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Chembiochem. 2017 Aug 4;18(15):1473-1476. doi: 10.1002/cbic.201700185. Epub 2017 Jun 7.

1,2,4-Triazine-Modified 2'-Deoxyuridine Triphosphate for Efficient Bioorthogonal Fluorescent Labeling of DNA.

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Institute of Organic Chemistry, Karlsruhe Institute of Technology (KIT), Fritz-Haber-Weg 6, 76131, Karlsruhe, Germany.


In order to establish the Diels-Alder reaction with inverse electron demand for postsynthetic DNA modification, a 1,2,4-triazine-modified 2'-deoxyuridine triphosphate was synthesized. The bioorthogonally reactive 1,2,4-triazine group was attached at the 5-position of 2'-deoxyuridine by a flexible alkyl linker to facilitate its acceptance by DNA polymerases. The screening of four DNA polymerases showed successful primer extensions, using a mixture of dATP, dGTP, dCTP, and the modified 2'-deoxyuridine triphosphate, by using KOD XL or Vent polymerase. The triazine moiety was stable under the conditions of primer extension, which was evidenced by labeling with a BCN-modified rhodamine at room temperature in yields of up to 82 %. Two or three modified bases could be incorporated in quantitative yields when the modification sites were separated by three base pairs. These results establish the 1,2,4-triazene group as a bioorthogonally reactive moiety in DNA, thereby replacing the problematic 1,2,4,5-tetrazine for postsynthetic labeling by the Diels-Alder reaction with inverse electron demand.


DNA polymerase; Diels-Alder reaction; click chemistry; nucleic acids; primer extension

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