Control of Gene Expression by Functional and Artificial Nucleic Acids

为了开发一种新的调控基因表达的策略，合成了一种寡核苷酸-功能分子偶联物，并对其生物学性质进行了研究。主要研究结果如下：1)设计、合成了一些杂环化合物，并将其融入到桥联核酸骨架中。将得到的含有非天然碱基的桥联核酸引入到寡核苷酸中，并考察了它们的三链形成能力。非常有趣的是，发现桥联核酸衍生物成功地识别了吡咪碱嘌呤中断位点。2)作为一种在寡核苷酸中引入官能团的简便方法，发展了一种基于Click化学的延伸后修饰方法。此外，还成功地制备了一些与转录因子结合位点(双链DNA)偶联的寡核苷酸。3)将以上制备的含有非天然碱基的寡核苷酸导入活细胞，并对其调控基因表达的能力进行了评估。含有非天然碱基的寡核苷酸单独或与本课题组开发的抗基因阻断策略一起，显示出对基因表达的有效调控。此外，与转录因子结合位点结合的寡核苷酸促进了靶基因的表达。4)详细研究了寡核苷酸类似物的化学结构与其三链形成能力之间的关系，并成功地开发出了一种新型桥联核酸，其三链形成能力高于PINTO-BNA。

In order to develop a novel strategy to control a gene expression, an olignnucleotidc-functional molecule conjugate has been synthesized and its biological properties were also studied. The results are summarized below.1) Some heterocyclic compounds were designed, synthesized and incorporated into a bridged nucleic acid skeleton. The obtained bridged nucleic acids bearing unnatural nucleobases were also introduced into oligonucleotides, and their triplex-forming ability was investigated. Very interestingly, it was found that the recognition of pyrimicline-purine interruption site was successfully achieved by the bridged nucleic acid derivatives /2) As a convenient method to introduce a functional group into oligonucleotides, a post-elongation modification method based on Click chemistry was developed. Furthermore, some oligonucleotides conjugated with a transcription factor-binding site (double stranded DNA) were also successfully prepared.3) The oligonucleotides bearing unnatrural nucleobases prepared above were transfected into living cells, and their ability to control gene expression was evaluated. The oligonucleotides bearing unnatural nucleobases, by themselves or together with the antigene-block strategy developed by our group, showed effective regulation of gene expression. Furthermore, the oligonucleotides conjugated with a transcription factor-binding site promoted the target gene expression. These results are of great interest and would be useful for further biological application.4) A relationship between chemical structures of oligonucleotide analogues and their triplex-forming ability was evaluated in detail, and the novel bridged nucleic acid, of which triplex-forming ability was higher than that of the pinto-type BNA, was successfully developed.