G-Quadruplex and Protein Binding by Single-Molecule FRET Microscopy
G-quadruplex (G4) is a non-canonical nucleic acid structure that arises from the stacking of planar G-tetrads, stabilized by monovalent cations. G4 forming sequences exist throughout the genome and G4 structures are shown to be involved in many processes
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Introduction G-quadruplex (G4) is a non-canonical nucleic acid structure that can form from a guanine-rich single strand. The core structure of G4 comprises stacks of guanine(G)-tetrad planes formed by Hoogsteen hydrogen bonds, stabilized by monovalent cations, especially potassium [1]. Putative G4 forming sequences are unevenly distributed throughout the human genome [2, 3]. G4 structures have been reported to be involved in DNA replication, gene regulation, genome instability, and human diseases [4–7]. One well-characterized G-quadruplex is the human telomeric 30 overhang of 50–200 nucleotide region containing a repeat sequence (TTAGGG)n. Four repeats of this sequence can fold into a G-quadruplex structure in the presence of monovalent cations, such as sodium or potassium [8, 9]. Additional repeat sequences can associate with the G4, but extra repeats can destabilize G4 in terms of thermostability and enthalpy [10]. The G4 structure can also influence the accessibility of the telomeric DNA to proteins such as telomerase and helicase [11]. However, determining how
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_18, © Springer Science+Business Media, LLC, part of Springer Nature 2019
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the telomeric overhang affects G-quadruplex formation and the accessibility of protein binding is difficult to monitor by traditional biochemical methods, especially when the process is dynamic. Single-molecule methods can offer an advantage of providing structural dynamics at the molecular level. Total internal reflection fluorescence (TIRF) microscopy is a popular single- molecule detection method which yields reduced background noise and enables collecting data from hundreds of molecules in one measurement [12, 13]. In our system, DNA samples are labeled with two dyes, Cy3 and Cy5, in the FRETsensitive distance. FRET is a distance-dependent energy transfer process used to monitor the interaction between the two dyes, donor and acceptor which report on structural dynamics of labeled molecules. For example, when two dyes are labeled on one molecule, such as in two positions within the same DNA, the FRET change induced by protein binding would report on how the protein changes the conformation of the DNA strand within the labeled position. We have previously reported single-molecule fluorescence study on the G4 folding of telomeric overhang and the interaction with POT1 binding [14, 15]. POT1 is a component of the telomere binding protein complex termed shelterin, which specifically associates with ten nucleotide of telomeric repeat sequence (TTAGGGTTAG). POT1 protects telomeric overhang by preventing DNA repair machinery from associating with nondamaged telomere DNA [16]. The binding of POT1 on telomeric G4 can unfold the structure. Such binding and unfolding of the target G4 can be probed and quantified by smFRET. Here, we describe detailed protocols of smFRET studies on telo
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