TARGET-SPECIFIC ARREST OF MESSENGER-RNA TRANSLATION BY ANTISENSE 2'-O-ALKYLOLIGORIBONUCLEOTIDES

We describe a novel experimental approach to investigate mRNA translat ion. Antisense 2'-O-allyl oligoribonucleotides (oligos) efficiently ar rest translation of targeted mRNAs in rabbit reticulocyte lysate and w heat germ extract while displaying minimal non-specific effects on tra nslation. Oligo/mRNA-hybrids positioned anywhere within the 5' UTR or the first similar to 20 nucleotides of the open reading frame block ca p-dependent translation initiation with high specificity. The thermody namic stability of hybrids between 2'-O-alkyl oligos and RNA permits t ranslational inhibition with oligos as short as 10 nucleotides. This i nhibition is independent of RNase H cleavage or modifications which re nder the mRNA untranslatable. We show that 2'-O-alkyl oligos can also be employed to interfere with cap-independent internal initiation of t ranslation and to arrest translation elongation. The latter is accompl ished by UV-crosslinking of psoralen-tagged 2'-O-methyloligoribonucleo tides to the mRNA within the open reading frame. The utility of 2'-O-a lkyloligoribonucleotides to arrest translation from defined positions within an mRNA provides new approaches to investigate mRNA translation .