Twisted intercalating nucleic acids form stable triplexes with polypurine tracts of double-stranded DNA. Their affinity depends on their length, primary structure and base contents, parallel or antiparallel orientation of oligonucleotides respectively to DNA, number of TINA residues and their relative positions. Basing on parallel CT, GT and antiparallel GT triplex-forming 16-mer oligonucleotides targeted to polypurine tract of HIV proviral DNA, we synthesized eleven different oligonucleotides with 2-4 TINA insertions in different positions. Studies of their interaction with target duplex by gel shift, fluorescence spectroscopy, circular dichroism and thermal denaturation demonstrated that antiparallel GT oligonucleotides form more stable triplexes than parallel TC or TG ones. Two best candidates were selected for the further studies. The first one (5'-AGGGxGGGTTTxTGTTTT-3', Kd = 219 nM) contains only two TINA insertions and does not aggregate in non-denaturing conditions, in contrast to majority of other oligonucleotides.