Putting a New Spin of G-Quadruplex Structure and Binding by Analytical Ultracentrifugation
Analytical ultracentrifugation is a powerful biophysical tool that provides information about G-quadruplex structure, stability, and binding reactivity. This chapter provides a simplified explanation of the method, along with examples of how it can be use
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Introduction
1.1 Analytical Ultracentrifugation
Analytical ultracentrifugation (AUC) is underappreciated by the G-quadruplex (G4) community. AUC is a venerable biophysical technique that has a (nearly) 100 year history. Theodor Svedberg invented the analytical ultracentrifuge in 1925, and won the Nobel Prize in Chemistry the next year for his research on colloids and proteins using his invention. AUC has since been widely used as a fundamental technique for the determination of macromolecular structure, reaction stoichiometry and ligand affinity [1–4]. AUC is based on first-principle physical theory, and can be used to determine absolute molecular weights of molecules, along with their hydrodynamic shapes. Our laboratory has found AUC useful for a variety of G4 structural studies [5–13]. The intent of this chapter is to provide a simplified overview of AUC and then to show its utility for characterizing G4 structure and binding. Figure 1 shows a schematic of the most basic AUC experiment, sedimentation velocity (SV). A sample is placed in one sector of the centerpiece within a sealed cell assembly with quartz windows (Fig. 1a). A reference solution is placed in the second sector. 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_5, © Springer Science+Business Media, LLC, part of Springer Nature 2019
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Fig. 1 Schematic representation of a sedimentation velocity experiment. The panel on the left represents a double sector centrifuge cell with a sedimenting sample in the upper sector and the reference buffer in the lower sector. The right panel shows four absorbance scans taken at four different times during sedimentation
cell assembly is then placed in a rotor and spun in an ultracentrifuge to produce a high centrifugal field. The centrifugal field is sufficient to cause the sedimentation of molecules within the sample cell in the direction of the field, toward the bottom of sector. The AUC instrument uses an optical system, typically absorbance, to scan the sample cell to monitor the concentration of molecules at each radial position in the cell, and to record their movement as a function of time at constant centrifugal force. As molecules move within the sample cell, a boundary is formed that changes with time. Figure 1b shows a schematic of an SV experiment, with intermittent scans showing the migration of a boundary toward the bottom of the sample cell. As the boundary migrates to the bottom it also broadens because of diffusion of the sedimenting molecule. Such primary data contain sufficient information to extract the structural properties of the sedimenting molecules. As any number of basic textbooks (see Note 1) derive and show, the sedimentation coefficient (s) is defined as the velocity (v) of the moving boundary (determined at the midpoint) divided by the centrifugal field strength (ω2r)(rpm in radians/sec squared times the radial distance from
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