We synthesized long, nucleobase-modified, single-stranded DNA (ssDNA) using terminal deoxynucleotidyl transferase (TdT) enzymatic polymerization. Specifically, we investigated the effect of unnatural nucleobase size and incorporation density on ssDNA resistance to exo- and endonuclease degradation. We discovered that increasing the size and density of unnatural nucleobases enhances ssDNA resistance to degradation in the presence of exonuclease I, DNase I, and human serum. We also studied the mechanism of this resistance enhancement using molecular dynamics simulations. Our results show that the presence of unnatural nucleobases in ssDNA decreases local chain flexibility and hampers nuclease access to the ssDNA backbone, which hinders nuclease binding to ssDNA and slows its degradation. Our discoveries suggest that incorporating nucleobase-modified nucleotides into ssDNA, using enzymatic polymerization, is an easy and efficient strategy to prolong and tune the half-life of DNA-based materials in nucleases-containing environments.

中文翻译：

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核酸碱基修饰的单链DNA的酶促合成可提供对核酸酶降解的可调抗性。

我们使用末端脱氧核苷酸转移酶（TdT）酶促聚合合成了长，经碱基修饰的单链DNA（ssDNA）。具体来说，我们调查了非天然核碱基大小和掺入密度对ssDNA抵抗外切和内切核酸酶降解的影响。我们发现增加非天然核碱基的大小和密度会增强ssDNA在核酸外切酶I，DNA酶I和人血清存在下对降解的抗性。我们还使用分子动力学模拟研究了这种电阻增强的机理。我们的结果表明，ssDNA中非天然核碱基的存在降低了局部链的柔韧性，并阻碍了核酸酶接近ssDNA主链，这阻碍了核酸酶与ssDNA的结合并减缓了其降解。