Fluorophore ligand binding and complex stabilization of the RNA Mango and RNA Spinach aptamers

Jeng, Sunny; Chan, Hedy H.Y.; Booy, Evan P.; McKenna, Sean Andrew; Unrau, Peter J.

doi:10.1261/rna.056226.116

Cited by 54 publications

(65 citation statements)

References 44 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Contrarily, YO3-biotin fluoresces with comparable intensities when bound in either the Mango or the Spinach construct (Supplementary Fig. 2d, e ), similarly to what has been observed previously for TO1-biotin 30 . We further examined the binding affinities (EC 50 ) of the fluorophores in the two aptamers (Supplementary Table 1 ), and found that DFHBI-1T has a significantly lower EC 50 in Spinach than YO3-biotin (~340 nM compared to ~4 µM).…”

Section: Resultssupporting

confidence: 87%

Development of a genetically encodable FRET system using fluorescent RNA aptamers

et al. 2018

View full text Add to dashboard Cite

Fluorescent RNA aptamers are useful as markers for tracking RNA molecules inside cells and for creating biosensor devices. Förster resonance energy transfer (FRET) based on fluorescent proteins has been used to detect conformational changes, however, such FRET devices have not yet been produced using fluorescent RNA aptamers. Here we develop an RNA aptamer-based FRET (apta-FRET) system using single-stranded RNA origami scaffolds. To obtain FRET, the fluorescent aptamers Spinach and Mango are placed in close proximity on the RNA scaffolds and a new fluorophore is synthesized to increase spectral overlap. RNA devices that respond to conformational changes are developed, and finally, apta-FRET constructs are expressed in E. coli where FRET is observed, demonstrating that the apta-FRET system is genetically encodable and that the RNA nanostructures fold correctly in bacteria. We anticipate that the RNA apta-FRET system could have applications as ratiometric sensors for real-time studies in cell and synthetic biology.

show abstract

Section: Resultssupporting

confidence: 87%

Development of a genetically encodable FRET system using fluorescent RNA aptamers

et al. 2018

View full text Add to dashboard Cite

show abstract

“…3d) and Mango-tagged 5S-RNA and U6-RNA foci (~2-3-fold fluorescence turn-on in fixed mammalian cells 25 ). The in vitroevolved dye-binding aptamers all feature G-quadruplex RNA folds 38,39 , and it has been shown that the dyes can bind other G-quadruplex RNAs non-specifically in cells 39 , possible contributing to diminished cellular contrast. It is important to note that other factors besides in vitro enhancement contribute to cellular contrast, including robust folding of the RNA tag in the cellular environment, temperature-and salt-dependent stability of the RNA-probe complex, and probe photobleaching properties.…”

Section: Discussionmentioning

confidence: 99%

“…In vitro evolved dye-binding aptamers contain a G-quadruplex fold 37,38 which has been shown to complicate RNA folding in mammalian cells 39 . Furthermore, ligands for evolved aptamers with the G-quadruplex fold may bind other RNAs with G-quadruplex folds 39 , have been shown to bind nucleic acids nonspecifically 25 and G-quadruplex structures may be disrupted by dedicated helicases 40 . Thus, to improve folding and stabilize against RNase degradation in cells, such aptamers often include a folding scaffold when fused to mammalian RNAs [25][26][27]41 .…”

Section: Visualization Of Mrna Dynamics In Live Mammalian Cellsmentioning

confidence: 99%

Riboglow: a multicolor riboswitch-based platform for live cell imaging of mRNA and small non-coding RNA in mammalian cells

Braselmann

et al. 2017

Preprint

View full text Add to dashboard Cite

RNAs directly regulate a vast array of cellular processes, emphasizing the need for robust approaches to fluorescently tag and track RNAs in living cells. Here, we develop an RNA imaging platform using the Cobalamin (Cbl) riboswitch as an RNA aptamer and a series of probes containing Cbl as a fluorescent quencher linked to a range of fluorophores. We demonstrate fluorescence turn-on upon RNA binding and proof of concept for tracking both mRNA and small U RNA in live cells. Main textThe complex spatiotemporal dynamics of messenger RNAs (mRNAs) and non-coding RNAs (ncRNAs) affect virtually all aspects of cellular function. These RNAs associate with a large group of RNA binding proteins that dynamically modulate RNA localization, processing and function 1,2 . Such interactions govern mRNA processing, export from the nucleus, and assembly into translationally competent messages, as well as association into large macromolecular granules that are not translationally active, including P-bodies and stress granules (SGs) [3][4][5][6] . Similarly, uridine-rich small nuclear RNAs (U snRNAs, the RNA components of the spliceosome) 7 dynamically associate with protein components to comprise the functional spliceosomal complex in the nucleus 8 . During different stresses including bacterial infection, the U snRNAs along with the splicing machinery can be transiently sequestered in cytosolic foci All rights reserved. No reuse allowed without permission.(which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.The copyright holder for this preprint . http://dx.doi.org/10.1101/199240 doi: bioRxiv preprint first posted online Oct. 10, 2017; 3 called U-bodies 7 . Given the intricate connection between RNA localization, dynamics and function, there has been a strong push to develop tools for visualization of RNA in live cells to elucidate mechanisms underlying the mRNA and ncRNA life cycle.While there is a broad spectrum of tools to fluorescently tag proteins in live cells, far fewer approaches for live cell imaging of RNA exist. The most common system uses a series of tandem hairpins that bind an RNA-binding protein (MS2 or PP7 coat protein) coupled to a fluorescent protein (FP) [9][10][11] . This system has been used successfully to interrogate mRNA dynamics over time at the single molecule level 9,10 . However, one limitation of this system is that 12-24 copies of the aptamer are required to enhance fluorescence contrast, and the large size of the tag can perturb localization, dynamics and processing 12 of the RNA. An alternative approach involves attaching an aptamer to the RNA that directly binds a small molecular fluorogenic dye [13][14][15][16] . Recent years have seen the application of Spinach 17 , Broccoli 18 , and Mango 19 aptamers to label and track RNAs in live mammalian cells. However, all these aptamers evolved in vitro contain a G-quadruplex 20 , which has been shown to complicate RNA folding and ligand selectivity in mammalian cells [21][22...

show abstract

“…Hence, very few literatures have shown the successful imaging in mammalian cells. In the comparison of fluorophore ligand binding and complex stabilization studies, the Mango has a large thermal stability and better discrimination from a broad range of small molecules [35] and the Corn shows improved photostability. Therefore, there are no doubt that the Mango and the Corn could be good tools to elucidate the working mechanism of cancers biology fields.…”

Section: New Mangomentioning

confidence: 99%

“…S. Yoon, J.J. Rossi / Advanced Drug Delivery Reviews 134 (2018)[22][23][24][25][26][27][28][29][30][31][32][33][34][35] …”

mentioning

confidence: 99%

Aptamers: Uptake mechanisms and intracellular applications

Yoon

Rossi

2018

Advanced Drug Delivery Reviews

119

View full text Add to dashboard Cite

The structural flexibility and small size of aptamers enable precise recognition of cellular elements for imaging and therapeutic applications. The process by which aptamers are taken into cells depends on their targets but is typically clathrin-mediated endocytosis or macropinocytosis. After internalization, most aptamers are transported to endosomes, lysosomes, endoplasmic reticulum, Golgi apparatus, and occasionally mitochondria and autophagosomes. Intracellular aptamers, or "intramers," have versatile functions ranging from intracellular RNA imaging, gene regulation, and therapeutics to allosteric modulation, which we discuss in this review. Immune responses to therapeutic aptamers and the effects of G-quadruplex structure on aptamer function are also discussed.

show abstract

Fluorophore ligand binding and complex stabilization of the RNA Mango and RNA Spinach aptamers

Cited by 54 publications

References 44 publications

Development of a genetically encodable FRET system using fluorescent RNA aptamers

Development of a genetically encodable FRET system using fluorescent RNA aptamers

Riboglow: a multicolor riboswitch-based platform for live cell imaging of mRNA and small non-coding RNA in mammalian cells

Aptamers: Uptake mechanisms and intracellular applications

Contact Info

Product

Resources

About