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

• Published in: Nature communications 2020-03, Vol.11 (1), p.1283-1283, Article 1283
• Main Authors: Cawte, Adam D., Unrau, Peter J., Rueda, David S.
• Format: Article
• Language: English
• Subjects: 13; 14; 14/34; 14/63; 631/57/2265; 631/57/2267; 631/57/2268; 631/80/389/2052; Actin; Actins - genetics; Actins - metabolism; Aptamers; Aptamers, Nucleotide - metabolism; Arrays; Bleaching; Cell Survival; Fibroblasts - cytology; Fibroblasts - metabolism; Fluorescence; Folding; HEK293 Cells; Humanities and Social Sciences; Humans; Imaging; Localization; Mangoes; Metabolism; Microscopy; mRNA; multidisciplinary; Non-coding RNA; Proteins; Reproducibility of Results; RNA - metabolism; RNA, Messenger - genetics; RNA, Messenger - metabolism; Science; Science (multidisciplinary); Signal to noise ratio; Single Molecule Imaging; Spinach
• ISSN: 2041-1723; 2041-1723
• DOI: 10.1038/s41467-020-14932-7

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. Fluorogenic RNA aptamers have been used for RNA imaging, but folding and fluorescence stability often limited their use in high resolution applications. Here the authors present an array of stably folding Mango II aptamers for imaging of coding and non-coding RNAs at single-molecule resolution, in both live and fixed cells.