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

反义策略是一种利用寡核苷酸对目标基因进行序列特异性破坏的策略，已成为基因治疗和基因分析的新工具。为了实施基因治疗策略，确定反义寡核苷酸的序列是至关重要和不可避免的。RNA三级结构的信息是必不可少的，然而，结构的多样性使我们无法看到整个图。在本研究中，我们重点建立了用荧光共轭寡核苷酸分析天然折叠rna三级结构的方法。我们开发了两种反义寡核苷酸。一种是2'-0-芘共轭的2'-0-甲基型寡核苷酸（OMUpy）。将OMUpy加入到天然折叠RNA中，荧光强度比单独加入OMUpy的荧光强度显著提高100倍。由于将OMUpy添加到失配序列的RNA上几乎不影响其强度，因此可以认为OMUpy是分析靶RNA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探针）。从荧光各向异性分析来看，添加天然折叠RNA后，ru探针的旋转运动发生了显著变化。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探针与靶RNA杂交。该方法的优点是对混合地层的评价不需要进行B/F分离。使用这两种荧光寡核苷酸探针，我们证明了在没有B/F分离的均匀介质中可以确定目标RNA上的可达位点。研究还表明，这些方法可用于监测活细胞中的遗传流动。

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.