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.
Shcherbakova
Inna
I
Department of Biochemistry, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461, USA.
Mitra
Somdeb
S
Beer
Robert H
RH
Brenowitz
Michael
M
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