Department of Chemistry, Chemistry Research Laboratory, University of Oxford , 12 Mansfield Road, Oxford OX1 3TA, U.K.
Chemistry Branch, Department of Science and Mathematics, Faculty of Petroleum and Mining Engineering, Suez University , Suez 43721, Egypt.
J Am Chem Soc. 2017 Feb 1;139(4):1575-1583. doi: 10.1021/jacs.6b11530. Epub 2017 Jan 18.
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.
磷酸二酯骨架的分子性质使其成为遗传信息酶复制的进化选择，但人们对此并不了解。为了解决这个问题，并开发用于组装生物相容修饰 DNA 的新化学连接策略，我们合成了含有几种结构和电子上不同的人工连接的寡核苷酸。这产生了一种新的很有前途的连接方法，基于酰胺骨架形成，与 CuAAC“点击”连接在化学上是正交的。通过引物延伸、PCR 和深度测序研究这些人工连接的复制动力学和保真度表明，骨架灵活性、空间因素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和准确的信息解码。即使是微小的硫代磷酸酯修饰也会损害复制过程，但一些激进的三唑和酰胺 DNA 骨架表现得非常好，这表明磷酸基团不是必需的。这些发现对合成生物学领域具有重要意义。
