Monitoring structural changes in nucleic acids with single residue spatial and millisecond time resolution by quantitative hydroxyl radical footprinting.
Hydroxyl radical (.OH) footprinting provides comprehensive site-specific quantitative information about the structural changes associated with macromolecular folding, interactions and ligand binding. 'Fast Fenton' footprinting is a laboratory-based method for time-resolved .OH footprinting capable of millisecond time resolution readily applicable to DNA and RNA. This protocol utilizes inexpensive chemical reagents (H2O2, Fe(NH4)2(SO4)2, EDTA, thiourea or ethanol) and widely available quench-flow mixers to reveal transient, often short-lived, intermediate states of complex biochemical processes. We describe a protocol developed to study RNA folding that can be readily tailored to particular applications. Once familiar with quench-flow mixer operation and its calibration, nucleic acid labeling and the conduct of a dose-response experiment, a single kinetic experiment of 30 time points takes about 1 h to perform. Sample processing and separation of the .OH reaction products takes several hours. Data analysis can take 45 min to several weeks depending on the depth of analysis conducted.
Shcherbakova
Inna
I
Department of Biochemistry, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, New York 10461, USA. ishcher@aecom.yu.edu
Brenowitz
Michael
M
D000818:Animals; D018983:DNA Footprinting / Q000379:methods; D005821:Genetic Techniques; D006861:Hydrogen Peroxide; D017665:Hydroxyl Radical / Q000737:chemistry; D007501:Iron; D007700:Kinetics; D009690:Nucleic Acid Conformation; D010759:Phosphorus Isotopes; D012313:RNA / Q000737:chemistry*; D016337:RNA, Catalytic / Q000737:chemistry; D016808:Tetrahymena thermophila / Q000737:chemistry / Q000235:genetics
C045076:Fenton's reagent; D010759:Phosphorus Isotopes; D016337:RNA, Catalytic; D017665:Hydroxyl Radical; D012313:RNA; D006861:Hydrogen Peroxide; D007501:Iron