Single-Molecule Investigations of G-Quadruplex
The genome-wide occurrence of G-quadruplexes and their demonstrated biological activities call for detailed understanding on the stability and transition kinetics of the structures. Although the core structural element in a G-quadruplex is simple and requ
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Introduction Intramolecular G-quadruplexes are non-canonical, four-stranded structures formed in DNA or RNA sequences consisting of at least four tandem repeats of guanine (G) rich residues [1, 2]. The central unit of G-quadruplexes is a stack of G-tetrads each made of four guanine residues cross-linked by Hoogsteen hydrogen bonds. These G-tetrads stack into a G-quadruplex by π–π interactions. Monovalent cations such as K+ and Na+ stabilize the G-quadruplex structure by coordination with the O6 in guanine residues. By specific arrangements of the loop sequences between consecutive runs of guanine repeats, G-quadruplexes can adopt different conformations such as parallel, antiparallel, hybrid, and chair forms [3–5]. Potential G-quadruplex forming sequences have been identified in both prokaryotic and eukaryotic organisms. Recent studies have revealed that more than 376,000 G-quadruplex forming sequences are present in the human genome [6]. The
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_16, © Springer Science+Business Media, LLC, part of Springer Nature 2019
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occurrence of these putative G-quadruplexes is disproportionally higher close to or inside the promoters, or in telomere regions. Under physiological conditions, G-quadruplexes can form in duplex or single-stranded DNA regions. Previous studies have shown that G-quadruplex structures play modulatory roles in replication, transcription, cell proliferation, genome recombination, and telomere maintenance, among others [7–10]. Among G-quadruplexes formed in different regions, the G-quadruplexes in the human telomere with the 50 -TTAGGG repeat sequences have been the subject of extensive biophysical and biological studies. Formation of G-quadruplex structures in telomere has shown evidence to impede the extension of human telomerase [11, 12]; therefore, these telomeric structures are considered as potential molecular targets for new anticancer agents. To understand the biological roles of G-quadruplex structures, it is essential to know the properties of these structures in physiological conditions. G-quadruplexes in various promoter regions and telomeres have been well characterized by various bulk and single molecule methods. Ensemble average methods include biochemical techniques [13–15] such as electrophoretic mobility shift assay (EMSA), dimethylsulfate (DMS) footprinting, and the DNA/RNA polymerase stop assay; spectroscopic techniques such as UV/Vis [16], circular dichroism (CD) [17, 18], fluorescence resonance energy transfer (FRET) [18], and NMR [19]; and calorimetric techniques such as isothermal titration calorimetry (ITC) [17] and differential scanning calorimetry (DSC) [17]. Single molecule methods include imaging techniques such as atomic-force microscopy (AFM) [20], fluorescence based techniques such as single-molecule fluorescence resonance energy transfer (smFRET) [21, 22], and force-base
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