Single Molecule Approaches to Understanding Biological Systems

Single molecule methods are powerful techniques for understanding how biological processes emerge from the reactions of individual molecules. They enable one to make mechanical measurements (such as force and extension), to look at changes in the conformations and interactions of macromolecules or to visualize complex reactions of single molecules and to determine their order and rates. Above all, they reveal the richness and diversity of molecular behaviour, demonstrating that populations of molecules, as of people, can be very diverse..

Most single molecule research has been based on the analysis of reactions between a limited number of pure components. At Leicester, we are among a small number groups worldwide who are pioneering the application of single molecule methods to complex systems. This began here with Clive Bagshaw, who built one of the first prism-based total internal reflection fluorescence (TIRF) microscopes in the UK for single molecule use.

The newest member of the sub-theme, Andrey Revyakin, developed methods while in the USA for using TIRF to study the reaction mechanisms of mammalian RNA polymerase II, a complex enzyme built from numerous sub-complexes that transcribes the protein-coding RNA in cells. This has led to new insights into its behaviour and the factors that determine its rate of movement along the DNA. This research nicely illustrates the ability of single molecule research to reveal the stochastic processes that underlie what appears in traditional ensemble experiments to be programmed behaviour.

We have pioneered the application of single molecule TIRF to look at mammalian pre-mRNA splicing, a critical process that is so complex that it cannot be reconstituted in vitro and can only be reproduced in crude nuclear extracts (Ian Eperon and Dmitry Cherny). In particular, Dmitry Cherny described the first experiments in which the number of a particular type of protein (In this case, a splicing repressor) in a complex was measured in crude extracts. This method is the core of a sustained programme to describe the RNA-protein complexes that dictate splice site selection in mammalian cells. We are using both the prism TIRF and an objective TIRF, built by Andrew Hudson, that is equipped with 5 lasers.

TIRF studies require the attachment of molecules to a surface. In some situations, this might interfere with the reactions being studied. As one approach to the problem of following freely moving single molecules over a period of time, Andrew Hudson has been developing new ways of producing small droplets that could contain freely moving fluorescent molecules. Robert Weinmeister, who works with Andrew Hudson and Ian Eperon, has built a convenient and cheap system for producing these, rapidly and on-demand. This method will also have wider uses for studies of directed evolution and drug screening.

We are also beginning to develop the use of magnetic and optical tweezers to make force measurements on single molecules. For example, the investigation of proteins involved in the maintenance of cell shape and adhesion to the extracellular matrix and the effects of force on their interactions.

The combination of technical expertise with the demands of complex systems make Leicester a good environment for innovation and originality. Our main aim in the near future is to expand the range of single molecule methods that we can apply to such systems.

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College of Life Sciences
University of Leicester
Maurice Shock Building
University Road