# Conformational Transitions of Double-Stranded DNA in Thin Films
## 2 Materials and Methods
### DNA Thin Films Obtained after 3 min and 5 min
The integrated intensity of mode 4 increased by factor two when 3-min and 10-min films  
Figure 4: Average FTIR spectra and standard deviations of different DNA thin films obtained by FTIR spectroscopy in the 1350–750 cm\({}^{-1}\) range: 3-min film (black line denotes average spectrum, purple fill patter denotes standard deviation), 5-min film (black dashed line denotes average spectrum, and yellow fill pattern denotes standard deviation). For comparison, standard deviations of the 10-min film (green fill pattern) and 40-min film (red fill pattern) are presented. Inset: standard deviations of the respective DNA thin films in the sugar–phosphate region (950–750 cm\({}^{-1}\)). Numbers and arrows denote identified bands for the 3-min film. Labels *, ** and *** denote regions of spectral reshaping. are compared (Figure 7a). However, when the 10-min film is compared to the 40-min film, no drastic changes of integrated intensity are observed on modes \(3\), \(4\), and \(5\) (Figure 7a). Conformational transition is thus complete in 10-min film, and additional time in the desicator chamber has no significant impact on the conformation. Finally, a drastic decrease in intensity of the 765 cm\({}^{-1}\) band, which is observed in the inset of Figures 4 and 5, is accompanied by an almost threefold decrease in integrated absorption of mode 9. Since mode 9 (765 cm\({}^{-1}\) band) experiences a continuous decrease in integrated intensity in 3-min, 5-min, 10-min, and 40-min spectra, it is more likely that the observed decrease reflects changes in hydration rather than conformation. Details of the spectral reshaping of the backbone region of symmetric PO2\({}^{-}\) vibration from 1150-1000 cm\({}^{-1}\) are disclosed by the fitting procedure presented in Figure 6. The entire region is successfully fitted by eight modes whose initial positions were extracted from the second derivative spectra (not shown). Upon close inspection of Figure 6a, modes 1-6 are easily detectable as they coincide with the inflections of the absorption curve, while the detection of modes 7 and 8 is less straightforward. Modes do not show any frequency shift with respect to the time spent in the vacuum chamber, nor does the absorption curve (obtained experimentally or by fit); only dissimilar intensity, integrated intensity, and bandwidth changes of the modes are observed. Thus, the spectral changes observed in the  
Figure 5: Absorption bands in the backbone region from 900–750 cm\({}^{-1}\) for representative DNA thin films: (**a**) 3-min, (**b**) 5-min, (**c**) 10-min, and (**d**) 40-min. Experimental data are presented by black squares and identified absorption bands are denoted by a number. The cumulative fit is presented by the red line, while ten constituent Gaussians are shown by the green lines and identified by numbers in italic from _1–10_. Important A-form markers (860 cm\({}^{-1}\) and 805 cm\({}^{-1}\)) are highlighted in yellow; complete loss of the 805 cm\({}^{-1}\) band (Gaussian 5) is observed in 10-min and 40-min films. 1150-1000 cm\({}^{-1}\) can be interpreted by an increase and a decrease in integrated intensities of constituent modes. Namely, the slight increase in absorption near 1135 cm\({}^{-1}\) and 1120 cm\({}^{-1}\) denoted by * in Figure 4, is a result of the strengthening of modes \(I\) and \(3\). Spectral reshaping denoted by ** in Figure 4 is more complex than suggested solely by inspection of the absorption band in that region. While the absorption spectra suggest symmetric PO2\({}^{-}\) vibration at 1089 cm\({}^{-1}\) and phosphodiester bands at 1069 cm\({}^{-1}\) and 1055 cm\({}^{-1}\), a large peak placed in that region is influenced by modes 3-7. In the 3-min, 5-min, and 10-min films absorption intensity of maximum at 1089 cm\({}^{-1}\) decreases mostly due to a decrease in integrated intensities of modes 4 and 5 (Figure 6a-c)).