| Summary: | Conformational transitions of double-stranded DNA in different environments have long been studied as vital parts of both in vitro and in vivo processes. In this study, utilizing Fourier transform infrared spectroscopy (FTIR), we provide detailed analysis of dynamics of A- to B-form transitions in DNA thin films of different hydrated states based on a statistical analysis of a substantial number of spectra and band shape analysis (peak fitting) in both the phosphate (1150–1000 cm<sup>−1</sup>) and sugar–phosphate (900–750 cm<sup>−1</sup>) region. Hydration of DNA thin films is systematically controlled by the time spent in the desiccator chamber (from 3 min to 40 min) allowing conformation and hydration signatures, in addition to variations due to ambient conditions, to be resolved in the spectra. Conformation transition from A-form to more ordered B-form is observed if sufficient time in the desiccator chamber is allowed and is confirmed by changes on the bands at ≈890, 860, 837, and 805 cm<sup>−1</sup>. Phosphate vibrations at ≈1230 cm<sup>−1</sup> and 1089 cm<sup>−1</sup>, and backbone vibrations at ≈1030 cm<sup>−1</sup> and 765 cm<sup>−1</sup> were found to be sensitive to changes in hydration rather than conformation. Additionally, we found that spectral variations caused by ambient conditions can be significantly reduced without inducing conformational changes, which serves as a good basis for quality assurance.
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