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Monitoring structural changes in nucleic acids with single residue spatial and millisecond time resolution by quantitative hydroxyl radical footprinting

Nature Protocols volume 3, pages 288–302 (2008)Cite this article

Abstract

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.

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Figure 1: General diagram of a Fast Fenton footprinting experiment.
Figure 2: Schematic representation of 3′-end labeling.
Figure 3: Schematic representation of the three syringe quench-flow mixer experimental setup used to conduct a dose–response experiment to determine the appropriate Fenton reagent concentrations and reaction time.
Figure 4: Analysis of a dose–response experiment.
Figure 5: Schematic representation of a fast Fenton footprinting experiment conducted in the quench-flow mixer.
Figure 6: Monitoring structural changes upon RNA folding.
Figure 7: Generation and analysis of time-progress curves from a fast Fenton autoradiogram.
Figure 8: Manual clustering of the time-progress curves shown in Figure 7.
Figure 9: An example of an analysis of a set of Fast Fenton time-progress curves.

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Acknowledgements

The writing of this article was supported by National Institutes of Health grant PO1–GM066275 from the Institute of General Medical Sciences. We thank Jörg Schlatterer, Somdeb Mitra, Alain Laederach, Rick Russell and Yaqi Wan for critically reading the manuscript and confirming the details of the presented protocol.

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  1. Department of Biochemistry, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, 10461, New York, USA

    Inna Shcherbakova & Michael Brenowitz

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Correspondence to Inna Shcherbakova or Michael Brenowitz.

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Shcherbakova, I., Brenowitz, M. Monitoring structural changes in nucleic acids with single residue spatial and millisecond time resolution by quantitative hydroxyl radical footprinting. Nat Protoc 3, 288–302 (2008). https://doi.org/10.1038/nprot.2007.533

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