Target-specific arrest of mrna tranlation by antisense 2’-o-alkyloligoribonucleotides

Abstract

We describe a novel experimental approach to investigate mRNA translation. Antisense 2a-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 5a UTR or the first A20 nucleotides of the open reading frame block cap-dependent translation initiation with high specificity. The thermodynamic stability of hybrids between 2a-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 2a-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 2a-O-methyloligoribonucleotides to the mRNA within the open reading frame. The utility of 2a-0- alkyloligoribonucleotides to arrest translation from defined positions within an mRNA provides new approaches to investigate mRNA translation. © 1994 Oxford University Press.