5-Ethynyl-2'-deoxyuridine explained

5-Ethynyl-2′-deoxyuridine (EdU) is a thymidine analogue which is incorporated into the DNA of dividing cells. EdU is used to assay DNA synthesis in cell culture and detect cells in embryonic, neonatal and adult animals which have undergone DNA synthesis. Whilst at high doses it can be cytotoxic, this molecule is now widely used to track proliferating cells in multiple biological systems.

EdU-labelling allows cells to be isolated without denaturing DNA, allowing researchers to determine the transcriptional profile of cells.^[1] This approach has been used to assess transcription in neuronal cells^[2] and tissues^[3] that have recently divided either in vitro or in vivo.

Detection

EdU is labeled and detected with an azide molecule (most commonly fluorescent azides) through Cu(I)-catalyzed azide–alkyne cycloaddition (CuAAC) click chemistry.^{[4] [5]} Unlike the commonly used bromodeoxyuridine (BrdU), EdU detection requires no heat or acid treatment.

Toxicity

EdU incorporated into DNA induces DNA damage through the formation of interstrand crosslinks.^[6] These are detected by the cell during DNA replication, which is reflected by phosphorylation of histone H2AX, arrest in the cell cycle progression, and apoptosis.^[7]

Notes and References

1. Endaya B, Cavanagh B, Alowaidi F, Walker T, de Pennington N, Ng JM, Lam PY, Mackay-Sim A, Neuzil J, Meedeniya AC . 6 . Isolating dividing neural and brain tumour cells for gene expression profiling . Journal of Neuroscience Methods . 257 . 121–133 . January 2016 . 26432933 . 10.1016/j.jneumeth.2015.09.020 . 44969376 .
2. Endaya BB, Lam PY, Meedeniya AC, Neuzil J . Transcriptional profiling of dividing tumor cells detects intratumor heterogeneity linked to cell proliferation in a brain tumor model . Molecular Oncology . 10 . 1 . 126–137 . January 2016 . 26388584 . 5528932 . 10.1016/j.molonc.2015.09.001 .
3. Ng HX, Lee EP, Cavanagh BL, Britto JM, Tan SS . A method for isolating cortical interneurons sharing the same birthdays for gene expression studies . Experimental Neurology . 295 . 36–45 . September 2017 . 28511841 . 10.1016/j.expneurol.2017.05.006 . 3377296 .
4. Chehrehasa F, Meedeniya AC, Dwyer P, Abrahamsen G, Mackay-Sim A . EdU, a new thymidine analogue for labelling proliferating cells in the nervous system . Journal of Neuroscience Methods . 177 . 1 . 122–130 . February 2009 . 18996411 . 10.1016/j.jneumeth.2008.10.006 . free . 21427874 . 10072/25744 .
5. Cavanagh BL, Walker T, Norazit A, Meedeniya AC . Thymidine analogues for tracking DNA synthesis . Molecules . 16 . 9 . 7980–7993 . September 2011 . 21921870 . 6264245 . 10.3390/molecules16097980 . free .
6. Ligasová A, Strunin D, Friedecký D, Adam T, Koberna K . A fatal combination: a thymidylate synthase inhibitor with DNA damaging activity . PLOS ONE . 10 . 2 . e0117459 . 2015-02-11 . 25671308 . 4324964 . 10.1371/journal.pone.0117459 . free . 2015PLoSO..1017459L .
7. Zhao H, Halicka HD, Li J, Biela E, Berniak K, Dobrucki J, Darzynkiewicz Z . DNA damage signaling, impairment of cell cycle progression, and apoptosis triggered by 5-ethynyl-2'-deoxyuridine incorporated into DNA . Cytometry. Part A . 83 . 11 . 979–988 . November 2013 . 24115313 . 3846616 . 10.1002/cyto.a.22396 .