Vinyl-DNADetect Probes to Assess DNA Proliferation in Parasites using Inverse-Electron-Demand Diels-Alder Click Reactions.

Small-molecule metabolic chemical probes are tailored chemical biology tools that are designed to detect and visualize biological processes within a cell or an organism. Nucleoside analogues are a subset of metabolic probes that enable the study of DNA synthesis, proliferation kinetics, and cell cycle progression. However, most available nucleoside analogue probes have been designed for use in mammalian cells, limiting their use in other species, where there are metabolic pathway differences. The current gold-standard probe for studies of DNA synthesis in mammalian cells, 5-ethynyl-2'-deoxyuridine (EdU), can be detected via a copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC) with a fluorescent azide. However, the use of EdU in malaria parasites is not possible as lacks thymidine kinase, the first enzyme in the sequential phosphorylation of thymidine needed for eventual incorporation of this probe into DNA. We previously demonstrated that a pronucleotide approach with modified EdU analogues (designated DNADetect probes) can be used to study DNA proliferation in . However, as cytotoxicity can be associated with the use of copper(I) ions as a catalyst for CuAAC, and cells require fixation, negating the potential for live cell imaging, alternative probes would help overcome these challenges in and other species lacking thymidine kinase. Herein, we report the development and synthesis of novel 5-vinyl-2'-deoxyuridine (VdU)-based chemical probes, designated vinyl-DNADetect. These probes were designed not only to bypass thymidine kinase requirements but also to have the advantage of detection via a catalyst-free and spontaneous inverse-electron-demand Diels-Alder (IEDDA) click reaction with a fluorogenic tetrazine that does not require parasites to first be fixed. Using flow cytometry and fluorescence microscopy, we quantified and visualized incorporation into DNA, with several probes demonstrating high efficiency labeling. In addition, we demonstrated that these VdU-based probes could label DNA in live -infected erythrocytes and show the first use of two orthogonal metabolic chemical probes, VdU- and EdU-based, in a pulse-chase experiment for DNA double staining in wild-type .