Study to develop novel genomic drugs by using superfunctional oligonucleotide analogues

反义和抗原策略是基因组药物开发的新方法。在活细胞中，反义寡核苷酸与靶mRNA杂交并选择性地抑制基因表达序列。另一方面，抗原寡核苷酸（三聚体形成寡核苷酸，TFOs）与基因组dsDNA结合，导致基因表达控制。我们以前开发了各种构象锁定核酸（桥接核酸，BNAs）。用BNA单体修饰的Ologonucleotides被广泛用于评价物理和生物特性。这些结果总结如下。1)在接近生理条件下，发现含有2',4'-BNA单体的tfo与同嘌呤-同嘧啶dsDNA靶点的三联体形成具有显著的稳定性。2)开发了含非天然核碱基的2',4'-BNA单体的高效合成路线。3)没有天然的核苷来识别CG碱基对，这给抗原策略带来了严重的问题。设计并合成了一种新的核苷类似物，2',4'-BNA，具有2-吡啶酮碱基单元，并被证明对CG碱基对具有良好的识别能力。4)对2',4'-BNA寡核苷酸的磷酸二酯链或糖段进行了另一次修饰。新修饰的寡核苷酸与dsDNA或ssRNA靶标具有较高的结合亲和力。还观察到这些寡核苷酸类似物具有良好的酶稳定性。5)反义2′，4′-BNA寡核苷酸抑制靶基因在活细胞中的表达，具有高序列选择性和非细胞毒性。

Antisense and antigene strategies are new methodology for genomic drugs discovery. An antisense oligonucleotide hybridizes with a target mRNA and inhibits gene expression sequence-selectively in a living cell. On the other hand, an antigene oligonucleotide (triplex-forming oligonucleotides, TFOs) binds with genomic dsDNA to result in gene expression control. We previously developed various kinds of conformationally locked nucleic acids (Bridged Nucleic Aclds, BNAs). Ologonucleotides modified with BNA monomers were widely used to evaluate physical and biological properties. These results are summarized below.1) TFOs containing the 2',4'-BNA monomers were found to stabilize significantly triplex formation with the homopurine-homopyrimidine dsDNA target under near physiological conditions.2) Efficient synthetic routes for the 2',4'-BNA monomers bearing unnatural nucleobases wore developed.3) There is no natural nucleoside to recognize a CG base pair, and this psoes a serious problem in antigene strategy. A novel nucleoside analogue, 2',4'-BNA with a 2-pyridone base-unit was designed, synthesized and proved to be excellent for recognition of a CG base pair.4) Another modification was made on a phosphodiester linkage or the sugar moiety of the 2',4'-BNA oligonucleotide. The newly modified oligonucleotides showed high binding affinity with dsDNA or ssRNA target. Excellent enzymatic stability of these oligonucleotide analogues was also observed.5) Antisense 2',4'-BNA oligonucleotides inhibited expression of the target gene in living cell with high sequence-selectivity and non-cytotxicity.