寡核苷酸
克莱诺碎片
DNA
底漆延伸
组合化学
计算生物学
德纳姆
寡核苷酸合成
生物
碱基对
底漆(化妆品)
化学
生物化学
聚合酶
基因
基序列
核酸外切酶
DNA甲基化
有机化学
基因表达
作者
Yun Fei Cao,Jingsi Bai,Jinrong Zou,Yuhui Du,Tingjian Chen
标识
DOI:10.1021/acssynbio.3c00258
摘要
The genetic alphabet of life has been dramatically expanded via the development of unnatural base pairs (UBPs) that work as efficiently as natural base pairs in the storage and retrieval of genetic information. Among the most predominant UBPs, dNaM–dTPT3 and its analogues have been successfully employed to build semisynthetic cells with a functional six-letter genome. With the rapidly growing applications of UBPs in vitro and in vivo, there is an ever-increasing demand for DNA oligonucleotides containing unnatural bases (UBs) at desired positions. Conventional solid-phase synthesis of oligonucleotides has intrinsic limitations and needs to use unstable unnatural phosphoramidites and a DNA synthesizer, so it does not meet the daily urgent requirement for a few UB-containing DNA oligonucleotides in the laboratory. In this work, we develop a one-pot enzymatic method for preparing dNaM- or dTPT3-containing DNA oligonucleotides via controlled pause and restart of primer extension mediated by Klenow fragment (exo–). By systematic optimization of the reaction conditions, high efficiencies and product purities have been achieved. The universality of this method for preparing DNA oligonucleotides containing dNaM or dTPT3 in different sequence contexts is also demonstrated. This method allows convenient production of an arbitrary UB-containing DNA oligonucleotide in a single test tube with only two natural DNA oligonucleotides, stable nucleoside triphosphates, Klenow fragment (exo–), and other common reagents in the laboratory, providing the lowest cost and the highest simplicity for the enzymatic preparation of UB-containing oligonucleotides. Clearly, this method has great potential to facilitate the in vitro and in vivo applications of the UBPs.
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