UBC Theses and Dissertations
Ultraviolet absorbance and circular dichroism analysis of DNA oligomers containing adenine tracts Lim, Yee Chee
"A-tract" is defined as phased runs of at least four consecutive adenines, i.e. (dA)n-(dT)n , where n ≥ 4. The B*-form of DNA characteristic of A-tracts is distinct from the canonical B-DNA, with high base propeller twist and a narrower minor groove. The B*-form of DNA was examined using UV absorption and circular dichroism (CD) spectroscopy in order to estimate the extent of B*-type conformation adopted by 12-mer DNA oligomers containing different A-tract lengths. The systematic variation aims to study how the propensity towards B*-DNA formation depends on different A-tract lengths and different base compositions flanking the A-tract. CD and UV melting experiments indicate that B*-form has distinctive spectral signatures. The structural formation o f B*-DNA increases with A-tract length, but can be affected by the location of the A-tract within the sequence as well as neighboring AA/TT, AT, and TA base pairs. The spectroscopic results generally correlate well with differential scanning calorimetry (DSC) data. The calorimetrically obtained results were compared with thermodynamic parameters predicted by the Santa Lucia nearest-neighbor (NN) model. Disagreements between experimental and predicted thermodynamic values exist particularly for mixed AT sequences and those with the same number of NN parameters. Such discrepancies may be caused by different stabilities resulting from various extent of B*-type formation within a given DNA sequence. Since NN estimates of the melting temperature do not adequately account for structural differences, the incorporation of additional structural information may have a pronounced impact on thermodynamic variables and will help to improve the NN model considerably. Consequently, this allows for a more accurate prediction of the stability of short DNA sequences (< 25 base pairs), often used in molecular biology applications involving sequence dependent hybridization reactions. In light of the increasing interest in the development of locked nucleic acids (LNA) for probe and primer design and theurapeutic applications, the thermodynamics and spectroscopic studies on the structural effects of the incorporation of LNA nucleotides on the A-tract structure will also be presented.
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