleotides A(225)( downward arrow)G(226) relative to the start codon of translation of the Tpl-2 kinase mRNA; this serine/threonine kinase activates the mitogen-activated protein kinase pathway implicated in cell proliferation in breast cancer. An adenine-dependent hairpin ribozyme 1 (ADHR1) was previously isolated using the Systematic Evolution of Ligands by EXponential enrichment procedure. Switch on/switch off ribozymes are particularly useful since high amounts of stable ribozyme can be produced in the absence of adenine and the ribozyme specifically cleaves its target in the presence of adenine. The ADHR1 target sequence was replaced by a sequence derived from the Tpl-2 kinase mRNA. The resulting Tpl-2 ribozyme is active in cis cleavage: kinetic studies have been performed as a function of Mg(2+) concentration, adenine concentration, as well as at different pH and with various cofactors. Finally, the Tpl-2 ribozyme was shown to cleave its target in trans successfully. These findings demonstrate that a potential therapeutic ribozyme can be produced by simple sequence modification.