Live imaging of mRNA using RNA-stabilized fluorogenic proteins

Wu, Jiahui; Zaccara, Sara; Khuperkar, Deepak; Kim, Hyaeyeong; Tanenbaum, Marvin E.; Jaffrey, Samie R.

doi:10.1038/s41592-019-0531-7

Cited by 82 publications

(85 citation statements)

References 18 publications

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“…Recently, two new aptamer-based RNA imaging technologies have emerged both naming their aptamers Pepper. The first using RNA-stabilised fluorescent proteins able to image single mRNAs in live cells 27 . The second, is a fluorogenic RNA aptamer that has been shown to enable RNA imaging in cells, but not at the single molecule level 26 .…”

Section: Discussionmentioning

confidence: 99%

“…However, both the folding and fluorescence stability of the Spinach aptamer have hindered high-resolution imaging of such arrays 20 . More recently, arrays of the Pepper aptamer have been used to image RNAs with improved resolution 26 , but the use of fluorescently labelled proteins is still required for singlemolecule detection 27 . The brightness, thermodynamic stability and high-affinity of Mango aptamers bodes well to the accurate detection of RNAs abundance in a cellular environment.…”

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confidence: 99%

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

2020

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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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Section: Discussionmentioning

confidence: 99%

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confidence: 99%

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

2020

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“…This necessitates a large number of hairpin loops to be inserted into the RNA, typically 24, but up to 96, in order to increase the fluorescence intensity over background. Another strategy that helps is subcellular compartmentalization, for example, using nuclear localization sequences on the CP‐FP to sequester it away from the compartment of interest, in this case the cytosol to monitor mRNA function (J. Wu et al, ; Yan, Hoek, Vale, & Tanenbaum, ).…”

Section: Recent Developments In Single Mrna Imaging In Vivomentioning

confidence: 99%

“…A mutant of the trans‐activation response element of the human immunodeficiency virus is used as a stabilizer of a fluorescence protein‐degron tag fusion (FP‐tDeg). This RNA appendage, confusingly also termed “Pepper aptamer” (although it was not in vitro selected or evolved as the basic definition of an aptamer), was shown to confer the ability to image mRNA in living cells (J. Wu et al, ). In the absence of the stabilizing RNA motif, the FP‐tDeg is degraded quickly.…”

Section: Recent Developments In Single Mrna Imaging In Vivomentioning

confidence: 99%

“…Only in the presence of its RNA stabilizer, the half‐life of the FP‐tDeg is sufficiently long to be detected as bound to the target RNA. Incorporation of multiple copies of this Pepper RNA into the target mRNA and co‐expression of the FP‐tDeg fluorogen allow the target mRNA to be robustly visualized (J. Wu et al, ).…”

Section: Recent Developments In Single Mrna Imaging In Vivomentioning

confidence: 99%

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Following the messenger: Recent innovations in live cell single molecule fluorescence imaging

et al. 2020

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Messenger RNAs (mRNAs) convey genetic information from the DNA genome to proteins and thus lie at the heart of gene expression and regulation of all cellular activities. Live cell single molecule tracking tools enable the investigation of mRNA trafficking, translation and degradation within the complex environment of the cell and in real time. Over the last 5 years, nearly all tools within the mRNA tracking toolbox have been improved to achieve high‐quality multi‐color tracking in live cells. For example, the bacteriophage‐derived MS2‐MCP system has been improved to facilitate cloning and achieve better signal‐to‐noise ratio, while the newer PP7‐PCP system now allows for orthogonal tracking of a second mRNA or mRNA region. The coming of age of epitope‐tagging technologies, such as the SunTag, MoonTag and Frankenbody, enables monitoring the translation of single mRNA molecules. Furthermore, the portfolio of fluorogenic RNA aptamers has been expanded to improve cellular stability and achieve a higher fluorescence “turn‐on” signal upon fluorogen binding. Finally, microinjection‐based tools have been shown to be able to track multiple RNAs with only small fluorescent appendages and to track mRNAs together with their interacting partners. We systematically review and compare the advantages, disadvantages and demonstrated applications in discovering new RNA biology of this refined, expanding toolbox. Finally, we discuss developments expected in the near future based on the limitations of the current methods. This article is categorized under: RNA Export and Localization > RNA Localization RNA Structure and Dynamics > RNA Structure, Dynamics, and Chemistry RNA Interactions with Proteins and Other Molecules > RNA–Protein Complexes

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CRISPR/Pepper‐tDeg: A Live Imaging System Enables Non‐Repetitive Genomic Locus Analysis with One Single‐Guide RNA

Chen,

Huang,

Shi

et al. 2024

Advanced Science

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CRISPR‐based genomic‐imaging systems have been utilized for spatiotemporal imaging of the repetitive genomic loci in living cells, but they are still challenged by limited signal‐to‐noise ratio (SNR) at a non‐repetitive genomic locus. Here, an efficient genomic‐imaging system is proposed, termed CRISPR/Pepper‐tDeg, by engineering the CRISPR sgRNA scaffolds with the degron‐binding Pepper aptamers for binding fluorogenic proteins fused with Tat peptide derived degron domain (tDeg). The target‐dependent stability switches of both sgRNA and fluorogenic protein allow this system to image repetitive telomeres sensitively with a 5‐fold higher SNR than conventional CRISPR/MS2‐MCP system using “always‐on” fluorescent protein tag. Subsequently, CRISPR/Pepper‐tDeg is applied to simultaneously label and track two different genomic loci, telomeres and centromeres, in living cells by combining two systems. Given a further improved SNR by the split fluorescent protein design, CRISPR/Pepper‐tDeg system is extended to non‐repetitive sequence imaging using only one sgRNA with two aptamer insertions. Neither complex sgRNA design nor difficult plasmid construction is required, greatly reducing the technical barriers to define spatiotemporal organization and dynamics of both repetitive and non‐repetitive genomic loci in living cells, and thus demonstrating the large application potential of this genomic‐imaging system in biological research, clinical diagnosis and therapy.

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Live imaging of mRNA using RNA-stabilized fluorogenic proteins

Cited by 82 publications

References 18 publications

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

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

Following the messenger: Recent innovations in live cell single molecule fluorescence imaging

CRISPR/Pepper‐tDeg: A Live Imaging System Enables Non‐Repetitive Genomic Locus Analysis with One Single‐Guide RNA

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