Hydrated films of the self-complementary B-type d(CGCGAATTCGCG)<sub>2</sub> dodecamer, with 13 or 14 water molecules per nucleotide, were studied by Fourier transform infrared (FT-IR) spectroscopy either as equilibrated samples between 290 and 260 K or, after quenching into the glassy state, as nonequilibrated films isothermally at 200 K. IR spectral changes on isothermal relaxation at 200 K revealed that difference spectra are caused by interconversion of B-DNA's conformer substates, B<sub>I</sub> converting into B<sub>II</sub>. Curve resolution of IR spectra in the spectral region containing the symmetric stretching vibration of the ionic phosphate group, with a composite band centered at 1087 cm<sup>-1</sup> for B<sub>I</sub> and at 1109 cm<sup>-1</sup> for B<sub>II</sub>, gives the B<sub>I</sub>/B<sub>II</sub> conformer substate population ratio and its dependence on temperature between 290 and 260 K, and on time for isothermal relaxation at 200 K. Curve resolution of the overlapping composite band profile was optimized by comparison of second-derivative and of difference curves, aiming thereby for optimal correspondence between experimental and curve-fitted bands as a criterion for goodness-of-fit. The spectroscopic aspects relevant for reliable curve resolution of the overlapping composite band profile are reported and discussed in detail.

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