Rapid screening of DNA-ligand complexes via 2D-IR spectroscopy and ANOVA-PCA

Two-dimensional infrared spectroscopy is well-established as a specialized, high-end technique for measuring structural and solvation dynamics of biological molecules. Recent technological developments now make it possible to acquire time-resolved 2D-IR spectra within seconds and this opens up the possibility of screening-type applications comparing large datasets spanning multiple samples. However, such applications bring new challenges associated with finding accurate, efficient methodologies to analyze the large datasets in a timely, informative manner. Here, we demonstrate such an application by screening 1728 2D-IR spectra of 12 double-stranded DNA oligonucleotides obtained in the presence and absence of minor groove binding therapeutic molecule Hoechst 33258. By applying analysis of variance combined with principal component analysis (ANOVA-PCA) we demonstrate the ability to efficiently retrieve the base composition of a DNA sequence and discriminate ligand-DNA complexes from unbound sequences. We further show accurate differentiation of the induced fit and rigid-body binding modes that is key to identifying optimal binding interactions of Hoechst 33258, while ANOVA-PCA results across the full sequence range correlate directly with thermodynamic measurements of ligand-binding strength that require significantly longer data acquisition times.