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 Brenowitz

doi:10.1093/nar/gkl055

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 Brenowitz

Biochemistry

Research output: Contribution to journal › Article › peer-review

85 Scopus citations

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 language	English (US)
Article number	e48
Journal	Nucleic acids research
Volume	34
Issue number	6
DOIs	https://doi.org/10.1093/nar/gkl055
State	Published - 2006

ASJC Scopus subject areas

Genetics

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

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.",

author = "Inna Shcherbakova and Somdeb Mitra and Beer, {Robert H.} and Michael Brenowitz",

note = "Funding Information: We thank Elizabeth Jamison for assistance conducting the experiments with DNA, Lois Polack, Rick Russell and Dan Herschlag for critically reading the manuscript and Donald Crothers, Shar-Yin Huang, Syun-Ru Yeh and Tsuyoshi Egawa for assistance in conducting stopped-flow studies. This work was supported by National Institutes of Health grants PO1–GM066275 and RO1-GM39929 from the Institute of General Medical Sciences. The acquisition of the synchrotron footprinting data shown in Figures 2 and 6 was supported by grant P41-EB0001979 from the Institute of Biomedical Imaging and Bioengineering of the National Institutes of Health. R.H.B. acknowledges financial support from Fordham University and the Research Corporation Cottrell College Science grant CC5650. Funding to pay the Open Access publication charges for this article was provided by The National Institutes of Health.",

year = "2006",

doi = "10.1093/nar/gkl055",

language = "English (US)",

volume = "34",

journal = "Nucleic acids research",

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AU - Brenowitz, Michael

N1 - Funding Information: We thank Elizabeth Jamison for assistance conducting the experiments with DNA, Lois Polack, Rick Russell and Dan Herschlag for critically reading the manuscript and Donald Crothers, Shar-Yin Huang, Syun-Ru Yeh and Tsuyoshi Egawa for assistance in conducting stopped-flow studies. This work was supported by National Institutes of Health grants PO1–GM066275 and RO1-GM39929 from the Institute of General Medical Sciences. The acquisition of the synchrotron footprinting data shown in Figures 2 and 6 was supported by grant P41-EB0001979 from the Institute of Biomedical Imaging and Bioengineering of the National Institutes of Health. R.H.B. acknowledges financial support from Fordham University and the Research Corporation Cottrell College Science grant CC5650. Funding to pay the Open Access publication charges for this article was provided by The National Institutes of Health.

PY - 2006

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N2 - '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.

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

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