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

Adam D Cawte,Peter J Unrau,David S Rueda

doi:10.1038/s41467-020-14932-7

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.

Highlights

RNA molecules play vital roles in many cellular processes
Despite new generations of MS2 stem loops (MS2-SL) (MS2v5 and MS2v6) that reduce effects on RNA processing[13,14], there remains an advantage to developing high contrast imaging strategies that do not rely on fluorescent fluorescent protein (FP)–MS2 coat protein (MCP) proteins and that improve imaging contrast within the cell
It has long been thought that aptamer arrays, similar to the MS2 cassettes, could enable single-molecule imaging with improved sensitivity

Summary

Introduction

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. Despite new generations of MS2-SLs (MS2v5 and MS2v6) that reduce effects on RNA processing[13,14], there remains an advantage to developing high contrast imaging strategies that do not rely on fluorescent FP–MCP proteins and that improve imaging contrast within the cell Since their development, fluorogenic RNA aptamers have been poised as a potential complementary alternative to the MS2 system[15,16,17,18,19,20,21,22,23,24]. It has long been thought that aptamer arrays, similar to the MS2 cassettes, could enable single-molecule imaging with improved sensitivity Both the folding and fluorescence stability of the Spinach aptamer have hindered high-resolution imaging of such arrays[20]. Exchange of the Mango fluorogenic dye in fixed samples extends imaging times, which benefits super-resolution techniques such as structured illumination microscopy (SIM)

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