We show the effects of triple-helix formation by assays of primer extension inhibition ire vitro using two systems (two-strand-system (FTFOs) or three-strand-system (TFOs)) targeted to the polypurine tract (PPT) of HIV-1. The FTFOs were more effective thats the TFOs. We found that the FTFOs containing phosphorothioate groups at the 3'- and 5'-ends, of inside the hairpin loop, exhibited higher inhibitory effects on cDNA synthesis and greater exonuclease resistance than the unmodified FTFOs and TFOs. The abilities of the FTFOs containing phosphorothioate groups at the antisense sequence sites to inhibit HIV-I replications were examined. The FTFOs containing phosphorothioate. groups at the antisense sequence sites inhibit the replication of HIV-1 more efficiently than the antisense oligonucleotides, indicating sequence-specific inhibition of HIV-1 replication. (C) 1997 Academic Press.