Chemistry, University of Southampton, Southampton, SO17 1BJ, UK.
Nucleic Acids Res. 2012 Nov 1;40(20):10567-75. doi: 10.1093/nar/gks756. Epub 2012 Aug 16.
The biocompatibility of a triazole mimic of the DNA phosphodiester linkage in Escherichia coli has been evaluated. The requirement for selective pressure on the click-containing gene was probed via a plasmid containing click DNA backbone linkages in each strand of the gene encoding the fluorescent protein mCherry. The effect of proximity of the click linkers on their biocompatibility was also probed by placing two click DNA linkers 4-bp apart at the region encoding the fluorophore of the fluorescent protein. The resulting click-containing plasmid was found to encode mCherry in E. coli at a similar level to the canonical equivalent. The ability of the cellular machinery to read through click-linked DNA was further probed by using the above click-linked plasmid to express mCherry using an in vitro transcription/translation system, and found to also be similar to that from canonical DNA. The yield and fluorescence of recombinant mCherry expressed from the click-linked plasmid was also compared to that from the canonical equivalent, and found to be the same. The biocompatibility of click DNA ligation sites at close proximity in a non-essential gene demonstrated in E. coli suggests the possibility of using click DNA ligation for the enzyme-free assembly of chemically modified genes and genomes.
已评估三唑类似物与大肠杆菌中 DNA 磷酸二酯键的生物相容性。通过含有编码荧光蛋白 mCherry 的基因的每条链中都具有点击 DNA 骨架连接的质粒，来探测对包含点击基因的选择性压力的要求。通过将两个点击 DNA 连接子放置在距离荧光蛋白编码区 4 个碱基处，还探测了点击连接子之间的接近度对其生物相容性的影响。在大肠杆菌中，发现含有点击的质粒以与规范等同物相似的水平编码 mCherry。通过使用上述带有点击的质粒，在体外转录/翻译系统中表达 mCherry，进一步探测了细胞机制通读点击连接 DNA 的能力，发现也与规范 DNA 相似。还比较了从点击连接的质粒表达的重组 mCherry 的产量和荧光，发现它们是相同的。在非必需基因中近距离的点击 DNA 连接位点的生物相容性在大肠杆菌中得到证明，这表明可以使用点击 DNA 连接来进行酶免费的化学修饰基因和基因组的组装。
