Visualizing RNA dynamics in live cells with bright and stable fluorescent RNAs

Chen, Xianjun; Zhang, Dasheng; Su, Ni; Bao, Bingkun; Xie, Xin; Zuo, Fangting; Liu, Yang; Wang, Hui; Jiang, Li; Lin, Qiuning; Fang, Mengyue; Li, Ningfeng; Hua, Xin; Chen, Zhengda; Bao, Chunyan; Xu, Jinjin; Du, Wenli; Zhang, Lixin; Zhao, Yuzheng; Zhu, Linyong; Loscalzo, Joseph; Yang, Yi

doi:10.1038/s41587-019-0249-1

Cited by 211 publications

(206 citation statements)

References 60 publications

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“…Both fluorogens are structurally similar, differing only by their substituents modifying the 4-hydroxybenzyl moiety (3-methyl vs. 3,5-dimethoxy) (Figure 1a). As in many fluorogenic hybrid systems, 19,20 these small substitutions allow the generation of various colors and thus many hybrid systems are comprised of a suite of structurally related fluorogens that interact with a single, genetically-encoded tag.…”

Section: Resultsmentioning

confidence: 99%

Orthogonal fluorescent chemogenetic reporters for multicolor imaging

Tebo

Moeyaert

Thauvin

et al. 2020

Preprint

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Fluorescence microscopy is an indispensable tool in biological research, allowing subsecond and sub-micrometer mapping of molecules or processes inside living cells.Moreover, using spectrally separated fluorophores, one can observe multiple targets simultaneously, leading to a deeper understanding of the dynamic molecular interplays that regulate cell function and fate. Chemogenetic systems, which combine a protein tag and a synthetic fluorophore, provide certain advantages over fluorescent proteins since there is no requirement for chromophore maturation. However, the fluorophore promiscuity of chemogenetic systems renders two-color applications challenging. Here, we present the engineering of a set of spectrally orthogonal fluorogen activating tags based on the Fluorescence Activating and absorption Shifting Tag (FAST), that are compatible with two-color, live cell imaging. The resulting tags, greenFAST and redFAST, demonstrate orthogonality not only in their fluorogen recognition capabilities, but also in their one-and two-photon absorption profiles. A two-color cell cycle sensor based on greenFAST and redFAST is capable of detecting very short, early cell cycles in zebrafish development which had previously been difficult to image. Furthermore, this pair of orthogonal tags can be developed into split complementation systems that are capable of detecting multiple protein-protein interactions by live cell fluorescence microscopy. 3

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Section: Resultsmentioning

confidence: 99%

Orthogonal fluorescent chemogenetic reporters for multicolor imaging

Tebo

Moeyaert

Thauvin

et al. 2020

Preprint

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“…Additionally, the improvement in resolution is not solely contained to the use of SIM, but also that the fluorogenic properties of Mango enable higher contrast imaging of RNAs as previously speculated 46 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 . RNA Mango is likely compatible with these technologies while enabling direct RNA imaging at single molecule resolution without the need for fluorescent proteins.…”

Section: Super-resolution Rna Imaging With Mango Arraysmentioning

confidence: 94%

“…However, both the folding and fluorescence stability of the Spinach aptamer have hindered highresolution 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 single-molecule 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.…”

Section: Introductionmentioning

confidence: 99%

Live Cell Imaging of Single RNA Molecules with Fluorogenic Mango II Arrays

Cawte

Unrau

Rueda

2019

Preprint

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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 fluorophore exchange. This property makes the arrays readily compatible with structured illumination super-resolution microscopy.

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“…So far, they have mostly been employed for conventional, diffraction-limited imaging. FLAPs for super-resolution imaging have been reported only last year, specifically, the SiRA aptamer 4 for stimulated emission depletion (STED) microscopy and the Pepper aptamer 5 for structured illumination microscopy (SIM). Further optimization of these RNA markers will facilitate their widespread use in super-resolution microscopy.…”

Section: Mainmentioning

confidence: 99%

RhoBAST - a rhodamine-binding aptamer for super-resolution RNA imaging

Sünbül

Lackner

Martin

et al. 2020

Preprint

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RhoBAST is a novel fluorescence light-up RNA aptamer (FLAP) that transiently binds a fluorogenic rhodamine dye. Fast dye association and dissociation result in intermittent fluorescence emission, facilitating single-molecule localization microscopy (SMLM) with an image resolution not limited by photobleaching. We demonstrate RhoBAST's excellent properties as a RNA marker by resolving subcellular and subnuclear structures of RNA in live and fixed cells by SMLM and structured illumination microscopy (SIM).

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Visualizing RNA dynamics in live cells with bright and stable fluorescent RNAs

Cited by 211 publications

References 60 publications

Orthogonal fluorescent chemogenetic reporters for multicolor imaging

Orthogonal fluorescent chemogenetic reporters for multicolor imaging

Live Cell Imaging of Single RNA Molecules with Fluorogenic Mango II Arrays

RhoBAST - a rhodamine-binding aptamer for super-resolution RNA imaging

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