Single molecule studies of ligand-DNA interactions using atomic force microscopy
This thesis describes the results of experiments into the intra and inter-molecular binding of various ligands with dsDNA via the mechanism of intercalation, principally using the technique of atomic force microscopy (AFM). Since the description of the first AFM in the mid 1980’s, AFM has emerged as a sensitive and versatile analytical tool, capable both of detecting and manipulating artefacts at picometer resolutions. In these studies, AFM imaging, supported by circular dichroism, reveals unusual conformational changes in DNA that occur as a result of the binding of ligands that incorporate the acridine chromophore. These changes are distinct from those observed following the binding of other intercalators such as doxorubicin and echinomycin. Direct measurement of the length of linear DNA strands bound to acridine based ligands reveals a shortening of the DNA at very low ligand concentrations. This observation suggests that the structural changes that occur in DNA following the intercalation of the acridine chromophore are more wide ranging than previously thought and support molecular modeling studies that have proposed that the intercalated DNA duplex exhibits characteristics of both B and A form DNA. Variations in the conformational changes that occur in DNA as a result of intercalation may have implications for the application of new intercalating ligands as chemotherapeutic agents. In addition, single molecule force spectroscopy has been used to examine the capacity of bisintercalators to bind to DNA in an inter-molecular fashion. By stretching individual strands of dsDNA, acridine dimers are shown to bind to separate strands of DNA. Intermolecular binding of this kind remains an unexplored cytotoxic mechanism that may yet find an application in vivo. This observation is supported by a novel assay that utilises the controlled aggregation of gold nanoparticles. These nanoparticles, functionalised with DNA, are shown to aggregate on addition of a bisintercalator. The aggregation is fully reversible with the addition of sodium dodecylsulphate. These force spectroscopy experiments have also uncovered a previously unobserved, intermolecular binding mode of the peptide antibiotics echinomycin and TANDEM. In certain circumstances, these ligands are revealed to bind exclusively to the termini of separate DNA strands in a sequence dependent fashion. This finding may have implications for the employment of these ligands in the nanosciences, as a tool for joining short pieces of DNA or improving the efficiency of the enzymatic, blunt-end ligation of DNA.
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