Study to develop novel genomic drugs by using superfunctional oligonucleotide analogues

反义和反基因策略是基因组药物发现的新方法。反义寡核苷酸与靶mRNA杂交并在活细胞中序列选择性地抑制基因表达。另一方面，反基因寡核苷酸（三链体形成寡核苷酸，TFO）与基因组dsDNA结合以导致基因表达控制。我们以前开发了各种构象锁定核酸（桥核酸，BNAs）。用BNA单体修饰的寡核苷酸被广泛用于评价其物理和生物学性质。这些结果总结如下：1）发现含有2 '，4'-BNA单体的TFO在接近生理条件下显著稳定了与高嘌呤-高嘧啶dsDNA靶的三链体形成。2）开发了携带非天然核碱基的2 '，4'-BNA单体的有效合成路线。3）没有天然核苷来识别CG碱基对，这是抗基因策略中的一个严重问题。设计、合成了一种新的核苷类似物2 '，4'-BNA，它具有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键或糖基进行了修饰。新修饰的寡核苷酸显示出与dsDNA或ssRNA靶标的高结合亲和力。5）反义2 '，4'-BNA寡核苷酸抑制目的基因在活细胞中的表达，具有高度的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.