Characterization of Quadruplex DNA Structure by Circular Dichroism

Abstract

Circular dichroism (CD) is a phenomenon that arises from the differential absorption of left- and right-handed circularly polarized light, and may be seen with optically active molecules. CD spectroscopy provides useful spectral signatures for biological macromolecules in solution, and provides low-resolution structural information about macromolecular conformation. CD spectroscopy is particularly useful for monitoring conformational changes in macromolecules upon environmental perturbations. G-quadruplex structures show unique CD spectral signatures, and CD is an important tool for characterizing their formation and global structure. This protocol offers step-by-step methods for determining reliable and reproducible CD spectra of quadruplex structures and normalizing the spectra for presentation. CD spectra properly normalized with respect to quadruplex concentration and path length are required to facilitate accurate comparison of results among laboratories. The standard operating procedures proposed are recommended to make such comparison accurate and informative. © 2017 by John Wiley & Sons, Inc.

Keywords: DNA; G-quadruplex; circular dichroism; concentration determination.

Figure 1

Spectral fetures of human telomer quadruplex conformations. A. Circular dichroism for the antiparallel “basket” form in sodium (blue), the antiparallel “hybrid” form in potassium (red) and the parallel “propeller” form in 50% aceonitrile (black). B. Absorbance spectra for the same samples with the same color coding. C. Calculated g-factor as a function of wavelength obtained using the data in panels A and B. Spectra were recorded at 20 °C in BPS buffer (6 mM Na₂HPO₄, 4 mM NaH₂PO₄, 15 mM NaCl, pH=7.1; blue curves), BPK buffer (6 mM K₂HPO₄, 4 mM KH₂PO₄, 15 mM KCl, pH=7.1; red curves) or BPK + 50% (v/v) acetonitrile (black curves).

Figure 2

CD spectra (solid lines) and their second derivatives (dashed lines) for the human telomere “hybrid” form (A), parallel form (B) and the antiparallel “basket” form (C). Note that the scale of the ordinate axes for the second derivative curves have been reversed to facilitate comparison to the CD spectra. Solution conditions for the different conformations are identical to those given in Figure 1.