The expansion of the genetic alphabet with additional, unnatural base pairs (UBPs) is an important and long-standing goal in synthetic biology. Nucleotides acting as ligands for the coordination of metal cations have advanced as promising candidates for such an expansion of the genetic alphabet. However, the inclusion of artificial metal base pairs in nucleic acids mainly relies on solid-phase synthesis approaches, and very little is known about polymerase-mediated synthesis. Herein, we report the selective and high yielding enzymatic construction of a silver-mediated base pair (dIm^C–Ag^I–dPur^P) as well as a two-step protocol for the synthesis of DNA duplexes containing such an artificial metal base pair. Guided by DFT calculations, we also shed light into the mechanism of formation of this artificial base pair as well as into the structural and energetic preferences. The enzymatic synthesis of the dIm^C–Ag^I–dPur^P artificial metal base pair provides valuable insights for the design of future, more potent systems aiming at expanding the genetic alphabet.

中文翻译：

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使用修饰的嘌呤核苷三磷酸酶促合成人工碱基对

利用额外的非天然碱基对（UBP）扩展遗传字母是合成生物学中一项重要且长期的目标。用作金属阳离子配位体的核苷酸已经成为遗传字母表扩展的有前途的候选者。然而，在核酸中包括人工金属碱基对主要依赖于固相合成方法，而对于聚合酶介导的合成知之甚少。本文中，我们报道了银介导的碱基对（dIm ^C –Ag ^I –dPur ^P的选择性和高产酶构建）。）以及用于合成包含此类人工金属碱基对的DNA双链体的两步方案。在DFT计算的指导下，我们还阐明了这种人工碱基对的形成机理，以及结构和能量偏好。dIm ^C –Ag ^I –dPur ^P人造金属碱基对的酶促合成为将来设计更强大的系统以扩展遗传字母提供了有价值的见识。