2‘-<i>O</i>-Methyl-RNA Hairpins Generate Loop−Loop Complexes and Selectively Inhibit HIV-1 Tat-Mediated Transcription

Abstract

The interaction of the TAR RNA element of human immunodeficiency virus type 1 (HIV-1) with a 2'-O-methyl analogue of an RNA hairpin aptamer previously identified by in vitro selection [Ducongé, F., and Toulmé, J. J. (1999) RNA 5, 1605-1614] was characterized by UV-monitored thermal denaturation and surface plasmon resonance experiments. The complex between TAR and this aptamer derivative displays stability (K(d) = 9.9 +/- 1.0 nM) and kinetic properties [k(on) = 9.0 +/- 0.3 M(-1) s(-1), k(off) = (8.9 +/- 0.6) x 10(-4) s(-1)] close to those of the parent RNA aptamer. The modified aptamer forms a "kissing" complex with TAR driven by the same key elements as the TAR-RNA aptamer one. In particular, the G and A residues closing the aptamer loop remain crucial for TAR-2'-O-methyl aptamer complexes. Moreover, the 2'-O-methyl aptamer analogue specifically inhibits Tat-mediated transcription in an in vitro assay more efficiently than the RNA aptamer. This is likely due to the increased lifetime of the former oligonucleotide in the cell-free extract. The 2'-O-methyl modification extends the range of molecules that can be used to target viral hairpin RNA through loop-loop interactions. More generally, this demonstrates the interest of SELEX for targeting RNA hairpins and understanding nucleic acid interactions.