Fast Fenton footprinting: A laboratory-based method for the time-resolved analysis of DNA, RNA and proteins

Inna Shcherbakova, Somdeb Mitra, Robert H. Beer, Michael D. Brenowitz

Research output: Contribution to journalArticle

72 Citations (Scopus)

Abstract

'Footprinting' describes assays in which ligand binding or structure formation protects polymers such as nucleic acids and proteins from either cleavage or modification; footprinting allows the accessibility of individual residues to be mapped in solution. Equilibrium and time-dependent footprinting links site-specific structural information with thermodynamic and kinetic transitions. The hydroxyl radical (·OH) is a particularly valuable footprinting probe by virtue of it being among the most reactive of chemical oxidants; it reports the solvent accessibility of reactive sites on macromolecules with as fine as a single residue resolution. A novel method of millisecond time-resolved ·OH footprinting has been developed based on the Fenton reaction, Fe(II) + H2O2 → Fe(III) + ·OH + OH-. This method can be implemented in laboratories using widely available three-syringe quench flow mixers and inexpensive reagents to study local changes in the solvent accessibility of DNA, RNA and proteins associated with their biological function.

Original languageEnglish (US)
Article numbere48
JournalNucleic Acids Research
Volume34
Issue number6
DOIs
StatePublished - 2006

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RNA
DNA
Syringes
Thermodynamics
Oxidants
Hydroxyl Radical
Nucleic Acids
Catalytic Domain
Polymers
Proteins
Ligands
hydroxide ion

ASJC Scopus subject areas

  • Genetics

Cite this

Fast Fenton footprinting : A laboratory-based method for the time-resolved analysis of DNA, RNA and proteins. / Shcherbakova, Inna; Mitra, Somdeb; Beer, Robert H.; Brenowitz, Michael D.

In: Nucleic Acids Research, Vol. 34, No. 6, e48, 2006.

Research output: Contribution to journalArticle

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