The transactivation responsive element (TAR) plays a crucial role in the transcription of the HIV-1 genome upon specific binding of the viral protein Tat and cellular proteins. We have previously identified a RNA hairpin aptamer forming a stable and specific kissing complex with TAR RNA (Ducongé, F., and Toulmé, J. J. (1999) RNA 5, 1605-1614). We chemically modified this aptamer with hexitol nucleic acid (HNA) residues. We demonstrate that a fully HNA-modified aptamer is a poor ligand but, in contrast, mixmers containing both HNA and unmodified RNA nucleotides display interesting properties. Two HNA-RNA mixmers bind to TAR with an equilibrium dissociation constant in the low-nanomolar range and show a reduced nuclease sensitivity. In addition, they show a moderate dependence on magnesium ions for binding to TAR. These HNA-RNA mixmers are able to inhibit transactivation of transcription in an in vitro assay.