Toulmé J J, Di Primo C, Moreau S
INSERM U 386, IFR Pathologies Infectieuses, Université Victor Segalen, Bordeaux, France.
Prog Nucleic Acid Res Mol Biol. 2001;69:1-46. doi: 10.1016/s0079-6603(01)69043-3.
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.
许多RNA结构参与调控过程，这要么是因为它们构成一种信号，如参与核糖体移码的发夹或假结，要么是因为它们是蛋白质的结合位点，例如人类免疫缺陷病毒的反式激活应答RNA元件，其与病毒蛋白Tat和细胞蛋白的结合可使逆转录病毒基因组进行全长转录。能够与这类RNA基序高亲和力结合的选择性配体可作为剖析其所涉及分子机制的工具。当RNA结构在功能失调基因的表达或病原体增殖中起作用时，这类配体也可能构成治疗剂的原型。不同类别的配体（氨基糖苷类、嵌入剂、肽）都与这一目标相关。然而，寡核苷酸值得特别关注。它们已在反义策略框架中得到广泛应用。乍一看，这种合理方法表面上的简单性非常有吸引力。的确，已经发表了许多成功的研究，描述了在无细胞系统、培养细胞或体内，与RNA区域互补的寡聚物对翻译、剪接或逆转录的有效抑制作用。然而，RNA结构限制了靶位点对反义序列的可及性：RNA区域分子内缔合之间的竞争会削弱甚至消除反义效应。人们已经开发出各种可能性来规避这一限制。这包括合理策略和组合策略。设计高亲和力寡聚物以侵入RNA结构。或者，三链形成寡核苷酸（TFO）和适体可能识别折叠的RNA基序。由于三链螺旋形成存在强烈的序列限制，TFO的使用相当有限，而体外筛选提供了一种探索大量寡核糖核酸或寡脱氧核糖核酸文库以鉴定强的、选择性寡核苷酸结合剂的方法。针对HIV-1的TAR RNA元件筛选出的候选物（适体）与靶标形成稳定的环-环（亲吻）复合物，通过三级相互作用的重要贡献，提供了识别功能性RNA结构的寡核苷酸的有趣实例。
