# Multivariate Analysis as a Tool for Quantification of Conformational Transitions in DNA Thin Films
## 4 Discussion
The main aim of this work was to provide an automated computerized model capable of determining %B form in DNA thin films based on the vibrational signatures in the 1800-935 cm\({}^{-1}\) region. The SVM classification proved highly successful in determining the level of B-form. SVM1, SVM2, and SVMS all had 100% success rate in 1800-935 cm\({}^{-1}\) region and, together, correctly determined the B-form level for 192 different spectra of DNA thin films. Furthermore, 100% classification success rate was obtained for the SVM1 and SVM2 models in the base region from 1800 to 1550 cm\({}^{-1}\) providing an insight that perhaps only this small part of the DNA spectrum could be used for the quantification of B-form. This is important for the studies where it might not be possible to collect any spectra below 1000 cm\({}^{-1}\), or if the phosphate groups exhibit spectral changes due to a presence of different reagents. Particularly interesting, the SVM6 model correctly determined the level of B-form in the DNA spectra obtained on a slightly different instrumental setting. An ideal model would, of course, be capable of determining levels of B-form from the DNA spectra obtained in different laboratories but in similar experimental conditions. However, as DNA spectral profiles in different reports are rarely the same, this effort seems challenging at best without the standardization of DNA sample preparation. The PCA analysis and SVM classification (mainly the SVM3 and SVM4 models) provided even further insight into b) the distinction of conformation- and hydration-related effects in DNA thin films. Firstly, PCA showed interesting subgrouping of the 10- and 15-min spectra versus the 20-, 25-, and 40-min spectra (Figure 3), showing that B-form DNA thin films of different hydration levels have distinct vibrational signatures. This was then further confirmed by the inability of the SVM3 model to correctly determine %B-form in 10-25 min films when trained on the 3-, 5-, and 40-min spectra. Distinct spectral characteristics of the 40 min films due to changes in hydration provided too much variability for the model, even though B-form level is the same as in the 10-40 min films. However, when the 15 min spectra were introduced in the same training, in the SVM4 model, the classification ability of the 3-, 5-, 15-, and 40-min calibration datasets drastically improved. This attests to how sensitive FTIR spectroscopy is when it comes to the secondary structure of DNA. Even though the conformation stays the same in 10-40 min films, small variations in the base and phosphate region are visible in the spectra with slight changes in hydration level. (Un)fortunately, slight changes in the dielectric medium surrounding DNA can induce a spatial anisotropy of electric fields, which induces changes in molecular electric dipoles which affect native DNA vibrations. The heterogeneity in the local microenvironments is, in our case, most likely caused by the changes in hydrated water dipoles found in the water layers in DNA atmosphere. This is most pronounced for the asymmetric phosphate vibration, found at \(\approx\)1232 cm\({}^{-1}\). In our previous work [20], it was found highly susceptible to the changes in hydration, exhibiting frequency oscillations and variations in intensity. In this work, the quantification of the B-form based on the vibrational signatures in the region (c), 1350-1155 cm\({}^{-1}\), where the asymmetric PO\({}_{2}\)\({}^{-}\) vibration dominates the spectrum, was by far the worst for both the SVM and PCR modeling (Tables 1 and 2). The symmetric phosphate vibration, on the other hand, was found less sensitive to environmental changes [33], exhibits no frequency shifts and, consequently, performs better in the SVM and PCR modeling. The vibrations in the base region seem to be the least affected, displaying only small variations in intensity once B-form is stable.