Single molecule studies of ligand-DNA interactions using atomic force microscopy

Rackham, Benjamin (2014) Single molecule studies of ligand-DNA interactions using atomic force microscopy. Doctoral thesis, University of East Anglia.

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Abstract

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.

Item Type: Thesis (Doctoral)
Faculty \ School: Faculty of Science > School of Pharmacy
Depositing User: Mia Reeves
Date Deposited: 13 Jun 2014 08:24
Last Modified: 13 Jun 2014 08:24
URI: https://ueaeprints.uea.ac.uk/id/eprint/48783
DOI:

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