Toeprinting assay

The toeprinting assay, also known as the primer extension inhibition assay,[1] is a method used in molecular biology that allows you to examine the interactions between messenger RNA and ribosomes or other RNA-binding proteins.[2] It is different from the more commonly used DNA footprinting assay. The toeprinting assay has been utilized to examine the formation of the translation initiation complex.[3] To do a toeprint assay, you need the mRNA of interest, ribosomes, a DNA primer, free nucleotides, and reverse transcriptase, among other reagents.[4] Reverse transcriptase is an enzyme that catalyzes the formation of DNA from RNA; it is used to generate cDNA because RNA is unstable and difficult to study experimentally. Normally, under these conditions, the reverse transcriptase would simply create a complete cDNA copy of the mRNA of interest. However, when the correct reagents and experimental conditions are used, reverse transcriptase will be blocked by any bound ribosomes, resulting in shorter cDNA fragments called toeprints when the results are observed on a sequencing gel. A schematic of a toeprinting assay can be found here: https://www.researchgate.net/figure/41124064_fig2_A-Schematic-of-the-primer-extension-inhibition-toeprint-assay-illustrating-how.

References

  1. ↑ Hartz, D.; McPheeters, D. S.; Traut, R.; Gold, L. (1988-01-01). "Extension inhibition analysis of translation initiation complexes". Methods in Enzymology 164: 419–425. ISSN 0076-6879. PMID 2468068.
  2. ↑ Shirokikh, Nikolay E.; Alkalaeva, Elena Z.; Vassilenko, Konstantin S.; Afonina, Zhanna A.; Alekhina, Olga M.; Kisselev, Lev L.; Spirin, Alexander S. (2010-01-01). "Quantitative analysis of ribosome–mRNA complexes at different translation stages". Nucleic Acids Research 38 (3): e15. doi:10.1093/nar/gkp1025. ISSN 0305-1048. PMC 2817456. PMID 19910372.
  3. ↑ Chang, J T; Green, C B; Wolf, R E (1995-11-01). "Inhibition of translation initiation on Escherichia coli gnd mRNA by formation of a long-range secondary structure involving the ribosome binding site and the internal complementary sequence.". Journal of Bacteriology 177 (22): 6560–6567. ISSN 0021-9193. PMC 177509. PMID 7592434.
  4. ↑ Kozak, M (1998-11-01). "Primer extension analysis of eukaryotic ribosome-mRNA complexes.". Nucleic Acids Research 26 (21): 4853–4859. ISSN 0305-1048. PMC 147915. PMID 9776744.
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