We are interested in the use and development of novel biophysical techniques to study biomolecular interactions including proteins, DNA and RNA at the level of individual molecules. The advantages of single-molecule detection are many, apart from the fascination of looking at individual biomolecules at work, single-molecule techniques can measure intermediates and follow time-dependent pathways of chemical reactions and folding mechanisms that are difficult or impossible to synchronize at the ensemble level. Thus, single-molecule techniques provide novel insights into how molecules and systems behave, having the advantage that spatial and temporal averaging is avoided, temporal synchronisation is not necessary and novel phenomena, which otherwise are averaged and remain hidden in ensembles, may be discovered. Our aim is to monitor, in real-time, the behaviour of individual biological molecules and complexes, in vitro and in live cells. By combining the dynamic information obtained at the single-molecule or single-complex level with structural and biochemical analyses, we hope to create "molecular movies" of biological processes and from them obtain a deeper understanding of these processes.

http://www.st-andrews.ac.uk/~singlemol/singlemol.html

Molecular basis of RNA-mediated gene regulation processes

Since the discovery that RNA can catalyze biochemical reactions and more recently that RNA elements, so-called riboswitches, built into mRNA can sense the concentration of a particular metabolite and turn gene expression on or off in response, RNA does not any longer play a passive role in cellular processes and RNA research has entered a phase in which its central importance as a bridge between genomics and proteomics has been emphasised. The main goal of this project is to dissect the molecular mechanisms involved in ligand- and ion-assisted folding of these RNA switches and understand how their highly dynamic nature is used to trigger gene expression modulation. Nowadays, that it is clear that bacteria and fungi are becoming more resistant to existing antibiotics, the emerging field of riboswitches is becoming a very promising ground to develop anti-bacterial drugs that target essential metabolic pathways in these organisms. Therefore, the results obtained from these studies not only will greatly expand the existing knowledge on RNA folding and riboswitch gene regulation mechanisms but also could be very valuable in the development of a new generation of novel antimicrobial drugs targeting riboswitches for use as antibiotics.

Single-molecule studies on protein-nucleic interactions

From the very beginning of life, there has been a challenge to maintain and replicate the genetic material DNA. The integrity of DNA is constantly challenged by the damaging effects of numerous chemical and physical agents, compromising the informational content. Every living organism devotes considerable resources to these vital tasks. Often this involves enzymes that detect aberrant or intermediate DNA structures, manipulate them, and process them, before passing the products on to other enzymes or proteins in a pathway. We are interested in the study of DNA processing pathways at single-molecule level. In particular, biochemical analysis has produced a common general outline of the nucleotide repair mechanism, which includes recognition of the DNA lesion, unwinding of the DNA double helix around the damage and, finally, excision of an oligonucleotide containing the damage bases. This process involves the ordered assembly of several proteins (endonucleases, helicases, single-strand binding proteins, PCNA) and undoubtedly requires complex intermediate structures. The exact composition and lifetime of the different intermediates, and thus, the precise reaction mechanism, is not known. Single-molecule fluorescence methods provide a way to target and identify distinct intermediate species dynamically exchanging (protein-protein and protein-DNA complexes), quantify their prevalence and determine their lifetime. This project is in collaboration with Prof. M. F. White.