title: Following molecular transitions with single residue spatial and millisecond time resolution.
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, respectively. The hydroxyl radical (*OH) is a uniquely insightful footprinting probe by virtue of it being among the most reactive 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 is presented based on the Fenton reaction, Fe(II) + H(2)O(2) --> Fe(III) + *OH + OH(-). It is implemented using a standard three-syringe quench-flow mixer. The utility of this method is demonstrated by its application to the studies on RNA folding. Its applicability to a broad range of biological questions involving the function of DNA, RNA, and proteins is discussed.
authors.affiliation: Department of Biochemistry, Albert Einstein College of Medicine, Bronx, New York 10461, USA.
authors.forename: Inna
authors.initials: I
authors.lastname: Shcherbakova
authors.forename: Somdeb
authors.initials: S
authors.lastname: Mitra
authors.forename: Robert H
authors.initials: RH
authors.lastname: Beer
authors.forename: Michael
authors.initials: M
authors.lastname: Brenowitz
mesh_terms: D000818:Animals; D001345:Autoradiography; D001681:Biological Assay / Q000379:methods*; D016000:Cluster Analysis; D017665:Hydroxyl Radical / Q000737:chemistry; D007700:Kinetics; D008958:Models, Molecular; D009696:Nucleic Acids / Q000737:chemistry; D011506:Proteins / Q000737:chemistry; D012313:RNA; D012260:Ribonucleases / Q000378:metabolism; D013768:Tetrahymena / Q000737:chemistry; D013997:Time Factors
chemical_list: D009696:Nucleic Acids; D011506:Proteins; D017665:Hydroxyl Radical; D012313:RNA; D012260:Ribonucleases