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Oligonucleotides complementary to RNA sequences interact poorly with folded target regions. In vitro selection of oligonucleotides carried out against RNA structures have led to aptamers that frequently differ from antisense sequences, but rather take advantage of non-double-stranded peculiarities of the target. Studies along this line provide information about tertiary RNA architectures as well as their interaction with ligand of interest. We describe here a genomic SELEX approach and its application to the recognition of stem-loop structures prone to the formation of kissing complexes. We also provide technical details for running a procedure termed 2D-SELEX that takes advantage of both in vitro selection and dynamic combinatorial chemistry. This allows selecting aptamer derivatives containing modified nucleotides that cannot be incorporated by polymerases. Last we present in vitro transcription conditions under which large amounts of RNA, suitable for NMR structural studies, can be obtained. These different aspects of the SELEX technology have been applied to the trans-activating responsive element of the human immunodeficiency virus type 1, which is crucial for the transcription of the retroviral genome.

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 degrees , 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.

We previously identified an RNA aptamer targeted to the trans-activating responsive (TAR) element of the HIV-1 genome [F. Ducongé, J.J. Toulmé, In vitro selection identifies key determinants for loop--loop interactions: RNA aptamers selective for the TAR RNA element of HIV--1. RNA 5 (1999) 1605--1614]. This hairpin aptamer binds to its target through loop-loop interactions. We derived chemically modified R06 aptamers that show improved nuclease resistance and affinity for TAR. We review here the results obtained with chimeric aptamers containing locked nucleic acid (LNA) residues. Chimeras containing 2 to 4 LNA residues in an RNA or 2'-O-methyl,RNA context display binding properties of interest and compete with the viral protein Tat for binding to TAR. NMR studies have shown that these properties are modulated by the conformation of the loop-loop helix depending on the presence of LNA residues.

The trans-activating responsive (TAR) RNA element located in the 5' untranslated region of the HIV-1 genome is a 57-nt imperfect stem-loop essential for the viral replication. TAR regulates transcription by interacting with both viral and cellular proteins. RNA hairpin aptamers specific for TAR were previously identified by in vitro selection [Ducongé,F. and Toulmé,J.J. (1999) In vitro selection identifies key determinants for loop-loop interactions: RNA aptamers selective for the TAR RNA element of HIV-1. RNA, 5, 1605-1614]. These aptamers display a 5'-GUCCCAGA-3' consensus apical loop, partially complementary to the TAR one, leading to the formation of a TAR-aptamer kissing complex. The conserved GA combination (underlined in the consensus sequence) has been shown to be crucial for the formation of a highly stable complex. To improve the nuclease resistance of the aptamer and to increase its affinity for TAR, locked nucleic acid (LNA) nucleotides were introduced in the aptamer apical loop. LNA are nucleic acids analogues that contain a 2'-O,4'-C methylene linkage and that raise the thermostablity of duplexes. We solved the NMR solution structure of the TAR-LNA-modified aptamer kissing complex. Structural analysis revealed the formation of a non-canonical G*A pair leading to increased stacking at the stem-loop junction. Our data also showed that the introduction of LNA residues provides an enhanced stability while maintaining a normal Watson-Crick base pairing with a loop-loop conformation close to an A-type.