Inhibition of HIV-1 Tat-dependent trans activation by steric block chimeric 2 '-O-methyl/LNA oligoribonucleotides

The HIV-1 trans-activation responsive element (TAR) RNA 59-residue stem-loo p interacts with the HIV trans-activator protein Tat and other cellular fac tors to stimulate transcriptional elongation from the viral long terminal r epeat (LTR). Inhibition of these interactions blocks full-length HIV transc ription and hence replication. We have found that three types of 12-residue oligonucleotide analogues, namely, a 2'-O-methyl oligoribonucleotide (OMe) , a chimeric oligonucleotide containing 7xOMe and 5 x 5-methyl C locked nuc leic acid (LNA) residues, and a peptide nucleic acid (PNA), inhibit Tat-dep endent in vitro transcription in HeLa cell nuclear extract equally efficien tly (50% inhibition at 100-200 nM) and sequence specifically. The results a re correlated with surprisingly similar binding strengths to a model 39-res idue TAR under transcription conditions. A 12-mer containing 11 contiguous LNA residues was less effective in both Tat-dependent transcription inhibit ion and TAR 39 binding. Anti-TAR 3'-carboxyfluorescein- (FAM-) labeled OMe and OMe/LNA chimeric 12-mers were also efficient Tat-dependent in vitro tra nscription inhibitors as were 3'-FAM-labeled OMe oligonucleotides containin g some phosphorothioate (PS) linkages. By use of a HeLa cell line containin g stably integrated plasmids expressing firefly luciferase under HIV-LTR/Ta t dependence as well as a Renilla luciferase constitutive control, we showe d submicromolar, selective, dose-dependent, and sequence-dependent intracel lular inhibition of Tat-TAR trans activation by the anti-TAR 3'-FAM 12-resi due 7xOMe/5xLNA oligonucleotide when delivered by cationic lipid. No intrac ellular activity was observed for the corresponding anti-TAR 3'-FAM OMe 12- mer. An alternating PS-containing 3'-FAM OMe 12-mer oligonucleotide exhibit ed partial inhibition of trans-activation activity, but this was correlated with a similar effect on control gene expression, suggesting nonspecific i nhibition.