Thymidine analogues for tracking DNA synthesis

Abstract

Replicating cells undergo DNA synthesis in the highly regulated, S-phase of the cell cycle. Analogues of the pyrimidine deoxynucleoside thymidine may be inserted into replicating DNA, effectively tagging dividing cells allowing their characterisation. Tritiated thymidine, targeted using autoradiography was technically demanding and superseded by 5-bromo-2-deoxyuridine (BrdU) and related halogenated analogues, detected using antibodies. Their detection required the denaturation of DNA, often constraining the outcome of investigations. Despite these limitations BrdU alone has been used to target newly synthesised DNA in over 20,000 reviewed biomedical studies. A recent breakthrough in "tagging DNA synthesis" is the thymidine analogue 5-ethynyl-2'-deoxyuridine (EdU). The alkyne group in EdU is readily detected using a fluorescent azide probe and copper catalysis using 'Huisgen's reaction' (1,3-dipolar cycloaddition or 'click 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. The bio-orthogonal detection of EdU allows its application in more experimental assays than previously possible with other "unnatural bases". These include physiological, anatomical and molecular biological experimentation in multiple fields including, stem cell research, cancer biology, and parasitology. The full potential of EdU and related molecules in biomedical research remains to be explored.

Scheme 1

EdU and click chemistry for labeling DNA synthesis.

Scheme 2

Copper-catalyzed click reaction of an alkyne and azide.

Figure 1

Mature (red) and immature (green) sensory neurons colocalise with EdU (white) introduced into the proliferative cells of adult rodents, one week prior to tissue harvest. The majority of cells which underwent DNA synthesis during the period of EdU exposure have differentiated into immature and mature neurons following 7 days. The fluorescence conjugated azides are commercially available. The fluorescence molecules are widely used probes of small molecular size and bright emission, ranging from the ultraviolet to infrared wavelengths. They are readily multiplexed with other fluorescence techniques including immunofluorescence. Thus, these techniques are robustly applied to track cell genesis and adult stem cells.