Mass spectrometric investigations of quadruplex DNA

DNA exists in a wide variety of structural forms besides the canonical, Watson-Crick duplex. One example is the quadruplex. Quadruplex DNA consists of multiple sets of guanine tetrads stacked together and are stabilized only by certain metal cations. These structures exhibit a variety of structures based upon loop orientation, strand polarity, and strand stoichiometry. While this form of DNA was discovered in the 1970s, the first strong evidence that this form of DNA exists in living cells was obtained in 2001. The purpose of this dissertation is to use cutting-edge mass spectrometry based techniques to study quadruplex DNA.;We developed a new method for using mass spectrometry to measure quadruplex-metal cation affinities and report this work. The method correlates the gas-phase mass spectrometry signals of bound and unbound quadruplex structures to the equilibrium binding constant. We also show an application of the method by measuring different quadruplex-metal cation binding affinity constants as a function of temperature. Using a classic van't Hoff-type analysis we determined the reaction enthalpy from this data.;Based upon recent work by the laboratory of Professor Woody Wright, we hypothesize that human telomeric DNA is modified in vivo to stabilize quadruplex DNA structure formation. To test this hypothesis, we designed and implemented a liquid-chromatography/mass spectrometry method to search for modified nucleosides. Unfortunately, analysis of isolates of human telomere DNA provided by our collaborators revealed a problem in their telomere isolation and purification protocol. Presented in this dissertation is the current status of this project.;To study the interaction of a protein and a quadruplex we extended our recently developed method for measuring binding affinity constants (PLIMSTEX) to the interaction of alpha-thrombin and its quadruplex DNA ligand. This protein is important in the mammalian blood-clotting cascade and selectively binds to a 15-mer DNA sequence that adopts a quadruplex structure in solution. In this dissertation, we determined the global hydrogen-deuterium exchange kinetic data for this interaction and then used the PLIMSTEX method to measure the binding affinity constant of this interaction. Our measured affinity constant matches the previously determined value within a small factor.