Live cell imaging of single RNA molecules with fluorogenic Mango II arrays.

Nature Communications
Adam D CawteDavid Rueda

Abstract

RNA molecules play vital roles in many cellular processes. Visualising their dynamics in live cells at single-molecule resolution is essential to elucidate their role in RNA metabolism. RNA aptamers, such as Spinach and Mango, have recently emerged as a powerful background-free technology for live-cell RNA imaging due to their fluorogenic properties upon ligand binding. Here, we report a novel array of Mango II aptamers for RNA imaging in live and fixed cells with high contrast and single-molecule sensitivity. Direct comparison of Mango II and MS2-tdMCP-mCherry dual-labelled mRNAs show marked improvements in signal to noise ratio using the fluorogenic Mango aptamers. Using both coding (β-actin mRNA) and long non-coding (NEAT1) RNAs, we show that the Mango array does not affect cellular localisation. Additionally, we can track single mRNAs for extended time periods, likely due to bleached fluorophore replacement. This property makes the arrays readily compatible with structured illumination super-resolution microscopy.

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Citations

Jun 17, 2020·Wiley Interdisciplinary Reviews. RNA·Fatu Badiane MarkeyMona Batish
Jul 8, 2020·Journal of the Royal Society, Interface·Ruben Perez-CarrascoRamon Grima
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Methods Mentioned

BETA
smFISH
super-resolution microscopy

Software Mentioned

FISH
Zeiss
NEB Hifi builder
ImageJ
SIMcheck ImageJ
Matlab
Igor Pro
FociPicker3D
TrackMate
TrackMate ImageJ

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