| Deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) play crucial roles in life. They carry and transmit genetic information, regulate and maintain gene expression, and in certain cases, catalyze biochemical reactions. In order to achieve these various functions, nucleic acids adopt complex and dynamic three-dimensional structures. The focus of this dissertation is to develop and apply the site-directed spin labeling (SDSL) method to study motions and conformations in nucleic acids. On the strategy of SDSL, the first step is to attach the spin label to the macromolecule. Research here provides a new approach of attaching nitroxides at the 5' terminus of nucleic acids using enzymatic reaction. A method was also developed to characterize and separate diasteromeric nitroxide-labeled nucleic acids formed from spin labeling via the modified backbone position. On the application of SDSL, research here successfully obtained two types of structural information in nucleic acids. First, specific duplex motions within a large RNA were investigated using CW-EPR experiments. These motions are critical for the catalytic activity of the group I ribozyme. Second, distances were measured at specific positions of adenosine-tract rich DNA using double double electron resonance spectroscopy (DEER). Distances measured are used to determine the sensitivity of DEER method in probing conformational variation across A-tract containing double-stranded DNAs. Studies here advanced the utility of site-directed spin labeling to obtain structure and motional information in nucleic acids where other technologies have difficulty. This is a step in unraveling the relationship of structure, dynamics, and function in DNA and RNA. |
