# Multivariate Analysis as a Tool for Quantification of Conformational Transitions in DNA Thin Films
## 1 Introduction
Due to a variety of local microenvironments, fluctuations, and dynamics of the DNA structure, the vibrational energy levels of individual functional groups in DNA can be greatly affected, resulting in variations in IR vibrational spectra even when standard materials, such as calf thymus or salmon testes DNA, are utilized. Consequently, the determination and quantification of DNA conformation in various types of samples is often not unambiguous but is subjected to a number of factors from the hydration of the sample--DNA concentration, temperature, etc.--to the researcher's experience. Inconsistencies in the reported spectral profiles of two DNA conformations (A- and B-form) and misinterpretation of the spectral features are, unfortunately, not the only obstacles needed to be overcome. Some of the issues hindering the application of FTIR DNA studies still remain unresolved and include the lack of computational methods capable of the quantification of DNA conformation in objective manner, automation of spectral analysis which would enable rapid handling of large numbers of spectra and computerized assessment of the quality of the spectra, which is paramount for the quality assurance in clinical studies and application of DNA-based functional material. Even though the automated procedure for the quantification of DNA concentration from FTIR spectra was attempted [16], no such methods were reported for the quantification of DNA conformation. Thus, the automation of DNA spectral analysis and development of computational methods capable of the quantification of DNA conformation remain an open field not yet fully explored that could help to overcome the drawbacks of the current methods in DNA spectral analysis. In our previous research [20], we provided a detailed report on the dynamics of A- to B-form transitions in DNA thin films of different hydrated states based on a statistical analysis of a large number of spectra in the backbone region from 1350 to 750 cm\({}^{-1}\). The hydration of DNA thin films was systematically controlled by the time spent in a desiccator chamber (from 3 to 40 min) and conformation- and hydration-related signatures were resolved. Based on band shape analysis (peak fitting), an estimate of %B-form for the 3-40 min films was calculated utilizing integrated intensities of the 860, 836, and 805 cm\({}^{-1}\) bands. However, due to a large number of samples (over 350 thin films), this type of DNAconformation analysis proved to be complex and time consuming as each spectrum had to be analyzed individually. The main aim of this work (in contrast to our previous one) is to facilitate and automate the spectral analysis of DNA thin films of different spectral profiles and conformations. In order to achieve this goal, we extend the previously presented spectral range to the DNA base region, 1800-1350 cm\({}^{-1}\), and exploit the spectral information obtained in the 900-750 cm\({}^{-1}\) region (namely, the calculated fraction of B-form) in order to develop automated computerized procedures (models) capable of the following: (a) the quantification of %B-form and (b) categorization of DNA thin films spectra of different conformations and hydration states based on spectral fingerprint. We utilize the well-known and widely accessible computer algorithms: the PCA decomposition, SVM classification, and PC regression, and show, to our knowledge, for the first time the ability of the machine-learning algorithms to determine DNA conformation in thin films. Furthermore, we assess the ability of models to correctly determine (a) and (b) in different DNA spectral regions: the base region from 1800 to 1350 cm\({}^{-1}\), region of asymmetric phosphate vibration from 1350 to 1155 cm\({}^{-1}\) and region of symmetric phosphate vibration from 1155 to 990 cm\({}^{-1}\). This allows us to understand the contributions of different spectral parts of DNA to overall conformation- and hydration-related vibrational signatures.