In Vivo Chemical Probing for G-Quadruplex Formation
While DNA inside the cells is predominantly canonical right-handed double helix, guanine-rich DNAs have potential to fold into four-stranded structures that contain stacks of G-quartets (G4 DNA quadruplex). Genome sequencing has revealed G4 sequences tend
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Introduction Rather than being a static helix, DNA possesses structural variability. Hydrogen bonding between nucleobases of the complementary DNA strands keeps DNA in the double-stranded right-handed helix: classical B-DNA form. However, DNA elements with special patterns of nucleotides sequence have potential for structural transitions into other DNA forms, non-B-DNA structures. These structures were extensively characterized by biophysical studies, using DNA oligonucleotides or plasmid DNA in various solution conditions. While non-B-DNA structures provide enormous potential for autoregulation of genome function [1], the extent and even existence of such unusual structures inside of living cells is still the matter of some debate. The study of the interplay between DNA conformation and genome biology has been hindered by
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_23, © Springer Science+Business Media, LLC, part of Springer Nature 2019
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experimental difficulties associated with detecting non-B-DNA structures and assessing their regulation in vivo, especially in eukaryotic cells [2, 3]. In this chapter, we describe our method to map non-B DNA genome-wide in the living cells, with a focus on G4 DNA quadruplexes. Other groups are currently adopting and adapting this approach allowing researchers to uncover the role of DNA conformational dynamics in genome function. G4 DNA quadruplexes are four-stranded DNA that stacks planar sets of four mutually Hoogsteen H-bonded guanine bases. Biophysical studies on synthetic oligonucleotide sequences delivered from the eukaryotic genomes indicate the broad variety of G4 structures depending on strand orientation, the size of the singlestranded DNA loops, and solution conditions, such as the selection and concentration of the prevailing cation [4]. These studies have enabled the search for sequences with quadruplex forming potential in genomes [5]. It was shown that DNA elements with predisposition for quadruplex formation (G4 DNA) often reside within regulatory regions, including a significant enrichment of G4 DNA in the promoters of oncogenes [6]. Chemical biological studies have provided crucial insight into G-quadruplex-binding ligands that exhibit pronounced anticancer activities in vivo, ability for transcriptional reprogramming, as well as locus-specific changes in epigenetic information [7, 8]. The potential importance of G4 DNA quadruplex formation within the genomes of living cells and the extensive literature correlating pharmacologic disturbance of quadruplex stability with perturbation of cellular programs stimulated experiments to search for the presence of these structures in vivo [5]. High-affinity G4 DNA quadruplex-recognizing antibodies were used to visualize these structures by immunostaining inside a range of cells [9, 10]. The high stability of G4 DNA quadruplex enabled immunoprecipitation of immun
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