# Multivariate Analysis as a Tool for Quantification of Conformational Transitions in DNA Thin Films
## 1 Introduction  
In the last 20 years, Fourier transform infrared spectroscopy (FTIR) has become a popular technique for studying different types of biological samples due to it being fast, inexpensive and non-destructive. The immense rise in research can be found in the field of biomedicine, with distinct goals of the development and use of (FT)IR techniques in standardized clinical applications [1]. Numerous reports utilizing FTIR spectroscopy extend it to various medical applications, from cancer detection and differentiating of normal and abnormal biomaterial to monitoring treatment efficacy [1, 2, 3, 4, 5, 6, 7]. Furthermore, the application of FTIR biospectroscopy aims to understand and characterize the changes in the secondary structure of biomolecules, prior to the histologic manifestation of the disease, i.e., changes on deoxyribonucleic acid (DNA) found inside the nucleus of the cell. In addition to the classical analysis of FTIR spectra (univariate analysis of frequencies, absorbance intensities, and integrated intensities of bands), many new studies in bioscience (including the ones listed above) deploy multivariate, chemometric techniques in order to extract the most information from the obtained FTIR data [8, 9]. Authors not only utilize multivariate statistical methods in order to enhance the detection ability of FTIR spectroscopy but also to automate and facilitate the analysis of a large number of spectra required for biomedical application in an objective manner. Thus, multivariate statistical methods--such as the principal component analysis (PCA), support vector machine (SVM) algorithm, principalcomponent regression (PCR), and partial least squares (PLS)--have become very popular and useful tools utilized to complement the classical spectral analysis. Ever since the three-dimensional double-helical model of DNA was proposed, the interest for this biomacromolecule has been increasing rapidly, and now it is considered the most important molecule of modern life-science [10]. Even though many secondary structures of DNA are known today, the helical double-stranded B-form and A-form DNA are researched extensively due to their important biological roles in gene expression and cellular functions. Importantly, it was found that inconsistencies in those structures can lead to the discovery of different malignancies and pathogens in cells and tissues and may hinder or facilitate progress towards treatment [11; 12]. Thus, the correct identification of DNA conformation is a relevant step for the studies where conformational transitions in DNA are used as the basis for cancer or damage detection in DNA samples [11; 13; 14; 15; 16]. However important, the biological aspect of DNA is not the only aspect researched extensively. Due to the DNA conformational and structural heterogeneity, the DNA molecule is an interesting bionanomaterial and valuable resource exhibiting a great potential for the future developments in material science and biotechnology. DNA-based sensors, colloidal crystal engineering with DNA, and various three-dimensional (3D) nano-structured functional materials, including DNA superconductors and nanowires, are only a few examples how the electric, magnetic, and optical properties of DNA can be tailored and utilized [17; 18; 19]. The DNA samples in which the helical structure assumes A-form or B-form can be obtained from various physical states of DNA, such as solutions, gels, thin films and crystals, and from different DNA types ranging from short oligonucleotides to genomic DNA. FTIR spectroscopy, a non-destructive and sensitive molecular technique, proved to be one of the few experimental methods capable of elucidating DNA structural features in a such diverse and heterogeneous milieu [11]. However, it is not without shortcomings.