Development of a ‘clickable’ non-natural nucleotide to visualize the replication of non-instructional DNA lesions

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.