Thymidine analogues for tracking DNA synthesis
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Replicating cells undergo DNA synthesis in the highly regulated, S-phase of the cell cycle. Several arms of biomedical research use analogues of the pyrimidine deoxynucleoside base thymidine to quantify or track cell proliferation. These "unnatural bases" may be inserted into replicating DNA, effectively tagging dividing cells during S-phase by evading multiple cellular safety mechanisms. Tritiated thymidine, targeted using autoradiography was technically demanding and superseded by 5-bromo-2-deoxyuridine (BrdU) and related halogenated analogues, which are detected using antibodies. The detection of these unique bases following their incorporation into the DNA backbone requires denaturation of the DNA, often constraining the outcome of investigations. Despite these limitations BrdU alone has been used to target newly synthesised DNA in over 20,000 biomedical studies in the reviewed literature. A recent breakthrough for targeting dividing cells is the application of the thymidine analogue 5-ethynyl-2'-deoxyuridine (EdU). An alkyne group is uniquely present in EdU which allows its ready detection by a fluorescent azide probe in the presence of copper using the well-established 䈵isgen?s reaction? also known as 1,3-dipolar cycloaddition or 䣬ick chemistry?. This rapid, two-step biolabelling approach allows the tagging and imaging of DNA within cells whilst preserving the structural and molecular integrity of the cells.
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