title: Labeling of Cellular DNA with a Cyclosal Phosphotriester Pronucleotide Analog of 5-ethynyl-2'-deoxyuridine.
abstract: DNA synthesis is a fundamental biological process central to all proliferating cells, and the design of small molecule probes that allow detection of this DNA is important for many applications. 5-Ethynyl-2'-deoxyuridine, known as EdU, has become a workhorse for metabolic labeling of DNA in mammalian cells, followed by bioconjugation to a small molecule fluorescent azide using copper-catalyzed azide-alkyne cycloaddition (CuAAC), click chemistry, to allow detection. In this study, we demonstrate that a cyclosal phosphotriester pronucleotide analog of EdU is suitable for metabolic incorporation into DNA of proliferating cells and subsequent labeling by CuAAC. This analog has two advantages over EdU; first, by delivering EdU with a preinstalled 5'-monophosphate moiety, it bypasses the need for thymidine kinase processing, and second, the increased lipophilicity compared to EdU may enable passive diffusion across the cell membrane and may circumvent the reliance on nucleoside active transport mechanisms for cellular uptake. These advantages pave the way for the development of additional novel pronucleotides to widen experimental opportunities for future bioconjugation applications involving cellular DNA.
authors.affiliation: Eskitis Institute for Drug Discovery, Griffith University, Don Young Road, Nathan, Qld, 4111, Australia.
authors.forename: Ngoc
authors.initials: N
authors.lastname: Huynh
authors.affiliation: Eskitis Institute for Drug Discovery, Griffith University, Don Young Road, Nathan, Qld, 4111, Australia.
authors.forename: Charlotte
authors.initials: C
authors.lastname: Dickson
authors.affiliation: Eskitis Institute for Drug Discovery, Griffith University, Don Young Road, Nathan, Qld, 4111, Australia.
authors.forename: Dusan
authors.initials: D
authors.lastname: Zencak
authors.affiliation: Eskitis Institute for Drug Discovery, Griffith University, Don Young Road, Nathan, Qld, 4111, Australia.
authors.forename: David H
authors.initials: DH
authors.lastname: Hilko
authors.affiliation: Eskitis Institute for Drug Discovery, Griffith University, Don Young Road, Nathan, Qld, 4111, Australia.
authors.forename: Alan
authors.initials: A
authors.lastname: Mackay-Sim
authors.affiliation: Eskitis Institute for Drug Discovery, Griffith University, Don Young Road, Nathan, Qld, 4111, Australia.
authors.forename: Sally-Ann
authors.initials: SA
authors.lastname: Poulsen
mesh_terms: D049109:Cell Proliferation / Q000502:physiology*; D002478:Cells, Cultured; D004247:DNA / Q000378:metabolism*; D003857:Deoxyuridine / Q000031:analogs & derivatives* / Q000138:chemical synthesis / Q000737:chemistry / Q000494:pharmacology; D006801:Humans; D013194:Staining and Labeling / Q000379:methods*; D013234:Stem Cells / Q000166:cytology / Q000378:metabolism*
chemical_list: D004247:DNA; C031086:5-ethynyl-2'-deoxyuridine; D003857:Deoxyuridine
keywords: DNA synthesis; bioconjugation; chemical reporter strategy; click chemistry; nucleoside transporter; prodrug; pronucleotide; thymidine