Structural interpretation of DNA-protein hydroxyl-radical footprinting experiments with high resolution using HYDROID
Hydroxyl-radical footprinting (HRF) is a powerful method for probing structures of nucleic acid-protein complexes with single-nucleotide resolution in solution. To tap the full quantitative potential of HRF, we describe a protocol, hydroxyl-radical footprinting interpretation for DNA (HYDROID), to q...
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Published in | Nature protocols Vol. 13; no. 11; pp. 2535 - 2556 |
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Main Authors | , , , , , , , , |
Format | Journal Article |
Language | English |
Published |
England
Nature Publishing Group
01.11.2018
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Subjects | |
Online Access | Get full text |
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Summary: | Hydroxyl-radical footprinting (HRF) is a powerful method for probing structures of nucleic acid-protein complexes with single-nucleotide resolution in solution. To tap the full quantitative potential of HRF, we describe a protocol, hydroxyl-radical footprinting interpretation for DNA (HYDROID), to quantify HRF data and integrate them with atomistic structural models. The stages of the HYDROID protocol are extraction of the lane profiles from gel images, quantification of the DNA cleavage frequency at each nucleotide and theoretical estimation of the DNA cleavage frequency from atomistic structural models, followed by comparison of experimental and theoretical results. Example scripts for each step of HRF data analysis and interpretation are provided for several nucleosome systems; they can be easily adapted to analyze user data. As input, HYDROID requires polyacrylamide gel electrophoresis (PAGE) images of HRF products and optionally can use a molecular model of the DNA-protein complex. The HYDROID protocol can be used to quantify HRF over DNA regions of up to 100 nucleotides per gel image. In addition, it can be applied to the analysis of RNA-protein complexes and free RNA or DNA molecules in solution. Compared with other methods reported to date, HYDROID is unique in its ability to simultaneously integrate HRF data with the analysis of atomistic structural models. HYDROID is freely available. The complete protocol takes ~3 h. Users should be familiar with the command-line interface, the Python scripting language and Protein Data Bank (PDB) file formats. A graphical user interface (GUI) with basic functionality (HYDROID_GUI) is also available. |
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Bibliography: | A.K.S. designed and developed the HYDROID package. A.R.P. supervised the project. A.K.S and A.R.P. tested the protocol and wrote the paper. H.X. performed experimental studies on 3’-labeled nucleosomes. D.A.G. performed experimental studies of 5’-labeled nucleosomes. G.A.A. developed gel simulation algorithms, HYDROID_GUI package and contributed to software packaging. G.A.K. tested the software and contributed to video tutorials. D.L., V.M.S., and C.W. contributed to the discussions and manuscript preparation. AUTHOR CONTRIBUTIONS |
ISSN: | 1754-2189 1750-2799 |
DOI: | 10.1038/s41596-018-0048-z |