Richard Owczarzy

Biopolymers, 2006, Vol. 82, No. 3, pp 199-221.

Studies of DNA dumbbells VIII. Melting analysis of DNA dumbbells with dinucleotide repeat stem sequences

Kathleen E. Mandell, Peter M. Vallone, Richard Owczarzy, Peter V. Riccelli, and Albert S. Benight
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Melting curves and circular dichroism spectra were measured for a number of DNA dumbbell and linear molecules containing dinucleotide repeat sequences of different lengths. To study effects of different sequences on the melting and spectroscopic properties, six DNA dumbbells whose stems contain the central sequences (AA)10, (AC)10, (AG)10, (AT)10, (GC)10, and (GG)10 were prepared. These represent the minimal set of 10 possible dinucleotide repeats. To study effects of dinucleotide repeat length, dumbbells with the central sequences (AG)n, n = 5 and 20, were prepared. Control molecules, dumbbells with a random central sequence, (RN)n, n = 5, 10, and 20, were also prepared. The central sequence of each dumbbell was flanked on both sides by the same 12 base pairs and T4 end-loops. Melting curves were measured by optical absorbance and differential scanning calorimetry in solvents containing 25, 55, 85, and 115 mM Na+. CD spectra were collected from 20 to 45oC and [Na+] from 25 to 115 mM. The spectral database did not reveal any apparent temperature dependence in the pretransition region. Analysis of the melting thermodynamics evaluated as a function of Na+ provided a means for quantitatively estimating the counterion release with melting for the different sequences. Results show a very definite sequence dependence, indicating the salt-dependent properties of duplex DNA are also sequence dependent. Linear DNA molecules containing the (AG)n and (RN)n, sequences, n = 5, 10, 20, and 30, were also prepared and studied. The linear DNA molecules had the exact sequences of the dumbbell stems. That is, the central repeat sequence in each linear duplex was flanked on both sides by the same 12-bp sequence. Melting and CD studies were also performed on the linear DNA molecules. Comparison of results obtained for the same sequences in dumbbell and linear molecular environments reveals several interesting features of the interplay between sequence-dependent structural variability, sequence length, and the unconstrained (linear) or constrained (dumbbell) molecular environments

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