# Conformational Transitions of Double-Stranded DNA in Thin Films
## 2 Materials and Methods  
Dry salmon testes lyophilized DNA threads obtained from Sigma-Aldrich Corp., St. Louis, MO, USA (DNA sodium salt) were dissolved for 48 h in 10 mM NaCl solution at 4 \({}^{\circ}\)C. Routinely performed sample characterization such as protein content, pH, and conductivity was performed and obtained as presented in our previous research [22]. The final DNA concentration of all solutions was 5 g/L (15 mM DNA phosphate and 7.5 mM DNA base pairs). In order to obtain thin films, 30 uL of the respective DNA stock solutions were deposited on optical grade silicon transmission windows and placed inside a desiccator chamber under an active vacuum (rotary vacuum pump) 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. FTIR spectra of thin films were recorded on a PerkinElmer Waltham, MA, USA Spectrum GX spectrometer equipped with a nitrogen-cooled Mercury-Cadmium-Telluride (MCT) detector and a KBr beam splitter. Spectra were measured at 25 \({}^{\circ}\)C and recorded in transmission mode with 4 cm\({}^{-1}\) resolution and 64 co-added scans and later processed in Kinetics add-on for Matlab 2010, MathWorks, Natick, MA, USA. Raw data were baseline-corrected and normalized using the band at 966 cm\({}^{-1}\) (deoxyribose C-C stretching mode) as an internal reference because it showed negligible spectral changes in position or intensity. All spectra obtained under the same conditions were averaged using an eFTIR software, EssentialFTIR, Monona, WI, USA and standard deviations were calculated using OriginPro 2019, OriginLab Corp., Northampton, MA, USA. For easier identification, average spectra obtained under different active vacuum times are abbreviated as 3-min film, 5-min film, etc. and, if not stated otherwise, are assumed to be obtained (b) \"after stabilization.\" Additional information on thin film preparation and statistical analysis (number of spectra etc.) can be found in Appendix A. Band shape analysis (peak fitting) of the phosphate (1150-1000 cm\({}^{-1}\)) and sugar-phosphate region (900-750 cm\({}^{-1}\)) was performed using OriginPro 2019 and eFTIR software (for details see Appendix B). The entire spectral range from 1350-750 cm\({}^{-1}\) can be split into three parts, i.e., 1350-1150 cm\({}^{-1}\) mostly dominated by asymmetric vibrations of PO\({}_{2}\)\({}^{-}\) groups, 1150-1000 cm\({}^{-1}\) dominated by symmetric PO\({}_{2}\)\({}^{-}\) vibrations and vibrations of sugar and diester linkage, and 900-750 cm\({}^{-1}\) region that contains important conformation-related markers whose vibrations can mainly be assigned to vibrations of deoxyribose. The average spectrum of 10-min films reflects several features that reveal the presence of both A- and B-form dsDNA structures presented in Figure 2. Main bands indicative of the presence of both forms are asymmetric PO\({}_{2}\)\({}^{-}\) vibration at 1232 cm\({}^{-1}\), a shoulder at 1055 cm\({}^{-1}\), and A- and B-form markers at 860 cm\({}^{-1}\) and 837 cm\({}^{-1}\), respectively. In a complete A-form, asymmetric PO\({}_{2}\)\({}^{-}\) vibration is found at \(\approx\)1240 cm\({}^{-1}\), a strong absorption band at 1062-1072 cm\({}^{-1}\) is present (instead of a shoulder at 1055 cm\({}^{-1}\)) and both A-form markers at 860 cm\({}^{-1}\) and 805-810 cm\({}^{-1}\) are present in the spectra [9; 15]. Note that all of these features are not observed in the 10-min film; however, some extent of A-form exists in the thin film. An estimate of %B and %A form for 10-min film, calculated according to %B = \((A_{837})/(A_{860}\)\(+\)\(A_{837})\) and %A = \((A_{860})/(A_{860}\)\(+\)\(A_{837})\), gives \(\approx\)75% B-form and \(\approx\)25% A-form; \(A_{860}\) and \(A_{837}\) denotes integrated intensities of the respective bands [5].