Mass spectrometric studies of single- and double-stranded oligodeoxynucleotides.

摘要

Investigation of DNA behavior in nonpolar media such as the gas phase allows for the study of the inherent reactivity of DNA molecules without the complicating effects of solvent. This thesis details efforts to examine the inherent reactivity of single- and double-stranded DNA, using mass spectrometric and computational methods, which represents the start of a comprehensive understanding of DNA behavior in nonpolar media.;We first conducted the gas-phase study of the charge distribution and fragmentation patterns of single-stranded oligodeoxynucleotides (ODN) using electrospray-mass spectrometry (ESI-MS). The reactivity of the ODN ions is closely related with the intrinsic properties (such as proton affinity and acidity) of the nucleobases, and such a correlation is supported by electrostatic potential and proton affinity calculations. Our results highlight the complexity of how the negative charges and their distribution along the phosphate backbone affect the initial fragmentation of ODN.;In an effort to understand the intrinsic interactions stabilizing the DNA duplex, we studied the gas phase behavior of a complete set of 9-mer DNA duplexes (5'-GGTTXTTGG-3'/3'-CCAAYAACC-5', X/Y = G, C, A or T) with and without single internal mismatches. The effects of base stacking and hydrogen bonding in the gas phase versus in solution are discussed. Our data indicate that in the gas phase, as in solution, duplex stability reflects both hydrogen bonding and base stacking interactions. However, unlike in solution, hydrogen bonding forces dominate in the gas phase.;Finally, we report a systematic mass spectrometric study of a series of DNA duplexes ranging in size from 5- to 12-mers. We have established that relative ion abundances of the electrosprayed duplexes correlate to solution phase stabilities for duplexes of similar size. We also characterized the behavior of the duplexes when subjected to CID. This direct comparison between the gas phase and solution phase stabilities allows us insight into how solvation and the ESI process may affect DNA stability. Last, we apply what we learn to the biological question of why the mutated nucleobase O6-methyl guanine is so carcinogenic by examining its intrinsic behavior in DNA duplexes.