Role of RNase H in hybrid-arrested translation by antisense oligonucleotides.

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Abstract
The mechanism of hybrid-arrested translation by antisense oligodeoxynucleotides has been investigated with the rabbit reticulocyte lysate system. The oligonucleotides studied were directed against different regions of mouse .alpha.- or .beta.-globin mRNAs. Freshly prepared reticulocyte lysates were found to contain 1-2% of the level of RNase H in nucleated cells. This level of activity was sufficient to cleave nearly 100% of the targeted mRNA at the site of hybridization with a complementary oligodeoxynucleotide in 1 hr under conditions of active translation. Using poly[rA)oligo(dT) as a competitive inhibitor of the enzyme, hybrid arrest by oligodeoxynucleotides complementary to the sequence spanning the initiation codon or to a sequence in the coding region was found to be due entirely to cleavage of mRNA by RNase H. Hybridization of oligodeoxynucleotides adjacent to the cap site of .beta.-globin mRNA, but not the .alpha.-globin mRNA, also inhibited protein synthesis directly. Even in this case, however, cleavage of the mRNA by RNase H was the predominant pathway of inhibition.

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