EPSRC Doctoral Training Grant - DTA, University of Strathclyde | Ramakers, Lennart

Project: Research Studentship - Internally AllocatedResearch Studentship (Internally Allocated)

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Description

The binding of proteins and small molecules to double-stranded (ds)-DNA is fundamental to biological function, but our comprehension of the underlying molecular physics of these interactions remains incomplete. Here, we focus on minor groove binding compounds (MGBs). These are small organic molecules that demonstrate sequence specificity favouring AT-rich tracts of dsDNA and which have potential applications as next-generation antibiotics. We utilize two dimensional infrared spectroscopy (2D-IR) to investigate MGB-DNA complexes that allow us to explore the interactions and dynamics of binding by exploiting azide-containing molecular probes based on the Hoechst DNA-binding dye family with the aim of providing bond-level spatial resolution within the minor groove on timescales approaching 100 fs. By characterizing the response of archetypical small organic molecules containing an azide moiety to their local environment and using this information with the insights gained by studying the vibrations of the DNA bases, these interactions can be explored.

Layman's description

This project has been focused on trying to understand the interactions between DNA and small molecules. The interaction of DNA with other molecules is fundamental to many biological processes involving DNA and therefore it is an important area of study. There are several different ways that small molecules can interact with a DNA molecule. In this project we are specifically interested in small molecules which interact with DNA through the so called minor groove of DNA, formed due to the fact that DNA is a double helix.

An archetypal small molecule, which is known to bind to DNA, is used in this project to explore these interactions. This molecule is a well-known fluorescent dye that has been used to stain DNA in biology. This molecule was chosen as it has been widely studied and the DNA binding sequence is also well-known. In order to gain a deeper understanding of the interaction between this molecule and DNA, a small DNA fragment with the molecule bound to it was studied using UV-visible, fluorescence, infrared and advanced infrared spectroscopies. Using both the original, commercially available Hoechst molecule and a custom version of this molecule, modified to contain a small infrared probe, the interactions underpinning the small molecule-DNA system can be studied on a sub 100 femtosecond timescale and at the spatial resolution of a chemical bond.

In order to achieve the aims of this project to gain a deeper understanding of the interactions, archetypical versions of the small infrared tag used to modify the Hoechst molecule are also investigated using both infrared and advanced infrared spectroscopic techniques. Using these techniques the response of the infrared probe group, in this case an azide group, to its local environment is explored. This can then be used to understand the response of the probe attached to a modified version of the binding molecule and so will allow the local environment in the DNA minor groove to be explored.

Key findings

The implementation of linear (FT-IR), ultrafast (IR pump-probe) and multidimensional (2D-IR) IR spectroscopies on both non-natural and natural IR probe modes has allowed both the calibration of aromatic and aliphatic azides to extract information about the probes local environment and DNA minor groove binding to be explored. The complex azide absorbance band of benzyl azide, an archetypal aromatic azide, was used to extract the electrostatic potential and hydrogen bonding strength of the surrounding environment. These parameters were successfully extracted from the changes to the line-shape observed in the FT-IR spectrum of the asymmetric azide absorbance band and from the changes in the vibrational lifetime observed as the dipole moments and hydrogen bonding strengths of the environment change. One drawback to these approaches was that either the changes in the line-shape correlated to the changes in the local environment were similar leading to difficulties with environments where the parameters changes simultaneously or that it is necessary to have prior knowledge of the overall nature of the environment. This was overcome by the correlation of changes in the 2D-IR response of the benzyl azide to the local environment which could be pin-pointed to a particular solvent, allowing the parameters of the local environment to be extracted without any prior knowledge of the environment or any issues related to simultaneous changes in multiple parameters.

The azide absorbance bands of two archetypal aliphatic azides, namely 3-aizdo-propylamine and 3-azido-propanoic acid, were studied as the parameters of the local environment changed. In the case of the aliphatic azides the spectral band has several distinct contributions arising from the presence of different isomers. The FT-IR response of these molecules were determined to be highly sensitive to the hydrogen bonding strength of the local environment. However there appears to be very little correlation between changes in the IR line-shape and the electrostatic potential of the environment, potentially due to the poor solubility of these molecules in such aprotic environments. Additionally it was found that beyond the FT-IR spectroscopy the ultrafast IR pump-probe and 2D-IR spectroscopic methods did not yield any further means to probe the azide moieties local environment.

The exploration of the interaction between DNA and minor groove binders was explored utilising the DNA base modes as natural IR probes. The binding-induced differences due to the binding of an archetypal minor groove binder provided base-specific information about the interaction of Hoechst33258 (1) and a novel azido-minor groove binding molecule with both an optimal and sub-optimal binding sequence. These studies utilising the DNA base vibrational modes as natural IR probes reveal significant differences between the mechanisms underlying the optimal and sub-optimal binding scenarios. The azide moiety on the novel binding molecule was found to be successfully incorporated into the minor groove upon binding, demonstrating the feasibility of the utilising such a non-natural IR probes to explore binding interactions. Finally it was determined to be possible to study the dissociation mechanism of short DNA oligonucleotides and how these mechanisms are altered by the presence of minor groove binders using the DNA base modes as natural IR probes.

(1) L. A. I. Ramakers, G. Hithell, J. J. May, G. M. Greetham, P. M. Donaldson, M. Towrie, A. W. Parker, G. A. Burley and N. T. Hunt, J. Phys. Chem. B, 2017, Article ASAP, DOI: 10.1021/acs.jpcb.7b00345.
StatusFinished
Effective start/end date1/10/131/04/17

Funding

  • EPSRC (Engineering and Physical Sciences Research Council): £7,896.00

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