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Professor Nathan Luedtke

Professor Nathan Luedtke

Metabolic incorporation of a bioorthogonal functional group (e.g., alkyne, azide, alkene) into cellular DNA can be achieved by simply adding a synthetic nucleoside to the nutritional media of cells or whole animals. Intracellular enzymes incorporate the modified nucleoside into new DNA molecules, and the non-native functional group can be detected using a bioorthogonal chemical reaction, such as the azide-alkyne "click" reaction, alkene-tetrazine ligation, or metal-catalyzed cross coupling. The resulting products can be used to decipher the timing and location of DNA synthesis in vivo. One limitation to this approach can be the genotoxicity of the modified nucleoside itself. For example, 5-ethynyl-2'-deoxyuridine 2 (EdU, T511285) can cause rapid and irreversible cell cycle arrest when added to cells, due to its ability to activate DNA damage response pathways. Modifications made to the 2'(S) position of EdU can dramatically decrease this type of toxicity. For example, (2'S)-2'-deoxy-2'-fluoro-5-ethynyluridine, (F-ara-EdU, T511293) is incorporated into cellular DNA, where has little or no impact on cell cycle progression or DNA damage response. In some cases, highly variable metabolic incorporation efficiencies are observed in various cell types and experimental setups. For example, 5-ethynyl-2'-deoxycytidine (EdC, T511307), exhibited superior labeling of new adenovirus particles as compared to both EdU and F-ara-EdU. By screening a small collection of nucleoside derivatives, labeling protocols can thereby be optimized. In some cases, the presence of the metal catalysis used in copper-catalyzed azide-alkyne cycloaddition is undesirable, since Cu(I) can degrade cellular components such as nucleic acids and proteins. Metal-free strain-promoted azide-alkyne click reactions can be accomplished by treating cells with 5-(azidomethyl)-2'-deoxyuridine (AmdU, T342254), followed by a cyclooctyne-containing probe.

Luedtke Group Website

Recent papers from the Luedtke Group

1.
Neef AB, Luedtke NW. 2011. Dynamic metabolic labeling of DNA in vivo with arabinosyl nucleosides. Proceedings of the National Academy of Sciences. 108(51):20404-20409. http://dx.doi.org/10.1073/pnas.1101126108
2.
Wang I, Suomalainen M, Andriasyan V, Kilcher S, Mercer J, Neef A, Luedtke N, Greber U. 2013. Tracking Viral Genomes in Host Cells at Single-Molecule Resolution. Cell Host & Microbe. 14(4):468-480. http://dx.doi.org/10.1016/j.chom.2013.09.004
3.
Neef AB, Luedtke NW. 2014. An Azide-Modified Nucleoside for Metabolic Labeling of DNA. ChemBioChem. 15(6):789-793. http://dx.doi.org/10.1002/cbic.201400037
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DISCIPLINES

  • Organic Synthetic Chemistry
  • Catalysis

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