C. Giovannangeli et al., EFFICIENT INHIBITION OF TRANSCRIPTION ELONGATION IN-VITRO BY OLIGONUCLEOTIDE PHOSPHORAMIDATES TARGETED TO PROVIRAL HIV DNA, Journal of Molecular Biology, 261(3), 1996, pp. 386-398
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.