前药型寡核苷酸（前药-ON）是一类设计用于在特定细胞内条件或外部刺激下激活的寡核苷酸。前药-ON可以在靶组织或细胞中被激活，从而降低不良反应的风险。在这项研究中，我们合成了由β-半乳糖苷酶激活的前药型寡脱氧核苷酸，β-半乳糖苷酶是一种在癌症和衰老细胞中过度表达的酶。这些寡脱氧核苷酸 (ODN) 含有通过 O4 位自毁连接体与半乳糖缀合的修饰胸苷。紫外熔解分析表明，与互补 RNA 杂交时，与未修饰的 ODN 相比，修饰降低了熔解温度 (Tm)。此外，β-半乳糖苷酶对糖苷键的裂解导致接头从核碱基部分自发去除，产生未修饰的 ODN。此外，将多个修饰的胸苷引入 ODN 中完全抑制了 RNase H 介导的互补 RNA 切割。这些发现表明开发前药-ON的可能性，该前药在β-半乳糖苷酶高表达的癌细胞或衰老细胞中被特异性激活。

Prodrug-type oligonucleotides (prodrug-ONs) are a class of oligonucleotide designed for activation under specific intracellular conditions or external stimuli. Prodrug-ONs can be activated in the target tissues or cells, thereby reducing the risk of adverse effects. In this study, we synthesized prodrug-type oligodeoxynucleotides activated by β-galactosidase, an enzyme that is overexpressed in cancer and senescent cells. These oligodeoxynucleotides (ODNs) contain a modified thymidine conjugated with galactose via a self-immolative linker at the O4-position. UV-melting analysis revealed that the modifications decreased the melting temperature (Tm) compared with that of the unmodified ODN when hybridized with complementary RNA. Furthermore, cleavage of the glycosidic bond by β-galactosidase resulted in the spontaneous removal of the linker from the nucleobase moiety, generating unmodified ODNs. Additionally, the introduction of multiple modified thymidines into ODNs completely inhibited the RNase H-mediated cleavage of complementary RNA. These findings suggest the possibility of developing prodrug-ONs, which are specifically activated in cancer cells or senescent cells with high β-galactosidase expression.