Doctor of Philosophy
Department of Chemistry
Kapur, Amit, Studies of non-covalent small molecule DNA interactions and protein-DNA interactions, Doctor of Philosophy thesis, Department of Chemistry, University of Wollongong, 2002. http://ro.uow.edu.au/theses/1178
Electrospray ionization mass spectrometry has been used to study the non-covalent interactions between Tus and TerB. The Tus-Ter complex was very stable using a spray solvent of 10 m M ammonium acetate at pH 8. Initial attempts to distinguish binding affinities of Tus and mutant Tus proteins for Ter DNA were unsuccessful. Increasing the ammonium acetate concentration in the elctrospray solvent (800 mM) increased the dissociation constants sufficiently such that relative orders of binding affinities for Tus and various mutant Tus proteins for various DNA sequences could be determined. A dissociation constant of 700 x 10-9 M for the binding of the mutant Tus protein A173T (where residue 173 is changed from alanine to threonine) or TerDNA was estimated, compared with a value of ≤2 x 10- 9 M for Tus where A173T was unchanged. These results were in agreement with solution studies and are the first example in which ESI-MS has been used to compare binding affinities for a DNA-binding protein with mutant proteins for specific D N A recognition sequences. Overall, this work demonstrates that ESI is a powerful technique for the observation of non-covalent DNA-drug and DNA-protein interactions.
In the second part of this work, the use of ESI-MS for studying the non-covalent interactions between the anti-tumor drugs daunomycin and nogalamycin to duplex DNA. Ions corresponding to the complex were most abundant relative to free DNA when prepared in the pH range 8-9 and acquired using gentle ESI-MS conditions on a triple quadrupole mass spectrometer. Titration experiments gave ESI-mass spectra in which the most intense ions correspond to three molecules of nogalamycin or 4 molecules of daunomycin dound to the duplex 8 mers (d(CGGCGCCG)2 and d(GGCTAGCC)2) and 4 molecules of nogalamycin or 6 molecules of daunomycin bound to the duplex 12 mer (d(TGAGCTAGCTCA)2). These data are consistent with the neighbour exclusion principle. Competition experiments involving a single drug in an equimolar mixture of two oligonucleotides (d(TGAGCTAGCTCA)2 with either d(CGGCGCCG)2 or d(GGCTAGCC)2) showed that the intercalators bound preferentially to d(CGGCGCCG)2 compared to d(GGCTAGCC)2 relative to the 12 mer. This shows that ESI-MS has the potential to detect differences in sequence selectivity. ESI-mass spectra from experiments in which both drugs were reacted with the same oligonucleotide were more complicated and as such, a clear preference for one drug could not be established. Similar studies were done using longer 16 mer self-complementary oligonucleotides (d(ATATATATATATATAT)2, d(ATATATACGTATATAT)2 and d(CCATATACGTATATGG)2). These data showed only small amounts of dsDNA-drug complex. Most ions observed were from single stranded oligonucleotides complexed to drug. Non-self-complementary oligonucleotides were subsequently used to avoid ambiguities in assigning dsDNA-drug peaks. Extensions of these studies using a quadrupole-time-of-flight instrument, showed no evidence of these non-specific single stranded DNA-drug complexes. Different CID experiments (cone voltage, capillary voltage, desolvation temperature and collision energy) all showed that the order of binding of the intercalators was ethidium>nogalamycin>daunomycin. Preliminary results on the observation of the retamycin-dsDNA complex were also presented.