Poster Presentation The 45th Lorne Conference on Protein Structure and Function 2020

Developing a FRET based RNA Biosensor (#519)

Brady Johnston 1 , Bishnu Paudel 2 , Jason Schmidberger 1 , Antoine van Oijen 2 , Ian Small 1 , Charlie Bond 1
  1. School of Molecular Sciences, The University of Western Australia, Crawley, WA, Australia
  2. Centre for Medical and Molecular Bioscience, University of Wollongong, Wollongong, NSW, Australia

Using single-molecule fluorescence microscopy we characterised the novel binding kinetics of a designer PPR protein for use as a FRET-based RNA biosensor.

The pentatricopeptide repeat family of proteins are made up of a series of 35 amino acid motifs that are repeated numerous times to form a larger protein structure.1 Each motif binds a single RNA base through the interactions of two amino acids - residues 5 and 35 of the motif.The amino acids present determine the binding target according to a binding code.2,3 

These proteins can be reprogrammed in designer PPR (dPPR) proteins that allow for binding to novel RNA targets.4 When binding their target, a large conformational change occurs within the proteins. This change can be analysed using a FRET reporter system that allows for live monitoring of the conformational state. This system holds potential for use as a sequence specific RNA biosensor both in-vivo and in-vitro. Using single-molecule fluorescence we were able to observe the dynamics and kinetics of this designer PPR protein when binding to its RNA target and how this changes with a mismatch or altered sequence.

  1. Lurin, C., Andrés, C., Aubourg, S., Bellaoui, M., Bitton, F., Bruyère, C., Caboche, M., Debast, C., Gualberto, J., Hoffmann, B., Lecharny, A., Ret, M., Martin-Magniette, M., Mireau, H., Peeters, N., Renou, J., Szurek, B., Taconnat, L., Small, I. (2004). Genome-wide analysis of Arabidopsis pentatricopeptide repeat proteins reveals their essential role in organelle biogenesis. The Plant Cell Online 16(8), 2089 2103. https://dx.doi.org/10.1105/tpc.104.022236
  2. Yagi, Y., Hayashi, S., Kobayashi, K., Hirayama, T., Nakamura, T. (2013). Elucidation of the RNA recognition code for pentatricopeptide repeat proteins involved in organelle RNA editing in plants. PLoS ONE 8(3), e57286 8. https://dx.doi.org/10.1371/journal.pone.0057286
  3. Barkan, A., Rojas, M., Fujii, S., Yap, A., Chong, Y., Bond, C., Small, I. (2012). A combinatorial amino acid code for RNA recognition by pentatricopeptide repeat proteins. PLoS Genetics 8(8), e1002910 8. https://dx.doi.org/10.1371/journal.pgen.1002910
  4. Gully, B., Shah, K., Lee, M., Shearston, K., Smith, N., Sadowska, A., Blythe, A., Bernath-Levin, K., Stanley, W., Small, I., Bond, C. (2015). The design and structural characterization of a synthetic pentatricopeptide repeat protein Acta Crystallographica Section D: Biological Crystallography 71(2), 196-208. https://dx.doi.org/10.1107/s1399004714024869