The intracellular mechanism of action of exogeneously administered antisense oligonucleotides is not known. Two mechanisms have been suggested based on studies in cell free systems. These are: 1) inhibition of ribosome attachment or movement along mRNA; and, 2) creating a substrate for RNAse H degradation of targeted mRNA. RNAse H is an enzyme which destroys the RNA portion of an RNA/DNA hybrid complex and is present in the cytoplasm of all proliferating cells. Its natural function in cells is unknown. We have obtained a full-length cDNA coding for bacterial RNAse H. We will place this cDNA into a mammalian expression vector which replicates episomally and thus attains high copy number. Cells transfected with this construct and sham-transfected cells will be compared for their responsiveness to antisense oligonucleotide addition. If RNAase H is involved in the intracellular functioning of antisense oligos, then cells with high levels of RNAse H should respond better to addition of these compounds. Likewise, the RNAse H cDNA in reverse orientation in the vector will be transfected in a similar experiment. In this case, endogenous RNAse production should be inhibited (due to this antisense vector) and response to other antisense oligos added extracellularly should be reduced.
