A curated library of circular dichroism spectra of 23 G-quadruplexes of known structure was built and analyzed. The goal of this study was to use this reference library to develop an algorithm to derive quantitative estimates of the secondary structure content of quadruplexes from their experimental CD spectra. Principal component analysis and singular value decomposition were used to characterize the reference spectral library. CD spectra were successfully fit to obtain estimates of the amounts of base steps in anti-anti, syn-anti or anti-syn conformations, in diagonal or lateral loops, or in other conformations. The results show that CD spectra of nucleic acids can be analyzed to obtain quantitative structural information about secondary structure content in an analogous way to methods used to analyze protein CD spectra.

Ac urated library of circular dichroism spectra of 23 G-quadruplexes of knownstructure was built and analyzed. The goal of this study was to use this reference library to develop an algorithm to derive quantitative estimates of the secondary structure content of quadruplexes from their experimental CD spectra. Principal component analysis and singular value decomposition were used to characterizet he reference spectral library.C Ds pectra were successfully fit to obtain estimates of the amounts of base steps in anti-anti, synanti or anti-syn conformations,indiagonal or lateral loops,or in other conformations.T he results show that CD spectra of nucleic acids can be analyzed to obtain quantitative structural information about secondary structure content in an analogous way to methods used to analyze protein CD spectra.

Circular dichroism (CD) arises from the differential absorption of left- and right-handed circularly polarized light, and may be seen for optically active molecules. CD provides useful spectral signatures for biological macromolecules in solution and provides low-resolution structural information about macromolecular conformation. CD is particular 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 the characterization of their formation and global structure. This protocol offers step-by-step methods for the determination of reliable and reproducible CD spectra of quadruplex structures, and how to properly normalize these spectra for presentation. CD spectra properly normalized with respect to quadruplex concentration and pathlength are required to facilitate accurate comparison of results among laboratories. The standard operating procedures proposed are recommended to make such comparison accurate and informative.

We describe a rapid fluorescence indicator displacement assay (R-FID) to evaluate the affinity and the selectivity of compounds binding to different DNA structures. We validated the assay using a library of 30 well-known nucleic acid binders containing a variety chemical scaffolds. We used a combination of principal component analysis and hierarchical clustering analysis to interpret the results obtained. This analysis classified compounds based on selectivity for AT-rich, GC-rich and G4 structures. We used the FID assay as a secondary screen to test the binding selectivity of an additional 20 compounds selected from the NCI Diversity Set III library that were identified as G4 binders using a thermal shift assay. The results showed G4 binding selectivity for only a few of the 20 compounds. Overall, we show that this R-FID assay, coupled with PCA and HCA, provides a useful tool for the discovery of ligands selective for particular nucleic acid structures.