Single molecule imaging
Single molecule microscopy approaches established by Rueda (Department of Medicine, Medicine) reveal the structural dynamics of individual molecules, otherwise hidden in ensemble-averaged experiments. This provides us with direct observations of key reaction intermediates, even when present at low levels or for short periods of time, allowing us to characterise reaction mechanisms. These approaches have allowed dynamic RNA splicing to be visualised and Activation-induced deaminase (AID) occupancy on a DNA template to be linked to mutation.
We use single-molecule microscopy to study four main areas:
- RNA folding: the fundamental principles that govern RNA folding from individual folding motifs to large, multidomain, catalytic RNAs. We also study how RNA helicases aid this process under physiological conditions.
- RNA splicing: the structure and dynamics of two small nuclear RNAs, U2 and U6, that form the active site of the spliceosome – a complex responsible for catalysing RNA splicing.
- DNA replication: the kinetic intermediates involved in proofreading DNA during replication have not been characterised. We investigate this process by monitoring the movement of E. coli DNA polymerase I on a DNA template during DNA synthesis with single base-pair resolution.
- ssDNA scanning and deamination: the APOBEC family of enzymes comprise single-stranded DNA cytosine deaminases that are important in eliminating retroviral infectivity and initiating somatic hypermutation. We investigate ssDNA scanning and motif-targeting mechanisms for the APOBEC enzymes, Apo3G and AID