My research concerns the sequence specific recognition of DNA by small molecules, oligonucleotides and proteins, and the formation of unusual DNA structures (triplexes and quadruplexes). Compounds that bind to DNA in a sequence specific fashion have potential for artificially controlling gene expression and may be used as anticancer or antiviral agents. Several DNA binding antibiotics are currently used in cancer chemotherapy, and we are seeking to understand the molecular mechanisms by which they bind to DNA with a view to designing new agents with improved selectivity. In all our studies we make extensive use of the footprinting technique, using both natural and synthetic DNA fragments, and have developed this assay as a powerful tool for measuring the specificity, stability and kinetics of ligand-DNA interactions. This has been used to determine the sequence selectivity of several DNA-binding small molecules.
For the past 15 years my work has focussed on triple helix formation as a means for targeting specific DNA sequences. Together with Professor Tom Brown (Chemistry) I have developed several nucleotide analogues, which are designed to form stable triplexes under physiological conditions. Using a combination of these we demonstrated the first example of four base-pair recognition of a DNA sequence by a triplex-forming oligonucleotide at pH 7. In order to facilitate our studies on the stability of triplexes, quadruplexes and small-molecule-DNA complexes we developed a high throughput fluorescence assay for determining DNA melting profiles.
DNA quadruplexes can be formed by G-rich DNA sequences, and these may play a role in telomere structure or controlling gene expression. We have studied their biophysical properties studies, examining their stability, structure, and competition with DNA duplexes.
Together with Professor Tom Brown (now in Chemistry, Oxford) we have developed a novel fluorescence assay for measuring the stability of DNA duplexes, triplexes and quadruplexes [Darby et al. (2002) High throughput measurement of duplex, triplex and quadruplex melting curves using molecular beacons and the LightCycler. Nucleic Acids Res. 29, e39]. This uses synthetic oligonucleotides to which are attached a fluorophore (fluorescein) and a quencher (methyl red). These are positioned so that these groups are close together in a folded DNA structure, such as a quadruplex or duplex, thereby quenching the fluorescence. On increasing the temperature the DNA melts, separating these fluorescence groups and there is a large increase in fluorescence. These experiments are performed on the Roche LightCycler, allowing us to examine 32 samples in parallel, using only small volumes (20 µL) of dilute oligonucleotides (0.25 µM).
Current MPhil/PhD students:
Interaction of novel pyrrolobenzodiazepines (PBDs) with DNA
Commonwealth Academic Staff Scholarship
Structure and properties of “sticky DNA” formed by GAA repeats.
Saudi Arabia Scholarship
DNA recognition by Triple helix formation
Thai Government Scholarship
Mutants of Uracil DNA Glycosylase for Detecting Modified Cytosine
Mayflower Studentship from the University of Southampton
Molecular and Cellular Biosciences
Affiliate research group(s)
Institute for Life Sciences (IfLS)
One means of achieving precise DNA sequence recognition over several base pairs involves the formation of intermolecular DNA triple helices.
We are studying the structure of DNA quadruplexes in linear and supercoiled DNA and are examining their effects on gene expression when they are located in promoters.
We are using the footprinting technique with natural and synthetic DNA substrates to study the sequence selectivity of novel DNA binding small molecules.
We are exploiting the formation of DNA triplexes as a means for generating new DNA nanostructures.
We are generating mutants of this DNA repair enzyme, which have altered recognition properties, and are using these as tools in biotechnological applications.
We are part of this BBSRC-funded sLoLa project, led by Prof Tom Brown (Chemistry), and are using click-chemistry to generate unusual DNA structures and examine their biological properties.
Professor Keith R Fox
Faculty of Natural & Environmental Sciences
Life Sciences Building 85
University of Southampton
Telephone:(023) 8059 4374