Abstract
Two-dimensional infrared spectroscopy (2D-IR) 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 spectra spanning multiple samples. However, such applications bring new challenges associated with finding accurate, efficient methodologies to analyze large data sets in a timely, informative manner. Here, we demonstrate such an application by screening 2016 2D-IR spectra of 12 double-stranded DNA oligonucleotides obtained in the presence and absence of binding therapeutic molecule Hoechst 33258. By applying analysis of variance combined with principal component analysis (ANOVA-PCA) to 2D-IR data for the first time, 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 indicators of ligand-binding strength that require significantly longer data acquisition times to obtain.
Highlights
M olecular interactions of proteins and DNA are central to biological function, but our ability to understand these interactions in ever greater detail is closely linked to the development of new analytical technologies
Analysis of factor interactions reveal sequence-specific differences in the 2D-IR spectroscopy of DNA-Hoechst 33258 (H33258) complexes that correlate with traditional thermodynamic measurements of binding affinity, showing that 2D-IR and ANOVA−PCA has the potential for use as a rapid and effective probe of DNA binding interactions
It has been shown that the ANOVA−PCA method is able to separate a large, highly dimensional data set into tangible subsets that can be analyzed in a step by step manner
Summary
M olecular interactions of proteins and DNA are central to biological function, but our ability to understand these interactions in ever greater detail is closely linked to the development of new analytical technologies. The advent of mid-IR pulse shaping enabled spectral acquisition within 70 s,13 while laser systems with pulse repetition rates of 100 kHz have further reduced spectral acquisition times to a few seconds.[14,15] This enhanced data collection efficiency opens up the possibility of applying 2D-IR in a more analytical context, for example, as a tool for screening novel bioactive compounds Such an application will produce large sets of data that need to be cross-compared to separate out and identify specific samples that fit the required category, meaning that currently employed data analysis approaches will be prohibitively slow and laborious. Analysis of factor interactions reveal sequence-specific differences in the 2D-IR spectroscopy of DNA-H33258 complexes that correlate with traditional thermodynamic measurements of binding affinity, showing that 2D-IR and ANOVA−PCA has the potential for use as a rapid and effective probe of DNA binding interactions
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