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.

Apoptosis is the process of an intracellular death program leading to characteristic biochemical and morphological changes within a cell that consequently result in cell death^[1]. A failure of cells to undergo apoptosis is a common feature of many cancers [2] thus investigation of apoptosis inducing mechanisms is of particular importance for cancer research and requires suitable detection methods.

A hallmark of late apoptosis is extensive genomic DNA fragmentation that generates a multitude of DNA double-strand breaks (DSBs) with accessible 3′-hydroxyl (3′-OH) groups. This characteristic forms the basis for a well-established apoptosis detection method: Terminal deoxynucleotidyl transferase dUTP Nick End Labeling (TUNEL) assay^[3].

TUNEL assays identify apoptotic cells by the terminal deoxynucleotidyl transferase (TdT)-mediated addition of labeled (X) deoxyuridine triphosphate nucleotides (X-dUTPs) to the 3’-OH end of DNA strand breaks that are subsequently visualized depending on the introduced label (Fig. 1), thus serving as parameter for the percentage of apoptotic cells within the analyzed cell population.

The assay sensitivity strongly depends on the incorporation efficiency of the modified dUTP that is influenced by size/bulkiness of the attached label. A number of fluorescently labeled dUTPs → Probes & Epigenetics, biotinylated dUTPs → Probes & Epigenetics or digoxigenylated dUTPs → Probes & Epigenetics are generally suitable substrates for TdT, but dUTP labeled with smaller labels such as bromine (BrdUTP) or an alkyne group (EdUTP) have been demonstrated to exhibit a higher incorporation efficiency and thus higher sensitivity in TUNEL assays probably due to minimal sterical hindrance^[3,4,5].

While incorporation of BrdUTP is detected by specific fluorophore or reporter enzyme labeled antibodies (Fig. 1A), detection of EdUTP is achieved by covalent conjugation of an azide-containing fluorophore → Click Chemistry via Cu(I) catalyzed alkyne-azide click chemistry (CuAAC) (Fig. 1B).

Figure 1: The principle of TUNEL assay relies on terminal deoxynucleotidyl transferase (TdT)-mediated addition of a modified dUTP (X-dUTP) to 3’-OH ends of DNA fragments that are generated as a result of apoptosis induction. To avoid the loss of fragmented DNA and to allow enzyme and nucleotide entrance, cells need to be fixed and subsequently permeabilized prior to the labeling reaction. Incorporated bromoylated dUTP (BrdUTP) is detected by specific antibody conjugates with a reporter enzyme or fluorescent dye (A). The incorporation of alkyne-containing dUTP (EdUTP) is visualized by Cu(I)-catalyzed alkyne-azide click chemistry (CuAAC) with an azide containing fluorphore → Click Chemistry (B).

A variety of cellular processes are triggered by sequence-specific binding of a protein to a nucleic acid (DNA or RNA) e.g. regulation of gene expression by transcription factor binding to specific DNA sequences or coordination of translation by RNA-binding proteins.

Electromobility shift assays (EMSA) are a prominent technique to detect the affinity of a protein to a known DNA or RNA sequence in vitro^[1]. Briefly, a labeled RNA or DNA probe is incubated with a protein source (crude cell extract or recombinant protein) and subsequently separated via non-denaturing polyacrylamide gel electrophoresis. End labeling of DNA and RNA probes is generally preferred over random labeling methods due to minimized interference with protein binding. Protein-nucleic acid complexes exhibit a slower mobility compared to free nucleic acid probes thus complex formation can be identified by the altered migration pattern (shift).

Visualization of a protein-nucleic acid complex depends on type of label used for DNA/RNA probe labeling. Traditional labeling approaches rely on the enzymatic incorporation of radioisotope labels however, several non-radioactively labeled nucleotides have been successfully used for EMSA probe labeling (Tab. 1). Further enzymatically incorporable labeled nucleotides are available within our DNA Labeling → Probes & Epigenetics and RNA Labeling → Probes & Epigenetics section.

Table 1: Nucleotide selection guide for nonradioactive EMSA probe labeling.

DIG: digoxigenin; ATTO680: fluorescent dye (exc.: 680 nm /em.: 700 nm); DY776: fluorescent dye (exc.: 771 nm /em.: 801 nm).

Epigenetics is the study of mechanisms that induce heritable changes in gene expression without changing the DNA sequence.

The most extensively studied epigenetic mechanisms so far are DNA methylation and histone modifications that regulate gene expression by chromatin remodeling which subsequently results in increased or decreased DNA accessibility for the transcriptional machinery depending on the specific epigenetic modification pattern^[1].

In addition, RNA methylation has most recently gained attention as an epigenetic modification in mammalian cells as well^[2].

Several nucleotide analogs (Tab. 1) are suitable for epigenetic modification analysis. Please refer to the corresponding references for detailed application data.

Table 1: Selection guide for epigenetic modification analysis.