Monday, 23 May 2005
327

This presentation is part of: Biological Chemistry Posters

Spin-Labeling and Characterization of DNA Oligonucleotides

Joseph J. Schramm III, Christopher Tuohy, Heather Skiff, and Dr. Donald J. Hirsh. The College of New Jersey, Ewing, NJ

Our goal is to examine spin-spin interactions between two sites with unpaired electrons using Electron Paramagnetic Resonance (EPR) spectroscopy at low temperatures. We propose the use of the DNA double helix as a “scaffold” to which paramagnetic groups are attached at varying bases and therefore varying distances. To determine the suitability of this system, a number of questions were addressed. 1) Could we attach a paramagnetic group to the DNA? 2) Could we accurately measure concentrations of the two single strands to ensure the formation of a 1:1 DNA duplex without excess of either strand? 3) Could we develop a buffered cryoprotectant solution that allowed DNA duplex formation and formed a good glass at low temperatures? 4) Finally, could we determine the conformation of the DNA duplex in the cryoprotectant solution? Knowledge of the conformation allows us to model the structure and estimate the distance between the sites. One of the DNA strands has been labeled at the 5'-end with a nitroxide group at over 90% yield, as measured by HPLC. Extinction coefficients of the single strands were determined analytically. A solution containing 55 % phosphate buffer, 30 % polyethylene glycol (6000), and 15 % ethylene glycol formed a good glass and depressed the melting temperature (Tm) for the DNA duplex only slightly, from ~60 °C in buffer to ~52 °C in cryoprotectant solution. A UV-melting curve demonstrated 30% hyperchromicity in phosphate buffer and 29% hyperchromicity in cryoprotectant/buffer solution. Circular Dichroism spectra of this duplex are consistent with B-form DNA.

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