RNA Mango Aptamer-Fluorophore: A Bright, High-Affinity Complex for RNA Labeling and Tracking

Dolgosheina, Elena; Jeng, Sunny; Panchapakesan, Shanker Shyam S.; Cojocaru, Razvan; Chen, Patrick S. K.; Wilson, Peter D.; Hawkins, Nancy; Wiggins, Paul A.; Unrau, Peter J.

doi:10.1021/cb500499x

Cited by 362 publications

(442 citation statements)

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“…These dyes bind either to a dedicated protein (for example, a SNAP tag 46 ) fused to a coat protein or directly to RNA aptamers (such as Spinach and RNA-Mango 47, 48 ). These dyes can be brighter and more photostable than fluorescent proteins.…”

Section: Visualizing the Messagementioning

confidence: 99%

In the right place at the right time: visualizing and understanding mRNA localization

Buxbaum

Haimovich

Singer

2014

Nat Rev Mol Cell Biol

514

489

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The spatial regulation of protein translation is an efficient way to create functional and structural asymmetries in cells. Recent research has furthered our understanding of how individual cells spatially organize protein synthesis, by applying innovative technology to characterize the relationship between mRNAs and their regulatory proteins, single-mRNA trafficking dynamics, physiological effects of abrogating mRNA localization in vivo and for endogenous mRNA labelling. The implementation of new imaging technologies has yielded valuable information on mRNA localization, for example, by observing single molecules in tissues. The emerging movements and localization patterns of mRNAs in morphologically distinct unicellular organisms and in neurons have illuminated shared and specialized mechanisms of mRNA localization, and this information is complemented by transgenic and biochemical techniques that reveal the biological consequences of mRNA mislocalization.

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Section: Visualizing the Messagementioning

confidence: 99%

In the right place at the right time: visualizing and understanding mRNA localization

Buxbaum

Haimovich

Singer

2014

Nat Rev Mol Cell Biol

514

489

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“…The Spinach system (Figure 1C) is an example of the first generation of RNA aptamers for live-cell imaging (52). Since then, new aptamer systems have been developed, including Spinach 2 (53), Mango (54), Broccoli (55, 56), and Corn (57). Repetitive Spinach aptamers have been shown to increase the brightness by a maximum of 17-fold (with 64 repeats) compared to a single Spinach monomer at a cost of a significant lengthening of the aptamer sequence (58).…”

Section: Approaches To Study Rna Localizationmentioning

confidence: 99%

RNA Localization in Bacteria

Fei

Sharma

2018

Microbiol Spectr

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Diverse mechanisms and functions of post-transcriptional regulation by small regulatory RNAs (sRNAs) and RNA binding proteins (RBPs) have been described in bacteria. In contrast, little is known about the spatial organization of RNAs in bacterial cells. In eukaryotes, subcellular localization and transport of RNAs play important roles in diverse physiological processes, such as embryonic patterning, asymmetric cell division, epithelial polarity, and neuronal plasticity. It is now clear that bacterial RNAs also can accumulate at distinct sites in the cell. However, due the small size of bacterial cells, localized RNA and translation are more challenging to study in bacterial cells, and the underlying molecular mechanisms of transcript localization are less understood. Here we review the emerging examples of RNAs localized to specific subcellular locations in bacteria, with indications that subcellular localization of transcripts might be important for regulatory processes. Diverse mechanisms for bacterial RNA localization have been suggested, including close association to their genomic site of transcription, or to the localizations of their protein products in translation-dependent or independent processes. We also provide an overview of the state-of-the-art of technologies to visualize and track bacterial RNAs, ranging from hybridization-based approaches in fixed cells to in vivo imaging approaches using fluorescent protein reporters and/or RNA aptamers in single living bacterial cells. We conclude with a discussion of open questions in the field and ongoing technological developments regarding RNA imaging in eukaryotic systems that might likewise provide novel insights into RNA localization in bacteria.

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“…Although many light-up strategies require that the aptamer be chemically modified with a fluorophore, which is not possible for in vivo reporting, a subcategory with in vivo application is the group of aptamers for which binding with the aptamer increases ligand fluorescence. These aptamers include those that bind triphenylmethanes [1], bisbenzimides [2], imidazoles [3], and the benzothiazolidene asymmetric cyanine dye, thiazole orange [4]. When linked to other aptamers that drive their ability to bind ligand, these light-up aptamers can report on the presence of analytes in vitro that range from metabolites to proteins depending on the linked aptamer’s specificity [5; 6].…”

Section: Introductionmentioning

confidence: 99%

“…Of the light-up aptamers with applications to intracellular signaling [1; 2; 3; 4; 10], the malachite green (MGA) and Spinach2 (SPN2A) aptamers have the highest signal/noise when assayed in vitro . This contrast is much higher than achieved with the MS2-based aptamer reporters that were developed for real-time monitoring of transcription using aptamers that recognize peptides fused to GFP [11; 12].…”

Section: Introductionmentioning

confidence: 99%

Light-up and FRET aptamer reporters; evaluating their applications for imaging transcription in eukaryotic cells

İlgü¹,

Ray²,

Bendickson

et al. 2016

Methods

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The regulation of RNA transcription is central to cellular function. Changes in gene expression drive differentiation and cellular responses to events such as injury. RNA trafficking can also have a large impact on protein expression and its localization. Thus, the ability to image RNA transcription and trafficking in real time and in living cells is a worthwhile goal that has been difficult to achieve. The availability of “light-up” aptamers that cause an increase in fluorescence of their ligands when bound by the aptamer have shown promise for reporting on RNA production and localization in vivo. Here we have investigated two light-up aptamers (the malachite green aptamer and the Spinach aptamers) for their suitability as reporters of RNA expression in vivo using two eukaryotic cell types, yeast and mammalian. Our analysis focused on the aptamer ligands, their contributions to background noise, and the impact of tandem aptamer strings on signal strength and ligand affinity. Whereas the background fluorescence is very low in vitro, this is not always true for cell imaging. Our results suggest the need for caution in using light-up aptamers as reporters for imaging RNA. In particular, images should be collected and analyzed by operators blinded to the sample identities. The appropriate control condition of ligand with the cells in the absence of aptamer expression must be included in each experiment. This control condition establishes that the specific interaction of ligand with aptamer, rather than nonspecific interactions with unknown cell elements, is responsible for the observed fluorescent signals. High background signals due to nonspecific interactions of aptamer ligands with cell components can be minimized by using IMAGEtags (Intracellular Multiaptamer GEnetic tags), which signal by FRET and are promising RNA reporters for imaging transcription.

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RNA Mango Aptamer-Fluorophore: A Bright, High-Affinity Complex for RNA Labeling and Tracking

Cited by 362 publications

References 50 publications

In the right place at the right time: visualizing and understanding mRNA localization

In the right place at the right time: visualizing and understanding mRNA localization

RNA Localization in Bacteria

Light-up and FRET aptamer reporters; evaluating their applications for imaging transcription in eukaryotic cells

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