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

Area of Science:

• Chemical Biology
• Parasitology
• Nucleoside Analogue Probes

Background:

• Metabolic chemical probes, particularly nucleoside analogues, are vital for studying DNA synthesis and cell proliferation.
• Existing probes like 5-ethynyl-2'-deoxyuridine (EdU) are limited to species with specific metabolic pathways (e.g., thymidine kinase), excluding organisms like *Plasmodium*.
• Copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC) used for EdU detection can cause cytotoxicity and requires cell fixation, hindering live-cell imaging.

Purpose of the Study:

• To develop novel metabolic chemical probes for studying DNA synthesis in organisms lacking thymidine kinase, such as *Plasmodium*.
• To create probes that utilize a catalyst-free click reaction for improved safety and live-cell imaging capabilities.
• To enable dual-labeling experiments using orthogonal probes.

Main Methods:

• Synthesis of novel 5-vinyl-2'-deoxyuridine (VdU)-based probes (vinyl-DNADetect).
• Detection of VdU incorporation via catalyst-free inverse-electron-demand Diels-Alder (IEDDA) click chemistry with tetrazines.
• Application of VdU probes in *Plasmodium* using flow cytometry and fluorescence microscopy, including live-cell imaging in infected erythrocytes.
• Demonstration of orthogonal labeling with VdU and EdU probes in a pulse-chase experiment.

Main Results:

• Successfully synthesized and characterized VdU-based metabolic probes.
• Demonstrated efficient labeling of *Plasmodium* DNA with VdU probes, bypassing the need for thymidine kinase.
• Achieved live-cell imaging of DNA synthesis in *Plasmodium falciparum*-infected erythrocytes using VdU probes.
• Successfully performed dual-labeling of *Plasmodium* DNA using orthogonal VdU and EdU probes.

Conclusions:

• VdU-based probes represent a significant advancement for studying DNA synthesis in *Plasmodium* and other species lacking thymidine kinase.
• The catalyst-free IEDDA click reaction offers a safer and more versatile alternative to CuAAC for metabolic labeling.
• These novel probes facilitate live-cell imaging and enable advanced techniques like orthogonal dual-labeling for comprehensive biological studies.