Determination of optimal sites of antisense oligonucleotide cleavage within TNF alpha mRNA

Antisense oligonucleotides provide a powerful too[ in order to determine the consequences of the reduced expression of a selected target gene and may include target validation and therapeutic applications. Methods of predicting optimum antisense sites are not always effective. We have compared the efficacy of antisense oligonucleotides, which were selected in vitro using random combinatorial oligonucleoticle libraries of differing length and complexity, upon putative target sites within TNF alpha mRNA. The relationship of specific target site accessibility and oligonucleotide efficacy with respect to these parameters proved to be complex. Modification of the length of the recognition sequence of the oligonucleoticle library illustrated that independent target sites demonstrated a preference for antisense oligonucleotides, of a defined and independent optimal length. The efficacy of antisense oligonucleoticle sequences selected in vitro paralleled that observed in phorbol 12-myristate 13-acetate (PMA)-activated U937 cells. The application of methylphosphonate:phosphodiester chimaeric oligonucleoticles to U937 cells reduced mRNA levels to up to 19.8% that of the untreated cell population. This approach provides a predictive means to profile any mRNA of known sequence with respect to the identification and optimisation of sites accessible to antisense oligonucleotide activity.