Exploring TAR–RNA aptamer loop–loop interaction by X-ray crystallography, UV spectroscopy and surface plasmon resonance

Abstract

In HIV-1, trans-activation of transcription of the viral genome is regulated by an imperfect hairpin, the trans-activating responsive (TAR) RNA element, located at the 5′ untranslated end of all viral transcripts. TAR acts as a binding site for viral and cellular proteins. In an attempt to identify RNA ligands that would interfere with the virus life-cycle by interacting with TAR, an in vitro selection was previously carried out. RNA hairpins that formed kissing-loop dimers with TAR were selected [Ducongé F. and Toulmé JJ (1999) RNA, 5:1605–1614]. We describe here the crystal structure of TAR bound to a high-affinity RNA aptamer. The two hairpins form a kissing complex and interact through six Watson–Crick base pairs. The complex adopts an overall conformation with an inter-helix angle of 28.1°, thus contrasting with previously reported solution and modelling studies. Structural analysis reveals that inter-backbone hydrogen bonds between ribose 2′ hydroxyl and phosphate oxygens at the stem-loop junctions can be formed. Thermal denaturation and surface plasmon resonance experiments with chemically modified 2′-O-methyl incorporated into both hairpins at key positions, clearly demonstrate the involvement of this intermolecular network of hydrogen bonds in complex stability.

Structure and stability of RNA/RNA kissing complex: with application to HIV dimerization initiation signal

“…In the predicted structures, both TAR-TAR*(GA) and TAR-R06 contain a 6-bp intermolecular kissing interactions. The predicted structures are the same as that of the experimental measured structures (Lebars et al 2008).…”

“…The unit of the entropies is (k B ). As a special case for the specific kissing complex formed in the TAR-TAR* complex (Lebars et al 2008), the loop lengths of L 1 and L 3 are zero and the length of H 2 is 6 bp. As an approximation, we fix the value of lnðv 6;0;0 Þ to 0 (not listed in the Table).…”

Direct evidence for RNA–RNA interactions at the 3′ end of the Hepatitis C virus genome using surface plasmon resonance

“…This conclusion is somehow surprising. Previous works have shown that stable kissing complexes can only be formed through Watson-Crick base-pairing between the partially or totally complementary sequences of apical loops, i.e., in a stem-loop context (Westhof et al 1988;Lee and Crothers 1998;Mujeeb et al 1998;Kim and Tinoco 2000;Ennifar et al 2001;Lebars et al 2008;Van Melckebeke et al 2008). The intermolecular loop-loop helix is stacked between the two stem helices.…”

“…SPR is widely used to investigate protein-protein, protein-small molecules, or protein-DNA interactions (for review, see Rich and Myszka 2011) but rarely for investigating RNA-RNA complexes, likely due to their susceptibility to hydrolysis. In previous studies, we have successfully investigated interactions between RNA oligonucleotides with structured RNA of human viruses or cells (Ducongé et al 2000;Aldaz-Carroll et al 2002;Lebars et al 2008;Dausse et al 2011).…”

Nucleotides and Nucleic Acids; Oligo- and Polynucleotides

“…340 The crystal structure of the HIV-1 TAR RNA aptamer binding to TAR showed that it existed as a kissing complex of a hairpin from the aptamer with the TAR RNA itself. 341 Aptamers have also been developed, in the interest of biosecurity, binding to the botulinium neurotoxin. 342 As well as aptamers against proteins, they have been targeted towards whole cells.…”