Fast Fenton footprinting: a laboratory-based method for the time-resolved analysis of DNA, RNA and proteins.
'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.
Department of Biochemistry, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461, USA.
Inna
I
Shcherbakova
Somdeb
S
Mitra
Robert H
RH
Beer
Michael
M
Brenowitz
D018983:DNA Footprinting / Q000379:methods*; D004492:Edetic Acid / Q000737:chemistry; D005296:Ferrous Compounds / Q000737:chemistry; D006861:Hydrogen Peroxide / Q000737:chemistry*; D017665:Hydroxyl Radical / Q000737:chemistry; D007501:Iron / Q000737:chemistry*; D007700:Kinetics; D008274:Magnesium / Q000737:chemistry; D009690:Nucleic Acid Conformation; D023302:Protein Footprinting / Q000379:methods*; D012313:RNA / Q000737:chemistry*; D016337:RNA, Catalytic / Q000737:chemistry
C045076:Fenton's reagent; D005296:Ferrous Compounds; D016337:RNA, Catalytic; C055526:Fe(II)-EDTA; D017665:Hydroxyl Radical; D012313:RNA; D004492:Edetic Acid; D006861:Hydrogen Peroxide; D007501:Iron; D008274:Magnesium