19F NMR Spectroscopy for the Analysis of DNA G-Quadruplex Structures Using 19F-Labeled Nucleobase
G-quadruplex structures have been suggested to be biologically important in processes such as transcription and translation, gene expression and regulation in human cancer cells, and regulation of telomere length. Investigation of G-quadruplex structures
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F NMR Spectroscopy for the Analysis of DNA G-Quadruplex Structures Using 19F-Labeled Nucleobase
Takumi Ishizuka, Hong-Liang Bao, and Yan Xu Abstract G-quadruplex structures have been suggested to be biologically important in processes such as transcription and translation, gene expression and regulation in human cancer cells, and regulation of telomere length. Investigation of G-quadruplex structures associated with biological events is therefore essential to understanding the functions of these molecules. We developed the 19F-labeled nucleobases and introduced them into DNA sequences for the 19F NMR spectroscopy analysis. We present the 19F NMR methodology used in our research group for the study of G-quadruplex structures in vitro and in living cells. Key words G-quadruplex structures, Human telomeres, Aptamer, 19F NMR spectroscopy
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Introduction
1.1 G-Quadruplex Structures
G-quadruplexes are four-stranded nucleic acid secondary structures formed in specific G-rich sequences [1, 2]. G-quadruplex structures have attracted attention because of their important roles in biological events such as gene regulation [3, 4], telomere length regulation and protection [5–9], transcription [10–13], and DNA replication [14–19], suggesting that G-quadruplex structures are viewed as promising molecular targets for therapeutics and diagnostics [4, 20–30]. Previous studies have shown that human telomere DNA form the different G-quadruplex topologies. A 22 nucleotide (nt) DNA with the sequence 50 -A(GGGTTA)3GGG-30 can form an antiparallel-stranded basket-type G-quadruplex in sodium ion solution [31] and a parallel-stranded propeller-type G-quadruplex in crystal containing potassium ion [32]. In potassium solution, the sequence forms a (3 + 1) hybrid G-quadruplex [33–35] and also adopts different topologies [36, 37]. A direct observation of long-telomeric-overhang DNA by atomic force microscopy (AFM) revealed that that telomeric-overhang DNA
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_26, © Springer Science+Business Media, LLC, part of Springer Nature 2019
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forms a higher-order DNA structure containing consecutive G-quadruplexes [6]. Additionally, an intramolecular G-quadruplex structure has also formed in thrombin binding aptamer (TBA) as a 15-nt DNA with the sequence 50 -GGTTGGTGTGGTTGG-30 , which has an ability of specific binding to human α-thrombin and exhibits anticoagulant properties [38, 39]. TBA has been widely used as model of G-quadruplex structure [40–43], which forms an antiparallel G-quadruplex with a chair-type conformation as reported by NMR and X-ray structural studies [44–46]. In addition to intramolecular G-quadruplexes, dimeric and tetrameric intermolecular G-quadruplexes have been reported to form by various DNA sequences. For instance, it has been suggested that a 12-nt human telomeric DNA with the sequence 50 -(TAGGGT)2-30 formed parallel- and a
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