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Nucleotides for Application in Protein-DNA/-RNA interaction (EMSA)

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).

Generation of...MethodLabeling siteEnzymeLabeled nucleotideIncorporated nucleotidesDetection
...DNA probe3'-End Labeling3'-OHTerminal Deoxynucleotidyl Transferase (TdT)Biotin-11-UTP[2]1-3Indirect via reporter enzyme or fluorophore conjugated streptavidin
DIG-11-ddUTP[3]1Indirect via reporter enzyme or fluorophore conjugated Digoxigenin antibody
5'-End Labeling5'-OHT4 Polynucleotide Kinase (T4 PNK)ATPγS[4]1Indirect via Iodacetamide modified biotin or fluorophores
...RNA probeIn vitro transcriptionrandomT7 RNA PolymeraseUTP-ATTO680[5,6]multipleDirect measurement of fluorescence
UTP-DY776[6]multipleDirect measurement of fluorescence
5'-End Labeling5'-OHT4 Polynucleotide Kinase (T4 PNK)ATPγS[4]1Indirect via Iodacetamide modified biotin or fluorophores

Selected References

[1] Hellmann et al. (2007) Electrophoretic mobility shift assay (EMSA) for detecting protein–nucleic acid interactions. Nature Protocols 2:1849.
[2] Yan et al. (2006) Characterization of the human intestinal CD98 promoter and its regulation by interferon-γ. Am. J Physiol. Gastrointest. Liver Physiol. 292:G535.
[3] Kass et al. (2000) Non-radioactive electrophoretic mobility shift assay using digoxigenin-ddUTP labeled
probes. Dros. Inf. Serv. 83:185.
[4] Pagano et al. (2011) Quantitative approaches to monitor protein-nucleic acid interactions using fluorescent probes. RNA 17:14.
[5] Besse et al. (2009) Drosophila PTB promotes formation of high-order RNP particles and represses oskar translation. Genes Dev. 23:195.
[6] Kohn et al. (2010) Near-infrared (NIR) dye-labeled RNAs identify binding of ZBP1 to the noncoding Y3-RNA. RNA 16 (7):1420.