DESIGN OF FLUORESCENT RNA-PROBE FOR THE ANALYSIS OF THE HIGH-ORDER STRUCTURE OF RNA AND ITS APPLICATION TO THE GENE DESTRUCTION BY ANTISENSE OLIGONUCLEOTIDES

反义策略是利用寡核苷酸序列特异性地破坏靶基因，使其成为基因治疗和基因分析的新工具。为了实现基因治疗的策略，反义寡核苷酸序列的确定是至关重要的，也是必然的。RNA三级结构的信息对于这个目的是必不可少的，然而，结构的多样性使我们无法看到整个图。本研究的目的是建立一种利用荧光标记寡核苷酸分析天然折叠RNA三级结构的方法。我们开发了两种类型的反义寡核苷酸。一种是2'-0-芘缀合的2'-0-甲基型寡核苷酸（OMUpy）。通过将OMUpy添加到天然折叠RNA中，与单独的OMUpy相比，荧光强度急剧增加100倍。由于向具有错配序列的RNA中添加OMUpy几乎不影响强度，因此得出结论，OMUpy可以是用于分析靶RNA上的结合位点的优异探针。另一种是钌配合物标记的寡核苷酸（Ru探针）。从荧光各向异性测定，Ru-探针的旋转运动显着改变后，除了天然折叠的RNA。旋转运动的变化与表观分子大小密切相关，因此，运动的限制表明Ru-探针与靶RNA杂交。该方法的优点是，B/F分离是不必要的混合形成的评价。利用这两种荧光寡核苷酸探针，我们证明了靶RNA上的可及位点可以在均相介质中确定，而无需B/F分离。研究还表明，这些方法可用于监测活细胞中的遗传流。

The antisense strategy, which can destruct the target gene by oligonucleotides in a sequence specific manner, has been established as the novel tool for the gene therapy and the gene analysis. To pursue the strategy toward the gene therapy, it is crucial and inevitable to decide the sequence of the antisense oligonucleotides. The information of the tertiary structure of the RNA is essential for the purpose, and, however, the diversity of the structure prevents us from viewing the whole figure. In this study, we focused in the establishment of the method to analyze the tertiary structure of native folded RNAs by fluorescence-conjugated oligonucleotides. We developed two types of antisense oligonucleotides. The one is the 2'-0-pyrene-conjugated 2'-0-Methl type oligonucleotide (OMUpy). By addition of OMUpy to the native folded RNA, the fluorescence intensity drastically increased 100 times compared with OMUpy alone. As the addition of OMUpy to the RNA with the mismatch sequence scarcely affected the intensity, it was concluded that OMUpy can be an excellent probe for the analysis of the binding site on the target RNA. The other is the Ru-complex-labeled oligonucleotide (Ru-probe). From the fluorescence anisotropy assay, the rotational motion of Ru-probe was remarkably changed upon addition to the native folded RNA. The change in rotational motion is tightly correlated with the apparent molecular size and, accordingly, the restriction of the motion suggests that Ru-probe hybridized with the target RNA. The advantage of the method is that the B/F separation is not necessary for the evaluation of the hybrid formation. Using these two fluorescent oligonucleotide probes, we demonstrated that the accessible sites on the target RNA could be decided in the homogeneous media without B/F separation. It was also demonstrated that these methods be used to monitor the genetic flow in living cells.