Go with the laminar flow : developing the lamina system for molecular interaction analysis.

Within the complex milieu of a cell, which comprises a large number of different biomolecules, interactions are critical for function. In this post-reductionist era of biochemical research, the ‘holy grail’ for studying biomolecular interactions is to be able to characterise them in native environments. While there are a limited number of in situ experimental techniques currently available, there is a continuing need to develop new methods for the analysis of biomolecular complexes that can cope with the additional complexities introduced by native-like solutions. This thesis describes the development of the ‘LaMInA system’ (Laminar flow-based Molecular Interaction Analysis system), which employs fluorescence microscopy to track the diffusion of fluorescently-labelled biomolecules in microfluidic devices. By exploiting the inherent size-dependence of diffusion coefficients and controlling the retention time of biomolecules in a microfluidic channel, the distance that a molecule of interest diffuses across the channel into an adjacent fluid can be used as a direct measure of its size. Utilising this principle, the LaMInA system can be used to measure the sizes of biomolecules and detect size-change-inducing molecular interactions. Included is an in-depth discussion of the experimental setup and considerations for fluorescent labelling, optimising the detection system, and methods of data processing and analysis. Proof-of-concept measurements of green fluorescent protein (GFP) were made using different microfluidic device designs and diffusion coefficients obtained showed good agreement between different measurements and with the size of this protein. The LaMInA system was able to determine diffusion coefficients for a series of proteins ranging from 14 – 670 kDa in size and these were consistent with theoretical values. The potential utility of this system for studying ligand-induced oligomerisation was demonstrated with two protein model systems, and proof-of-concept measurements of a protein-protein and a protein-DNA interaction, were also carried out. The thesis concludes with preliminary steps taken towards analysis in a complex solution, with measurements of GFP overexpressed and measured in a bacterial cell lysate. Overall, the LaMInA system developed herein presents a simple, inexpensive method for determining the diffusion coefficients of biological molecules as a measure of their size and means of detecting a variety of biomolecular interactions, with the potential for analysis in complex solutions