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

反义策略可以被寡核苷酸以序列特异性的方式破坏靶基因，已成为基因治疗和基因分析的新工具。为了寻求基因治疗的策略，确定反义寡核苷酸的序列是至关重要和不可避免的。RNA的三级结构信息对于这一目的是必不可少的，然而，结构的多样性使我们无法看到整个图。在这项研究中，我们重点建立了用荧光标记的寡核苷酸分析天然折叠RNA的三级结构的方法。我们开发了两种类型的反义寡核苷酸。一种是2‘-0-芘偶联的2’-0-甲基型寡核苷酸(OMUpy)。在天然折叠的RNA中加入OMUpy后，其荧光强度比单独使用OMUpy时增加了100倍。由于在错配序列的RNA中加入OMUpy对结合强度几乎没有影响，因此OMUpy可以作为分析靶RNA上结合位点的一种很好的探针。另一种是Ru-络合物标记的寡核苷酸(Ru-Probe)。从荧光各向异性分析来看，Ru-探针的旋转运动在加入天然折叠的RNA后发生了显著的变化。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-探针与目标RNA杂交。该方法的优点是在评价混杂地层时不需要进行B/F分离。使用这两个荧光寡核苷酸探针，我们证明了在均相介质中可以确定靶RNA上的可访问位置，而不需要B/F分离。这也证明了这些方法可以用来监测活细胞中的基因流。

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.