Re-evaluation of analytical chemistry techniques in studying DNA structures

Abstract

This work describes the use of analytical chemistry techniques to examine the structural changes that DNA adopts when subjected to a number of external/internal factors. A self-complementary sequence, d(CG)9, and a non-self-complementary sequence (mixed sequence) were used to study the conformational effects displayed by each type of oligonucleotide sequence. The structural changes adopted by DNA was examined using a variety of analytical techniques, such as: nuclear magnetic resonance imaging (NMR), differential scanning calorimetry (DSC), ultra violet visible (UV-Vis) spectroscopy, circular dichroism (CD) spectroscopy, and high-performance liquid chromatography (HPLC). 1) d(CG)9 and a mixed sequence in the B- and Z-DNA conformation was examined by CD and UV-Vis at a concentration of 1mM using a home-made cuvette called a Flexicell with a minimum pathlength of 0.129± 0.015 mm. The CD and UV-Vis spectra’s produced were found to be reliable when compared to commercial cuvettes with a pathlength of 1 cm and sample concentration of 10 µM. 2) d(CG)9 was lyophilized and reconstituted using either water or buffer to determine if d(CG)9 adopts a different structure when reconstituted using different conditions. It was determined that lyophilized d(CG)9 adopts a hairpin conformation when reconstituted with water, and a B-DNA duplex when reconstituted with a buffer containing NaCl. 3) d(CG)9 was thermally denatured using DSC to determine if DSC can be a viable method to study oligonucleotides. It was determined that d(CG)9 undergoes a two-state unfolding pathway. 4) Nuclear Overhauser Effect spectroscopy (NOESY) and correlation spectroscopy (COSY) were used to examine the conformational differences of 2’-deoxyadenosine when incubated in water. From the distance and torsion angle constraints obtained from NOESY and COSY respectively, and from existing crystal structures, it was found the structures that were determined by NMR spectroscopy were misleading because of spectral artifacts. 5) A mixed sequence was treated with organic modifying agents to determine the minimal condition required for DNA denaturation when different modifiers were used. It was determined that urea at a concentration of 8 M and at a pH of 12.5 is sufficient to denature the mixed sequence duplex.