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Nucleic Acids Research, 2000, Vol. 28, No. 21 4059-4067
© 2000 Oxford University Press

HIV-1 LTR as a target for synthetic ribozyme-mediated inhibition of gene expression: site selection and inhibition in cell culture

Birgit Bramlage, Ettore Luzi and Fritz Eckstein*

Max-Planck-Institut für experimentelle Medizin, Hermann-Rein-Straße 3, D-37075 Göttingen, Germany

A library of three synthetic ribozymes with randomized arms, targeting NUX, GUX and NXG triplets, respectively, were used to identify ribozyme-accessible sites on the HIV-1 LTR transcript comprising positions –533 to 386. Three cleavable sites were identified at positions 109, 115 and 161. Ribozymes were designed against these sites, either unmodified or with 2'-modifications and phosphorothioate groups, and their cleavage activities of the transcript were determined. Their biological activities were assessed in cell culture, using a HIV-1 model assay system where the LTR is a promoter for the expression of the reporter gene luciferase in a transient expression system. Intracellular efficiency of the ribozymes were determined by cotransfection of ribozyme and plasmid DNA, expressing the target RNA. Modified ribozymes, directed against positions 115 and 161, lowered the level of LTR mRNA in the cell resulting in inhibition of expression of the LTR-driven reporter gene luciferase of 87 and 61%, respectively. In the presence of Tat the inhibitions were 43 and 25%. The inactive variants of these ribozymes exhibited a similar inhibitory effect. RNase protection revealed a reduction of RNA which was somewhat stronger for the active than the inactive ribozymes, particularly for ribozyme 115. Unmodified ribozymes showed no inhibition in the cell. The third ribozyme, targeting a GUG-triplet at position 109, possessed only low cleavage activity in vitro and no inhibitory effect in cell culture.

* To whom correspondence should be addressed. Tel: +49 551 3899 274; Fax: +49 551 3899 388; Email: eckstein@mail.em.mpg.de Present address: Ettore Luzi, Department of Experimental Pathology and Oncology, University of Firenze, Viale G.B. Morgagni 50, I-50134 Firenze, Italy


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