| Description |
The intramolecular DNA i-motif is a non-canonical, tetraplex structure that is formed from 2′-deoxycytidine-rich short sequences of DNA. These sequences are thought to be potential gene expression regulatory elements intrinsic to DNA itself and respond to pH changes within the nucleus of human cells. The isothermal pH-response characteristics, structure, and folding profiles are all important to understanding the potential role i-motif-forming regions of DNA may play in gene expression. Here, we have identified and studied various i-motif-forming oligodeoxynucleotide sequences, some found in human DNA repair gene promoters, in an effort to understand their pH-dependent response characteristics. In the first part of this dissertation, we characterized and screened the pH-dependent and thermal stabilities of 25 unique i-motif-forming DNA sequences found in human DNA repair gene promoter regions. From those, four were identified as high-potential sequences that could fold under physiological, intracellular conditions to regulate gene expression. Our work includes characterization of their pH-dependent thermal melting profiles, isothermal pH-dependent folding profiles, and UV difference spectra. We determined the likelihood of folding for each sequence and the likelihood of in vivo activity. In the second part of this dissertation, we studied in detail the isothermal folding and unfolding characteristics of one candidate i-motif-forming sequence from the iv screen found within the promoter of the human DNA repair gene RAD17. An unusual isothermal hysteresis that was dependent on the rate of solution pH change was observed and compared to two previously studied i-motif-forming sequences: dC19 and the human telomere sequence. Folding transitions in some cases were found to be multiphasic but occurring only when samples were prepared by injection into a low pH-buffered solution, simulating a rapid drop in pH. For the final portion of this dissertation, we probed model i-motif-forming oligodeoxynucleotides using a chemical bromination and synthetically incorporated substituent modifications in an effort to identify key nucleotides underlying the observed isothermal hysteresis phenomenon. |
