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Nucleotides for Application in Cell Cycle & Proliferation

Cell proliferation is characterized by de novo DNA synthesis during the S-Phase of the cell cycle as well as increased transcriptional activity accompanied by de novo RNA synthesis.

A common approach for monitoring global DNA and RNA synthesis relies on the incorporation of 5-labeled (deoxy) uridine analogs instead of their structural analogs (RNA: uracil; DNA: thymidine) into nascent DNA or RNA strands by cellular DNA and RNA polymerases, respectively. Non-phosphorylated labeled (deoxy)uridines (5-X-(d)U) are cell permeable and thus suitable for in vivo labeling (living cells)[1-5]. On the contrary, labeled (deoxy)nucleotide triphosphates (5-X-(d)UTP) are not cell permeable and can be used for in vitro DNA/RNA synthesis analysis (cell free extracts)[6,7] or needs to be delivered into cells for in vivo studies e.g. by lipofection or microinjection[8].

Traditional approaches rely on the incorporation of Bromine-labeled (deoxy)uridine (5-Br(d)U) or (deoxy)uridine triphosphates (5-Br(d)UTP) that are subsequently detected by BrdU specific antibodies (Tab. 1). There are however harsh permeabilization procedures required to achieve a sufficient staining efficiency which is due to the high molecular weight of the antibody (around 150.000 kDa) that limits the rate of antibody diffusion into the cell.

The incorporation of ethynyl-labeled (deoxy)uridine (5-E(d)U) provides a novel alternative for in vivo monitoring of DNA and RNA synthesis. The detection of 5-E(d)U is achieved by covalent conjugation of an azide-containing fluorophore → Click Chemistry via Cu(I) catalyzed alkyne-azide click chemistry (CuAAC) (Tab. 1) that are readily incorporated into cells under mild permeabilization conditions due their significantly lower size (<1000 kDa).

Table 1: Nucleotide selection guide for monitoring of global DNA and RNA synthesis.

Measurement of de novo...Cell permeable nucleosidesNon cell permeable nucleotidesDetection
...DNA synthesis5-BrdU[1]5-BrdUTP[2]BrdU specific antibody
Azides of fluorescent dyes → Click Chemistry
...RNA synthesis5-BrU[5]5-BrUTP[6,7]BrdU specific antibody
Azides of fluorescent dyes → Click Chemistry

Further alkyne modified uridine analogs → Click Chemistry such as 5-EdUTP or EUTP are available as well.

Selected References

[1] Vogel et al. (1986) Detection of bromodeoxyuridine-incorporation in mammalian chromosomes by a bromodeoxyuridine-antibody. Human Genetics 72 (2):129.
[2] Salic et al. (2008) A chemical method for fast and sensitive detection of DNA synthesis in vivo. Proc. Natl. Acad. Sci. USA 105:2415.
[3] Li et al. (2010) Fluorogenic "click" reaction for labeling and detection of DNA in proliferating cells. Biotechniques 49 (1):525.
[4] Halicka et al. (2010) Segregation of RNA and separate packaging of DNA and RNA in apoptotic bodies during apoptosis. Exp. Cell Res. 269:248.
[5] Jao et al. (2008) Exploring RNA transcription and turnover in vivo by using click chemistry. Proc. Natl. Acad. Sci. USA 105 (41):15779.
[6] Pasero et al. (1999) In vitro DNA replication in yeast nuclear extracts. Methods 18 (3):368.
[7] Javed et al. (2004) In situ immunofluorescence analysis analyzing RNA synthesis by 5-Bromouridine-5’-Triphosphate labeling. Methods in Molecular Biology 285 (1):29.
[8] Haukenes et al. (1997) Labeling of RNA transcripts of eukaryotic cells in culture with BrUTP using a liposome transfection reagent (DOTAP). Biotechniques 22:308.