Electron paramagnetic resonance (EPR) spectroscopy was applied extensively in studies of nucleic acid structures and dynamics. Two modified 2'-deoxyuridine triphosphates were synthesized bearing a spin label linked to the base by a rigid linker to ensure a tight coupling of spin label dynamics. The incorporation of both spin-labeled nucleotides could be shown in primer extension reactions in presence of DNA polymerases from eukaryotic, prokaryotic, and archaic origin. In further experiments we were able to introduce multiple spin labels during primer extension reactions.