Single molecule microscopy reveals 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. 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
G Senavirathne, JG Bertram, M Jaszczur, KR Chaurasiya, P Pham, CH Mak, MF Goodman and D Rueda (2015) Activation-induced deoxycytidine deaminase (AID) co-transcriptional scanning at single-molecule resolution. Nature Communications 6:10209
MRG Taylor, M Špírek, KR Chaurasiya, JD Ward, R Carzaniga, X Yu, EH Egelman, LM Collinson, D Rueda, L Krejci and SJ Boulton (2015) Rad51 paralogs remodel pre-synaptic Rad51 filaments to stimulate homologous recombination. Cell 162 (2), 271-286
BP Paudel, D Rueda (2014) Molecular crowding accelerates ribozyme docking and catalysis. Journal of the American Chemical Society 136 (48), 16700-16703
Karunatilaka, K. S., Solem, A., Pyle, A. M. M., & Rueda, D. (2010). Single-molecule analysis of mss116-mediated group II intron folding. Nature, 467(7318), 935–939.
Christian, T. D., Romano, L. J., & Rueda, D. (2009). Single-molecule measurements of synthesis by DNA polymerase with base-pair resolution. Proceedings of the National Academy of Sciences, 106(50), 21109–21114.