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Related Concept Videos

Proteomics01:33

Proteomics

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A proteome is the entire set of proteins that a cell type produces. We can study proteomes using the knowledge of genomes because genes code for mRNAs, and the mRNAs encode proteins. Although mRNA analysis is a step in the right direction, not all mRNAs are translated into proteins.
Proteomics is the study of proteomes' function. It involves the large-scale systematic study of the proteome to denote the protein complement expressed by a genome. Scientist Mark Wilkins coined the term...
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Related Experiment Video

Updated: Apr 20, 2026

Monitoring Equilibrium Changes in RNA Structure by 'Peroxidative' and 'Oxidative' Hydroxyl Radical Footprinting
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Mapping RNA-protein interactions using hydroxyl-radical footprinting.

Timothy W Nilsen

    Cold Spring Harbor Protocols
    |December 3, 2014
    PubMed
    Summary

    This study details a hydroxyl radical footprinting method to map RNA-protein interactions. This technique identifies RNA regions protected by bound proteins, offering a sensitive approach for studying molecular binding sites.

    Area of Science:

    • Molecular Biology
    • Biochemistry
    • Structural Biology

    Background:

    • Protein binding to RNA protects specific sequences from cleavage.
    • Identifying these protected regions, or "footprints," is crucial for understanding RNA-protein interactions.
    • Existing methods like RNase cleavage can lack specificity.

    Purpose of the Study:

    • To present a chemical footprinting protocol for mapping RNA-protein binding sites.
    • To utilize hydroxyl radicals for probing RNA accessibility.
    • To establish a sensitive method for analyzing RNA-protein interactions.

    Main Methods:

    • End-labeled RNA molecules were incubated with and without proteins.
    • Cleavage was induced using hydroxyl radicals generated by Fe(II)-EDTA and a reducing agent.

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  • RNA fragments were separated and analyzed using denaturing polyacrylamide gel electrophoresis.

    • Main Results:

    • Hydroxyl radicals cleaved exposed ribose groups in the RNA backbone.
    • Regions protected by bound proteins showed reduced cleavage.
    • The method allowed for the identification of solvent-exposed sites of RNA-protein contact.

    Conclusions:

    • Hydroxyl radical footprinting is a feasible and sensitive method for mapping RNA-protein interactions.
    • This technique offers advantages over RNase-based methods due to the smaller size and less specific cleavage of hydroxyl radicals.
    • The protocol provides a valuable tool for studying the structural basis of RNA-protein recognition.