Studies on stereoregulated phosphorothioates for their gene-regulatory ability

反义技术是严重病毒性疾病最有前途的诊断方法之一。具有硫代磷酸键的硫代寡脱氧核苷酸（S-oligos）已被专门研究。然而，要将该方法应用于临床，在每个硫代磷酸盐链的立体异构所产生的众多立体异构s -寡聚物中识别并制备真正有效的特异性结构一直是一个关键的未解决的问题，因此，在本研究中，我们进行了以下研究来克服上述问题。(1)我们对硫代磷酸盐Rp和Sp构型与S-oligo RNA/DNA双工结构之间的关系进行了2D-NMR分析并结合分子动力学计算。研究表明，RNA双链的结构与野生型DNA-RNA双链的结构相似，但Rp连锁产生更高的Tm，这表明d…更多的双链稳定性取决于这种因素而不是双链结构，可能是水化程度。另一方面，发现双相结构的差异是S-oligo-DNA杂化稳定性的原因。这些新发现表明，合适的结构取决于靶标，并且该规则的机制也取决于靶标。（2）. 对S-oligo polyC的非特异性抗病毒活性也进行了研究，发现这是由于其独特的四链溶液结构i-motif，其稳定性取决于其Rp或Sp构型。此外，从结构上研究了S-oligo polyC可能的靶基因——端粒C-repeat，发现存在同分异构体C-repeat四分体。这一新发现不仅为了解S-oligo非特异性活性的整个机制，而且为了解端粒拓扑结构在癌症和衰老方面的生物学功能提供了线索。(3)立体选择性合成S-oligos是另一个尚未解决的重要课题，研究发现在亚磷酸盐法中使用间氯甲酚作为离去基，在基本条件下具有高立体选择性和高酯交换收率。少

In the antisense technique, one of the most promising diagnostic methods for severe viral diseases, . phosphorothioate oligodeoxynucleotides (S-oligos) which has a thiophosphate linkage have been investigated exclusively. To apply this method in clinic, however, there has been a crucial unsolved problem that one should identify and prepare the truly effective specific structure among numbers of stereoisomeric S-oligos arising from the stereoisomerism of each thiophosphate linkage, and in the present study, therefore, we have performed following investigation s to overcome the above problem. (1) We have carried out 2D-NMR analysis combined with molecular dynamic calculation on the relationship between the thiophosphate Rp and Sp configurations and the S-oligo RNA/DNA duplex structure. It has been elucidated that the structure of the duplexes with RNA is similar with each other as well as with that of wild type DNA-RNA duplex, although Rp linkage produced higher Tm, indicative that the d … More uplex stability is dependent such a factor other than the duplex structure, possibly as extent of hydration. On the other hand, difference of the duplex structure was found to be responsible for S-oligo-DNA hybrid stability. These new findings indicate that the, suitable configuration depends on the target, and that the mechanism of this rule also on it. (2). Nonspecific antiviral activity of S-oligo polyC has also been studied, and found to be due to its unique four-stranded solution structure, i-motif, whose stability is dependent on its Rp or Sp configuration. Also, a possible target gene for S-oligo polyC, telomere C-repeat, has been investigated in terms of the structure, and it has been found that there are isomeric C repeat tetrads. This new discovery may give suggestion not only to understand whole mechanism for nonspecific activity of S-oligo but biological functions of telomere topologies in relation to cancer and aging. (3) Stereoselective synthesis of S-oligos, another unsolved essential subject, has been studied and use of m-chlorocresol as a leaving group in the phosphite method was found to lead to high stereoselectivity and high yield of transesterification in the basic conditions. Less