Munnur, Deeksha Ganesh; (2012) Structural studies of DNA complexes with minor groove-binding drugs. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Targeting the minor groove of DNA with small molecules is an important recognition strategy in biology. A wide range of minor groove binding ligands (MGBLs) with good sequence discrimination ability are of interest as potential therapeutic agents in a variety of human diseases such as cancer, along with anti-bacterial and/or anti-parasitic activities. Whilst the detailed mechanism of action of some of these MGBLs is still unproven, they are known to be effective inhibitors of a number of minor and major groove binding protein-DNA interactions. This thesis reports on crystallographic studies to determine the molecular structure of MGBLs bound to DNA sequences, in order to better understand the details of their molecular recognition by DNA. Several interesting MGBLs differing in their structural features were crystallised with A-T rich oligonucleotides for neutron and high-resolution X-ray data collection. Phases for the X-ray crystal structures were determined using molecular replacement, with diffraction data up to 1.2 A resolution. The crystal structure revealed the MGBLs bound in the central AATT or AAATTT rich region of the minor groove of the DNA. The ligands form hydrogen bonds with the bases of the DNA at the floor of the minor groove directly or mediated via water molecules depending on the shape of the ligand. Several oligonucleotide-MGBL complexes were crystallised in the presence of deuterium oxide (heavy water) with the aim of studying the water network around the minor groove in the presence of ligand using neutron crystallography. In order to further our understanding of the biological mechanism of action of MGBLs, biophysical studies were undertaken with the DNA major groove binding transcription factor, NF-κB. This transcription factor binds to the continuous guanine and cytosine bases of the major groove leaving the minor groove exposed to other molecules. Surface plasmon resonance (SPR) and small angle X-ray scattering (SAXS) studies were undertaken to study the effects on MGBLs on NF-κB-DNA binding. It was revealed that MGBLs had significant effect on the protein-DNA interactions which was further dependent on the shape of the MGBLs.

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