RNA Fluorescence with Light-Up Aptamers

Ouellet, Jonathan

doi:10.3389/fchem.2016.00029

Cited by 141 publications

(117 citation statements)

References 44 publications

(64 reference statements)

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“…An alternative approach involves fluorogenic dye-binding aptamers that give rise to a turn-on fluorescence signal when the dye binds the aptamer [16][17][18][19][20] . While numerous proof-ofprinciple aptamers have been developed 21 , only the Spinach 22 , Broccoli 23 and Mango 24,25 aptamers have been used in live mammalian cells. These dye-binding aptamers have been used to visualize highly expressed RNA polymerase III-dependent transcripts such as 5S and U6 RNA [25][26][27] .…”

Section: Introductionmentioning

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

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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 No reuse allowed without permission.(which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.The copyright holder for this preprint .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...

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Section: Introductionmentioning

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

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“…Light-up RNA aptamers have been selected to induce a fluorescence emission upon binding specific small molecules allowing a protein-free RNA tagging [55]. Since their development, fluorescent aptamers have been used in vitro and in vivo as a valid alternative to other imaging strategies (e.g.…”

Section: Light-up Rna Origami and In-gel Imagingmentioning

confidence: 99%

“…Since their development, fluorescent aptamers have been used in vitro and in vivo as a valid alternative to other imaging strategies (e.g. fluorescent in situ hybridization and molecular beacons, [55,56]). Malachite green, Mango, Spinach and Broccoli aptamers have been inserted in the design of different RNA nanostructures to successfully demonstrate the self-assembly and folding through their functional activation and fluorescence emission [19,24,29,53].…”

Section: Light-up Rna Origami and In-gel Imagingmentioning

confidence: 99%

Co-transcriptional folding of a bio-orthogonal fluorescent scaffolded RNA origami

Torelli

Kozyra

Shirt-Ediss

et al. 2019

Preprint

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The scaffolded origami technique has provided an attractive tool for engineering nucleic acid nanostructures. This paper demonstrates scaffolded RNA origami folding in vitro in which all components are transcribed simultaneously in a single-pot reaction. Double-stranded DNA sequences are transcribed by T7 RNA polymerase into scaffold and staple strands able to correctly fold in high yield into the nanoribbon. Synthesis is successfully confirmed by atomic force microscopy and the unpurified transcription reaction mixture is analyzed by an in gel-imaging assay where the transcribed RNA nanoribbons are able to capture the specific dye through the reconstituted split Broccoli aptamer showing a clear green fluorescent band. Finally, we simulate the RNA origami in silico using the nucleotide-level coarse-grained model oxRNA to investigate the thermodynamic stability of the assembled nanostructure in isothermal conditions over a period of time.Our work suggests that the scaffolded origami technique is a valid, and potentially more powerful, assembly alternative to the single-stranded origami technique for future in vivo applications. KEYWORDSCo-transcriptional folding, scaffolded RNA origami, bio-orthogonal, split Broccoli aptamer, oxRNA simulation 1 Introduction RNA plays sophisticated roles with different and essential coding and noncoding functions. mRNAs, tRNAs, rybozymes, aptamers and CRISPR RNAs are just few examples of RNA species in a vast functional repertoire. Considering the diversity in functional and structural motifs, the emerging field of RNA nanotechnology has been developing rapidly over the past years. As a consequence, a variety of RNA nanostructures with different functionalities, sizes and shapes have

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“…Since the discovery that an RNA aptamer can specifically interact with a fluorogen to generate a module that makes the fluorogen much brighter than its counterpart, free in solution 4 , many RNA-fluorogen complexes have been developed and used for fluorescent detection of RNA in cells [5][6][7][8][9][10][11] . Such an RNA aptamer which can specifically interact with a fluorogen to elicit fluorescence is known as a "light-up" aptamer 12,13 .…”

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confidence: 99%

Nanomechanics and co-transcriptional folding of Spinach and Mango

Mitra

2019

Preprint

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Recent advances in fluorogen-binding RNA aptamers known as "light-up" aptamers provide an avenue for protein-free detection of RNA in cells. Crystallographic studies have revealed a G-Quadruplex (GQ) structure at the core of light-up aptamers such as Spinach, Mango and Corn.Detailed biophysical characterization of folding of such aptamers is still lacking despite the potential implications on their in vivo folding and function. We used single-molecule fluorescence-force spectroscopy that combines fluorescence resonance energy transfer with optical tweezers to examine mechanical responses of Spinach2, iMangoIII and MangoIV.Spinach2 unfolded in four discrete steps as force is increased to 7 pN and refolded in reciprocal steps upon force relaxation. Binding of DFHBI-1T fluorogen preserved the step-wise unfolding behavior although at slightly higher forces. In contrast, GQ core unfolding in iMangoIII and MangoIV occurred in one discrete step at forces > 10 pN and refolding occurred at lower forces showing hysteresis. Binding of the cognate fluorogen, TO1, did not significantly alter the mechanical stability of Mangos. In addition to K + , which is needed to stabilize the GQ cores, Mg 2+ was needed to obtain full mechanical stability of the aptamers. Co-transcriptional folding analysis using superhelicases showed that co-transcriptional folding reduces misfolding and allows a folding pathway different from refolding. As the fundamental cellular processes like replication, transcription etc. exert pico-Newton levels of force, these aptamers may unfold in vivo and subsequently misfold.

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RNA Fluorescence with Light-Up Aptamers

Cited by 141 publications

References 44 publications

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

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

Co-transcriptional folding of a bio-orthogonal fluorescent scaffolded RNA origami

Nanomechanics and co-transcriptional folding of Spinach and Mango

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