Single Molecule Imaging

“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

Newton MD, Taylor BJ, Driessen RP, Roos L, Cvetesic N, Allyjaun S, Lenhard B, Cuomo ME & Rueda DS. (2019). DNA stretching induces Cas9 off-target activity. Nature Structural & Molecular Biology, 26, 185-192.

Paudel BP, Fiorini E, Borner R, Sigel RKO, Rueda DS. (2018). Optimal molecular crowding accelerates group II intron folding and maximizes catalysis. Proceedings of the National Academy of Sciences of the United States of America, 115, 11917-11922.

Willhoft O, Ghoneim M, Lin C-L, Chua EYD, Wilkinson M, Chaban Y, Ayala R, McCormack EA, Ocloo L, Rueda DS, Wigley DB. (2018). Structure and dynamics of the yeast SWR1-nucleosome complex Science 362 (6411), eaat7716. DOI: 10.1126/science.aat7716.

Autour A, C Y Jeng S, D Cawte A, Abdolahzadeh A, Galli A, Panchapakesan SSS, Rueda DS, Ryckelynck M, Unrau PJ. (2018). Fluorogenic RNA Mango aptamers for imaging small non-coding RNAs in mammalian cellsNature Communications 9(1), 656.

Rueda FO, Bista M, Newton MD, Goeppert AU, Cuomo ME, Gordon E, Kroner F, Read JA, Wrigley JD, Rueda DS and Taylor BJM. (2017). Mapping the sugar dependency for rational generation of a DNA-RNA hybrid-guided Cas9 endonuclease. Nature Communications, DOI: 10.1038/s41467-017-01732-9