# Conformational Transitions of Double-Stranded DNA in Thin Films
## 2 Materials and Methods
### DNA Thin Films Obtained after 3 min and 5 min  
Average FTIR spectra and standard deviations of the 3-min and 5-min films are presented in Figure 4. For comparison, standard deviations of 10-min and 40-min films are also presented. Two important observations can be made: the absorption spectrum of the 3-min film indicates changes in the conformation compared to 10-min and 40-min films, while a comparison of their respective standard deviations indicates that observed changes are statistically relevant. Important conformational markers that appear in the 3-min spectra are the appearance of a band at 1188 cm\({}^{-1}\), shift and intensity increase in 893 cm\({}^{-1}\) band to 897 cm\({}^{-1}\) (inset of Figure 4), intensity increase in 860 cm\({}^{-1}\) band, and the appearance of a band at 805 cm\({}^{-1}\). All of the above are indicators of an increase in %A-form; indeed, calculation of the %A using extended equation %A = (A\({}_{860}+A_{805}\))/(A\({}_{860}+A_{805}+A_{837}\)) estimates about 60% of A-form. In addition to conformation-related markers, intensity changes suggesting increased hydration include decreased intensity of the asymmetric \(\text{PO}_{2}{}^{-}\) vibration, increased intensity of the symmetric \(\text{PO}_{2}{}^{-}\) vibration, and reshaping of a 1055 cm\({}^{-1}\) band [9]. Figure 3: Average FTIR spectra of different DNA thin films in the 1350–750 cm\({}^{-1}\) range: 10-min film (light green), 20-min film (dark cyan), 40-min film (red). Inset: enlarged view of the respective DNA thin films in the sugar–phosphate region (950–750 cm\({}^{-1}\)). Numbers and arrows denote identified bands for the 40-min film. Two more important changes that remained unaddressed so far can be identified in the 3-min and 5-min films; a strong decrease in intensity in the 1030 cm\({}^{-1}\) region (Figure 4, ***) and a strong increase in 765 cm\({}^{-1}\) band (inset of Figure 4). In order to better the interpretation of the spectra and this particular issue, we performed peak fitting of the absorption bands in the backbone region, i.e., the conformation-related region from 900-750 cm\({}^{-1}\) (Figure 5) and region of symmetric PO\({}_{2}\)\({}^{-}\) vibration from 1150-1000 cm\({}^{-1}\) (Figure 6). Important information obtained by peak fitting is summarized in Figure 7, in which relative integrated intensities of respective constituent Gaussians for 3-min, 5-min, 10-min, and 40-min films are presented. The absorption spectrum in the backbone region from 900-750 cm\({}^{-1}\) that contains important conformation-related bands can be successfully fitted by ten constituent Gaussians (modes); no frequency shift of the constituent Gaussians is observed, and only changes of the integrated intensities and bandwidths are observed (Figure 5). In Figure 5, absorbance maximum shows a red-shift from 897 to 891 in 3-min and 40-min film, respectively. However, this shift is a result of dissimilar intensity changes of modes \(1\) and \(2\) rather than frequency shift of the respective modes; the integrated intensity of mode 1 decreases by \(\approx\)60 % when 3-min and 40-min films are compared (from 4.3 to 1.75), while the integrated intensity of mode 2 shows a slight increase (from 2.3 to 2.65). Similar reshaping of the absorption spectra at about 890 cm\({}^{-1}\) was found in [23]; intensity increase in higher frequency mode (in our spectra mode _1_) denotes thin film with a higher fraction of A-form. This is confirmed further by the decrease in integrated intensities of modes \(3\) and \(5\) that correspond to A-form markers situated at 860 cm\({}^{-1}\) and 805 cm\({}^{-1}\), respectively. Note that the 805 cm\({}^{-1}\) band completely vanishes from the absorption spectra of 10-min and 40-min film; consequently, so does mode \(5\) (Figure 7a). On the other hand, the signature of a B-form, i.e., 836 cm\({}^{-1}\) band that corresponds to mode 4, experiences intensity increase.