Ratiometric Fluorogenic RNA-Based Sensors for Imaging Live-Cell Dynamics of Small Molecules

Wu, Rigumula; Mudiyanselage, Aruni P. K. K. Karunanayake; Sun, Zhining; Tian, Qian; Zhao, Bin; Bagheri, Yousef; Lutati, David; Keshri, Puspam; You, Mingxu

doi:10.1021/acsabm.9b01237

Cited by 18 publications

(6 citation statements)

References 42 publications

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“…The tRNA–DF30 scaffold has the potential to display two different aptamers, as has been shown previously for the F30 scaffold − (Figure , Figure S4). The two aptamers HBC620–Pepper and DFHBI-1T–Spinach2 represent orthogonal dye–aptamer pairs that should be spectrally resolvable.…”

Section: Resultssupporting

confidence: 57%

Investigating the Effect of RNA Scaffolds on the Multicolor Fluorogenic Aptamer Pepper in Different Bacterial Species

Mumbleau,

Chevance,

Hughes

et al. 2024

ACS Synth. Biol.

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RNA synthetic biology tools have primarily been applied in E. coli; however, many other bacteria are of industrial and clinical significance. Thus, the multicolor fluorogenic aptamer Pepper was evaluated in both Grampositive and Gram-negative bacteria. Suitable HBC−Pepper dye pairs were identified that give blue, green, or red fluorescence signals in the E. coli, Bacillus subtilis, and Salmonella enterica serovar Typhimurium (S. Typhimurium). Furthermore, we found that different RNA scaffolds have a drastic effect on in vivo fluorescence, which did not correlate with the in vitro folding efficiency. One such scaffold termed DF30−tRNA displays 199-fold greater fluorescence than the Pepper aptamer alone and permits simultaneous dual color imaging in live cells.

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Section: Resultssupporting

confidence: 57%

Investigating the Effect of RNA Scaffolds on the Multicolor Fluorogenic Aptamer Pepper in Different Bacterial Species

Mumbleau,

Chevance,

Hughes

et al. 2024

ACS Synth. Biol.

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“…By rationally designing RNA nanostructures of different geometries, it will be possible to explore the full range of dipole orientations. Apta-FRET systems can be used to create RNA devices that detect small conformational changes and be used to develop ratiometric biosensors 35 . The apta-FRET system that we introduce in this study can also serve as basis for designing intracellular sensors, since the Broccoli and Pepper aptamers have both been independently verified to activate the fluorescence of their cognate fluorophores inside cells 31,33,36 .…”

Section: Discussionmentioning

confidence: 99%

RNA origami scaffolds as a cryo-EM tool for investigating aptamer-ligand binding of a Broccoli-Pepper FRET pair

Vallina

McRae

Hansen

et al. 2022

Preprint

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RNA nanotechnology uses motifs from nature as well as aptamers from in vitro selection to construct nanostructures and devices for applications in RNA medicine and synthetic biology. The RNA origami method allows cotranscriptional folding of large RNA scaffolds that can position functional motifs in a precise manner, which has been verified by Förster Resonance Energy Transfer (FRET) between fluorescent aptamers. Cryogenic electron microscopy (cryo-EM) is a promising method for characterizing the structure of larger RNA nanostructures. However, the structure of individual aptamers is difficult to solve by cryo-EM due to their low molecular weight. Here, we place aptamers on the RNA origami scaffolds to increase the contrast for cryo-EM and solve the structure of a new Broccoli-Pepper FRET pair. We identify different modes of ligand binding of the two aptamers and verify by selective probing. 3D variability analysis of the cryo-EM data show that the relative position between the two bound fluorophores on the origami fluctuate by only 3.5 Angstrom. Our results demonstrate the use of RNA origami scaffolds for characterizing small RNA motifs by cryo-EM and for positioning functional RNA motifs with high spatial precision. The Broccoli-Pepper apta-FRET pair has potential use for developing advanced sensors that are sensitive to small conformational changes.

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“…RNA can be anchored to specific locations via interactions with proteins, and the problem of probe diffusibility we expect will be surmounted. The introduction of ratiometry into Spinach technology promises to be the next leap for quantitative chemical imaging in the cytosol . We envisage that quantitative chemical imaging applied to ratiometric small-molecule-based probes and biomacromolecule-based scaffolds that provide stable spatial localization within the cell will enable the study of diverse biological processes at an entirely new level of molecular detail.…”

Section: Discussionmentioning

confidence: 99%

Quantitative Imaging of Biochemistry in Situ and at the Nanoscale

2020

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Biochemical reactions in eukaryotic cells occur in subcellular, membrane-bound compartments called organelles. Each kind of organelle is characterized by a unique lumenal chemical composition whose stringent regulation is vital to proper organelle function. Disruption of the lumenal ionic content of organelles is inextricably linked to disease. Despite their vital roles in cellular homeostasis, there are large gaps in our knowledge of organellar chemical composition largely from a lack of suitable probes. In this Outlook, we describe how, using organelle-targeted ratiometric probes, one can quantitatively image the lumenal chemical composition and biochemical activity inside organelles. We discuss how excellent fluorescent detection chemistries applied largely to the cytosol may be expanded to study organelles by chemical imaging at subcellular resolution in live cells. DNA-based reporters are a new and versatile platform to enable such approaches because the resultant probes have precise ratiometry and accurate subcellular targeting and are able to map multiple chemicals simultaneously. Quantitatively mapping lumenal ions and biochemical activity can drive the discovery of new biology and biomedical applications.

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Ratiometric Fluorogenic RNA-Based Sensors for Imaging Live-Cell Dynamics of Small Molecules

Cited by 18 publications

References 42 publications

Investigating the Effect of RNA Scaffolds on the Multicolor Fluorogenic Aptamer Pepper in Different Bacterial Species

Investigating the Effect of RNA Scaffolds on the Multicolor Fluorogenic Aptamer Pepper in Different Bacterial Species

RNA origami scaffolds as a cryo-EM tool for investigating aptamer-ligand binding of a Broccoli-Pepper FRET pair

Quantitative Imaging of Biochemistry in Situ and at the Nanoscale

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