# Multivariate Analysis as a Tool for Quantification of Conformational Transitions in DNA Thin Films
## 4 Discussion  
The analysis of bands in three distinct DNA spectral regions: the base region from 1800 to 1350 cm\({}^{-1}\), phosphate region from 1350 to 935 cm\({}^{-1}\) and sugar phosphate region from 900 to 750 cm\({}^{-1}\), showed that the bands in the sugar-phosphate region, mostly undergo one-directional spectral changes with respect to the time spent in a vacuum chamber (frequency shifts and changes in the absorption intensity and integrated intensity). On the other hand, the base and phosphate vibrations tend to display dissimilar spectral changes with respect to the desiccation time, which greatly impedes the spectral analysis. From the inset of Figure 1, it is evident that the major absorption bands situated at 1710, 1661, and 1609 cm\({}^{-1}\) do not exhibit frequency shifts, but rather dissimilar changes in the intensities that result in general reshaping of the absorption curve in the 1720-1620 cm\({}^{-1}\) interval. Thus, 3 min film, which corresponds to \(\approx\)40% B-form, attains an interesting and distinct shape in the base region with A-like conformation mostly due to the low absorption intensity near 1695 cm\({}^{-1}\). As argued previously, 3 min film is obtained just after it was formed from the DNA solution, thus preventing attainment of equilibrium during the transition and finally resulting in more disordered A-like conformation. Similar behavior, a low intensity near the 1695 cm\({}^{-1}\) vibration in A-form DNA and sensitivity to changes in hydration was observed in [11]; however, no further interpretation was given by the authors. Thus, it appears that the intensity of vibration near 1695 cm\({}^{-1}\) is highly susceptible to both conformation transitions and changes in hydration [32]. This is further confirmed by the large intensity increase of the named band in the case of 40 min films  
Figure 5: Results for the regression model PCR2 calculated for the 1800–935 cm\({}^{-1}\) region. Data were trained against their respective class on the 3-, 5-, 10-, and 40-min spectra and then validated by the 15-, 20-, and 25-min spectra. For details, see Table 2.  
observed in Figure 1. Even though the conformational markers are stable in 40 min films and reflect \(\approx\)75% B-form, the increased absorption intensity in the 1720-1620 cm\({}^{-1}\) interval, confirmed by the integrated intensity increase (see Figure 2), points to a decreased number of water molecules situated in the outer-hydration shells of DNA, as argued in [20]. To conclude, the univariate analysis of the base bands, the same as the previous analysis of the bands in the phosphate region, proved that spectral signatures of the two respective forms are not as easily resolved without the information on conformational markers (the 860, 837, and 805 cm\({}^{-1}\) bands) since they exhibit dissimilar spectral changes depending on hydration conditions. Furthermore, conformational variations in the spectra were only disentangled due to the information being obtained from different DNA thin films, with the 3-, 5-, 10-, and 40-min films being the cornerstone of this analysis. This means that the determination of DNA conformation by means of reading the frequencies and intensities of individual base and phosphate vibrations in different DNA samples, i.e., univariate analysis is highly unlikely to be rapid and accurate. It is more likely that the accurate conformation quantification for a large number of different DNA samples will require special skill and will be time-consuming. This is especially important in cases where a large number of spectra have to be processed in a short time interval, such as different medical applications, indicating that some level of multivariate analysis should be used instead. The PCA analysis, SVM classification and PC regression of data proved useful techniques in (a) the quantification of %B-form and (b) categorization of DNA thin films spectra of different conformation and hydration states based on spectral fingerprints in several regions.