Thymidine kinase-independent click chemistry DNADetect™ probes as an EdU alternative for mammalian cell DNA labelling

Nucleoside analogues have been powerful tools to study DNA synthesis, cell cycle progression and cellular fate for decades. EdU (5-ethynyl-2'-deoxyuridine), which contains an alkyne handle that allows copper-catalyzed azide-alkyne cycloaddition (CuAAC) to a fluorescent azide in a "click" chemistry reaction, is one of the most frequently utilised nucleoside analogue probes. EdU is transported into cells by nucleoside transporters followed by phosphorylation by thymidine kinase, the first step in thymidine metabolism leading to DNA synthesis. As some organisms like malaria parasites lack the thymidine kinase enzyme and cannot be labelled by EdU or related thymidine analogue probes, we previously designed and validated DNADetect EdU analogues as chemical probes. These probes comprise a masked monophosphate on the 5'-hydroxyl group of the nucleoside sugar moiety that is metabolised directly to EdU monophosphate, bypassing the need for thymidine kinase. Here, we demonstrate that DNADetect probes can be used to label DNA in mammalian cells. DNADetect probes are incorporated into proliferating HeLa cells as efficiently as EdU and outcompeted by thymidine. Additionally, we implement a protocol for best practice use of metabolic chemical probes by using a specifically designed inactive control probe for each active probe. While this approach is commonly applied with chemical probes that modulate protein function, it is yet to be commonly applied with metabolic chemical probes.