# Conformational Transitions of Double-Stranded DNA in Thin Films
## 4 Discussion
However, radical shifts of asymmetric and symmetric vibrations reported in [9] are not found in our research, supporting less drastic changes to the conformation and the coexistence of both forms in thin films. Unfortunately, the authors do not provide any information on the bands below 950 cm\({}^{-1}\); therefore, the region that contains important A- and B-form markers [34] cannot be compared to their study. Our results further support the interpretation of spectral changes with respect to two competing effects (hydration and conformation), although with several subtleties. Our study does not cover DNA transitions in extreme conditions but rather in-between and, more importantly, extends interpretation of the spectra below 950 cm\({}^{-1}\) so that correlation with the sugar vibrations can be established. As substantial shifts of asymmetric and symmetric PO\({}_{2}\)\({}^{-}\) vibrations are missing in our spectra, phosphate bands alone cannot be used in monitoring conformational transitions; asymmetric PO\({}_{2}\)\({}^{-}\) vibration is found in the 1230-1234 cm\({}^{-1}\) interval reflecting only \"fine-tuning\" of the conformation, while symmetric PO\({}_{2}\)\({}^{-}\) vibration remains at 1089 cm\({}^{-1}\). On the other hand, conformation-related markers at 860 cm\({}^{-1}\), 836 cm\({}^{-1}\), and 805 cm\({}^{-1}\) track conformational transition more effectively; a decrease in A-form from \(\approx\)60% to \(\approx\)25% in 3-min and 10-min films, respectively, is observed. Additionally, backbone vibration at 893 cm\({}^{-1}\) is also found to be sensitive to conformational transitions because it experiences severe spectral reshaping due to dissimilar integrated intensity changes of constituent modes. The band at 893 cm\({}^{-1}\) is thus a valuable conformation-related marker, especially if the information on the lower frequency modes is missing in the spectra. Finally, note that changes in 893, 860, 836, and 805 cm\({}^{-1}\) eventually saturate, indicating the end of conformational transitions after approximately 10 min in the desiccation chamber. Hydration-related effects, on the other hand, do not seem to saturate. Asymmetric and symmetric PO\({}_{2}\)\({}^{-}\) vibration at \(\approx\)1234 cm\({}^{-1}\) and 1089 cm\({}^{-1}\), respectively, and backbone vibrations near 1030 cm\({}^{-1}\) and 765 cm\({}^{-1}\) exhibit continuous changes without saturation and consequently show great sensitivity to hydration conditions near PO\({}_{2}\)\({}^{-}\) groups rather than conformational transitions. Vibrations of the phosphate groups were also found to be sensitive to ambient conditions, which is best evident from the analysis of 180 samples of 5-min DNA thin films. The 5-min films were obtained in two ways--(a) immediately after their removal from the desiccator chamber and (b) 5 min after their removal from the desiccator chamber, i.e., \"before\" and \"after\" stabilization (Figure 9 and Figure 11). Average spectra of 5-min films, obtained by either (a) or (b), are in the same conformation as evident by the lack of significant frequency shifts in the entire spectral region from 1350-750 cm\({}^{-1}\) and lack of changes in the intensities of conformation related markers in the 900-750 cm\({}^{-1}\) region (Figure 9). Only differences in absorption intensities of asymmetric and symmetric PO\({}_{2}\)\({}^{-}\) vibration and backbone vibrations near 1030 cm\({}^{-1}\) are evident in the spectra and are most likely caused by the atmospheric changes that happen as films transition from a vacuum atmosphere to an ambient atmosphere. Furthermore, note that spectral differences between films obtained by (a) and (b) are greatly reduced in 40-min films (inset of Figure 9), i.e., the influence of ambient conditions can be reduced by increasing the desiccation time.