Nucleic Acids Research, 2000, Vol. 28, No. 2 438-445
© 2000 Oxford University Press
Inhibition of in vitro and ex vivo translation by a transplatin-modified oligo(2'-O-methylribonucleotide) directed against the HIV-1 gag-pol frameshift signal
INSERM U.386, IFR Pathologies Infectieuses, Université Victor Segalen Bordeaux 2, 146 Rue Léo Saignat, 33076 Bordeaux Cedex, France, 1UMR CNRS 2225, Institut de Génétique et Microbiologie, Bâtiment 400, Université Paris-Sud, 91405 Orsay Cedex, France and 2Centre de Biophysique Moléculaire CNRS, Rue Charles Sadron, 45071 Orléans Cedex 2, France
A 2'-O-methylribooligonucleotide containing a G1·U·G3 triad modified by trans-diamminedichloro-platinum(II) was targeted to the RNA region responsible for the gag–pol frameshifting during translation of the HIV-1 mRNA. The binding of the platinated oligonucleotide to its target RNA induced a rearrangement of the (G1,G3)-intrastrand crosslink, leading to the formation of an intermolecular oligonucleotide–RNA G–A crosslink. This resulted in the selective arrest of translation of a luciferase gene placed downstream of the HIV-1 frameshift signal both in a cell-free extract (rabbit reticulocyte lysate) and in RNA-transfected cells. A specific inhibition of luciferase activity was still observed when the oligonucleotide–RNA complex was not pre-formed prior to either translation or transfection. Moreover, a selective inhibition was also observed when the oligonucleotide and the plasmid DNA encoding the luciferase and bearing the RNA gag–pol frameshifting signal were co-transfected in NIH 3T3 cultured cells. Therefore the intrastrand
interstrand conversion of the platinum crosslink kinetically competes with the translation machinery and blocks the polypeptide elongation. These transplatin-modified oligonucleotides which operate within a live cell on a ‘real-time’ basis and do not need an external triggering signal constitute a promising new class of selective reactive probes.
* To whom correspondence should be addressed. Tel: +33 (0)5 57 57 10 14; Fax: +33 (0)5 57 57 10 15; Email: jean-jacques.toulme@bordeaux.inserm.fr
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