EFFICIENT INHIBITION OF TRANSCRIPTION ELONGATION IN-VITRO BY OLIGONUCLEOTIDE PHOSPHORAMIDATES TARGETED TO PROVIRAL HIV DNA

Tripler-forming oligophosphoramidates containing thymines and cytosine s or 5-methyl cytosines (5' T(4)CT(4)C(6)T3') bind strongly to a 16 ba se-pair oligopurine-oligopyrimidine sequence of HIV proviral DNA even at neutral pH. These triple-helical complexes formed with oligonucleot ide analogues with N3'-->P5' phosphoramidate linkages are remarkably s table compared to oligonucleotides with natural phosphodiester linkage s. In transcription assays the (T,C)-containing phosphoramidate oligom ers induce an efficient arrest of both bacteriophage and eukaryotic tr anscriptional machineries under conditions where the isosequential pho sphodiesters have no inhibitory effect. In both cases the RNA polymera se (SP6, T7 or pol II) is physically blocked by the non-covalent tripl er and RNA synthesis is stopped at the tripler site. However the eukar yotic transcription machinery is blocked more efficiently (at submicro molar concentration) than the bacteriophage polymerases. The analysis of the 3'-ends of the truncated transcripts provides evidence for diff erences in the termination patterns induced by the tripler barrier for the bacteriophage and the eukaryotic systems. This in vitro comparati ve study provides the basis for the rational design of strong transcri ptional inhibitors. The efficient in vitro inhibition obtained using t he phosphoramidate oligomers in the eukaryotic transcription assay mak es them good candidates for the development of sequence-specific antig ene agents. (C) 1996 Academic Press Limited.