Electrophoretic Mobility Shift Assay and Dimethyl Sulfate Footprinting for Characterization of G-Quadruplexes and G-Quad
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 functio
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Introduction G-quadruplexes are formed in single-stranded guanine rich nucleic acid sequences and assembled by Hoogsteen hydrogen bonding of four guanines arranged within a planar tetrad and further stabilized by monovalent cations such as K+ and Na+ [1]. G-quadruplex formation has been observed in synthetic oligonucleotide sequences isolated from the human genome such as telomeres and gene promoter regions and more recently there has been significant number of advances in G-quadruplex detection that
Danzhou Yang and Clement Lin (eds.), G-Quadruplex Nucleic Acids: Methods and Protocols, Methods in Molecular Biology, vol. 2035, https://doi.org/10.1007/978-1-4939-9666-7_11, © Springer Science+Business Media, LLC, part of Springer Nature 2019
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supports the existence of G-quadruplex structures in the genome of human cells significantly in gene regulatory regions and hot spots of genomically unstable regions of human chromosomes [2]. The association of G-quadruplexes to telomere biology, transcription regulation, and genomic instability undoubtedly suggest existence of proteins that modulate G-quadruplex conformation or serve as a platform for protein-protein interactions. The biological relevance of G-quadruplexes is linked to several G-quadruplex interacting proteins: shelterin complex proteins are known to function in telomere hemostasis [3]; and some proteins involve in G-quadruplex unfolding processes such as helicases [4] or G-quadruplex stabilization such as nucleolin [5]. In this paper, we will provide a detailed protocol for electrophoretic mobility shift assay (EMSA) and dimethyl sulfate (DMS) footprinting assay for investigating G-quadruplex and protein-G-quadruplex interactions. Expected outcomes and references to extensions of the method will be further discussed. The established EMSA experiment has been applied to investigate many G-quadruplex-protein complex processes [6–10]. EMSA assay was originally described in 1981 by Fried and Crother [11] and Garner and Rezin [12] and became a widely used, robust method to elucidate crucial information for protein-nucleic acid interactions [6, 13, 14]. The underlying mechanism of the assay is that molecules in different size and charge show different electrophoretic mobilities when resolved on a native polyacrylamide or agarose gel [15]; the complex formed between nucleic acid and protein generally generate slower migrating species than free nucleic acids and DNA-protein complexes with lifetimes exceed the duration of the electrophoresis can be detected as distinct bands (Fig. 1). This technique poses several advantages. The assay is highly sensitive especially when using radioisotope labeled nucleic acids allowing the assay to be performed using nanomolar concentration of the protein and nucleic acids [16]. If high-sensitivity is not required, covalent or non-covalent fluorophores [17, 18] and biotin [19] labeled probes have also been successfully used and reported. Although the assay is often used for qualitative purposes, under a
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