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Nucleic Acids Research 2005 33(1):114-125; doi:10.1093/nar/gki155
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Published online 7 January 2005

© 2005, the authors Nucleic Acids Research, Vol. 33 No. 1 © Oxford University Press 2005; all rights reserved
The online version of this article has been published under an open access model. Users are entitled to use, reproduce, disseminate, or display the open access version of this article for non-commercial purposes provided that: the original authorship is properly and fully attributed; the Journal and Oxford University Press are attributed as the original place of publication with the correct citation details given; if an article is subsequently reproduced or disseminated not in its entirety but only in part or as a derivative work this must be clearly indicated. For commercial re-use permissions, please contact journals.permissions{at}oupjournals.org.

Article

Translation of stable hepadnaviral mRNA cleavage fragments induced by the action of phosphorothioate-modified antisense oligodeoxynucleotides

Peter Hasselblatt*, Birgit Hockenjos, Christian Thoma, Hubert E. Blum and Wolf-Bernhard Offensperger

Department of Medicine II, University of Freiburg Hugstetter Strasse 55, D-79106 Freiburg, Germany

*To whom correspondence should be addressed. Tel: +43 1 797 30; Fax: +43 1 798 71 53; Email: Hasselblatt{at}imp.univie.ac.at

Received November 15, 2004. Revised December 9, 2004. Accepted December 9, 2004.

Phosphorothioate-modified antisense oligodeoxynucleotides (ASOs) are used to suppress gene expression by inducing RNase H-mediated cleavage with subsequent degradation of the target mRNA. However, previous observations suggest that ASO/RNase H can also result in the generation of stable mRNA cleavage fragments and expression of truncated proteins. Here, we addressed the underlying translational mechanisms in more detail using hepadnavirus-transfected hepatoma cells as a model system of antisense therapy. Generation of stable mRNA cleavage fragments was restricted to the ASO/RNase H pathway and not observed upon cotransfection of isosequential small interfering RNA or RNase H-incompetent oligonucleotides. Furthermore, direct evidence for translation of mRNA fragments was established by polysome analysis. Polysome-associated RNA contained cleavage fragments devoid of a 5' cap structure indicating that translation was, at least in part, cap-independent. Further analysis of the uncapped cleavage fragments revealed that their 5' terminus and initiation codon were only separated by a few nucleotides suggesting a 5' end-dependent mode of translation, whereas internal initiation could be ruled out. However, the efficiency of translation was moderate compared to uncleaved mRNA and amounted to 13–24% depending on the ASO used. These findings provide a rationale for understanding the translation of mRNA fragments generated by ASO/RNase H mechanistically.

Correspondence may also be addressed to Wolf-Bernhard Offensperger. Tel: +49 781 471 12 22; Fax: +49 781 471 16 02; Email: Offensperger{at}josefsklinik.de

Present addresses: Peter Hasselblatt, Research Institute of Molecular Pathology, Dr Bohr-Gasse 7, A-1030 Vienna, Austria

Christian Thoma, EMBL, Gene Expression Program, Meyerhofstrasse 1, D-69117 Heidelberg, Germany

Wolf-Bernhard Offensperger, St Josefsklinik, Weingartenstrasse 70, D-77654 Offenburg, Germany


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