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Nucleic Acids Research, 2001, Vol. 29, No. 17 3664-3673
© 2001 Oxford University Press

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

B. H. Lloyd*, R. V. Giles1, D. G. Spiller2, J. Grzybowski2, D. M. Tidd2 and D. R. Sibson

Clatterbridge Cancer Research Trust, J. K. Douglas Research Laboratories, Clatterbridge Hospital, Bebington, Wirral CH63 4JY, UK, 1Department of Haematology and 2School of Biological Sciences, University of Liverpool, Liverpool L69 7BZ, UK

Antisense oligonucleotides provide a powerful tool 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 oligonucleotide 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 oligonucleotide library illustrated that independent target sites demonstrated a preference for antisense oligonucleotides of a defined and independent optimal length. The efficacy of antisense oligonucleotide sequences selected in vitro paralleled that observed in phorbol 12-myristate 13-acetate (PMA)-activated U937 cells. The application of methylphosphonate:phosphodiester chimaeric oligonucleotides 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.

* To whom correspondence should be addressed. Tel: +44 151 343 4304; Fax: +44 151 343 1820; Email: bryonyl{at}ccrt.co.uk


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