# Multivariate Analysis as a Tool for Quantification of Conformational Transitions in DNA Thin Films
## 3 Results
### Vibrational Signatures of DNA Thin Films in Base Region  
In Figure 1, the average spectra of 3, 5-, 10-, 15-, and 40-min films in the 1800-970 cm\({}^{-1}\) range are presented (for details see Materials and Methods). The vibrational bands in the spectral region from 1800 to 1350 cm\({}^{-1}\) are mostly due to the C=O, C=N, and C=C vibrations of purine and pyrimidine rings of DNA (base vibrations), while the vibrations in the range from 1350 to 970 cm\({}^{-1}\) belong to the vibrations of the DNA backbone (for a more detailed assignment of backbone vibrations see our previous work [20]). Specifically, the absorption maximums observed near 1710 and 1661 cm\({}^{-1}\) are dominantly due to the C=O vibrations of guanine and thymine, the maximum near 1609 cm\({}^{-1}\) is mostly due to the C=N vibrations of adenine, while the band at \(\approx\)1490 cm\({}^{-1}\) originates from the C=N vibrations in cytosine and guanine [26; 27; 28; 29; 30]. Several weaker bands also appear in the spectra; however, we leave them unassigned as the details of their changes are beyond the scope of this work.  
The spectral changes observed in the base region between different films presented in the main panel of Figure 1 can be rated as changes in the shape of the absorption curve and changes in the absorption intensity of the bands. Namely, the region between 1715 and 1685 cm\({}^{-1}\), that contains the C=O band at 1710 cm\({}^{-1}\), experiences spectral reshaping, while in the rest of the base region the bands retain their shape and show only an increase in the absorption intensity. Spectral reshaping in the 1715-1685 cm\({}^{-1}\) region is most evident in the inset of Figure 1: the 3 min film reflects a distinct shape compared to the 10-40 min films mostly due to the lower absorption intensity near 1695 cm\({}^{-1}\). In 10-40 min films, the absorption curve shape in the 1730-1610 cm\({}^{-1}\) region is mostly unaffected by an increase of desiccation time, only a steady increase in the absorption intensity is evident. Namely, the bands at 1661, 1609, and 1490 cm\({}^{-1}\), but other weak base bands as well, show a continuous absorption intensity increase as the desiccation time increases (see Figure 1). The intensity increase of the base bands is also followed by the intensity increase of the  
Figure 1: The average FTIR spectra of DNA thin films of different hydration levels in the 1800–970 cm\({}^{-1}\) range: 3 min film (purple), 5 min film (black), 10 min film (light green), 15 min film (dark green), 40 min film (red). Inset: enlarged view of the respective DNA thin film in the base region (1730–1610 cm\({}^{-1}\)). Numbers denote identified bands of 10 min film.  
asymmetric PO\({}_{2}\)\({}^{-}\) vibration (Figure 1) and integrated intensity increase of those bands. The integrated intensity increase of the large absorption band in the 1800-1550 cm\({}^{-1}\) region and asymmetric and symmetric PO\({}_{2}\)\({}^{-}\) vibrations in the 1320-1155 and 1155-990 cm\({}^{-1}\) region, respectively, are presented in Figure 2 and are more evident for 20-40 min films. Note that 10-40 min films all have roughly the same %B-form (\(\approx\)75%), so the observed intensity increase and integrated intensity increase for 20-40 min films are more likely the result of slight changes in hydration rather than changes in conformation.  
Note that no frequency shifts of the bands are observable in the entire base region. The changes in the shape of the absorption curve in the 1720-1620 cm\({}^{-1}\) region are most likely caused by the dissimilar intensity changes of bands, i.e., the variations in the intensity of closely overlapped bands, with their individual frequencies unchanged. Similar behavior, spectral reshaping without frequency shifts of constituent modes, was found for phosphate vibrations in the 1350-990 cm\({}^{-1}\) region [20]. We give more detailed interpretation of the changes in the base and phosphate region in Discussion.