Modulation of RNA function by oligonucleotides recognizing RNA structure.

Abstract

Numerous RNA structures are responsible for regulatory processes either because they constitute a signal, like the hairpins or pseudoknots involved in ribosomal frameshifting, or because they are binding sites for proteins such as the trans-activating responsive RNA element of the human immunodeficiency virus whose binding to the viral protein Tat and cellular proteins allows full-length transcription of the retroviral genome. Selective ligands able to bind with high affinity to such RNA motifs may serve as tools for dissecting the molecular mechanisms in which they are involved. Such ligands might also constitute prototypes of therapeutic agents when RNA structures play a role in the expression of dysfunctional genes or in the multiplication of pathogens. Different classes of ligands (aminoglycosides, interacalating agents, peptides) are of interest to this aim. However, oligonucleotides deserve particular consideration. They have been extensively used in the frame of the antisense strategy. The apparent simplicity of this rational approach is, at first sight, very attractive. Indeed, numerous successful studies have been published describing the efficient inhibition of translation, splicing, or reverse transcription in cell-free systems, in cultured cells, or in vivo by oligomers complementary to an RNA region. However, RNA structures restrict the access of the target site to the antisense sequence: The competition between the intramolecular association of RNA regions weakens or even abolishes the antisense effect. Various possibilities have been developed to circumvent this limitation. This includes both rational and combinatorial strategies. High-affinity oligomers were designed to invade the RNA structure. Alternatively, triplex-forming oligonucleotides (TFO) and aptamers may recognize the folded RNA motif. Whereas the use of TFOs is rather limited owing to the strong sequence constraints for triple-helix formation, in vitro selection offers a way to explore vast oligoribo or oligodeoxyribo libraries to identify strong, selective oligonucleotide binders. The candidates (aptamers) selected against the TAR RNA element of HIV-1, which form stable loop-loop (kissing) complexes with the target, provide interesting examples of oligonucleotides recognizing a functional RNA structure through an important contribution of tertiary interactions.