# Conformational Transitions of Double-Stranded DNA in Thin Films
## 4 Discussion  
In the previous section, the main differences between DNA thin films were detected and ascribed to either conformational changes or changes in hydration. All thin films studied, 3, 5, 10, 15, 20, 25, and 40 min, can be roughly classified into two groups with respect to their vibrational signature in the backbone region, i.e., 3-min and 5-min films in the first, and 10-40 min films in the second. In the first group, 3-min and 5-min film showed similar spectral features reflecting A-like dsDNA conformation (Figure 4). On the other hand, 10-40 min films showed no significant variations in B-like conformation and only a small variation in the phosphodieser bands with respect to the time spent in the vacuum chamber (Figure 3). Importantly, 10-40 min films have a different spectral signature in the 900-750 cm\({}^{-1}\) region than 3-min and 5-min films (i.e., compare panels of Figure 5). Band shape analysis presented in Figures 4-6 has proven very illuminating in resolving bands and constituent modes sensitive to changes in conformation and hydration. The band at 897 cm\({}^{-1}\) (3 -min film) was found very sensitive to desiccation time; dynamics of modes \(1\) and \(2\) resulted in spectral reshaping that can easily be tracked in the spectra and thus might serve as a new spectral signature of conformational transitions. On the other hand, a shoulder near 1030 cm\({}^{-1}\) and a band at 765 cm\({}^{-1}\) showed great sensitivity to changes in hydration conditions without saturation, making them good candidates for tracking changes on the backbone that might have taken place as a result of changes in DNA hydration shells. In this section, we focus our discussion on two important issues-- why the 3-min film reflects more A-like conformation than the 10-min film, and why almost no conformational changes are observed in the spectra after 10 min. It was previously found that the conformational B \(\rightarrow\) A and A \(\rightarrow\) B transitions are related to the changes in hydration, but authors also highlight the importance of dynamics of conformational transitions [2, 15, 27]. Due to the slow kinetics of the transition, attainment of equilibrium between B- and A-forms caused by rapid changes in hydration may be prevented [27]. This is why most conformation studies that use specially designed chambers in which hydration is controlled by the use of special salts require slow and step-wise protocols in which samples are maintained from several up to 24 h [9]. In our study, the opposite is achieved in the desiccator chamber. DNA liquid samples are \"shocked\" within the first 2 min with the rapid changes in the atmosphere where pressure changes from 1000-10 mbar in the desiccator chamber (left inset of Figure 1). Approaching 3 min, the formation of the thin film is visible in the chamber and constant pressure of 10 mbar is established. The change of DNA aggregate state from solution to the thin film is thus stopped and 3-min film spectra are taken; DNA spectra reflect more disordered A-like conformation. The level of disorder in the DNA structure has previously been calculated with respect to integrated intensity ratios of the deoxyribose bands at 1069 cm\({}^{-1}\) and 1055 cm\({}^{-1}\)[20]. Because thin films are allowed more  
Figure 7: Relative integrated intensity as a function of time in the active vacuum of respective constituent Gaussians for 3-min, 5-min, 10-min, and 40-min films in **(a)** 900–750 cm\({}^{-1}\) region and **(b)** 1150–900 cm\({}^{-1}\) region obtained by peak fitting. The relative integrated intensity was calculated according to \(A_{\rm{smin}}/A_{\rm{3 min}}\). Frequencies of constituent Gaussians (in cm\({}^{-1}\)) and their respective labels (modes _1–10_ and _1–8_) are also presented. time in the desiccator chamber, the integrated intensity ratio 1069/1055 decreases from 0.42 in the 3-min film to 0.28 in the 40-min film, indicating transition into a more ordered state.