Chemical Stabilization of Unnatural Nucleotide Triphosphates for the in Vivo Expansion of the Genetic Alphabet.
Chemical Stabilization of Unnatural Nucleotide Triphosphates for the in Vivo Expansion of the Genetic Alphabet.
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DOI：
10.1021/jacs.6b12731
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发表时间：
2017-02-15
影响因子：
15
通讯作者：
Romesberg FE
中科院分区：
文献类型：
--
作者：
Feldman AW;Dien VT;Romesberg FE
我们已经开发了一种非天然碱基对（UBP）和一种半合成生物（SSO），该生物导入非天然核苷三磷酸组分并使用它们复制含有UBP的DNA。然而，UBP的繁殖至少部分地受到非天然三磷酸盐的稳定性的限制，所述非天然三磷酸盐被细胞和分泌的磷酸酶降解。为了解决这个问题，我们现在报告了非天然三磷酸盐的合成和评价，其β，γ-桥氧被二氟亚甲基部分取代，产生dNaMTPCF 2和dTPT 3 TPCF 2。我们发现，虽然dNaMTPCF 2不能支持体内复制，可能是由于聚合酶识别差，dTPT 3 TPCF 2可以，而且，它的稳定性增加可以有助于增加UBP保留。这些数据表明，这种化学方法的SSO优化的承诺，并建议，应寻求其他修改，赋予磷酸酶抗性，而不干扰聚合酶识别。
We have developed an unnatural base pair (UBP) and a semi-synthetic organism (SSO) that imports the constituent unnatural nucleoside triphosphates and uses them to replicate DNA containing the UBP. However, propagation of the UBP is at least in part limited by the stability of the unnatural triphosphates, which are degraded by cellular and secreted phosphatases. To circumvent this problem, we now report the synthesis and evaluation of unnatural triphosphates with their β,γ-bridging oxygen replaced with a difluoromethylene moiety, yielding dNaMTPCF2 and dTPT3TPCF2. We find that while dNaMTPCF2 cannot support in vivo replication, likely due to poor polymerase recognition, dTPT3TPCF2 can, and moreover, its increased stability can contribute to increased UBP retention. The data demonstrate the promise of this chemical approach to SSO optimization, and suggest that other modifications should be sought that confer phosphatase resistance without interfering with polymerase recognition.