Single-Cell Biophysics: Measurement, Modulation, and Modeling

Single-Cell Biophysics: Measurement, Modulation, and Modeling

Sunday Speaker Abstracts

Imaging Small Cellular RNAs with Fluorescent Mango RNA Aptamers

Adam Cawte

1,2

, Sunny Jeng

, Alexis Autour

, Michäel Ryckelynck

, Peter Unrau

David

Rueda

1,2

Imperial College London, London, United Kingdom,

MRC London Institute of Medical

Sciences, London, United Kingdom,

Simon Fraser University, Burnaby, BC,

Canada,

Université de Strasbourg, Strasbourg, France.

In recent years, there has been an explosion of fluorescent RNA aptamers that have been isolated

using SELEX. Since their discovery, fluorogenic RNA aptamers, such as Spinach and

Mango

1,2

have held great potential to enable the visualisation of RNA molecules in cells.

However, their applicability has been limited primarily to bacterial cells.

Evolving new RNA

aptamers with improved physicochemical properties (i.e., thermal stability, fluorescence

brightness and ligand affinity) should better their use in cellular imaging.

Three new Mango-like aptamers have recently been evolved using microfluidic-assisted in vitro

compartmentalization, mutagenesis and fluorescent selection,

which improved fluorescence

brightness, ligand binding affinity and thermal stability. We show that these aptamers are readily

useable to image small non-coding RNAs (such as 5S rRNA and U6 snRNA) in both live and

fixed human cells with improved sensitivity and resolution. The imaging data show that the

Mango tagged RNAs sub-cellular localisation pattern is conserved, as validated using

immunofluoresence. Our data show that these new aptamers are vastly improved for cellular

imaging over previous variants, and can in principle be incorporated into a wide range of coding

and non-coding RNAs. We anticipate that these new aptamers will drastically improve RNA

imaging in cells.

1. Paige, J. S., Wu, K. Y. & Jaffrey, S. R. RNA mimics of green fluorescent protein. Science.

333, 642–646 (2011).

2. Dolgosheina, E. V. et al. RNA Mango aptamer-fluorophore: A bright, high-affinity complex

for RNA labeling and tracking. ACS Chem. Biol. 9, 2412–2420 (2014).

3. Zhang, J. et al. Tandem Spinach Array for mRNA Imaging in Living Bacterial Cells. Sci. Rep.

5, 17295 (2015).

4. Autour, A., Westhof, E., and Ryckelynck, M. (2016). iSpinach: a fluorogenic RNA aptamer

optimized for in vitro applications. Nucleic Acids Research 44, 2491-2500.