Molecular Requirements of High-Fidelity Replication-Competent DNA Backbones for Orthogonal Chemical Ligation
Molecular Requirements of High-Fidelity Replication-Competent DNA Backbones for Orthogonal Chemical Ligation
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DOI：
10.1021/jacs.6b11530
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发表时间：
2017-02-01
影响因子：
15
通讯作者：
Brown, Tom
中科院分区：
文献类型：
--
作者：
Shivalingam, Arun;Tyburn, Agnes E. S.;Brown, Tom
磷酸二酯骨架的分子特性使其成为遗传信息酶促复制的进化选择，但其分子特性尚未得到很好的理解。为了解决这个问题，并开发新的化学连接策略，用于组装生物相容性修饰的DNA，我们合成了含有几个结构和电子变化的人工连接的寡核苷酸。这产生了一种新的非常有前途的连接方法，其基于酰胺骨架形成，其与CuAAC“点击”连接在化学上正交。通过引物延伸、PCR和深度测序对这些人工连接的复制动力学和保真度的研究表明，骨架灵活性、空间因子和与聚合酶氢键结合的能力之间的微妙相互作用调节了快速准确的信息解码。即使是微小的硫代磷酸酯修饰也会损害复制过程，但一些自由基三唑和酰胺DNA骨架表现得令人惊讶地好，表明磷酸基团不是必需的。这些发现在合成生物学领域具有重要意义。
The molecular properties of the phosphodiester backbone that made it the evolutionary choice for the enzymatic replication of genetic information are not well understood. To address this, and to develop new chemical ligation strategies for assembly of biocompatible modified DNA, we have synthesized oligonucleotides containing several structurally and electronically varied artificial linkages. This has yielded a new highly promising ligation method based on amide backbone formation that is chemically orthogonal to CuAAC "click" ligation. A study of kinetics and fidelity of replication through these artificial linkages by primer extension, PCR, and deep sequencing reveals that a subtle interplay between backbone flexibility, steric factors, and ability to hydrogen bond to the polymerase modulates rapid and accurate information decoding. Even minor phosphorothioate modifications can impair the copying process, yet some radical triazole and amide DNA backbones perform surprisingly well, indicating that the phosphate group is not essential. These findings have implications in the field of synthetic biology.