title: Development of a 'clickable' non-natural nucleotide to visualize the replication of non-instructional DNA lesions.
abstract: The misreplication of damaged DNA is an important biological process that produces numerous adverse effects on human health. This report describes the synthesis and characterization of a non-natural nucleotide, designated 3-ethynyl-5-nitroindolyl-2'-deoxyriboside triphosphate (3-Eth-5-NITP), as a novel chemical reagent that can probe and quantify the misreplication of damaged DNA. We demonstrate that this non-natural nucleotide is efficiently inserted opposite an abasic site, a commonly formed and potentially mutagenic non-instructional DNA lesion. The strategic placement of the ethynyl moiety allows the incorporated nucleoside triphosphate to be selectively tagged with an azide-containing fluorophore using 'click' chemistry. This reaction provides a facile way to quantify the extent of nucleotide incorporation opposite non-instructional DNA lesions. In addition, the incorporation of 3-Eth-5-NITP is highly selective for an abasic site, and occurs even in the presence of a 50-fold molar excess of natural nucleotides. The biological applications of using 3-Eth-5-NITP as a chemical probe to monitor and quantify the misreplication of non-instructional DNA lesions are discussed.
authors.affiliation: Department of Chemistry, Case Western Reserve University, Cleveland, OH 44106, USA.
authors.forename: Edward A
authors.initials: EA
authors.lastname: Motea
authors.forename: Irene
authors.initials: I
authors.lastname: Lee
authors.forename: Anthony J
authors.initials: AJ
authors.lastname: Berdis
mesh_terms: D057930:Click Chemistry; D004247:DNA / Q000737:chemistry; D004249:DNA Damage; D004261:DNA Replication; D007211:Indoles / Q000138:chemical synthesis / Q000737:chemistry*; D007700:Kinetics; D009705:Nucleosides / Q000138:chemical synthesis / Q000737:chemistry; D009711:Nucleotides / Q000138:chemical synthesis / Q000737:chemistry*
chemical_list: C571884:3-ethynyl-5-nitroindolyl-2'-deoxyriboside triphosphate; D007211:Indoles; D009705:Nucleosides; D009711:Nucleotides; D004247:DNA
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