# Conformational Transitions of Double-Stranded DNA in Thin Films
## Appendix A  
In this section, we provide details of DNA thin film preparation and present average spectra of all thin films obtained in this study. DNA thin films were obtained by placing 30 \(\upmu\)L of the respective DNA stock solutions on optical grade silicon transmission windows and placed inside a desicator chamber where the vacuum effect was achieved and maintained (active vacuum) by Leybold S1 rotary vacuum pump equipped with Leybold Thermovac TTR91N vacuum gauge (Leybold GmbH, Kein, Germany). The time spent in the desicator chamber varied from 3 min to 40 min after which thin films were recorded (a) immediately after their removal from the desiccator chamber and (b) 5 min after their removal from the desiccator chamber, thus allowing the samples to come to ambient conditions (atmospheric pressure, room temperature and humidity), i.e., to stabilize. We would like to stress the importance of this sample preparation. First, by placing samples inside a chamber with an active vacuum, one has more control over thin film formation than in standard air-dried procedures usually associated with attenuated total reflectance (ATR) and other IR techniques; thin films are prepared in equal conditions such as pressure,temperature, and time, resulting in less variation in the spectra. Second, by recording spectra (a) \"before stabilization\" and (b) \"after stabilization,\" it is possible to distinguish between hydration conditions that induce conformational transitions and those that do not. According to the stated protocols, 369 DNA thin films were prepared in total and one spectrum per thin film was obtained. Thus, spectra of DNA thin films with respect to time in active vacuum were collected as follows: 3 min (54 spectra), 5 min (180 spectra), 10 min (54 spectra), 15 min (18 spectra), 20 min (9 spectra), 25 min (9 spectra), and 40 min (45 spectra). All spectra obtained under the same conditions were averaged using eFTIR software and standard deviations were calculated using OriginPro 2019. For easier identification, average spectra of DNA thin films obtained under different active vacuum times are abbreviated as 3-min film, 5-min film, etc. In Figure 11, average spectra of 3-min, 5-min, 10-min, 15-min, 20-min, 25-min, and 40-min films obtained (a) immediately after their removal from the desiccator chamber and (b) five minutes after their removal from the desiccator chamber are presented. In Figure 11, it is evident that there is no difference in conformation between the spectra obtained by (a) and (b) in any of the panels (a)-(g), as evident by the lack of changes in conformation related bands in the 900-750 cm\({}^{-1}\) region. In other words, after the thin film is obtained and removed from the desiccator chamber, regardless of the desiccation time, conformational changes are locked and cannot be significantly changed by ambient conditions. The only differences observed between the spectra obtained by (a) and (b) in each panel are variations in absorption intensities of asymmetric and symmetric PO\({}_{2}\)\({}^{-}\) vibration and backbone vibration near 1030 cm\({}^{-1}\). These small variations in absorption intensities, along with changes in the spectral shape in the 1070-1050 cm\({}^{-1}\) region are more expressed in 3-min and 5-min films (e.g., see Figure 11b). By increasing the time spent in the desiccation chamber, observed differences in absorption intensities between the spectra obtained by (a) and (b) are reduced and differences in spectral shape vanish; the 40-min film shows only negligible differences in the entire spectral range studied, 1350-750 cm\({}^{-1}\). Spectral behavior of 3-min and 5-min films highlights the sensitivity of asymmetric and symmetric PO\({}_{2}\)\({}^{-}\) vibration and backbone vibrations at \(\approx\)1030 cm\({}^{-1}\) to ambient conditions; even though no frequency shifts and no conformational changes are observed, variations in intensity might occur.