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Monitoring Equilibrium Changes in RNA Structure by 'Peroxidative' and 'Oxidative' Hydroxyl Radical Footprinting
Published on: October 17, 2011
Hydroxyl-radical footprinting to probe equilibrium changes in RNA tertiary structure
Inna Shcherbakova1, Somdeb Mitra
1Department of Biochemistry, Albert Einstein College of Medicine, Bronx, New York, USA.
Methods in Enzymology
|October 16, 2010
Summary
Hydroxyl-radical footprinting reveals nucleic acid structural changes using reactive species. This method, combined with nucleotide resolution, provides detailed insights into folding and ligand binding dynamics.
Area of Science:
- Biochemistry
- Molecular Biology
- Structural Biology
Background:
- Hydroxyl-radical footprinting probes nucleic acid structure by cleaving solvent-accessible regions.
- Changes in nucleic acid structure can be monitored over time or by varying reagent concentration.
Purpose of the Study:
- To present protocols for equilibrium hydroxyl-radical footprinting using Fenton chemistry.
- To guide the adjustment of Fenton reagent concentrations for specific experimental conditions.
- To discuss product separation techniques and data analysis for equilibrium footprinting.
Main Methods:
- Utilizes hydroxyl radicals to probe solvent-accessible regions of nucleic acids.
- Employs Fenton chemistry (peroxidative and oxidative) for radical generation.
- Integrates techniques for single nucleotide resolution of cleaved fragments.
Main Results:
- Provides detailed information on local tertiary structure changes.
- Enables probing of nucleic acid folding and ligand binding.
- Offers two protocols for equilibrium hydroxyl-radical footprinting.
Conclusions:
- Equilibrium hydroxyl-radical footprinting is a powerful technique for studying nucleic acid structure.
- The presented protocols facilitate detailed structural analysis of nucleic acids.
- Methodology allows for precise investigation of dynamic structural changes.
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