# Multivariate Analysis as a Tool for Quantification of Conformational Transitions in DNA Thin Films
## 3 Results
### Principal Component Analysis  
In order to develop multivariate models capable of determining %B-form in DNA thin films, we first utilized PCA for the decomposition of all spectra in the principal component space. Figure 3 shows the score plot of all 369 spectra in the 1800-650 cm\({}^{-1}\) range built with the first four principal components: in the PC1 versus PC2 score plot a strong clustering of the spectra with respect to the time spent in a vacuum chamber is evident. PC1 accounts to \(\approx\)15% of total variance, with the 3 and 5 min spectra obtaining only negative PC1 scores, while the 10-40 min spectra obtain only positive PC1 scores. This strong separation along the PC1 axis correlates well with the previously established grouping with respect to conformation with the 3 and 5 min films reflecting more A-like conformation, while the 10-40 min films all show mostly B-form. However, as the 10 and 15 min spectra score slightly different on the PC1 axis than the 20-40 min films, it can be reasoned that PC1 variance accounts for conformational and other differences in the spectra. Strong contenders are hydration-related features as the 20-40 min films obtain similar PC1 scores, different from the 10 and 15 min films. Note that in Figure 2 the integrated intensities of the base and phosphate bands of 20-40 min films showed similar grouping, which was correlated to hydration effects as after 10 min there are no more conformational changes.  
Figure 2: Integrated intensity as a function of time in active vacuum for three distinct spectral regions: the base region from 1800 to 1550 cm\({}^{-1}\) (red circles), asymmetric phosphate vibration from 1320 to 1155 cm\({}^{-1}\) (blue upright triangles) and symmetric phosphate region from 1155 to 990 cm\({}^{-1}\) (inverted green triangles). The increased integrated intensity of the 2040 min films compared to the 3–15 min films indicates hydration-related changes in thin films for all three regions rather than conformational changes.  
To conclude, the PC analysis revealed that DNA films associated with the lower level of B-form, 40-60%, have negative PC1 scores (3 and 5 min films), while the films associated with the high level of B-form, \(\approx\)75%, all have positive PC1 scores (10-40 min films). Additionally, further (sub)grouping of the 10-40 min spectra based on the PC1 and PC2 scores was observed: the 10- and 15-min films in the first cluster have similar PC1 and PC2 scores, opposed to the 20-40 min films in the second cluster. Such decomposition of data in the PC score plot served as a good indication how two different types of SVM and PCR models can be trained and validated. The first type of model would be based on the classification of spectra with respect to desiccation time, i.e., entire data can be classified into seven respective classes (3-40 min films) and the model would be validated on how well it can predict spectral signatures related to each desiccation time. The second type of model is based on the classification of spectra with respect to %B-form: entire data would be classified into three respective classes, 3, 5, and 10-40 min spectra, all representing different levels of B-form, as determined in our previous work.