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Transcription of click-linked DNA in human cells.

Birts CN, Sanzone AP, El-Sagheer AH, Blaydes JP, Brown T, Tavassoli A - Angew. Chem. Int. Ed. Engl. (2014)

Bottom Line: An unequivocal requirement for this approach is the biocompatibility of the resulting triazole-linked DNA.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.

View Article: PubMed Central - PubMed

Affiliation: Cancer Sciences, Faculty of Medicine, University of Southampton, Southampton, SO16 6YD (UK).

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Assembly of the click-linked pmCherry plasmid. a) Oligonucleotides functionalized with a 5′ azide are ligated to oligonucleotides with a 3′ alkyne through the CuAAC reaction, thereby resulting in a triazole backbone linker. 5-Methylcytosine (5-MedC) was used as the 5′ nucleobase of the C–triazole–C linkage for synthetic convenience; the 3′ propargyl-5-MedC is derived from thymidine. b) The click-linked primers used to construct the click-linked pmCherry plasmid by SDM contain a readily identifiable BamHI watermark.
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fig01: Assembly of the click-linked pmCherry plasmid. a) Oligonucleotides functionalized with a 5′ azide are ligated to oligonucleotides with a 3′ alkyne through the CuAAC reaction, thereby resulting in a triazole backbone linker. 5-Methylcytosine (5-MedC) was used as the 5′ nucleobase of the C–triazole–C linkage for synthetic convenience; the 3′ propargyl-5-MedC is derived from thymidine. b) The click-linked primers used to construct the click-linked pmCherry plasmid by SDM contain a readily identifiable BamHI watermark.

Mentions: To date, there has been no report of an unnatural DNA-backbone linker[1,2] that is functional in human cells (or other eukaryotic 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. Second, it would illustrate that the cellular machinery tolerates variations in the backbone of canonical DNA, which would have a significant impact on current approaches to the assembly of large DNA fragments; no longer bound by the need for a phosphodiester linker, chemists would be free to explore and develop more efficient chemical DNA-ligation reactions. Third, the self-templating property of DNA, combined with a highly efficient chemical ligation, would potentially allow one-pot gene synthesis. We have recently reported click DNA ligation: the use of copper-catalyzed alkyne–azide cycloaddition (CuAAC) for joining DNA strands (Figure 1 a)[1] and the biocompatibility of the resulting triazole-linked DNA in Escherichia coli.[1,3] We next sought to probe the biocompatibility of click-ligated DNA in human cells.


Transcription of click-linked DNA in human cells.

Birts CN, Sanzone AP, El-Sagheer AH, Blaydes JP, Brown T, Tavassoli A - Angew. Chem. Int. Ed. Engl. (2014)

Assembly of the click-linked pmCherry plasmid. a) Oligonucleotides functionalized with a 5′ azide are ligated to oligonucleotides with a 3′ alkyne through the CuAAC reaction, thereby resulting in a triazole backbone linker. 5-Methylcytosine (5-MedC) was used as the 5′ nucleobase of the C–triazole–C linkage for synthetic convenience; the 3′ propargyl-5-MedC is derived from thymidine. b) The click-linked primers used to construct the click-linked pmCherry plasmid by SDM contain a readily identifiable BamHI watermark.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4016740&req=5

fig01: Assembly of the click-linked pmCherry plasmid. a) Oligonucleotides functionalized with a 5′ azide are ligated to oligonucleotides with a 3′ alkyne through the CuAAC reaction, thereby resulting in a triazole backbone linker. 5-Methylcytosine (5-MedC) was used as the 5′ nucleobase of the C–triazole–C linkage for synthetic convenience; the 3′ propargyl-5-MedC is derived from thymidine. b) The click-linked primers used to construct the click-linked pmCherry plasmid by SDM contain a readily identifiable BamHI watermark.
Mentions: To date, there has been no report of an unnatural DNA-backbone linker[1,2] that is functional in human cells (or other eukaryotic 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. Second, it would illustrate that the cellular machinery tolerates variations in the backbone of canonical DNA, which would have a significant impact on current approaches to the assembly of large DNA fragments; no longer bound by the need for a phosphodiester linker, chemists would be free to explore and develop more efficient chemical DNA-ligation reactions. Third, the self-templating property of DNA, combined with a highly efficient chemical ligation, would potentially allow one-pot gene synthesis. We have recently reported click DNA ligation: the use of copper-catalyzed alkyne–azide cycloaddition (CuAAC) for joining DNA strands (Figure 1 a)[1] and the biocompatibility of the resulting triazole-linked DNA in Escherichia coli.[1,3] We next sought to probe the biocompatibility of click-ligated DNA in human cells.

Bottom Line: An unequivocal requirement for this approach is the biocompatibility of the resulting triazole-linked DNA.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.

View Article: PubMed Central - PubMed

Affiliation: Cancer Sciences, Faculty of Medicine, University of Southampton, Southampton, SO16 6YD (UK).

Show MeSH
Related in: MedlinePlus