“We develop quantitative single-molecule approaches to investigate mechanisms behind complex biochemical systems”
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
Single Molecule Imaging

Selected Publications

Mundigala, H., Michaux, J. B., Feig, A. L., Ennifar, E., & Rueda, D. (2014). HIV-1 DIS stem loop forms an obligatory bent kissing intermediate in the dimerization pathway. Nucleic Acids Research, 42(11), 7281–7289.

Senavirathne, G., Jaszczur, M., Auerbach, P. A., Upton, T. G., Chelico, L., Goodman, M. F., & Rueda, D. (2012). Single-stranded DNA scanning and deamination by APOBEC3G cytidine deaminase at single molecule resolution. The Journal of Biological Chemistry, 287(19), 15826–15835.

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.

Lamichhane, R., Daubner, G. M., Thomas-Crusells, J., Auweter, S. D., Manatschal, C., Austin, K. S., Valniuk, O., Allain, F. H.-T. H., & Rueda, D. (2010). RNA looping by PTB: Evidence using FRET and NMR spectroscopy for a role in splicing repression. Proceedings of the National Academy of Sciences, 107(9), 4105–4110.

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.

Guo, Z., Karunatilaka, K. S., & Rueda, D. (2009). Single-molecule analysis of protein-free U2-U6 snRNAs. Nature Structural & Molecular Biology, 16(11), 1154–1159.

Steiner, M., Karunatilaka, K. S., Sigel, R. K. O., & Rueda, D. (2008). Single-molecule studies of group II intron ribozymes. Proceedings of the National Academy of Sciences, 105(37), 13853–13858.

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