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Updated: May 21, 2026

Exploring Sequence Space to Identify Binding Sites for Regulatory RNA-Binding Proteins
Published on: August 9, 2019
RNase footprinting of protein binding sites on an mRNA target of small RNAs
Yi Peng1, Toby J Soper, Sarah A Woodson
1T. C. Jenkins Department of Biophysics and CMDB Program, Johns Hopkins University, Baltimore, MD, USA.
Abstract:
Endoribonuclease footprinting is an important technique for probing RNA-protein interactions with single nucleotide resolution. The susceptibility of RNA residues to enzymatic digestion gives information about the RNA secondary structure, the location of protein binding sites, and the effects of protein binding on the RNA structure. Here we present a detailed protocol for using RNase T2, which cleaves single stranded RNA with a preference for A nucleotides, to footprint the protein Hfq on the rpoS mRNA leader. This protocol covers how to form the RNP complex, determine the correct dose of enzyme, footprint the protein, and analyze the cleavage pattern using primer extension.
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