Abstract
AbstractClick DNA ligation promises an alternative to the current enzymatic approaches for DNA assembly, with the ultimate goal of using efficient chemical reactions for the total chemical synthesis and assembly of genes and genomes. Such an approach would enable the incorporation of various chemically modified bases throughout long stretches of DNA, a feat not possible with current polymerase‐based methods. An unequivocal requirement for this approach is the biocompatibility of the resulting triazole‐linked DNA. The correct function of this unnatural DNA linker in human cells is demonstrated here by using a click‐linked gene encoding the fluorescent protein mCherry. Reverse transcription of mRNA isolated from these cells and subsequent sequencing of the mCherry cDNA shows error‐free transcription. Nucleotide excision repair (NER) is shown to not play a role in the observed biocompatibility by using a NER‐deficient human cell line. This is the first example of a non‐natural DNA linker being functional in a eukaryotic cell.
Highlights
Click DNA ligation promises an alternative to the current enzymatic approaches for DNA assembly, with the ultimate goal of using efficient chemical reactions for the total chemical synthesis and assembly of genes and genomes
Backbone linker[1,2] that is functional in human cells
Such a linker would be significant for several reasons; first, it would open up the possibility of the purely chemical synthesis and assembly of heavily modified genes and genomes, which would enable informative experiments in cell biology
Summary
Click DNA ligation promises an alternative to the current enzymatic approaches for DNA assembly, with the ultimate goal of using efficient chemical reactions for the total chemical synthesis and assembly of genes and genomes. To probe the biocompatibility of triazolelinked DNA in human cells, the products of SDM with click-linked or normal mutagenic primers were dialyzed against water to remove buffer salts and microinjected into a breast cancer cell line (MCF-7).
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