Departments of Chemistry and Biology and Konstanz Research School Chemical Biology, Universität Konstanz, Universitätsstrasse 10, 78464, Konstanz, Germany.
Institute of Bioengineering and Nanotechnology, 31 Biopolis Way, The Nanos, #09-01, 138669, Singapore, Singapore.
Angew Chem Int Ed Engl. 2017 Sep 18;56(39):12000-12003. doi: 10.1002/anie.201704190. Epub 2017 Jun 27.
Hydrophobic artificial nucleobase pairs without the ability to pair through hydrogen bonds are promising candidates to expand the genetic alphabet. The most successful nucleobase surrogates show little similarity to each other and their natural counterparts. It is thus puzzling how these unnatural molecules are processed by DNA polymerases that have evolved to efficiently work with the natural building blocks. Here, we report structural insight into the insertion of one of the most promising hydrophobic unnatural base pairs, the dDs-dPx pair, into a DNA strand by a DNA polymerase. We solved a crystal structure of KlenTaq DNA polymerase with a modified template/primer duplex bound to the unnatural triphosphate. The ternary complex shows that the artificial pair adopts a planar structure just like a natural nucleobase pair, and identifies features that might hint at the mechanisms accounting for the lower incorporation efficiency observed when processing the unnatural substrates.
疏水人工核苷酸碱基对没有氢键配对的能力，是扩展遗传密码子的有前途的候选物。最成功的核苷酸碱基替代品彼此之间以及与天然对应物几乎没有相似之处。因此，令人困惑的是，这些非天然分子是如何被 DNA 聚合酶处理的，这些聚合酶已经进化到能够有效地与天然构建块一起工作。在这里，我们报告了结构上的深入了解，即一种最有前途的疏水非天然碱基对，dDs-dPx 对，由 DNA 聚合酶插入到 DNA 链中。我们解决了 KlenTaq DNA 聚合酶与修饰模板/引物双链体结合到非天然三磷酸的晶体结构。三元复合物表明，人工碱基对采用平面结构，就像天然核苷酸碱基对一样，并确定了可能暗示解释处理非天然底物时观察到的较低掺入效率的机制的特征。
