Toeprinting assay

The toeprinting assay, also known as the primer extension inhibition assay, is a method used in molecular biology that allows you to examine the interactions between messenger RNA and ribosomes or other RNA-binding proteins. 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. To do a toeprint assay, you need the mRNA of interest, ribosomes, a DNA primer, free nucleotides, and reverse transcriptase, among other reagents. 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.