Synthesis of photolabile dUTP analogues and their enzymatic incorporation for DNA labeling

Two new photolabile nucleotide analogues with furan-fused deoxyuridine were synthesized through Sonogashira coupling. Their enzymatic incorporation into DNA was evaluated with two DNA polymerases (Taq and Deep vent exo-) by polymerase chain reaction (PCR). Deep vent exo-recognized both nucleotides as substrates for primer extension, while Taq was much less proficient. Light irradiation of PCR products released the amino and carboxyl moieties of DNA. Further labeling with fluorescein isothiocyanate for a long DNA construct with F-dUnTP incorporation was successfully achieved.