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Electrophoretic Mobility Shift Assay and Dimethyl Sulfate Footprinting for Characterization of G-Quadruplexes and G-Quadruplex-Protein Complexes

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G-Quadruplex Nucleic Acids

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2035))

Abstract

DNA G-quadruplexes are globular nucleic acid secondary structures which occur throughout the human genome under physiological conditions. There is accumulating evidence supporting G-quadruplex involvement in a number of important aspects of genome functions, including transcription, replication, and genomic stability, and that protein and enzyme recognition of G-quadruplexes may represent a key event to regulate physiological or pathological pathways. Two important techniques to study G-quadruplexes and their protein interactions are the electrophoretic mobility shift assay (EMSA) and dimethyl sulfate (DMS) footprinting assay. EMSA, one of the most sensitive and robust methods for studying the DNA-protein interactions, can be used to determine the binding parameters and relative affinities of a protein for the G-quadruplex. DMS footprinting is a powerful assay for the initial characterization of G-quadruplexes, which can be used to deduce the guanine bases involved in the formation of G-tetrads under physiological salt conditions. DMS footprinting can also reveal important information in G-quadruplex-protein complexes on protein contacts and regional changes in DNA G-quadruplex upon protein binding. In this paper, we will provide a detailed protocol for the EMSA and DMS footprinting assays for characterization of G-quadruplexes and G-quadruplex-protein complexes. Expected outcomes and references to extensions of the method will be further discussed.

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Acknowledgments

This research was supported by the National Institutes of Health (R01CA122952 (DY), R01CA177585 (DY), and P30CA023168 (Purdue Center for Cancer Research)). We thank Dr. Megan Carver for her thoughtful suggestions and proofreading this chapter.

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Authors and Affiliations

  1. Medicinal Chemistry and Molecular Pharmacology, College of Pharmacy, Purdue University, West Lafayette, IN, USA

    Buket Onel, Guanhui Wu & Clement Lin

  2. University of Arizona, College of Pharmacy, Tucson, AZ, USA

    Daekyu Sun

  3. BIO5 Institute, Tucson, AZ, USA

    Daekyu Sun

  4. Arizona Cancer Center, Tucson, AZ, USA

    Daekyu Sun

  5. Department of Medicinal Chemistry and Molecular Pharmacology, College of Pharmacy, Purdue University, West Lafayette, IN, USA

    Danzhou Yang

  6. Purdue Center for Cancer Research, West Lafayette, IN, USA

    Danzhou Yang

  7. Purdue Institute for Drug Discovery, West Lafayette, IN, USA

    Danzhou Yang

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  1. Buket Onel
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  3. Daekyu Sun
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  4. Clement Lin
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  5. Danzhou Yang
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Correspondence to Danzhou Yang .

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Editors and Affiliations

  1. Department of Medicinal Chemistry and Molecular Pharmacology, College of Pharmacy, Purdue University, Purdue Center for Cancer Research, Purdue Institute for Drug Discovery, West Lafayette, IN, USA

    Danzhou Yang

  2. Medicinal Chemistry and Molecular Pharmacology, College of Pharmacy, Purdue University, West Lafayette, IN, USA

    Clement Lin

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Onel, B., Wu, G., Sun, D., Lin, C., Yang, D. (2019). Electrophoretic Mobility Shift Assay and Dimethyl Sulfate Footprinting for Characterization of G-Quadruplexes and G-Quadruplex-Protein Complexes. In: Yang, D., Lin, C. (eds) G-Quadruplex Nucleic Acids. Methods in Molecular Biology, vol 2035. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-9666-7_11

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  • DOI: https://doi.org/10.1007/978-1-4939-9666-7_11

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