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Nucleic Acids Research, 1994, Vol. 22, No. 22 4591-4598
© 1994

RNA

Target-specific arrest of mRNA tranlation by antisense 2'-O-alkyloligoribonucleotides

Hans E. Johansson+, Graham J. Helsham2, Brian S. Sproat1 and Matthias W. Hentze*

Gene Expression Programme 1Biochemical Instrumentation Programme, European Molecular Biology Laboratory Meyerhofstrasse 1, D-69117 heidelberg, Germany 2BBSRC Institute for Animal Health, Pirbright Laboratory Pirbright, Working, Surrey GU24 0NF, UK

*To whom correspondence should be addressed

Received August 22, 1994. Revised October 10, 1994. Accepted October 10, 1994.

We describe a novel experimental approach to investigate mRNA translation. Antisense 2'-O-allyl oligoribonucleotides (oligos) efficiently arrest translation of targeted mRNAs in rabbit reticulocyte lysate and wheat germ extract while displaying minimal non-specific effects on translation. Oligo/mRNAhybrids positioned anywhere within the 5' UTR or the first ~20 nucleotides of the open reading frame block cap-dependent translation initiation with high specificity. The thermodynamic stability of hybrids between 2'-O-alkyl oligos and RNA permits translational inhibition with oligos as short as 10 nucleotides. This inhibition is independent of RNase H cleavage or modifications which render the mRNA untranslatable. We show that 2'-O-alkyl oligos can also be employed to interfere with cap-independent internal initiation of translation and to arrest translation elongation. The latter is accomplished by UV-crosslinking of psoralentagged 2'-O-methyloligoribonucleotides to the mRNA within the open reading frame. The utility of 2'-0- alkyloligoribonucleotides to arrest translation from defined positions within an mRNA provides new approaches to investigate mRNA translation.

+Present address: Department of Chemistry and Biochemistry, University of Colorado, Boulder, CO 80309-0215, USA


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